Covid-19 Metabolomics and Diagnosis: Volume 2 3031279212, 9783031279218

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Frank N. Crespilho   Editor

Covid-19 Metabolomics and Diagnosis Volume 2

Covid-19 Metabolomics and Diagnosis

Frank N. Crespilho Editor

Covid-19 Metabolomics and Diagnosis Volume 2

Editor Frank N. Crespilho University of São Paulo São Paulo, Brazil

ISBN 978-3-031-27921-8 ISBN 978-3-031-27922-5 (eBook) https://doi.org/10.1007/978-3-031-27922-5 © The Editor(s) (if applicable) and The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

Contents

Fourier-Transform Infrared Spectroscopy and Spectromicroscopy Studies for Diagnosis of Covid-19 Infection . . . . . . . . . . . . . . . . . . . . . . . . . . Giovana Rosso Cagnani, Lucyano J. A. Macedo, Thiago da Costa Oliveira, and Frank N. Crespilho Point-of-Care Devices with Electrochemical Detection for COVID-19 Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Luiz R. G. Silva, Jéssica S. Stefano, Tiago A. Silva, Marcio F. Bergamini, Luiz H. Marcolino-Junior, and Bruno C. Janegitz Carbon Nanomaterials for Electrochemical Detection of SARS-CoV-2 Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thiago da Costa Oliveira, Giovana Rosso Cagnani, and Frank Nelson Crespilho Use of Metallic Nanostructures in Electrochemical Biosensing of SARS-CoV-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Luiz Otávio Orzari, Jéssica Rocha Camargo, Rodrigo Vieira Blasques, Luiz Humberto Marcolino-Junior, Marcio Bergamini, and Bruno Campos Janegitz

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3D Printing for Virus Diagnosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Jéssica S. Stefano, Luiz Ricardo G. Silva, Vinicius A. O. P. Silva, Marcio F. Bergamini, Luiz H. Marcolino-Junior, Juliano A. Bonacin, and Bruno C. Janegitz

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Post-COVID-19 Metabolomics: Pursuing the Sequels of a Pandemic . . . . Leonardo Santos Alexandre and Emanuel Carrilho

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Metabolic Behavior of Covid-19 Infection Severity . . . . . . . . . . . . . . . . . . . . 113 Vinícius G. Ferreira, Mariana B. Almeida, and Emanuel Carrilho

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Contents

“Pandemics-on-a-Chip”: Organ-on-a-Chip Models for Studying Viral Infections . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133 Amanda Maciel Lima, Jéssica Freire Feitor, Vinícius Guimarães Ferreira, Mariana Bortholazzi Almeida, Laís Canniatti Brazaca, Daniel Rodrigues Cardoso, and Emanuel Carrilho

Fourier-Transform Infrared Spectroscopy and Spectromicroscopy Studies for Diagnosis of Covid-19 Infection Giovana Rosso Cagnani, Lucyano J. A. Macedo, Thiago da Costa Oliveira, and Frank N. Crespilho

1 Introduction Non-invasive techniques such as Fourier-transform infrared (FTIR) spectroscopy and spectromicroscopy are to investigate the vibrational modes of various molecules, which provide information about bond types, molecular conformations and functional groups [23]. In recent years, spectroscopy has emerged as an analytical tool for biomedical applications, with a prominent role in clinical evaluation. FTIR is currently used in COVID-19 research to identify biochemical changes in biological samples [22] in the presence of new Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). The SARS-CoV-2 is a positive single-stranded RNA virus with a circular phospholipid membrane formed by membrane protein (M), envelope protein (E), and spike glycoprotein (S) [9, 15, 17]. The spike glycoprotein forms trimers in the form of spicules that project out of the membrane and have lengths ranging from 9 to 12 nm, giving coronaviruses the characteristic crown appearance [32]. The spike protein is known to have specific compatibility with human airway epithelial cells that express the angiotensin-converting enzyme 2 (ACE2), the receptor used by SARS-Cov-2 viruses to enter human cells. The viruses enter cells via the binding of the spike S1 portion, which contains the receptor-binding domain (RDB), to receptors on the cell surface, followed by virus membrane [20, 31]. Once in the human body, the class M immunoglobulins (IgM) provide the first line of defense during viral infections. As the condition progresses, IgM levels decrease, immunoglobulin G (IgG) levels increase rapidly, with peak detection after the 14th day of infection [13, 27]. Hence, IgM antibody detection indicates the first stage of viral exposure, followed by IgG antibody detection in the latter stage, implying infection with SARSCoV-2 [16]. Since SARS-CoV-2 has a high rate of transmission and rapid mutation, testing is fundamental for identifying infected people and risk areas [25]. Reverse G. R. Cagnani (B) · L. J. A. Macedo · T. da Costa Oliveira · F. N. Crespilho São Carlos Institute of Chemistry, University of São Paulo, São Carlos 13560-970, Brazil e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 F. N. Crespilho (ed.), Covid-19 Metabolomics and Diagnosis, https://doi.org/10.1007/978-3-031-27922-5_1

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transcription polymerase chain reaction (RT-PCR) is the gold standard technique for COVID-19 diagnosis. It is an expensive method that takes a few days to report due to the lack of infrastructure in some countries, requiring the transportation of samples from remote areas to distant laboratories [28]. As many infected patients spread the disease while waiting for PCR test results, there is a need for a rapid COVID-19 diagnostic technique [26]. Therefore, it is still necessary to create COVID-19 test methods that can provide results immediately and on-site [5]. In this context, reagentfree, non-invasive, and reliable vibrational spectroscopy techniques with little or no sample preparation have been proposed for COVID diagnosis and disease. This chapter outlines the basic concepts of FTIR techniques and studies the application of infrared (IR) spectroscopy in evaluating the biochemical properties of samples, saliva, serum, and nasopharyngeal fluid from individuals infected with SARS-CoV-2. Furthermore, we present statistical tools and SARS-CoV-2 virus fingerprint regions that contribute to positive and negative COVID-19 infection.

2 Principles of Infrared Spectroscopy and Spectromicroscopy Molecular vibrations are useful for characterization of matter as the nature of the chemical bond is translated into the identity of the vibrating atoms. In this sense, IR spectroscopy, along with Raman spectroscopy, that allows scientists to investigate how the electromagnetic radiation promotes molecular vibrations and further identify these vibrating entities [29]. Although many chemical bonds vibrate naturally, it is not all of them that interact with, or absorb, IR radiation. The selection rule of IR spectroscopy states that the vibration must change the molecule’s electric dipole moment, or the moiety that leads the vibration [1]. If this rule is not satisfied, there may be a vibrational mode even if there is no active signal in the IR spectrum. For example, although CO2 is a nonpolar molecule with some vibrational modes active in the IR spectrum; there is a change in electric dipole moment of the O=C=O structure changes due to unequal atom. IR spectrometers are used to record the region of the spectrum absorbed by the sample. Dispersive and FTIR spectrometers are the two main types of instruments [18]. Dispersive instruments work by illuminating monochromatic radiation on a sample, which necessitates the use of a monochromator and a slit to select an appropriate radiation wavelength (Fig. 1a). The transmittance is evaluated at each wavelength. On the other hand, after the introduction of the Fourier-transform spectrometers in the early 1970’s [11], although using a different optical setup with an interferometer, much advantage was obtained using these new spectrometers, as a polychromatic beam is irradiated on the sample and evaluated as an interference pattern called interferogram, which later is processed through a Fourier-transform procedure to gather an IR spectrum (Fig. 1b). Thus, Fourier-transform techniques are

Fourier-Transform Infrared Spectroscopy and Spectromicroscopy …

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Fig. 1 Schematic representation of a dispersive and b Fourier-transform-based IR spectrometers

the most widely used technique due to their rapid analysis and high signal-to-noise ratio. The coupling of microscope into sample compartment of an FTIR spectrometer (Fig. 2) is a breakthrough innovation in how a focused IR beam analyzes small samples [2]. A set of microscopic lenses are used to focus the IR beam that nearly reaches the IR radiation diffraction limit to spot a sample with a size as small as 2 μm [14]. All modes of analysis, such as transmission, reflection, and attenuated total reflection (ATR), that are used in traditional benchtop equipment can also be performed in FTIR spectromicroscopy. Grazing angle incidence can also be made with special lenses called “grazing angle objectives” [6]. These lenses are perfect for analyzing thin films immobilized on gold and platinum metal substrates. On the other hand, ATR mode of analysis is ideal for samples whose transmission and reflection properties are not suitable for FTIR measurements. This analysis mode takes advantage of internal reflection, which allows any material in contact with the surface of the reflecting element to interact with the evanescent IR wave. There is also the possibility of analyzing samples in liquid. Single-point detectors are used in microscopes because they can obtain high resolution and spectral information from a single point on a sample, effectively measuring

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Fig. 2 Schematic representation of a FTIR microscope. Reprint from Baker et al. [3]

the composition of the entire microscopic sample. One such example is mercurycadmium-telluride (MCT), widely used in FTIR microscopes as one of the most photosensitive materials, with a sensitivity as low as 600 cm−1 , making it ideal for mid-IR measurements. Equipping microscopes with multi-element detectors, such as focal plane arrays (FPA), increased FTIR spectromicroscopy’s potential for spatial resolution. This type of detector is made up of several individual detector elements arranged in a matrix pattern. A 64 × 64 FPA detector, for example, would allow one to collect spectra from 4096 different regions of the sample in a single spectroscopic scan [10, 19]. The simultaneous collection of several spectra divided into individual microscopic regions of the sample enabled the creation of images based on a specific absorption signal in the spectra. This spatial resolution advancement opened up a new area of IR spectroscopy known as spectromicroscopy. It is now feasible to analyze samples with heterogeneous microscopic regions individually.

3 FTIR for COVID-19 Studies Biological samples are scrutinized at wave numbers ranging from 4000 to 200 cm−1 . However, the spectral region between 1800 and 900 cm−1 is often called fingerprint region, where absorptions of different molecular constituents occur, such as lipids (C=O symmetric stretching at ∼1750 cm−1 ), carbohydrates (CO−O−C symmetric stretching at ∼1155 cm−1 ), and nucleic acid (asymmetric phosphate stretching at ∼1225 cm−1 , and symmetric phosphate stretching at ∼1080 cm−1 ) [7].

Fourier-Transform Infrared Spectroscopy and Spectromicroscopy …

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In addition, proteins exhibit characteristic vibrational modes in wavenumbers around ∼1650 cm−1 (80% C=O stretching, 10% C−N stretching and 10% C−N bending), at ∼1550 cm−1 (60% N−H bending and 40% C−N stretching); and at ∼1260 cm−1 (C−N stretching) [7, 21], known as the amide I, amide II and amide III bands, respectively. These spectral regions establish methodologies for detecting COVID19 in serum samples, oral or pharyngeal cell smears, and saliva using FTIR. Recently, Barauna and coworkers [5] used ATR-FTIR spectroscopy to detect the SARS-CoV2 virus in oral and pharyngeal samples. The generic algorithm-linear discriminant analysis (GA-LDA) was employed to discriminate between positive and negative samples. The data was separated into 50 positive and negative samples to train the algorithm, while 20 positive and 61 negative samples were used for validation. For the SARS-Cov-2 detection, wavenumbers of RNA viruses are associated with the organism’s inflammatory responses. Figure 3a shows the selected variables by GALDA to which the molecular constituents such as polysaccharides (~1429 cm−1 ), asymmetric PO2 stretching in RNA and DNA (~1220 cm−1 ), symmetric PO2 stretching in nucleic acids (~1084 cm−1 ), C–O stretching in ribose (~1069 cm−1 ), and symmetric PO2 stretching in nucleic acids (~1041 cm−1 ). Figure 3b presents the sample results of GA-LDA validation, in which we observe some false positives and one false negative. Hence, analysis method achieved a blind sensitivity of 95% and specificity of 89%. The work of Nascimento and coworkers [23] also evaluated the use of statistical methods to predict COVID-19 positive and negative patients. However, ATRFTIR spectra (4000–650 cm−1 ) were not only examined by GA–LDA, but also by successive projection algorithm (SPA–LDA), partial least squares (PLS–DA), and

Fig. 3 Clinical examinations of pharyngeal swabs using ATR-FTIR spectroscopy. a Variables chosen by GA-LDA. Higher absorbance in the COVID-19-positive class, indicated by the arrow. High absorbance in the COVID-19-negative class, indicated by the arrow. A MANOVA test with all five GA-LDA-selected variables was used to determine the P-value between all negative and positive samples. b The validation set’s GA-LDA score plot (n = 61 negatives and 20 positives). Reprint from Barauna et al. [5]

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a combination of dimension reduction and variable selection methods by particle swarm optimization (PSO–PLS–DA). The calculations were carried out by adopting a data set of 237 saliva samples obtained from symptomatic patients (A total of 138 COVID-19 infections diagnosed via RT-qPCR) in the biofingerprint shown in Table 1. The results of the analyses indicate that the individual models performed well; on the other hand, the consensus class (GA-LDA, PLS-DA and PSO-PSL-DA) improved the validation performance to 85% accuracy, 93% sensitivity, 83% specificity, as shown in Table 2. A similar statistical approach was applied by Bandeira and coworkers [4] to investigate the presence of the SARS-CoV-2 virus in blood serum. The study analyzed the spectra obtained by FTIR reflectance spectromicroscopy (micro-FTIR) of samples collected from healthy and COVID-19 infected individuals. The data were treated using partial least squares: discriminant analysis. The results showed that the 1702– 1785 cm−1 range is a spectral marker for the degree of IgG glycosylation, allowing to probe of distinctive sub-populations of COVID-19 patients, depending on their Table 1 Principal mid-infrared (MIR) bands of the data set and chemical assignments

Wavenumber (cm−1 )

Tentative assignment

~3275

Stretching O–H symmetric

~3200–3550

Symmetric and asymmetric vibrations attributed to water

~2930

Stretching C–H

~2800–3000

C–H lipid region

~2100

Combination of hindered rotation and O–H bending (water)

~1750

Lipids: ν(C–C)

~1650

Amide I: ν(C=O)

~1550

Amide II: δ(N−H) coupled to ν(C−N)

~1450

Methyl groups of proteins: δ[(CH3 )] asymmetric

~1400

Methyl groups of proteins: δ[(CH3 )] symmetric

~1260–1250

Amide III: ν(C−N)

~1155

Carbohydrates: ν(C−O)

~1225

DNA and RNA: νas (PO− 2)

~1080

DNA and RNA: νs (PO− 2)

~1030

Glycogen vibration: νs (C−O)

~971

Nucleic acids and proteins: n(PO4 )

~966–960

C−O, C−C, deoxyribose

νs = symmetric stretching; νas = asymmetric stretching; and δ = bending Reprint from Nascimento et al. [23]

97 41

Train Test

Train Test

Second derivative

Second derivative

Second derivative

Second derivative/mean-centered

–

–

SPA-LDA

GA-LDA

PLS-DA

PSO-PSL-DA

Consensus class

41

Test 31

68

31

68

31

68

31

68

2

7

2

7

2

7

2

7

2

7

93.0

82.5

82.9

79.4

75.6

70.0

95.1

86.6

87.8

69.8

74.0

75.0

74.2

76.5

74.2

76.5

70.9

67.6

67.7

63.2

83.0

82.0

80.9

82.8

79.5

80.9

81.2

79.2

78.3

72.8

PREC CL.1 (%)

88.0

75.0

76.7

72.2

69.7

64.2

91.7

77.9

80.8

58.9

PREC CL.2 (%)

SENS = sensitivity; SPEC = specificity; PREC CL.1 = precision of class 1; PREC CL.2 = precision of class 2; and ACC = accuracy Reprint from: Nascimento et al. [23].

41

97

97

41

Test Train

97

41

Test Train

97

Train

31

68

SPEC (%)

Quality parameters Outlier

SENS (%)

NEG

Samples/Class POS

Set

Preprocessing

Model

Table 2 Models for classification’s quality parameters

85.0

79.0

79.2

78.2

75.0

72.7

84.7

78.8

79.8

66.7

ACC (%)

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degree of severity. In addition, the β-sheet structure of amide I of proteins (1689– 1698 cm−1 ) and deoxyribose from DNA (840–856 cm−1 ) bands presented the most significant contributions to positive and negative discrimination with a specificity of 87.5% and sensibility of 100%. From the wavenumber range in the between 1300 and 800 cm−1 , characterized as RNA biofingerprint of the SARS-CoV-2 virus, Wood and coworkers [30] report on a new transflection infrared-based saliva test for COVID19. The results showed a 93% sensitivity and 82% specificity using the Monte Carlo Double Cross-Validation (MCDCV) modeling approach. Nogueira and coworkers [24] investigated the applicability of ATR-FTIR spectroscopy associated with machine learning in oropharyngeal swab suspension fluid to evaluate COVID-19 positive samples. Samples were collected from symptomatic patients in two distinct states of Brazil, Espírito Santo and Bahia, which were called Liquid 1 and Liquid 2, respectively. To generate the classification model each spectrum was considered as an independent sample measurement to be subsequently included in each dataset. The classification model was carried out using the Partial Least Squares (PLS) analysis associated with the K-nearest Neighbours (KNN) classifier implemented in a MATLAB routine. The PLS analysis was considered using the second, third and fourth components, which are correlated to the wavenumbers shown in Table 3 (Liquid 1) and Table 4 (Liquid 2) of the PLS components (PLSCs). The first PLS was discarded from the analysis due to the heterogeneity displayed by the sample both Liquid 1 and Liquid 2. Already the KNN classifier was set with 10 neighbors and the calculations were based on the cosine distance metric with no distance weight. To build the classification Table 3 Main vibrational modes present in the fingerprint region between 650 and 1800 cm−1 and the wavenumber region between 2800 and 3000 cm−1 of the PLS loading spectra of PLSC2, PLSC3 and PLSC4 for the analysis of the LIQUID 1 dataset PLSC2

PLSC3

PLSC4

Wavenumber (cm−1 )

Vibrational mode

Structural component

921(924)

?

Membrane lipids(phospholipids

1092

Stretching PO2 symmetric (phosphate II) Nasym (C–O–C)

(polysaccharides-cellulose)

2878(2880)

Asymmetric stretch of –CH2 Methylene

1299

Deformation of N–H

Cytosine

1146

C–O bond

Phosphate and oligosaccharides

2838

Stretching C–H

Methoxy

1552(1550)

CN stretch and NH bend

Amide I

867

?

Left-handed helix DNA (Z form)

2941

?

?

Reprint from Nogueira et al. [24]

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Table 4 Main vibrational modes present in the fingerprint region between 650 and 1800 cm−1 and the high wavenumber region between 2800 and 3000 cm−1 of the PLS loading spectra of PLSC2, PLSC3 and PLSC4 for the analysis of the LIQUID 2 dataset PLSC2

PLSC3

PLSC4

Wavenumber (cm−1 )

Vibrational mode

Structural component

896

?

?

1359(1358)

Stretching C–O, deformation of ? C–H, deformation of N–H

2820

– CH2 and –CH3

Lipids

1442

d(CH2 )

Lipids, fatty acids (polysaccharides, pectin)

1524

vCN, vCC proteins, tyrosinase

Amide II

2913

?

?

1088

Stretching PO2 symmetric vibration in B-form DNA

Phosphate I

1497

C=C, deformation of C–H

Proteins

2855

Asymmetric CH2 stretching mode of the methylene chains in membrane lipids

Lipids

Reprint from Nogueira et al., [24]

model the collected data from 151 positive patients and 92 negative patients for both data sets, i.e., from Espírito Santo and Bahia. The model was validated by using kfold cross-validation employing data randomly separated into training (80% of data collected) and test sets (20% of data collected). Once the classification model was established using the training set, it is applied to classification and validation of the test set. By applying the second derivative to the FTIR spectra, Nogueira and coworkers [24] calculated the PSL components intending to distinguish positives and negatives groups of the COVID-19 for the liquid 1 and liquid 2 data sets. For the Liquid 1 set, it was observed that most differentiation between positive and negative samples using combinations including PLSC2, as shown in Fig. 4. On the other hand, in the liquid 2 group, the PLSC 2 had less contribution to the differentiation between negative and positive samples. Nevertheless, the combining scores of PLSC2, PLSC3, and PLSC4 still present good discrimination of the samples, as shown in Fig. 5. Using a PLS-cosine KNN statistical tool, the model developed was capable of discriminating between COVID-19 positive and negative patients with 84% and 87% sensitivity, 66% and 64% specificity, and 76.9% and 78.4% accuracy for samples of liquids 1 and 2, respectively. In contrast, the methodology developed by Kitane and coworkers [12] “used machine learning” to achieve 97.8% of accuracy, 97% of sensitivity, and 98.3% of specificity. To validate and train the model were collected saliva samples from 280

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Fig. 4 PLS score plots of a PLSC2 × PLSC3, b PLSC2 × PLSC4, c PLSC3 × PLSC4, d PLSC2 × PLSC3 × PLSC4 for COVID-19 positive and negative groups of the LIQUID 1 dataset. Reprint from Nogueira et al. [24]

Fig. 5 PLS score plots of a PLSC2 × PLSC3, b PLSC2 × PLSC4, c PLSC3 × PLSC4, d PLSC2 × PLSC3 × PLSC4 for COVID-19 positive and negative groups of the LIQUID 2 dataset. Reprint from Nogueira et al. [24]

volunteers (100 tested positive for COVID-19 and 180 negative), on which the ATRFTIR spectra were analyzed at wavenumbers as 600–1350 cm−1 , at 1500–1700 cm−1 , and at 2300–3900 cm−1 , which are attributed to the RNA fingerprint, the phosphate backbone vibrations (νP-O), the νC-O stretching vibrations of the ribose sugar and the specific RNA nucleobases and, stretching vibrations of OH, NH, and CH group. All the previously cited studies used machine learning or mathematical tools to predict patients infected with COVID-19 based on the difference between the

Fourier-Transform Infrared Spectroscopy and Spectromicroscopy …

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spectra collected from samples of a healthy person and a person infected with the SARS-CoV-2 virus. On the other hand, it is also possible to employ biorecognition bindings among the antibodies and targets molecule to diagnose COVID-19 by Fourier-transform infrared spectromicroscopy (μ-FTIR) (Fig. 6a) combined with grazing angle objective (GAO) [8]. In this case, it is necessary to develop a probe, as shown in Fig. 6b, that contains the biorecognition element and enables the analysis by infrared. To analyze serum samples of infected patients with SARS-CoV-2, a binding element the Receptor-Binding Domain (RBD) portion of the SARS-CoV-2 spike protein was used. The RBD protein is immobilized at the probe by a series of preparation steps and, after that, is available to recognize the specific virus antibody present in the serum sample. Generally, the procedure in this type of detection requires that spectrums will be collected after each step of protein immobilization to ensure the correct data interpretation. First, the IR spectrum of the RBD is collected to assure that protein was effectively immobilized. After that, the spectra of the substance used as a blocking agent of active sites of the probe are collected, which can be bovine serum albumin, ethanolamine, or glycine. The last step is the detection of the specific SARS-CoV-2 antibody present in the positive sample. The detection is verified due to the increase of absorbance in the amide I region—the region in which proteins are normally studied. The increase in the signal must be more evident in the positive samples than in the negative samples. Figure 6c shows the complete spectrum of a blank (RBD + BSA protein), SARS-CoV-2 negative, and positive samples collected after interaction with the probe. Figure 6d presents the amide I region (1730–1590 cm−1 ) of blank (RBD + BSA protein), SARS-CoV-2 negative, and positive samples. From the information presented in Fig. 6, i.e., due to the difference between absorbances or by integration of spectrum area of positive and negative samples, it is possible to develop a method to diagnose COVID-19 based on biorecognition bindings with excellent responses.

4 Summary FTIR spectroscopy has intrigued the scientific community’s interest over the years as it allows the identification of various chemical structures through their respective vibrational modes. As a result, many research groups are allocating time and resources (both financial and human) to use FTIR spectroscopy for various objectives and methodologies. Given the scenario of COVID-19 pandemic and the search for new diagnosis strategies, researchers recently identified an opportunity in infrared spectroscopy that could be used as screening technique to control the spread of the virus As demonstrated in this chapter, a variety of ways to analyze the infrared spectra of samples obtained from contaminated patients (saliva, oropharyngeal swabs, and blood serum). In general, these studies used statistical methods in conjunction with machine learning or biorecognition bindings to provide responses about negative and positive SARS-CoV-2 virus infection with high sensitivity and specificity.

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Fig. 6 Fourier-transform infrared spectromicroscopy combined with grazing angle objective (GAO) (a). Probe that contains the binding and biorecognition elements (b). IR spectra of blank (BSA + RBD) (blue line), negative (black line) and positive (red line) samples to SARS-CoV-2 (c). Amide I region (1730–1590 cm−1 ) of blank (RBD + BSA protein), SARS-CoV-2 negative, and positive samples (d)

Abbreviations ACE2 ATR DNA FPA FTIR IgG IgM IR KNN

Angiotensin-converting enzyme 2 Attenuated total reflection Deoxyribonucleic acid Focal plane arrays Fourier transform infrared Immunoglobulin G Immunoglobulin M Infrared K-nearest Neighbors classifier

Fourier-Transform Infrared Spectroscopy and Spectromicroscopy …

LDA MCDCV MCT PLS PSO RBD RNA RT-PCR SARS-CoV-2 SPA-LDA

13

Linear discriminant analysis Monte Carlo Double Cross-Validation Mercury-cadmium-telluride Partial least squares Particle swarm optimization Receptor-binding domain Ribonucleic acid Reverse transcription–polymerase chain reaction Severe Acute Respiratory Syndrome Coronavirus 2 Successive projection algorithm with linear discriminant analysis

References 1. Aroca RF, Ross DJ, Domingo C (2004) Surface-enhanced infrared spectroscopy. Appl Spectrosc 58(11):324A-338A 2. Bailly R (1948) A new tool for infrared studies. Science 108(2797):143 3. Baker MJ et al (2014) Using Fourier transform IR spectroscopy to analyze biological materials. Nat Protoc 9(8):1771–1791. https://doi.org/10.1038/nprot.2014.110 4. Bandeira CCS et al (2022) Micro-Fourier-transform infrared reflectance spectroscopy as tool for probing IgG glycosylation in COVID-19 patients. Sci Rep 12(1):1–13 5. Barauna VG et al (2021) Ultrarapid on-site detection of SARS-CoV-2 infection using simple ATR-FTIR spectroscopy and an analysis algorithm: high sensitivity and specificity. Anal Chem 93(5):2950–2958 6. Bensebaa F et al (2005) Grazing angle infrared microspectroscopy of micropatterned selfassembled monolayers. Appl Surf Sci 243(1–4):238–244 7. Butler HJ et al (2018) Optimised spectral pre-processing for discrimination of biofluids via ATR-FTIR spectroscopy. Analyst 143(24):6121–6134 8. Cagnani GR et al (n.d.) SARS-CoV-2 infections detection using surface-enhanced infrared absorption spectromicroscopy combined with grazing angle objective. Unpublished results 9. Cui J, Li F, Shi Z-L (2019) Origin and evolution of pathogenic coronaviruses. Nat Rev Microbiol 17(3):181–192 10. Hassan A, Macedo LJA, Crespilho FN (2020) Recognizing conductive islands in polymeric redox surfaces using electrochemical-coupled vibrational spectromicroscopy. Chem Commun 56(71):10309–10312 11. Ismail AA, van de Voort FR, Sedman J (1997) Fourier transform infrared spectroscopy: principles and applications. In: Techniques and instrumentation in analytical chemistry. Elsevier, pp 93–139 12. Kitane DL et al (2021) A simple and fast spectroscopy-based technique for Covid-19 diagnosis. Sci Rep 11(1):1–11. https://doi.org/10.1038/s41598-021-95568-5 13. Lee HK et al (2010) Production of specific antibodies against SARS-coronavirus nucleocapsid protein without cross reactivity with human coronaviruses 229E and OC43. J Vet Sci 11(1):165– 167. https://doi.org/10.4142/jvs.2010.11.2.165 14. Levenson E, Lerch P, Martin MC (2006) Infrared imaging: synchrotrons vs. arrays, resolution vs. speed. Infrared Phys Technol 49(1–2):45–52 15. Li G et al (2020) Coronavirus infections and immune responses. J Med Virol 92(4):424–432. https://doi.org/10.1002/jmv.25685 16. Li Z et al (2020) Development and clinical application of a rapid IgM-IgG combined antibody test for SARS-CoV-2 infection diagnosis. J Med Virol 92(9):1518–1524. https://doi.org/10. 1002/jmv.25727

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17. Lu R et al (2020) Genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding. The lancet 395(10224):565–574 18. Macedo LJA et al (2022) Non-destructive molecular FTIR spectromicroscopy for real time assessment of redox metallodrugs. Anal Methods 14(11):1094–1102. https://doi.org/10.1039/ D1AY01198G 19. Macedo LJA, Crespilho FN (2018) Multiplex infrared spectroscopy imaging for monitoring spatially resolved redox chemistry. Anal Chem 90(3):1487–1491 20. Millet JK, Whittaker GR (2018) Physiological and molecular triggers for SARS-CoV membrane fusion and entry into host cells. Virology 517:3–8 21. Morais CLM et al (2020) Tutorial: multivariate classification for vibrational spectroscopy in biological samples. Nat Protoc 15(7):2143–2162 22. Movasaghi Z, Rehman S, ur Rehman DI (2008) Fourier transform infrared (FTIR) spectroscopy of biological tissues. Appl Spectrosc Rev 43(2):134–179 23. Nascimento MHC et al (2022) Noninvasive diagnostic for COVID-19 from saliva biofluid via FTIR spectroscopy and multivariate analysis. Anal Chem 24. Nogueira MS et al (2021) Rapid diagnosis of COVID-19 using FT-IR ATR spectroscopy and machine learning. Sci Rep 11(1):1–13. https://doi.org/10.1038/s41598-021-93511-2 25. Olivier V, Delphine M, Olivier R, van Belkum A, Kozlakidis Z (2020) Considerations for diagnostic COVID-19 tests. Nat Rev Microbiol 26. Pokhrel P, Hu C, Mao H (2020) Detecting the coronavirus (COVID-19). ACS sensors 5(8):2283–2296 27. Racine R, Winslow GM (2009) IgM in microbial infections: taken for granted? Immunol Lett 125(2):79–85. https://doi.org/10.1016/j.imlet.2009.06.003 28. Steel JJ et al (2020) Empowering academic labs and scientists to test for COVID-19. Biotechniques:245–248 29. Stuart BH (2004) Infrared spectroscopy: fundamentals and applications. John Wiley & Sons 30. Wood BR et al (2021) Infrared based saliva screening test for COVID-19. Angew Chem 133(31):17239–17244 31. Zhu C et al (2021) Molecular biology of the SARs-CoV-2 spike protein: a review of current knowledge. J Med Virol 93(10):5729–5741 32. Zhu N et al (2020) A novel coronavirus from patients with pneumonia in China, 2019. New England J Med

Point-of-Care Devices with Electrochemical Detection for COVID-19 Diagnosis Luiz R. G. Silva, Jéssica S. Stefano, Tiago A. Silva, Marcio F. Bergamini, Luiz H. Marcolino-Junior, and Bruno C. Janegitz

1 Point-of-Care Devices Point-of-care (POC) testing can be described as analytical procedures performed for diagnostic and prognostic tests near to the patient (outside conventional laboratory), often at the patient’s bedside [1, 2]. These procedures contribute to immediate clinical decision making, improving patient outcomes and reducing the chances of complications of the patient’s condition as well as mortality, once it facilitates rapid diagnosis [3]. Hence, POC tests have to be sensitive, specific, easy to use, and provide fast results, comparable to laboratory-based methods to establish the appropriate treatment, leading to an improved clinical and/or economical outcome [4]. Conventional medical analyses performed in the laboratory have several disadvantages, such as being time-consuming, expensive, laborious, and requiring highly trained professionals [5–7]. Thus, there is a need to develop alternative methods of clinical analysis that can circumvent these disadvantages and be applied in a simple way [8]. Therefore, POC devices are presented as an ideal clinical analysis tool to overcome such challenges, which are simple, fast, easy to handle, can provide real-time diagnoses, and often do not require a highly specialized operator [8–10]. To be considered POC, an analytical device must primarily have high sensitivity and good detection capability [8, 11]. However, the great challenge that these devices face is to achieve reliable results in short times, and without sample pretreatment. L. R. G. Silva · J. S. Stefano · B. C. Janegitz (B) Laboratory of Sensors, Nanomedicine, and Nanostructured Materials, Federal University of São Carlos, Araras, São Paulo, Brazil e-mail: [email protected] T. A. Silva Department of Chemistry, Federal University of Viçosa, Viçosa, Minas Gerais, Brazil M. F. Bergamini · L. H. Marcolino-Junior Laboratory of Electrochemical Sensors (LabSensE), Department of Chemistry, Federal University of Paraná (UFPR), Curitiba, Paraná 81531-980, Brazil © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 F. N. Crespilho (ed.), Covid-19 Metabolomics and Diagnosis, https://doi.org/10.1007/978-3-031-27922-5_2

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They need to be portable, as small as possible, able to be used by inexperienced patients (user-friendly), and provide specific, sensitive, and accurate results [11, 12]. In this way, they can enable immediate or remote clinical decisions to improve patient care, even in poor countries without laboratories and other infrastructure that demand high investments [11]. The need for POC devices became more evident with the global SARS-CoV-2 virus pandemic, due to rapid contagion [13]. In this way, clinical tests are immediate, mainly as a form of control and prevention. However, due to the massive number of contaminated people, there was an overload of the tests available on the market, leading to a rapid lack of testing platforms [14, 15]. The main tests with greater reliability require long analysis times, taking days to get the result and other tests often present erroneous answers, such as false positives and negatives [15, 16]. Thus, the development and improvement of new tests have become urgent, especially tests of the POC type. In such situations, electrochemical methods become highly attractive, mainly through electrochemical biosensors, since they are fast, inexpensive, have good sensitivity and detectability. On the other hand, they require small amounts of samples, and it is not necessary for a specialized operator, can be miniaturized, portable, and present great possibilities for on-site application [12, 17]. Different types of fully portable and miniaturized electrochemical platforms for different applications can be seen in Fig. 1. It shows why electrochemical platforms are a highly viable alternative for POC application. These devices, when coupled to laptops, smartphones, and/or smartwatches, can perform analysis anywhere, whether in medical clinics or at patients’ homes, for analysis of different types, from simple biomarkers to viruses, such as SARS-CoV-2 [5, 18]. Therefore, this chapter aims to explore and present the different types of electrochemical devices developed for POC application in the detection and monitoring of the SARS-CoV-2 virus.

2 Electrochemical Devices for SARS-CoV-2 Electrochemical biosensors are analytical devices integrated with a recognition agent, which is commonly a biomolecule, which binds selectively to the analyte of interest (biological compound) [17, 28–30]. Due to the mechanism of binding between the recognition agent and the biological analyte, it is possible to obtain a measurable electrical signal and thus detect the analytes of interest quickly, simply, selectively, and in an accessible way [17, 28, 30]. Electrochemical biosensors can be classified into some categories, such as genosensors or RNA/DNA sensors, aptamer-based biosensors, and immunosensors. The categorization is based on their main recognition compounds and means of action to detect the specific target [17]. The genosensors are produced using specific DNA/RNA probes (they vary according to the target to be detected) and immobilized as a recognition element, allowing hybridization reactions to be carried out, which generally occur by molecular recognition of DNA-DNA or DNA-RNA [17, 31, 32]. In this context, Kashefi-Kheyrabadi and co-authors [33] reported a genosensor, in

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Fig. 1 Point of care electrochemical devices employing a portable potentiostat coupled to a laptop, b–i smartphones, and j smartwatches. a Reprinted of [19] with permission of ELSEVIER. b Reprinted of [14] with permission of ELSEVIER. c Reprinted of [20] with permission of ELSEVIER. d Reprinted of [21] with permission of ELSEVIER. e Reprinted of [22] with permission of ELSEVIER. f Reprinted of [23] with permission of ELSEVIER. g Reprinted (adapted) with permission from [24] Copyright (2022) American Chemical Society. h Reprinted of [25] with permission of ELSEVIER. i Reprinted of [26] with permission of ELSEVIER. j Reprinted of [27] with permission of ELSEVIER

which was employed a gold nanoneedle screen-printed electrode (SPGE) to design a DNA biosensor for the detection of SARS-CoV-2. The SPGE biosensor was based on four-way junctional hybridization, for modification with a universal DNA-Hairpin probe. The proposed method allowed the simultaneous analysis of Spike S1 protein from the SARS-CoV-2 virus and open reading frame (Orf1ab) genes in just 1 h. Figure 2 presents a scheme of the steps necessary for the detection of the SARS-CoV2 virus using the proposed biosensor. To build the biosensor, a thiolated universal DNA-Hairpin probe was immobilized on a gold nanoneedle screen-printed electrode through a gold-sulfur chemical interaction, and then filled with 6-mercapto-1-hexanol to avoid non-specific adsorption. The biosensor was then incubated with a mixture of two adapter sequences and the RNA target. In presence of all biosensor components, the 4-WJ configuration is formed, making the redox mediator-labeled adapter tape to approach the electrode surface, facilitating electron transfer and producing an electrochemical signal. Aptamer-based sensors are coated with artificial single-stranded nucleotides (DNA or RNA), these single strands have the same function as the previously explained genosensors. They have similarities with genosensors; however, these devices are composed of a few tens to hundreds of nucleotides and are produced by a technique called systematic evolution of ligands by exponential enrichment,

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Fig. 2 Illustration of a complete SARS-CoV-2 virus analysis steps and construction of the detection platform. A workflow for detecting SARS-CoV-2 RNA sequences from clinical samples using the electrochemical biosensor for detection of S1 protein and Orf1ab genes. Reprinted of [33] with permission of ELSEVIER

performed by iterative cycles of ligation, washing, and amplification [17, 34, 35]. For example, Rahmati and co-authors [36] have used a screen-printed carbon electrode (SPCE) to develop a biosensor based on copper hydroxide nanorods. In the presence of SARS-CoV-2 glycoprotein (target), a decrease in peak current (referring to copper hydroxide nanorods response) was observed due to the formation of target-aptamer complexes, thus, blocking the transfer of electrons from the electrode surface. Figure 3 presents an illustration of the developed (bio)sensors as well as the steps for analyzing the SARS-CoV-2 virus.

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Fig. 3 Schematic illustration of the aptasensor preparation steps. Reprinted of [36] with permission of ELSEVIER

Finally, immunosensors employ an antibody or antigen as a biorecognition element immobilized on the surface of the sensor, which converts the information associated with an immunochemical reaction into a measurable signal proportional to the concentration of the analyte [17, 37, 38]. In an interesting example, Stefano and co-authors [39] have developed an electrochemical immunosensor employing 3D-printed electrodes made of ready-to-use graphite-based lab-made filaments (no surface activation required) for the first time for SARS-CoV- detection. Figure 4 presents the procedure for producing the immunosensor and sensing the COVID19 virus. The 3D printed sensor was initially modified with EDC:NHS in which the mixture can be used due to the presence of carboxyl groups on the electrode surface. Then, the EDC:NHS acts as a crosslink to anchor the antibody (specific for SARS-CoV-2) on the surface of the electrochemical sensor. Finally, non-specific sites are blocked with BSA, this blocking has the same purpose as the procedure explained for the aptamer sensors previously. Thus, for virus analysis, the spike S1 protein was detected after incubation on the electrode surface for 30 min. According to the authors, the use of 3D printed sensors from lab-made filaments is a potential alternative for the production of biosensors, due to the ease of production of the sensors. Thus, it can be seen that the use of electrochemical biosensors is presented as a great alternative for SARS-CoV-2 detection, being able to apply different approaches in the construction of the biosensors. Since the beginning of the global pandemic caused by the SARS-CoV-2 virus, several studies have been developed in the area of

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Fig. 4 Representative scheme of the involved steps in the fabrication of the immunosensor. Reprinted of [39] with permission of ELSEVIER

electrochemical biosensors for the detection of the virus that causes COVID-19. In a database search using the keywords: “electrochemical device” and “SARS-CoV2”, 54 publications can be found over the last years of the pandemic (2020–2022) reporting the development of electrochemical devices with the most different types of arrangement and materials for the detection of the virus that causes COVID-19. Figure 5 shows the relationship between the year of publication and the number of published articles. In Fig. 1 is observed that in 2020 (the year of the emergence of the pandemic), only 4 scientific papers were reported regarding the production of electrochemical devices for the detection of the SARS-CoV-2 virus. Regarding the number of articles, 4 articles may seem few, however, these were pioneering and essential to demonstrate the potential of biosensors as tools for virus detection. This fact can be observed in the number of publications in the following year. In this scenario, 39 scientific articles were published, demonstrating the great effort of researchers to improve and provide high-quality electrochemical devices. Currently, in 2022, 11 articles have already been published presenting the manufacture of new biosensors and it is worth mentioning that this research was carried out before the end of the first quarter of the year. This demonstrates that the efforts for improvement and development of these analysis tools are continuing, and can match or exceed the number of publications in 2021.

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Fig. 5 Relation between the year of publication and the number of articles published on the development of electrochemical devices for the detection of the SARS-CoV-2 virus. The search was carried out in the “Web of Science” database with the keywords: “electrochemical device” and “SARS-CoV-2”

3 Point-of-Care Electrochemical Devices for SARS-CoV-2 Detection Although in the literature there are approximately 50 scientific articles that report the development of electrochemical sensors for the detection of SARS-Cov-2, only 12 are self-declared POC devices. The present chapter will present such devices, providing information about their materials, designs, and characteristics that framed them as POC. For better visualization, Table 1 summarizes the developed biosensors, as well as their analytical characteristics. The work of Karaman and co-author [44] has presented a glassy carbon electrodebased electrochemical immunosensor for the detection of SARS-CoV-2 NP in saliva. According to the authors, the developed immunosensor showed good selectivity for SARS-CoV-2 when tested against interferents such as MERS-CoV, SARS-CoV, and H1N1 viruses. In this way, the immunosensor was classified as fast, sensitive, and specific, and maybe an alternative to conventional tests and with the possibility of POC application.

Spike protein CR3022 antibody

SWVp

EISq

SWV

EIS

DPV

DPV

SWV

LEADa

Paper-based ZnO-NW-enhancedb biosensors

Electrochemical immunosensor

CNTs/WO3 -SPEsc

MV-gal1/SCPE-GNPd

FPCB-implemented DNA sensori

AuNPs-SPCEj

On-chip N-protein biosensing

ncovS1-MIP

Coronavirus proteins

CAo

Sensor

sensorl

RNA

DPVn

Biosensor

ncovS1 N-protein

SWV

EIS

N antigen

ssDNA

Spike S1

SARS-CoV-2 particles

SARS-CoV-2 NP

Target

Technique

Device

–

Nasopharyngeal

Nasopharyngeal

–

Swab

Nasopharyngeal or oropharyngeal

Saliva

Human serum

Nasopharyngeal or oropharyngeal

Saliva

Saliva

Samples

Table 1 Analytical characteristics of POC electrochemical devices for the sensing of SARS-CoV-2

1.59 × 10−6

200 copies mL−1

LODr (pg mL−1 )

0.4

3.1

1.0 to 1.0 × 105 0.03 to 0.20

0.4 1.12

0.1 to 1.0 × 106

[49]

[25]

[48]

[47]

[46]

4.57 × 102 copies mL−1 0.1

[45]

[44]

[43]

[42]

[41]

[40]

(continued)

References

50.0

101 to 105 copies mL−1

7.0 to 320.0

0.01 to 1.0

1.0 × 104 to 1.0 × 0.003 106

0.1 to 1000.0

5.0 × 105 to 2.0 × 0.23 106

10−17 to 10−12 mol L−1

(pg mL−1 )

Linear range (pg mL−1 )

22 L. R. G. Silva et al.

SARS-CoV-2 IgG and IgM

Target Human sera

Samples 10 to 1000

Linear range (pg mL−1 ) 1000

LODr (pg mL−1 ) [14]

References

Electrochemical Advanced Diagnostic; b Paper-based EIS biosensors featuring zinc oxide nanowires; c Screen-printed carbon electrode modified with carbon nanotubes and tungsten trioxide; d Screen-printed gold nano particle-carbon electrodes modified with Microparticle Vesicle-Galactins1; i Flex printed circuit board; j Screen-printed carbon electrodes with gold nanoparticles; l ncovS1 sensor armed with a molecularly imprinted polymer synthetic receptor; m Electrochemical paper-based analytical device; n Differential pulse voltammetry; o Chronoamperometry; p Square wave voltammetry; q Electrochemical impedance spectroscopy; r Limit of detection

SWV

COVID-19 ePADm

a Low-cost

Technique

Device

Table 1 (continued)

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Ramanujam and co-authors [41] developed a POC electrochemical system that allows the detection of the SARS-CoV-2 virus in a 2 mL volume (saliva) sample. In this way, the presented system allows extremely fast, the reliable analysis applied in loco, contributing to greater control of patients contaminated by the SARS-CoV-2 virus. With the growing number of developed electrochemical sensors capable of detecting the SARS-CoV-2 virus in a POC form, screen-printed electrodes still are highlighted as alternatives with great potential for employability. Such disposable sensors can be purchased commercially at an affordable cost, are miniaturized, and have excellent performance. That said, Zhao and co-authors [40], demonstrated a simple and elegant way for a system employing a mini-potentiostat with disposable electrodes. They used graphene oxide functionalized with calixarene to target SARS-CoV-2 RNA for determination of the virus causing COVID-19. The POC system was also based on the analysis performed on a smartphone with a potentiostat attached, such a system allows real-time analysis. Figure 6 shows (a) the sensor modification procedure and (b) the detection method of the SARS-CoV-2 virus, as well as the POC system employed. Furthermore, tests were performed on patients (25 individuals) and the results showed superior performance to RT-qPCR kits. In the seek to develop a new platform for rapid, sensitive, selective, and application POC analysis, Eissa and co-authors [48] proposed a biosensor based on an SPCE coated with gold nanoparticles and later modified with an antibody specific for SARSCoV-2. The developed sensor presented an excellent LOD, comparable to the test kits available in the market. Moreover, electrode selectivity was tested against several other viruses, demonstrating that the sensor is specific for the SARS-CoV-2 virus. Following in the same line as screen-printed electrodes, other types of 2D electrodes are also used as POC electrochemical platforms, due to their size and ease of use. In this sense, Ayankojo and co-authors [25] reported the fabrication of an electrochemical sensor based on a synthetic molecular imprint polymer receptor for the quantitative detection of the spike protein ncovS1. In this way, detection became possible due to the covalent interaction between 1,2-diols of the highly glycosylated protein and the boronic acid group of 3-aminophenylboronic acid. For a better understanding, Fig. 7 shows an illustrative scheme of the detection method as well as a conception of the type of sensor and POC electrochemical platform used in the work. According to the authors, the sensor responds in 15 min. In addition, the sensor is compatible with portable potentiostats which makes it a great choice for POC tests. The SPEs continue to demonstrate that they are highly employable sensors to develop electrochemical platforms to be applied as POC devices. For this, Hussein and co-authors [45] presented a new biosensor using screen-printed carbon electrodes modified with nanomaterials (CNTs and WO3 ) with subsequent anchoring of viral particles (SARS-CoV-2) within the polymer matrix. Such modification allows the creation of specific sites that bind to complementary sequences of the target virus. Also, this screen-printed carbon electrodes modified modification strategy provided high selectivity for the electrochemical sensor in the presence of several other viruses, such as H1N1, H5N1, MERS-CoV, and Influenza, among others. Moreover, the

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Fig. 6 Schematic representation of SARS-CoV-2 detection using the electrochemical biosensor. a Preparation of the premix A and B; b Electrochemical detection using a smartphone. Reprinted of [40] with permission of ELSEVIER

authors emphasize that the electrochemical platform for POC application presented sensitivity 27 times greater than the RT-PCR test in a time of approximately 5 min. In search of innovative and multi-purpose platforms using screen-printed electrodes modified, the development of universal tests is an interesting path with great potential. Ghazizadeh and co-authors [46] developed an electrochemical platform capable of identifying any virus with a spike protein, in addition to being able to

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Fig. 7 The general scheme of the electrochemical platform developed and the working principle of the biosensor in the diagnosis of COVID-19. Reprinted of [25] with permission of ELSEVIER

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specifically determine the SARS-CoV-2 virus. The electrochemical platform developed was based on the use of screen-printed gold nano particle-carbon electrodes modified with microparticle Vesicle-Galactins1. Such modification allowed the sensor to track any virus. The detection method was based on the capture of the virus by the modifier (Microparticle Vesicle-Galactins1). However, it is emphasized that the modifier can capture any virus present. Thus, it is necessary to add Au/AntibodySARS-CoV-2 that will specifically bind to the SARS-CoV-2 virus, giving specificity at the time of analysis. The platform can be described as universal since the modifier can connect to any virus and with the addition of Au/antibody (specific to the target to be analyzed). For better visualization of the action mechanism, Fig. 8 presents a complete illustration of the method. To develop an inexpensive and accessible device, Lima and co-authors [42] proposed an electrochemical advanced diagnostic sensor capable of detecting the SARS-CoV-2 virus in approximately 7 min for US$ 1.50 per test unit produced. The electrochemical device is composed of a graphite electrode modified with gold nanoparticles-cysteamine linked to ACE2. The electrochemical device, as well as its modification and detection step of the SARS-CoV-2 virus, can be seen in Fig. 9. According to the authors, the sensor presented an excellent performance in the performed tests, proving the ability to detect the UK variant B.1.1.7. In this context, the device developed for POC application demonstrates great potential for employability and may in the future become a test used for the control and monitoring of the virus that causes COVID-19. Paper-based sensors have gained prominence to produce easy-to-handle and inexpensive miniaturized electrochemical systems. Furthermore, the use of such a substrate in the manufacture of sensors provides the possibility of developing POC application systems. In this sense, Yakoh and co-authors [14] developed a paperbased immunosensor, specific and sensitive, for the detection of immunoglobulin produced against SARS-CoV-2. Figure 10 shows the entire electrochemical platform in detail. The electrochemical sensor proved to be effective in real clinical sera from patients, with satisfactory results. Thus, the use of paper-based sensors presents a low-cost alternative for the future development of POC test platforms. Paper-based sensors with microfluidic methods are presented as an extremely promising tool for POC analysis. In this regard, Li and authors [43] developed paperbased analytical devices modified with zinc oxide nanowires and subsequently colinked probe molecules specific to the COVID-19 antibody. Overall, the cost of each test was estimated at $ 0.1, based on the amounts of reagents involved in each test and the price of paper substrates. In addition, the developed devices demonstrate that it is possible to build sensors to detect the virus that causes COVID-19 from a substrate that is easy to acquire, widely available, and of extremely low cost. The search for new detection platforms and specific targets has gained strength in several areas of research, especially when we talk about electrochemical slides for POC applications. Promising strategies based on the integration of electrodes on printed circuit boards are presented as an alternative to improve electrochemical systems and the performance of the analysis. Therefore, Damiati and co-authors [47] pursued for the first time the development of a flex printed circuit board-implemented

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Fig. 8 Schematic representation for the stages of screening and specific detection of COVID-19 based on the electrochemical biosensor. a Immobilization of MV-gal1 on the screen-printed gold nano particle-carbon electrodes. b Dropping the SARS-CoV-2 on the MV-gal1screen-printed gold nano particle-carbon electrodes for the screening of the spiked COVID-19. c Dropping the Au/AntiSARS-CoV-2 spike on the MV-gal1@SARS-CoV-2 Antigen to specific detection of COVID-19. Reprinted of [46] with permission of SPRING NATURE

DNA sensor for hybridization-based detection of SARS-CoV-2. To facilitate the use and handling of the samples, the developed biosensor was incorporated into the authors’ development platform and an easy-to-use reservoir, which allows the analysis without any loss of samples. It can be seen from these works that electrochemical devices are very versatile in the matter of materials and designs, requiring mostly low-cost materials, such as paper, for their development. These characteristics prove their high potential to be employed in the development of POC platforms, which can be employed in complex

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Fig. 9 Low-cost electrochemical biosensor proposed by Lima and co-authors. a Schematic representation of graphite electrodes used in LEAD. b Gold nanoparticles-cysteamine functionalization on graphite electrodes after modification with glutaraldehyde. c Modification of gold nanoparticles-cysteamine with ACE2 using EDC and NHS to enable amide bond formation and BSA for surface blockage and the analytical response of LEAD in the presence of SARS-CoV-2. Reprinted of [42] with permission of PNAS

analysis, such as in the detection of viruses. The ability to develop simply provided the development and proposal of several types of electrochemical POC devices quickly, following the emergency need for new analytical devices on a large scale, due to the emergence of a new pandemic.

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Fig. 10 Schematic illustration of the a device components, b detection principle, and c detection procedure of the COVID-19 Paper-based sensor. Reprinted of [14] with permission of ELSEVIER

4 Conclusion and Future Perspectives The development of new sensing platforms, especially those of POC application to detect the SARS-CoV-2 virus, as well as others, are of great importance. These new devices have the challenge of overcoming existing problems in patient monitoring, especially for patients who are unable to perform tests in clinics or who have difficulties accessing commercial tests. Therefore, electrochemical devices are presented as a potential tool for POC analysis, due to the relatively low costs, the low number

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of samples required, fast analysis, and mainly due to the ease of portability when coupled to smartphones or watches. Given the aspects discussed in this book chapter, electrochemical devices are being increasingly used for monitoring the SARS-CoV-2 virus, especially in a POC manner. This growing application has become possible due to the evolution of electrochemical sensors over the years, employing new designs, with different platforms, whether printed, manufactured in the laboratory, or produced on paper. These sensor/biosensors can assist the handling and employability of diagnostics in a simple and fast way. In this scenario, regarding the advancement of new technologies, there is a demand for the production of robust electrochemical sensors, with greater specificity, and sensitivity, which can be miniaturized. Acknowledgements The authors thank the financial support from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) (2013/22127-2), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (303338/2019-9) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)—Finance Code 001, 88887.504861/2020-00 and 88887.517992/2020-00.

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Carbon Nanomaterials for Electrochemical Detection of SARS-CoV-2 Infections Thiago da Costa Oliveira, Giovana Rosso Cagnani, and Frank Nelson Crespilho

1 Introduction The word “nanotechnology” is extensively used to define sciences and techniques applied at the nanoscale level, that is, extremely small measurements, that allow molecular structures and their atoms to function and be manipulated [1]. Their properties differ from those of the same macroscopic-sized material because of quantum confinement and increased specific area in the nanomaterial. Therefore, nanotechnology aims to develop materials and devices on a nanometer scale leading to the possible manufacture of materials and machines by the rearrangement of atoms and molecules [2]. Nanotechnology is the study, design, creation, synthesis, manipulation, and application of materials, devices, and functional systems through the control of matter at the nanoscale, and the exploitation of phenomena and properties of matter at the nanoscale level [3]. When matter is manipulated on the scale of atoms and molecules, it demonstrates entirely new phenomena and properties. Therefore, scientists use nanotechnology to create novel and inexpensive materials, devices, and systems with unique properties. Nanotechnology is one of the most promising areas of modern science and technology with great economic and social impacts [4]. Nanomaterials are materials with morphological properties with at least one dimension smaller than one micrometer. Although there is no consensus on the minimum or maximum size of a nanomaterial, some authors restrict their size between 1 and 100 nm, a logical definition would place the nanoscale between the microscale (1 μm) and the atomic/molecular scale (approximately 0.2 nm) [5]. T. da Costa Oliveira (B) · G. R. Cagnani · F. N. Crespilho (B) São Carlos Institute of Chemistry, University of São Paulo (USP), São Carlos, São Paulo 13560-970, Brazil e-mail: [email protected] F. N. Crespilho e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 F. N. Crespilho (ed.), Covid-19 Metabolomics and Diagnosis, https://doi.org/10.1007/978-3-031-27922-5_3

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The concept of nanomaterial is simple, the material properties are dependent on the behavior of moving electrons within them and the arrangement of atoms in the matter. In a nanometric material, the movement of electrons are limited by the dimensions of the material itself. Furthermore, the ratio of atoms on the surface to the interior is significantly higher than in bulk materials. Therefore the reduction in material dimensions results in a modification of its properties and consequently materials with unique properties can be designed [6, 7]. Carbon nanomaterials make up an extensive group of materials with characteristics that are widely applicable in various areas, from technology to clothing. Graphene and carbon nanotubes (CNTs) are fundamental materials for the development of works in the areas of nanoscience and nanotechnology [8]. In simple terms, the structure of graphene can be described as a 2D crystal lattice formed by carbon atoms that corresponds to a single atomic layer of graphite. The structure of the CNT can be easily understood by considering the graphene sheet. In graphene, the carbon atoms are all sp2 hybridized and arranged in a hexagonal arrangement, exhibiting a lamellar structure. The structure of single-walled nanotubes can be described as cylinders formed from a sheet of graphene rolled up on itself. The angle at which the graphene sheet is rolled defines the electrical and mechanical properties of the nanotube. The structure of multi-walled nanotubes can be described as multi-layered concentric cylinders. Both types of nanotubes have exceptional physical, chemical, and biological properties. Because of the strong carbon–carbon chemical interaction, these materials are extremely mechanically resistant. A CNT can be metallic or a semiconductor depending on how the graphene sheet is folded to create a cylinder [9, 10]. Graphene and CNTs are used in different technological applications. They may be blended with other materials, including polymers, ceramics, cement, and fibers, to transfer their outstanding electrical, mechanical, thermal, and chemical capabilities, transferring their outstanding properties to the composite [10, 11]. Graphene and CNTs are also utilized to create prototypes for novel electrical devices such transistors, display-related electron emitters, and gas and biological sensors [12]. They also promise to be significant players in the development of nanoelectronics [13]. From a biological point of view, carbon nanomaterials have a high area for functionalization and rapid response to external radiation stimuli and integration with potential for application in nanomedicine [14–16].

2 Carbon Nanomaterials: Types and Classification Carbon nanomaterials have different properties depending on the structural form they acquire. Ellipsoidal or spherical carbon nanomaterials are known as fullerenes, whereas cylindrical ones are called nanotubes. These materials have many applications, including the development of improved coatings and films, lighter and more resistant materials, and various applications in the field of electronics. Some carbon allotropes are shown in Fig. 1.

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Fig. 1 Schematic representation of carbon allotropes: (a) graphite, (b) diamond, (c) fullerene, (d) single-walled carbon nanotube, (e) multi-walled carbon nanotube, and (f) graphene. Reprinted from Zarbin and Oliveira [17] with permission

2.1 Carbon Nanotubes CNTs, like diamond, graphite, or fullerenes, are an allotropic form of carbon. The structure of a CNT consists of a rolled-up cylindrical sheet of graphite. Nanotubes of different diameters and internal geometries are formed depending on the degree of winding and the way in which the original sheet was formed [10]. Nanotubes can be shaped as if the corners of a single graphite sheet layer were joined at their ends forming a tube, called single-walled nanotubes (SWCNTs) (Fig. 2a). Nanotubes whose structure resembles that of a series of concentric tubes with some inside others increasing the thicknesses from the center to the periphery are the most common type of double (DWCNTs) and multi-walled nanotubes (MWCNTs) (Fig. 2b, c) [5]. Most SWNTs have a diameter of approximately 1 nm and tube lengths that can be thousands of times longer. The structure of a SWNT may be seen by flawlessly wrapping an atom-thick sheet of graphite around a cylinder. The single layer of graphene in CNTs can be rolled in a variety of ways. The CNTs are classified as zigzag, armchair, or chiral, based on the number of unit vectors in the graphene crystal lattice along two directions in the honeycomb structure, as seen in Fig. 2a. The way in which the graphene sheet wraps is represented by a pair of indices (n, m), called the chiral vector. The integers m and n indicate the number of unit vectors along two directions in the crystal honeycomb graphene lattice. If m = 0, the nanotubes are called “zigzag”. If n = m, the nanotubes are called “armchair”. Otherwise, they are called “chiral” [1, 19].

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Fig. 2 CNTs structure and models based on the number of walls. (A) Structures with single-wall carbon nanotubes (SWCNTs) based on chirality (zigzag, armchair, and chiral), (B) double-walled carbon nanotubes (DWCNTs), and (C) multi-walled carbon nanotubes (MWCNTs) composed of numerous concentric shells. Reprinted from Tîlmaciu and Morris [18] with permission

SWCNTs are a very important variety of carbon nanomaterials because they exhibit important electrical properties not found in the numerous variants of MWCNTs. SWCNTs are the candidates for electronic downsizing beyond the microelectromechanical scale, which is the foundation of contemporary electronics.

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SWCNTs were important in the construction of the first intramolecular field effect transistors (FETs) [20, 21].

2.2 Graphene Graphene was discovered in 2004 when a thin sheet (so thin that it is one atom thick) came out of graphite by chance with the help of ordinary insulating tape. However, science does not understand coincidences, and the material was characterized, and its properties and applications determined. Fiber optics, faster computers, and various types of solar panels or sensors of all kinds are some of the possibilities offered by this thin, resistant, flexible, transparent and superconducting material, to name a few of its properties [22]. As a result of the discovery of this allotropic form of carbon, researchers Andre Geim and Kostya Novoselov were awarded the Nobel Prize in Physics in 2010 [10]. The structure of graphite can be considered a stack of many superimposed graphene sheets. The bonds between the stacked graphene layers result from Van der Waals forces and interactions between the π orbitals of the carbon atoms. In graphene, the carbon–carbon bond length is approximately 1.42 Å [23]. It is the basic structural component of all other graphitic elements including graphite, carbon nanotubes, and fullerenes. This structure can also be considered an extremely extensive aromatic molecule in both directions of space, that is, it would be the limiting factor in a family of flat molecules of polycyclic aromatic hydrocarbons called graphenes [24, 25]. Graphene is a two-dimensional material that is only one atom thick. The flat lamellar structure of graphite is composed of carbon atoms that form a hexagonal network. Its appearance may seem fragile and delicate because at first glance graphene is similar to a transparent and flexible fabric [26]. However, graphene is an extremely resistant material that also serves as a conductor of electricity. The discovery of graphene was, without a doubt, surprising. Until that point, both theoretical and experimental knowledge indicated that the existence of two-dimensional crystal structures detached from the volumetric crystal were impossible. Calculations indicated that such a structure would be unstable and would have to collapse to form a normal three-dimensional (3D) structure [27]. Therefore, the most relevant and manifest properties to date highlight the elevated hardness and resistance of graphene. Graphene it is 300 times stronger than steel and diamonds and much lighter. Its high elasticity distinguishes it from other crystals and allows it to relax considerably more. Graphene is often compared to carbon fiber because of its lightness; however, it is undoubtedly more flexible. This, together with its resistance, makes it withstand enormous deformation stresses, allowing it to hold atoms of gold, nickel, and other heavy metals unchanged, as proven by previous tests. Graphene also reacts chemically with other elements, giving rise to compounds with different characteristics, and behaves as a semiconductor [10, 28]. Based on its peculiarities, graphene is a material whose properties position it between metals and semiconductors. Graphene has high thermal and electrical conductivity. Specifically,

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in several tests, graphene was observed to consume less electricity than silicon when doing the same work and operate as a better electrical conductor than copper [29]. The devices reported in this study are based on two types of technologies, FETs and electrochemical (bio)sensors; both devices have the advantage of requiring a minimum amount of graphene to be deposited in the channel and on the working electrode, respectively. Moreover, it should be noted that monolayer graphene is currently more expensive to obtain and synthesize because high-vacuum equipment is needed to deposit or grow the graphene monolayer on certain substrates, and clean rooms with strict and rigorous cleaning protocols are necessary for the manufacture of semiconductors [5, 6, 30]. This is where the value of the monolayer graphene sheet increases and makes it difficult to scale up to an industrial level, although it should be noted that the amounts required for the electrodes and detection surfaces in these biosensors are minimal [31, 32]. Furthermore, electrochemical sensors usually use nanomaterials derived from graphene, such as graphene oxide (GO) reduced graphene oxide (rGO), because its various functional groups makes it easier for affinity between linkers such as 1-pyrenebutyric acid (PBA) and 1-pyrenebutyric acid N-hydroxysuccinimide ester (PBASE) to occur [33]. This provides a significant advantage over biosensors based on field effect transistors, because the functionalization step on the graphene monolayer would no longer be carried out.

3 Carbon Nanomaterials in Sensing A biosensor is a sensitive device that has a biological element acting as the conformation component (enzymes, antibodies, nucleic acids, cells, tissues, microorganisms), allowing a biomolecular recognition reaction to exist [34]. This device allows the detection of biological molecules, whose output signal corresponds to the presence of a certain biomolecule, as well as the amount of this present in the sample [35]. Because of this biological element, the main characteristics of biosensors are their high selectivity, because they can react selectively to many analytes contained in a substrate, and their potential to reduce the size of the sensors, resulting from their spatial integration [36]. In addition, biosensors make it possible to carry out in situ measurements and as precise measurements on samples with a fast response speed and capable of working in real time [5, 37–39]. Biosensors can be classified by the recognition element (enzyme, organelle, whole tissue or cell, antibody, biological receptor, nucleic acids, aptamers, etc.), method and type of interaction (biocatalytic sensors, bio affinity sensors, direct or indirect detection) [40] and type of transducer employed (optical, electrochemical, piezoelectric, thermal, or nanomechanical). Electrochemical devices have the greatest application in the development of biosensors [39]. Voltammetry is the main method in electrochemical detection, and divided into: Square-wave voltammetry (SWV), differential pulse voltammetry (DPV), and electrochemical impedance spectrometry (EIS) [41–43].

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Advantages offered by an electrochemical device are the ease of converting and interpreting an electrical signal; high degree of sensitivity for the detection of samples (low amount of sample-analyte is required for the detection of a certain biomolecule, however, low selectivity compared to other types of devices) [8, 9]; on-site detection; and ease of operation and low cost compared to other methods such as polymerase chain reaction (PCR). One of the challenges concerning the construction and design of biosensors is the immobilization of the biomolecule, which must conserve its properties and biological activity, against interactions with other biological species. Good output signal, precision, reproducibility, and lifetime characteristics are also important for the identification of a biomolecule. The biosensor should also have a constant behavior, and it would be desirable if the biosensors could detect different analytes without the need to change the internal structure of the sensor; this could be achieved by directly integrating the signal transduction of the biomolecule [8, 44]. Moreover, the immobilization method of the biosensor is of paramount importance for the design and development of a biosensor since this allows the interactions to exist between the analyte and sensor for subsequent signal processing by the transducer through molecular recognition [45, 46]. For this, different immobilization techniques have been developed, and divided into two categories: Physical Immobilization and Chemical Immobilization [47]. To avoid unnecessary lengthening of this study, for a more detailed discussion on the functionalization methods of biosensors is recommended in the following bibliographic sources [7, 9, 18, 48–52]. Graphene and its oxidized derivatives, such as GO, have emerged as biosensor materials because of the presence of multiple oxygen functional groups (hydroxyl, carboxyl, and epoxy functional groups). Because of the presence of these functional groups, GO sheets are highly hydrophilic, allowing the incorporation of various types of inorganic nanoparticles, such as noble metals, metal oxides, semiconducting nanoparticles, quantum dots (QDs), and nanoclusters (NCs), to improve the performance of sensors based on them [53, 54]. Furthermore, the reduction of GO into reduced GO (rGO) generates a high density of defects, resulting in higher electrochemical activity than chemical vapor deposition (CVD)-grown graphene; this is especially beneficial for developing electrochemical biosensors. Graphene-based nanocomposites also have distinct morphological features and sensing characteristics [55]. Figure 3 shows a schematic illustration of the synthesis of chemically modified graphene.

4 COVID-19 In the last two years, the international scientific community has joined efforts to contain the advance of the COVID-19 pandemic, induced by SARS coronavirus 2 (SARS-CoV-2), that has infected more than 574 million people worldwide and caused 6,393,345 deaths, since December 2019. Until the development and start of vaccination, at the end of 2020, the main strategy used to contain new infections

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Fig. 3 Schematic representation of chemically modified graphene synthesis. Reprinted from Khalil et al. [56] with permission

was to perform serological tests or PCR to isolate the contaminated and reduce the contagion. The rapid spread of airborne contagious pathogenic viruses like SARS-CoV-2, as well as their severe negative effects on various aspects of human society, combined with significant limitations in traditional diagnostic platforms, raised the global demand for the development of precise, sensitive, and rapid point-of-care systems capable of detecting viral illnesses with almost no false-negative results. Although PCR is the standard method for confirming the presence of the virus, high demand, combined with operational complexity, and long analysis time, led to the search and development of faster and more accessible methods for performing mass testing of the population. Thus, several electrochemical methods for detecting both antibodies and antigens have been developed utilizing carbon nanomaterials as transducers.

4.1 CNMS for SARS-Cov-2 Antigen Detection Laser-scribed graphene (LSG)-based biosensing platforms have received significant attention lately as small electrochemical systems with immense promise as pointof-care (POC) diagnostic tools. Beduk et al. [57] provided a miniaturized LSGbased electrochemical sensing system for coronavirus disease 2019 (COVID-19) detection using 3D gold nanostructures (Fig. 4). The electrode was modified with the SARS-CoV-2 spike protein antibody by applying the correct surface modifications as demonstrated by X-ray photoelectron spectroscopy (XPS), and scanning electron

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Fig. 4 In a disposable electrochemical immunoassay, the production of laser-scribed graphene (LSG) and electrodeposited gold nanostructures (AuNSs) is shown. Reprinted from Beduk et al. [57] with permission

microscopy (SEM), and electrochemical methods. The system was incorporated into a portable POC detection device that was handled via a proprietary smartphone application, because of its simplicity of operation, accessibility, and systematic data storage, giving a user-friendly diagnostic platform. The analytical properties of the electrochemical immunoassay were evaluated using a standard solution of S-protein in the range of 5.0–500 ng mL−1 with a detection limit of 2.9 ng mL−1 . A clinical study was conducted on blood serum samples of 23 patients with a positive COVID-19 diagnosis, and the findings of commercial RT-PCR, antibody blood, and ELISA IgG, and IgA tests were compared. When compared to commercial diagnostic equipment, the presented device gives faster results and represents a potential alternative solution for next-generation POC applications. A photoelectrochemical aptasensor for quantifying the severe acute respiratory syndrome coronavirus-2 receptor-binding domain (SARS-CoV-2 RBD) was described for the first time by Tabrizi et al. [58]. First, cadmium sulfide (CdS) and graphitic carbon nitride (gC3 N4 ) quantum dots were produced and evaluated. Subsequently, gC3 N4 and CdS were thoroughly combined. SEM was used to characterize the nanomaterials that were created. The CdS QDs-gC3 N4 nanocomposite was then mixed with chitosan, an amine-rich polymer, to form a Chitosan/CdS-gC3 N4 nanocomposite and used to coat the ITO surface. Following that, glutaraldehyde was used as an amine–amine crosslinker to immobilize the amine-terminal aptamer probes on the surface of the Chitosan/CdS QDs-gC3 N4 /ITO electrode (Fig. 5). The performance of the electrode was investigated utilizing cyclic voltammetry (CV),

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(a)

(b)

Fig. 5 Schematic of the separation mechanism of photogenerated electron hole pairs between gC3 N4 and CdS QD on the proposed aptasensor before (a) and after (b) incubation of SARS-CoV-2 RBD with aptamer probes. Reprinted from Tabrizi et al. [58] with permission

EIS, and photo-electrochemistry (PEC). The surface coverage of the immobilized aptamer probe was determined to be 26.2 pmol.cm−2 . These findings demonstrated that the proposed photo-electrochemical aptasensor may be used to quantify SARSCoV-2 RBD within 0.5–32.0 10–9 mol L−1 . The LOD was determined to be 1.2 10–10 mol L−1 (at a 3σ/slope). The proposed photo-electrochemical aptasensor was used to assess spiking SARS-CoV-2 RBD in human saliva samples at two different concentration levels. Additionally, the aptasensor showed good selectivity in the presence of HIgG, HIgA, HIgM, and HSA. Mojsoska et al. [59] developed a unique proof-of-concept label-free electrochemical immunoassay for the rapid identification of the SARS-CoV-2 through the Spike

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Fig. 6 Illustration of the LIG-based electrode fabrication using the direct laser writing process on PI sheets. Reprinted from Oliveira et al. [60] with permission

protein. The sensor was created by coating graphene with PBASE, that can bind to antibodies against the SARS-CoV-2 Spike protein. The detection range of the spike protein was determined by measuring the absolute change in the [Fe(CN)6 ]3−/4− current as antigen concentrations on the immunosensor surface increased after 45 min of incubation time. The device was used to analyze SARS-CoV-2 at three different concentrations (34.38 × 103 , 13.75 × 104 , and 5.50 × 105 PFU mL−1 ), and was able to detect a specific signal of spike S1 proteins above 260 10–9 mol L−1 and SARS-CoV-2 at a concentration of 5.5 × 105 PFU mL−1 . Oliveira et al. [60] created a ‘Diagnostic-on-a-Chip’, a disposable electrochemical biosensor that incorporates laser-induced graphene and the specific antigen for SARS-CoV-2 as a platform for detecting SARS-CoV-2 antibodies, as shown in Fig. 6. Direct laser printing and CO2 infrared laser cutting were used to manufacture the biosensors on sheets of commercial polyimide. The effectiveness of clinical sample testing of patient serum was demonstrated by the production of distinctive and concentration-dependent electrochemical signals in the presence of specific antibodies reacting with the antigen and K3 [Fe(CN)6 ] redox probe. The mean current values were 9.68 and 8.18 μA for the reactive and non-reactive samples, respectively (from patients infected and uninfected with SARS-CoV-2). To confirm the applicability of the proposed device, the findings were compared with the ELISA method established by UFPel (patent deposit registration number BR1020210020105) and the qRT–PCR method. Additionally, the three-electrode disposable electrochemical

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Fig. 7 Schematic of the functioning concept of the COVID-19 electrochemical sensing platform. Samples will be taken from the patients’ nasal swabs or saliva, and the virus SARS-CoV-2 RNA (if present) will be isolated and added to the graphene-ssDNA-AuNP platform. The digital electrochemical output will be recorded after 5 min of incubation. Reprinted from Alafeef et al. [61] with permission

biosensor and portable potentiostat device demonstrated excellent detection adaptability for COVID-19 POC tests in a rapid and user-friendly manner, opening the possibility of expanding population access to quick and efficient diagnosis. Alafeef et al. [61] created a graphene-based biosensor to detect the SARS-CoV-2 N gene, in which ssDNA probes were constructed to target several locations simultaneously inside the same viral N protein; this improved electrochemical performance over individual ssDNA, as shown in Fig. 7. The sensitivity of the biosensor was further increased by using thiol-modified ssDNA-capped gold nanoparticles (AuNPs) on the surface of the Au electrode as opposed to ssDNA alone with no AuNP conjugation. Within 5 min of incubation, the sensitivity of the proposed method was 6.9 copies/μL with 231 (copies/μL)−1 . The reaction was verified further against RNA samples obtained from Vero cells infected with SARS-CoV-2, with MERS-CoV and SARSCoV RNA acting as negative controls. This sensor effectively discriminated positive COVID-19 samples from negative ones with 100% accuracy, specificity, and sensitivity, with no substantial change in the output response for samples missing a virus viral target segment. The proposed chip was tested using 48 clinical samples from 26 healthy, asymptomatic individuals and 22 patients who tested positive for SARSCoV-2 using an FDA-approved Au-standard SARS-CoV-2 detection kit (LabGun COVID-19 RT-PCR diagnostic kit). The proposed technique for detecting COVID19 was highly selective and sensitive, relying on digital monitoring of the electrochemical reaction produced by the graphene-ssDNA-AuNP surface. The benefits of the proposed biosensor over previously reported tests include a higher detection limit,

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no requirement for an extra redox medium for electron exchange, quicker response to produce reliable data, outstanding shelf life, and a feasible economic manufacturing [61]. Fan et al. [62] developed an entropy-driven amplified electrochemiluminescence (ECL) approach to detect the SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) gene known as RdRp-COVID, which as a target for SARS-CoV-2 plays an important role in the diagnosis of COVID-19. For the sensors, a DNA tetrahedron (DT) was modified on the electrode’s surface to provide stable and programmable scaffold materials. An efficient target DNA-participated entropy-driven amplified reaction is executed on top of the scaffold materials to link the [Ru(bpy)3 ]2+ modified S3 to the linear ssDNA at the vertex of the tetrahedron and ultimately create an “ECL on” state. The rigid tetrahedral structure of the DT probe improves test sensitivity by increasing the ECL intensity while preventing cross-reactivity between the single-stranded DNA. This enzyme-free entropy-driven process eliminates the need for expensive enzyme reagents and enables the large-scale screening of SARS-CoV2 patients. The DT-based ECL sensor exhibited great specificity and high sensitivity for SARS-CoV-2 with a limit of detection (LOD) of 2.67 × 10–15 mol L−1 . This operational approach also provides a reliable and useful sensing platform for clinical bioanalysis by effectively detecting RdRp-COVID in human serum samples [62]. Ang et al. [63] proposed that graphene oxide nanocolloids (GONCs) be used as electroactive nanocarbon materials that simultaneously act as a transducing platform and the electroactive label for the detection of SARS-CoV-2 genomic sequences. The capacity of GONC to provide an inherent electrochemical signal resulting from the reduction of the electrochemically reducible oxygen functions present on its surface enables it to be employed as a simple and sensitive biosensing platform. Varying intrinsic electroactivity of the material was produced at each phase of the genosensing procedure, commencing with the immobilization of a short-stranded DNA probe, and followed by incubation with varied concentrations of the target SARS-CoV-2 DNA strand. The target analyte could be measured over a broad dynamic range of 10–10 to 10–5 mol L−1 by monitoring these fluctuations in electroactivity. Furthermore, the suggested genosensor showed great selectivity for the proposed geno-assay with low interference for the SARS-CoV target, a virus from the same coronavirus family as SARS-CoV-2 [63]. Hashemi et al. [64] established a stepwise procedure for designing and producing label-free 1D/2D carbonaceous immuno-sensors loaded with monoclonal IgG antibodies against the S1 portion of the S spike glycoprotein of SARS-CoV-2 to detect the SARS-CoV-2 antigen in biological/non-biological fluids. To determine the optimal substrate for effective antibody loading, the activation and antibody mounting capabilities of 1D-MWCNT with outer diameters (OD) of 20–30 and 50–80 nm, respectively, as well as 2D GO and rGO carbonaceous nanomaterials were evaluated using electrochemical methods (Fig. 8). Furthermore, the impact of common amplification agents, such as Au nanostars (AuNSs) and Ag nanowires (AgNWs), on the overall performance of the preferred carbonaceous structure was carefully evaluated, and the relevant detecting mechanism thoroughly examined. The results demonstrated the superiority of rGO over other carbonaceous materials in terms of active surface area,

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Fig. 8 (a) EIS parameters of (I) MWCNTs with ODs of 20–30 nm and (II) MWCNTs with ODs of 50–80 nm before and after modification with NHS/EDC and NHS/EDC-AB complexes, (b) EIS parameters of (I) GO and (II) rGO before and after modification with NHS/EDC and NHS/EDC-AB complexes, and (c) antibody mounting capability of MWCNTs vs. graphene by-products. Reprinted from Hashemi et al. [64] with permission

electrical conductivity, and electrocatalytic activity. The rGO-based immunosensor showed antibody loading rate of 6.33 × 10−9 mol cm−2 , LOD of 0.001 fg mL−1 , and sensitivity of 59.28 μA (fg/mL)−1 cm−2 . In addition, the evaluation of the effect of AuNSs and AgNWs conjugations with the rGO-based immunosensor, revealed the potential of AuNSs for enhancing the LOD of the immunosensor and the negative influence of Ag NWs on both the sensitivity and LOD of the final platform. Compared with previous 1D/2D carbonaceous nanomaterials, the coupled rGO-based immunosensor with AuNSs exhibited higher performance toward specific and rapid (1 min) detection of SARS-CoV-2 antigen in biological fluids. Ali et al. [65] created a biosensor capable of detecting SARS-CoV-2 virus by nano-printing 3D electrodes coated with rGO nanoflakes, and specific viral antigens were fixed onto these rGO nanoflakes modified electrodes. In this system, electrodes were combined with a microfluidic device and used in a typical electrochemical cell. When the antibodies were applied to the surface of the electrode, they preferentially bonded with antigens, thus modifying the measured impedance. Antibodies of the spike S1 protein and RBD antigen of SARS-CoV-2 virus were then detected at LODs of 2.8 10−15 and 16.9 10−15 mol L−1 respectively. The biosensor can be refreshed by 1 min of washing with a solution of formic acid (1.0 mol L−1 , pH 2.5), eluting the antibody from the antigen and allowing up to ten reliable readings from the same sensor, without compromising selectivity [65]. Zhao et al. [66] demonstrated the ultrasensitive detection of RNA from SARSCoV-2 using a modified electrochemical method that did not require amplification or reverse transcription; this is a significant step toward reducing the time and cost

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(a)

(b)

Fig. 9 Schematic depiction of SARS-CoV-2 detection using an electrochemical biosensor. (a) Preparation of premix A and B; (b) electrochemical detection process utilizing a smartphone. Reprinted from Zhao et al. [66] with permission

for diagnosing viral infections with the potential for PoC-based SARS-CoV-2 detection (Fig. 9). The bioassay detects target RNA by surface functionalizing graphene oxide with calixarene. The super sandwich-type bioassay in question employs (a) Au@Fe3 O4 nanoparticles coupled with capture probes (CP) and (b) graphene functionalized with p-sulfocalix[8]arene (SCX8-RGO), that enhances electrochemical mediator toluidine blue (TB) for ultrasensitive RNA detection from SARS-CoV-2.

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Initially, the target RNA of the virus is captured by CP/Au@Fe3 O4 , which, when mixed with previously conjugated Au@CX8-RGO-TB with a label probe (LP) and an auxiliary probe (AP), yields a complex of CP-target-LP that allows ultrasensitive electrochemical detection of the virus. An examination of 88 RNA extracts from 25 virus-positive patients and 8 recovery individuals was executed, with detectable ratios of 85.5% and 46.2%, respectively, which was higher than that of RT-qPCR (56.5% and 7.7%). In clinical samples, each test utilizing this approach had a detection limit of approximately 200 copies mL−1 for SARS-CoV-2 [66]. Using a mass-produced laser-etched graphene electrode, Rodriguez et al. [67] developed the SARS-CoV-2 RapidPlex, a portable wireless electrochemical system for the ultra-fast analysis of COVID-19 that could detect IgM and IgG antibodies, viral antigen N protein, and the inflammatory biomarker C-reactive protein (Fig. 10). COVID-19 negative saliva and blood samples were effectively detected using the SARS-CoV-2 RapidPlex technology. This technology has the benefit of being simple to use for SARS-CoV-2 home testing as well as telemedicine diagnosis and monitoring.

(a)

(b)

(c)

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Fig. 10 (a) Diagram of the SARS-CoV-2 RapidPlex multisensor telemedicine platform for detecting viral proteins, antibodies (IgG and IgM), and the inflammatory biomarker C-reactive protein. Wireless data transmission to a mobile user interface is possible. WE: working electrode; CE: counter electrode; and RE: reference electrode. (b) Laser-engraved graphene sensor arrays that are mass produced. (c) Image of a disposable, flexible graphene array. (d) Illustration of a SARS-CoV-2 RapidPlex system with a graphene sensor array and a printed circuit board for signal processing and wireless communication. Reprinted from Rodriguez et al. [67] with permission

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4.2 Field Effect Transistors With low concentrations of target analyte molecules, FET-type sensors provide high sensitivity and fast readings. Recently, FET-based electrochemical biosensors for detecting viruses in human nasopharyngeal swap specimens were created. Seo et al. [68] presented a field-effect transistor (FET)-based biosensing device for detecting SARS-CoV-2 in clinical samples during the first months of the pandemic. The sensor was created by covering the FET graphene sheets with an antibody specific to the SARS-CoV-2 spike protein (Fig. 11). The performance of the sensor was evaluated using antigen protein, cultured virus, and nasopharyngeal swab samples from COVID-19 patients. The PBASE-honeycomb-like pyrene structure of the linker is systematically stacked onto the FET graphene surface, thus eliminating any interference with the electron flow between the source and gate electrodes, enhancing the ultra-sensitivity of the device. Virus detection is proportional to the change in current caused by the interaction between the of antibody and antigen on the graphene surface. The LOD of this FET biosensor for the spike protein was 1 and 100 fg mL−1 in the PBS and transport medium, respectively. This sensitivity is significantly lower than that indicated by a typical ELISA test. Furthermore, the LOD obtained using cultured SARS-CoV-2 virus was 16 PFU mL−1 (plaque forming units) in the growth medium and 242 copies mL−1 in the clinical sample, and the capacity to discriminate between SARS-CoV-2 and MERS-CoV antigens was exhibited [68]. Shao et al. [69] demonstrated the use of a high-purity semiconducting (sc) singlewalled carbon nanotube (SWCNT)-based field-effect transistor (FET) with a unique binding chemistry for detecting SARS-CoV-2 antigens in clinical nasopharyngeal

Fig. 11 Systematic depiction of the COVID-19 FET sensor operating process. Graphene is used as a sensing medium, and SARS-CoV-2 spike antibody is attached to the graphene sheet using the probe linker, 1-pyrenebutyric acid N-hydroxysuccinimide ester. Reprinted from Seo et al. [68] with permission

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Fig. 12 Detection of SARS-CoV-2 Ag using SWCNT-based FET biosensors. (a) Schematic structure of SARS-CoV-2 to demonstrate the targeting proteins. (b) Schematic representation of a liquidgated SWCNT FET for detecting SARS-CoV-2 SAg and NAg. Interdigitated gold electrodes are used as the source (So) and drain (Dr) (yellow blocks). Source-drain bias has a 50 mV VSD. To apply the gate voltage (Vg), a reference electrode made of Ag/AgCl is used. Insets demonstrate SAb- and NAb-functionalized SWCNTs for SAg and NAg detection, respectively. Reprinted from Shao et al. [69] with permission

samples, as shown in Fig. 12. The functionalization of the anti-SARS-CoV-2 spike protein (SAb) and anti-nucleocapsid protein antibodies, enables the SWCNT FET sensors to recognize the S (SAg) and N antigens (NAg), with a limit of detection of 0.55 and 0.016 fg mL−1 for SAg and NAg, respectively, in calibration samples. As a proof of concept for its application as a rapid COVID-19 antigen detection instrument with high analytical sensitivity and specificity at low cost, SAb-functionalized FET sensors also demonstrated superior sensing performance in differentiating between positive and negative clinical samples [69]. Piccinini et al. [70] presented the biofunctionalization of graphene field-effect transistors (GFETs) via a vinyl sulfonated-polyethyleneimine (VS-PEI) nanoscaffold for enhanced biosensing of SARS-CoV-2 spike protein and human ferritin, two critical targets for the rapid diagnosis and monitoring of COVID-19 patients. The heterobifunctional nanoscaffold enables the covalent immobilization of binding proteins and antifouling polymers, whereas the entire design is held together by multivalent π–π interactions with graphene. First, concanavalin A was used for glycoprotein detection to enhance the sensing platform. Thereafter, monoclonal antibodies specific to the SARS-CoV-2 spike protein and human ferritin were attached, resulting in biosensors with detection limits of 0.74 and 0.23 10–9 mol L−1 , respectively, and apparent affinity constants (K DG F E T ) of 6.7 and 8.8 10–9 mol L−1 , respectively. Both biosensing technologies exhibited high specificity, rapid reaction time, and large dynamic range (0.1–100 10–9 mol L−1 ). Furthermore, the SARS-CoV-2 spike protein was detected in spiked nasopharyngeal swab samples. The GFET response was compared to the surface plasmon resonance (SPR) measurements to thoroughly evaluate this biosensing technique, revealing linear correlations (2–100 ng cm−2 ) and good agreement in terms of K D values. Finally, the performance of the biosensors, created using the nanoscaffold procedure, was compared to that of the commonly used monopyrene approach, revealing improved sensitivity. The sensors recognized all

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Fig. 13 (a) Schematic illustration of SARS-CoV-2 with spike protein (S) composition, membrane glycoprotein (M), nucleocapsid protein (N), and envelope protein (E). (b) The LIG-FET fabrication technique. The device was passivated using SU-8 photoresist at the conclusion of the process to reduce leakage voltage and define the location of the liquid gate dielectric layer. (c) An optical picture of the LIG-FET detector array demonstrating its great uniformity and flexibility. (d) LIGFET image with the channel area patterned by an 840 mW laser and the source/drain region patterned by a 900 mW laser. Reprinted from Cui et al. [71] with permission

nasopharyngeal swab samples (from a healthy volunteer) spiked with the S1 SARSCoV-2 antigen as “positive test “, and no false-positive test results were obtained from the control samples lacking the SARS-CoV-2 S1 antigen [70]. Cui et al. [71] described a laser-induced graphene field-effect transistor (LIG-FET) for SARS-CoV-2 detection (Fig. 13). To enhance the binding site between the viral protein and sensing area, a porous graphene channel resembling an oyster reef was combined with various LIG reduction degrees to generate the FET. The sensor was able to detect the SARS-CoV-2 spike protein in the PBS solvent and real human serum with high sensitivity of up to 0.2 V dec−1 and an LOD of 1 pg mL−1 . Additionally, there was no discernible cross-reactivity with the nucleocapsid protein antigen. The suggested sensors enable the rapid production of COVID-19 rapid testing because each LIG-FET can be created using a laser platform in a matter of seconds. The first viral sensing FET without sample pretreatment or labeling was created by LIG, paving the way for speedy and inexpensive COVID-19 detection.

5 Conclusion RT-PCR assays are widely accepted because they offer numerous advantages, making them potentially life-saving diagnostic methods. However, these methods might not be appropriate for large-scale monitoring of numerous samples because of their high

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cost, time-consuming procedures, several processes, and demand for highly experienced employees. The great versatility exhibited by carbon nanomaterials allows them to be applied in numerous ways, enabling the construction of different electrochemical sensors. The emergence and subsequent exacerbating of the pandemic has caused an increase in demand for rapid and reliable testing for the early detection of SARS-COV-2. Recently, quick, affordable, sensitive, and selective electrochemical sensor-based methods for SARS-CoV-2 detection have been established. Although neither the electrochemical detection of SARS-CoV-2 sensors/biosensors nor the serological and RT-PCR assays are the best techniques for COVID-19 identification, they might be seen as complementary. There is a lot of hope that electrochemical sensors/biosensors can provide better solutions for rapid and low-cost point-of-care diagnostics for the deadly SARS-CoV-2 virus. Additionally, the technology developed and used to combat SARS-COV-2 can be adapted to deal with other infectious outbreaks that may occur in the future.

Abbreviations AgNWs AUC AuNPs AuNS CdS-QDs CNM CV CVD DNA DPV DWCNT EIS ELISA FDA FET gC3 N4 GFET GO GONC (H)IgG (H)IgM HSA ITO IUPAC LIG LOD

Silver nanowires Area under the ROC Curve Gold nanoparticles Gold nanostars Cadmium sulfide quantum dots Carbon nanomaterials Cyclic voltammetry Chemical vapor deposition Deoxyribonucleic acid Differential pulse voltammetry Double-walled carbon nanotube Electrochemical impedance spectroscopy Enzyme linked immunosorbent assay United States Food and Drugs Administration Field effect transistors Graphitic carbon nitride Graphene-field effect transistors Graphene oxide Graphene oxide nanocolloids Human immunoglobulin G Human immunoglobulin M Human serum albumin Indium tin oxide International Union of Pure and Applied Chemistry Laser-induced graphene Limit of detection

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LOQ MERS-CoV MWCNT PBA PBASE PFU PoC RBD RdRp rGO RNA ROC RT-PCR S1 SAb SARS-CoV-2 SPR ssDNA SWCNT VS-PEI

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Limit of quantification Middle East respiratory syndrome-related coronavirus Multiwalled carbon nanotubes Pyrene butyric acid 1-Pyrene butyric acid N-hydroxy succinimide ester Plaque forming units Point-of-care Receptor-binding domain RNA-dependent RNA polymerase Reduced graphene oxide Ribonucleic acid Receiver operating characteristic curve Real-time polymerase chain reaction Spike Protein subunit 1 Anti-spike antibody Severe acute respiratory syndrome coronavirus 2 Surface plasmon resonance Single strand deoxyribonucleic acid Single walled carbon nanotubes Vinyl sulfonated-polyethyleneimine

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Use of Metallic Nanostructures in Electrochemical Biosensing of SARS-CoV-2 Luiz Otávio Orzari, Jéssica Rocha Camargo, Rodrigo Vieira Blasques, Luiz Humberto Marcolino-Junior, Marcio Bergamini, and Bruno Campos Janegitz

1 Introduction Accompanying the rapid technological growth of the modern information era, worldwide scientists are constantly in the search for novel, fast, and more effective ways of detecting both common and uncommon diseases, especially with an early diagnosis, when the possibility emerges [1–3]. Among these technologies, electrochemical sensors and biosensors are advantageous devices, encompassing different fields of science, such as chemistry, biotechnology, and material science. These are commonly referred to as having a considerably faster response and simpler operation while maintaining reliable outcomes [4–6]. Electrochemical sensors are based on the translation of different variables resulting from a redox reaction occurring on the device surface, being it a voltammetric (electric current generation) [7, 8], potentiometric (different potentials shifts and other interactions) [9, 10] or impedimetric signal (resistance and capacitance terms of electrochemical impedance correlations) [11, 12]. Biosensors of this class operates in similar manners, but with the addition of a transducer in the system architecture, that is responsible for converting a biological process, such as enzyme activity or antigen–antibody interaction, into one of the previously mentioned signals.

L. O. Orzari · J. R. Camargo · R. V. Blasques · B. C. Janegitz (B) Department of Nature Sciences, Mathematics and Education, Federal University of São Carlos, Araras, São Paulo 13600-970, Brazil e-mail: [email protected] L. O. Orzari · J. R. Camargo · R. V. Blasques Department of Physics, Chemistry and Mathematics, Federal University of São Carlos, Sorocaba, São Paulo 18052-780, Brazil L. H. Marcolino-Junior · M. Bergamini Laboratory of Electrochemical Sensors (LabSensE), Department of Chemistry, Federal University of Paraná, Curitiba, Paraná 81531-990, Brazil © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 F. N. Crespilho (ed.), Covid-19 Metabolomics and Diagnosis, https://doi.org/10.1007/978-3-031-27922-5_4

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Among already published works (Fig. 1) it is possible to note a considerable growth of electrochemical biosensors over the past 12 years, more than doubling the number of publications per year nowadays, with a substantial amount of new scientific papers being presented. Data was collected with the keywords Electrochemical biosensor with and without SARS-CoV-2, year by year, in the Web of Science database, April 2022. In the year 2020, there was an expected decrease in research being done, as the world was assailed by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), causing the World Health Organization to declare a pandemic state [13, 14]. This virus presents a quite fast human-to-human transmission and an estimated basic reproduction number of 2.2 [15]. This virus-provoked state has promoted the alteration of human behavior throughout the globe, favoring more healthy activities and increasing the use of transmission prevention methods. However, the spread of the virus was and still is considerably difficult to contain, leading to the development of technologies for its detection, preferably with rapid diagnosis, since the viral attack occurs in little more than a week. Therefore, it is of little surprise that electrochemical biosensors (Fig. 1 inset) for the determination of SARS-CoV-2 have emerged in the years following the outbreak of this danger. In the year 2021, nearly 3% of all electrochemical biosensors published were about the determination of this virus, which is an impressive feat for humankind, considering the range of diseases that can be investigated by electroanalytical techniques and the small-time invested to uncover a feasible device. By the start of this very year (2022),

Fig. 1 Publications from the last 12 years in the electroanalytical field. Keywords: Electrochemical biosensor, SARS-CoV-2. Data from the Web of Science database

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the scientific community has already surpassed the number of papers published, in this same regard, in 2019. Among these SARS-CoV-2 electrochemical biosensors, some materials were more present than others in the literature, with emphasis on metallic nanoparticles. These nanoparticles differ not only in size but also in the properties they have [16]. Currently, nanoparticles are widely used for the development of nanotechnology, with potential application in several areas [17]. In electroanalysis, there are two most common ways of metallic nanoclusters synthesis, both from a bottom-up approach: the first are the wet chemical methods: the reduction of metal salts in solution, resulting in the aggregation of metal atoms in condition defining shaped colloids, that are suspended in the solution, and can be further extracted [16, 18]. As examples, the casting [19] and Langmuir–Blodgett techniques can be employed to generate film layers over the desired surface with such suspensions [20]. These methods can also present precursor particle seeds, which is a small cluster that will grow inside the solution. If a seed is present, the reduction of the metal atoms will occur over its surface, increasing the size of the seed [16]. Without it, seeds will be generated on higher-energy surfaces of the system. Fu et al. [21] demonstrated the synthesis of a heterogeneous nanoshell of Au and Pt, with the addition of trisodium citrate to a HAuCl4 ·3H2 O, resulting in an immediate reduction of Au atoms and formation of clusters. The solution was separated by centrifugation and the supernatant was added to the H2 PtCl6 ·6H2 O solution, which was further reduced by ascorbic acid. This second reduction provoked the Pt atoms to reduce over the Au cluster surface, creating a particle with enhanced peroxidase-like behavior for SARS-CoV-2 colorimetric detection. The second consists of the electrodeposition of metal atoms over the electrode surface, by the application of reduction-inducing potential energy (or range of). This causes the metal atoms to adsorb inactive surface sites, such as defects [18, 22]. For instance, Rafatmah and Hemmateenejad [23] explored the electrodeposition of Au nanoparticles (AuNPs) over paper fibers surface in 1.0 mol L−1 HClO4 solution. The paper data reveals that both the potentials explored (− 0.08 and − 0.2 V) created dendrite-like particles over the fibers when in presence of 1.0 mmol L−1 HAuCl4 , while a higher concentration of the salt (4.0 mmol L−1 ) resulted in the production of more sphere-like particles. Both methods depend on the cluster growth, controlled by several different properties, such as the diffusion of the metals in solution, the capping agents added, and the nature of the metals themselves. All these can cause different final structures to be produced, with various shapes and electronic behaviors [18, 24]. As an example, Qin et al. [25] highlighted the effects of ascorbic acid pH on the Ag particles synthesis. The group studied the procedure in a pH range from 6.0 to 10.5 and highlighted that the lower the pH, the bigger and less active the Ag nanoparticle was (Fig. 2). For all these factors, the methodologies for their synthesis can result in different behaviors that can be implemented for the development of biosensors. Several metallic particles present interesting properties of binding to biomolecules, such as antibodies and enzymes [26]. Also, they can be used as catalysts and amplifiers for the signals of interest [27]. The most used metals for the development of

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Fig. 2 Transmission electron microscopy images of prepared AgNPs at pH 6.0, 7.0, 8.0, 9.0, 10.0 and 10.5 by using ascorbate as reductant. Reprinted from Qin et al. [25], with permission of Elsevier

nanoparticles are Au [14], Ag [28], Cu [29], Fe [30], Pt [21] and Pd [31]. In this context, the most widely used for making biosensors for SARS-CoV-2 are AuNPs that present several synthesis methodologies, and are commonly stable in colloidal form. These nanostructures have been extensively researched in the literature. This consistent use of metals can be associated with the biocompatibility and toxicity that these type of nanoparticle shows. This behavior emerges from different properties between the particles and biologically produced structures, such as immune cells, proteins, and enzymes. According to Yoshioka et al. [32], the toxic or non-toxic effect of a nanoparticle can be observed by the length of exposition time, the particle concentration and the effects generated by these strange bodies in the organism. These effects are controlled by different variables, such as particle shape and size distribution, surface charge and activity, as well as agglomeration state and purity. This topic reveals several binding mechanisms of metallic nanoparticles to biological products that can work as transducers for electrochemical biosensors. For instance, Tenzer et al. [33] demonstrated that 30 s after nanoparticles enters the blood plasma, a protein corona is formed by coordination around the particle, demonstrating the stability of such system. Therefore, this chapter aims to highlight the most recent studies of SARS-CoV-2 electrochemical biosensors, that also employed metallic nanoparticles, to elucidate the topic and inform research paths for fellow readers and scientists.

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2 Electrochemical Determination of SARS-CoV-2 with Metallic Nanostructures The rapid and reliable detection of SARS-CoV-2 in humans is necessary for adequate control of the infection since it has high levels of contagion [34]. In this sense, many studies have focused on the development of more sensitive devices, with reduced detection limits, to minimize erroneous results [35]. As already mentioned, among the biosensors that use metals for the construction of structures, that proposes the detection of SARS-CoV-2, Au-based ones are the most explored in the literature. The selection of suitable sensory surfaces, which allows the integration of metallic nanostructures, in line with the use of different biomolecules immobilization strategies, is essential since this integration will play an important role in the development and performance of electrochemical biosensors [36]. Thus, biosensors based on metallic nanostructures can be promising alternatives, as these can promote catalytic activities, contain a better surface area, greater electrical conductivity, and electrochemical activity [37–39]. In addition, various types of molecules can be used to detect viral agents in biological samples, for example, a specific viral protein, antibodies, viral nucleic acid, or some specific biomarkers [40]. Thus, to design sensory systems that link the metal particles and their properties with covid-19 related biomolecules, various immobilization mechanisms have been employed in electrochemical biosensors, such as direct adsorption- or covalent binding-based methods [41]. Knowing that rapid and accurate detection is very important for the control of the pandemic, also to detecting SARS-CoV-2, the biosensors must be selective, like other viral diseases such as influenza A, MERS-CoV and Streptococcus pneumoniae. These illnesses present very similar symptoms, requiring tests to define the disease. In this way, the work of Karaku¸s et al. demonstrates a probe (AuNP-mAb) that exhibits a dual-sensing mode for the detection of SARS-CoV-2 spike antigen (S–Ag). The electrochemical detection was achieved by the probe solution developed on the commercially available and disposable screen-printed Au electrode without requiring any electrode preparation and modification. The AuNP-mAb was prepared following the procedure in (Fig. 3). The AuNPs-mAb conjugate was prepared by adding SARSCoV-2 spike antibodies and AuNPs activated with 11-mercaptoundecanoic acid. After incubation time, the mAb (SARS-CoV-2 spike monoclonal antibody) reacted with AuNPs by covalent bond formation via EDC-NHS cross-linking agents. The developed system has been successfully applied to saliva samples and also offers simplicity, cost-effectiveness, and speed. Lastly, this system did not exhibit crossreactivity with other viral proteins (i.e. influenza A, MERS-CoV, and Streptococcus pneumoniae). According to the authors, the created AuNP–mAb conjugates, in the presence of the SARS-CoV-2 peak antigen present the colorimetric effect. This interaction causes the AuNPs to aggregate quickly and irreversibly, by antibody-antigen interaction, changing the color from red to purple, which is possible to observe even with the naked eye.

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Fig. 3 Schematic illustration of the formation of AuNP–mAb conjugates. Reprinted from Karaku¸s et al. [14], with permission of Elsevier

One of the mechanisms of SARS-CoV-2 in the human body is the interaction with the cell surface angiotensin-converting enzyme 2 (ACE2), via spike protein, severely affecting the II alveolar cells [42]. Such fact implies that this interaction could be an immune system stimulator, and the work of Kiew et al. [31] aimed to explore it further. The researchers developed electrochemical impedance spectroscopy (EIS) based device, consisting of the interaction of a recombinant ACE2 and a Pd thin film over the electrode surface. The electrode was immersed in a [Fe(CN6 )]3− rich electrolyte solution (Fig. 4) and following the SARS-CoV-2 spike protein and ACE2 interaction, the technique revealed alterations in the impedance behavior of the system, aided by the production of [Fe(CN6 )]4− at the electrode surface. The authors also reported several in vitro screening tests of different pharmaceutical drugs, which suppress the SARS-CoV-2-ACE2 interaction, such as ramipril and perindoprilat. In this work, Pd nano film was covalently bonded to ACE2, with direct interactions with S atom. This mechanism normally occurs with 11 group metals [43, 44] and is a commonly employed strategy in the field, being one of the main reasons Au is widely used in biosensors. In addition to the general detection of the virus, it is interesting to verify the stage of the disease. This can be essential to determine the time that the patient must remain out of society so that this virus is no longer transmitted. In this way, Avelino et al. [45] developed a structure based on electrodes of tin-doped indium oxide modified with polypyrrole and AuNPs. The assay was evaluated through biodetection using recombinant plasmids containing the nucleocapsid protein gene of SARS-CoV-2. The biological part has been added using cysteamine, glutaraldehyde, and BSA, as a covalent bonding method (exploring the previously described Au–S interaction) as observed in Fig. 5. The tests were performed using cyclic voltammetry and EIS. Also, interfering molecules (glucose, glycine, ascorbic acid, and cholesterol) were added to evaluate the selectivity of the biosensor. The authors reported that the results suggest that the platform can differ the data obtained between analyte and contaminants, allowing the application of the biosensor in clinical trials. Despite being a promising alternative for the detection of COVID-19 cases, this device has not been tested on samples without pre-treatments.

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Fig. 4 Schematic preparation of the EIS-based biosensing platform with ACE2-Pd-NTF electrode as biosensing probe against SARS-CoV-2’s S-protein. Reprinted from Kiew et al. [31], with permission of Elsevier

Fig. 5 Schematic representation of the assembly principle of the COVID-19 electrochemical sensing platform. Reprinted from Avelino et al. [45], with permission of Elsevier

3 Conclusions and Perspectives The development of new device architectures for SARS-CoV-2 biosensing is of paramount importance, especially sensors that aim a rapid and reliable detection and overcome the high cost and long processing time problems of conventional methods of analysis. In this way, the use of metallic nanostructures is a fundamental tool

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to increase the electrochemical signals and aid immobilization of DNAs, proteins, enzymes, and SARS-CoV-2 related aptamers, supporting the production of a new generation of electrochemical devices. Electroanalytical technologies, coupled with the use of metallic nanostructures, bring several advantages in the development of biosensors, such as large-scale production and cost and time reduction. In addition, with the advancement of disease control technologies, the use of SARS-CoV-2 biomolecules correlated strategies applied to the development of new electrochemical systems, promote the creation of robust devices that can be used quickly and accurately as needed. Given the aspects discussed in this book chapter, biosensors fabricated with metallic nanostructure are interesting alternatives for the detection and monitoring of SARS-CoV-2. In the next years, there is a demand for new designs and fabrication methods, facilitating the handling and employability of these devices. Due to these factors, the authors believe that in the coming years, great efforts will be made for the research around the detection and viruses and their mutations, including the development of new sensors and biosensors. In this scenario, there is an expectation of worldwide governments and researchers to increase investments of time and money, making these devices more accessible to the population. Acknowledgements The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES, financial code 001 and CAPES 09/2020 Epidemias 88887.504861/2020-00), and Fundação de Amparo à Pesquisa do Estado de São Paulo, (FAPESP, 2019/23342-0, 2019/23177-0, 2017/21097-3) for the financial support.

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3D Printing for Virus Diagnosis Jéssica S. Stefano, Luiz Ricardo G. Silva, Vinicius A. O. P. Silva, Marcio F. Bergamini, Luiz H. Marcolino-Junior, Juliano A. Bonacin, and Bruno C. Janegitz

1 Viral Disease and Pandemics: A Brief Contextualization A viral disease is a health condition or illness caused by a virus. Viruses are infectious agents with diameters varying from 16 to 300 nm, composed of infectious particles containing proteins and, in some cases, surrounded by a lipid membrane (the envelope). These infectious particles, or virions, contain only one type of nucleic acid, which can be either DNA or RNA [1]. Viruses are capable of replicating and spreading the virions by the encoding of highly efficient proteins, optimizing the replication process, and initiating an infectious process [2]. RNA viruses are more pathogenic, being rapidly adapted to environment changes, in addition, mutations in the genetic material of RNA viruses can occur, giving rise to hybrid viruses different from the parental ones [3]. Many of the most well-known diseases are caused by viruses, including Ebola, HIV/AIDS, influenza, and, more recently, COVID-19 [4]. Acute respiratory virus infections are highly contagious diseases that cause human mortality and morbidities, being responsible for, together, causing more deaths than any other type of infectious disease [5, 6]. Respiratory viruses affect the respiratory tract and are transmitted direct (physical contact) or indirectly, by contact with respiratory secretions (droplets and aerosols) of an infected person and surfaces, thus they are of easy spread and transmission, presenting a high potential to cause global pandemics [5, 7]. The high J. S. Stefano · L. R. G. Silva · V. A. O. P. Silva · B. C. Janegitz (B) Department of Nature Sciences, Mathematics and Education, Federal University of São Carlos, Araras, São Paulo 13600-970, Brazil e-mail: [email protected] M. F. Bergamini · L. H. Marcolino-Junior Chemistry Department, Federal University of Paraná, Curitiba, Paraná 81531-980, Brazil J. A. Bonacin Institute of Chemistry, University of Campinas (UNICAMP), Campinas, São Paulo 13083-859, Brazil © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 F. N. Crespilho (ed.), Covid-19 Metabolomics and Diagnosis, https://doi.org/10.1007/978-3-031-27922-5_5

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mutability potential of respiratory viruses hinders the obtention of effective vaccines, which can become a huge global problem in the control of a breakdown, affecting societies and economies, as witnessed in the last years with the emergence of the new coronavirus [6]. In December 2019, the emergence of a new virus caused by severe acute respiratory syndrome coronavirus 2, the SARS-CoV-2, was reported. This virus, which causes COVID-19 disease, proved to be highly contagious and rapid spreading, thus reaching a global pandemic outbreak [8]. Several cases have been reported until the moment, and the number is still increasing. After 2 years, around 6 million deaths have been reported, with a total number of infected people reaching 480 million [9]. The SARS-CoV-2 has shocked the world, however, it is noteworthy to mention that this is not an isolated case. Since the beginning of human history, respiratory viral infections have been a problem [3, 10]. Several cases of epidemic and pandemic situations have been witnessed by humankind, including the Spanish influenza H1N1 (1918–1919) which caused around 50 million deaths, Hong Kong influenza H3N2 (around 1 million deaths, from 1968 to 1970), Asian influenza H2N2 (1957–1959, causing 2 million deaths), Russian influenza H3N8 (1889–1893, 1 million deaths), among others [11]. Many of them were caused by pathogens transmitted to humans by contact with animals, and disseminated globally, demanding some precautions to avoid or control spread. In this aspect, quarantine, isolation, and traveling control are very common measures, besides, the use of masks and social distancing are important measures when facing respiratory viral infections. In addition, the detection of infections in the early stages is of great importance for the control of circulating people, helping to avoid propagation [11]. In this scenario, innovative and reliable tools for the diagnosis of viral diseases are of paramount importance. Given the low cost, simplicity, miniaturization, and reliability of electroanalytical systems, the electrochemical technique plays an important role in the development of biosensors. Different types of electrochemical biosensors for the detection of viruses have been reported in the literature [4], and the search for new platforms is very attractive in the development of point-of-care analytical systems. The use of 3D technology is on the rise for the development of electrochemical systems and has been playing an important role in the combat of the COVID-19 pandemic. In the literature, many approaches have been exploited using 3D printing, either providing protective materials, or testing platforms produced on large scale by this technology. These approaches are discussed in the next sections, after an introduction regarding the main 3D printing techniques employed in the fight against pandemics, and their use as sensing platforms.

2 3D Printing Technology 3D printing, also known as additive manufacturing (AM), is a technology capable to provide three-dimensional physical objects in an automated layer-based process. The objects are obtained from a model generated using CAD (Computer-Aided Design)

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software. The technology of 3D printing allows the operator to obtain complex 3D objects with high versatility regarding material employed, and design [12, 13]. The first approaches of AM were provided in the market in 1987, labeled as rapid prototyping [12]. Since its creation, 3D printing has presented a varied range of applications, being employed in industries of automobile, aerospace, clothing, pharmaceutical, and biomedical [14]. Recently, this technology has been successfully applied in chemistry research areas, as in the fabrication of a great diversity of analytical systems [15]. The versatility of this technique could be exemplified with the emergency of the COVID-19 pandemic, in which 3D printing has attracted great attention due to the great range of applications in the fight against the virus, from the obtention of materials and equipment for personal protection to the development of analytical systems or parts of it. This wide range of applications in 3D printing stems from its versatility and its seven different categories of printing: material extrusion, material jetting, binder jetting, sheet lamination, vat photopolymerization, powder bed fusion, and directed energy dispersion [16], in which each category comprehends different techniques. Some of them are more employed for application in the combat of COVID-19, notably Fused deposition modeling (FDM; a technique of material extrusion), stereolithography (SLA; a technique of vat photopolymerization) and selective laser sintering (SLS; a technique of powder bed fusion). FDM printing consists of melting a filament stored next to the printer and releasing this material by extrusion under a construction table, where the object will be created layer by layer (Fig. 1). After the printing is finished, the table undergoes cooling, making the printed material dilate to facilitate its removal. Several materials can be used as filaments for this type of printing, the most common are biodegradable polymers such as polylactic acid or petroleum derivatives such as acrylonitrile butadiene styrene. Also, many variations of filaments use materials such as steel, copper, superalloys, and carbon nanomaterials. FDM printers are the best known and used because of their relatively low cost, diversity of materials to be employed, and easy operation [17, 18]. Entry-level printers to get started with FDM 3D printing can be found on the market for around $ 300.00. SLA 3D printing is a technique based on the use of laser light, responsible for curing a liquid resin point by point. This type of printer consists of a base with a set of mirrors and a laser, with a top made of glass allowing the light path to reach the resin placed on top of the glass in a container with a transparent and flexible base. The printer has a downward-facing printing base, which at the beginning of printing is dipped in resin and contact with the bottom of the conditioning container (Fig. 2a) [20]. When the first layer of printing is obtained, the cured resin adheres to the printing table, which is then lifted, and the next layers have adhered to the already cured resin. After the printing is finished, the object needs to be washed to remove residues of liquid resin remain on the surface, preventing them from being cured with the light emitted by the environment. After washing, the pieces are exposed to UV light so that the curing process of the object is completed [21]. SLA printers using current technologies can be found with prices approaching $3499.00.

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Fig. 1 Representative scheme of FDM 3D printer, adapted from [19], with permission of MDPI

Fig. 2 Representative scheme of a SLA 3D printer and b DLP 3D printer, adapted from [19], with permission of MDPI

Another type of photopolymerization 3D printing is the Digital Light Processing (DLP), presented in Fig. 2b. This printing technique is performed similarly to SLA. However, instead of using a laser, DLP 3D printers relies on the use of a projector containing a powerful LED-emitting diode. It produces a blue light with a narrow wavelength range, and optical assembly is responsible to scatter this light [20]. This

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Fig. 3 Representative scheme of SLS 3D printer, adapted from Ref. [19], with permission of MDPI

device, called micro-mirror, is able to create an appropriate light pattern. Several tiny mirrors reflect and focuses the ultraviolet on light, passing through the tray and curing the resin. This process allows the curing to be carried out layer by layer and no longer point to point as in SLA printers [20, 22]. 3D printers based on the SLS technique are composed of three container tanks placed side by side, which will be used for supply, construction, and excess disposal of the powder material, used as the material base for the obtention of the object (Fig. 3). The supply and construction tanks have a mobile table controlled by a gas piston, and at the formation of each layer, the supply table is raised, and the construction table is lowered, in the same proportion. The printer also has a roller located above the tanks, which will be responsible for carrying the material from one container to another, taking the material from the supply compartment to the construction one, and all the surplus to the third tank. Previous to each layer, more powder is distributed over the bed [23]. The print is performed using a laser that directs its high-power light beam to a reflective mirror, which, with the aid of a motorized system, directs the focused light beam to the powder, in the area corresponding to that layer. The material is heated by the light beam to a temperature above its glass transition point, close to its liquid temperature, which when cooled selectively solidifies again in the desired shape, layer by layer [24]. This type of 3D printing is not often used due to its high cost, where the most basic models cost around $10,000.00.

3 3D Printed as a New Tool in the Combat of Viral Diseases During the ongoing COVID-19 outbreak, a great demand for essential medical equipment was created. The unexpected pandemic of SARS-CoV-2 surprised the globe and thus, no resources to deal with the situation were available. The need for various

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essential medical equipment was increasing, including special personal protective equipment (PPE), exposing both patients and caregivers to the disease [25]. Given the problematic situation, many institutions, including companies, universities, and even individuals started to produce protective items, such as face masks, valves, test swabs, and detection devices, and the use of 3D printing for this was essential [26]. In this section, the role of 3D printing in the construction of tools in the fight against COVID-19 is reported.

3.1 3D Printed Accessories It is known that the SARS-CoV-2 virus can contaminate the air and surfaces through droplet transmission, and the virus can remain on contaminated surfaces for up to 72 h. In this case, physical barriers are a very effective measure, which can include facial masks, face shields, and goggles [26]. One of the main concerns was with health professionals, who needed to have direct contact with other people, either contaminated or not with COVID-19. From March 2020, there was a worldwide shortage of PPE for health professionals such as doctors and dentists, this shortage occurred due to the greater demand for these products, in addition to the decrease in the productive capacity of industries, and lack of raw material. Given this scenario, 3D printing played an important role in the fabrication of PPEs. Around the world, companies and people who owned 3D printers mobilized in the manufacture of face shields, goggles, ventilator assembly parts, and nasal swabs. This movement took global proportions, as countries with greater production capacity for 3D printing of PPEs started to help other countries that also suffered from shortages without the possibility of expanding production [27]. Wesemann and co-authors [27] developed four types of face shields using FDM 3D printing technology with Green-TEC PRO Black filaments. Figure 4 presents four different 3D printed face shield models and their use in practice. For clinical suitability, the proposed designs comply with sufficient space for eyewear and protective mask. To validate the best face shield model, the weight, printing time, and tools needed for assembly were evaluated. Subsequently, the clinical evaluation of the face shields was performed, after their use by 10 health professionals for 1 h. Filament weight (21–42 g) and print time (1:40–3:17 h) varied for the four proposed designs. According to the authors, the design presented in “D” (Fig. 4) showed the best characteristics, however, in terms of scalable production, the design proposed in “B” was more efficient. Furthermore, it is worth mentioning that all designs successfully demonstrated the ability of 3D printing to automatically produce face shields, capable of being used in centers to combat the pandemic. In addition to the manufacturing of face shields, the use of 3D printing has also been reported for the manufacture of face masks. Swennen et al. [28] developed a 3D-printed individual protective face mask in conjunction with a filter membrane (FFP2/3), based on reusable polyamide (PA11-SX 14) employing the SLS printing technique. Figure 5 presents the parts involved in the construction of the protective

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Fig. 4 Different 3D printed face shield models, proposed to be applied by health professionals for protection against COVID-19 during their activities, reprinted from Ref. [27], with permission of MDPI

masks, as well as an illustration of the design used in the printing. The prototype consists of a face mask and a filtering membrane holder and two disposable components, a headband, and a filtering membrane. According to the authors, 3D printing of individualized 3D face masks in combination with FFP2/3 filter membranes is feasible and could be a valid alternative resource for shortages of personal protective equipment during the fight against the COVID-19 pandemic. Also, the production of goggles for protection has been reported, as well as the use of 3D printed structures for the composition of ventilator parts, such as valves, among other applications that significantly contributed as forms to deal safely with the pandemic [25, 26]. Furthermore, the printing of accessories was not the only way found to deal with the pandemic. The next sections will report the use of 3D printing to aid in the development of tests and its applications against COVID-19.

3.2 3D Printing of Nasopharyngeal Swabs One of the most important strategies in the fight against COVID-19 is to obtain an increased level of testing, playing a vital role in the detection of infected people, and enabling the appropriate decision making to mitigate the disease, either by isolating the infected person or tracking the possible contacts, avoiding the spread [14]. As COVID-19 is a respiratory disease, in general, respiratory secretions are used as the

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Fig. 5 3D printed individual protective face mask developed for use in conjunction with FFP2/3 filter membrane, reprinted from Ref. [28], with permission of Elsevier

sample. The reference method for SARS-CoV-2 detection is the reverse transcriptasepolymerase chain reaction (RT-PCR), which relies on the use of nasopharyngeal (NP) swabs for sample collection [29]. NP swabs are used to collect nasal and pharyngeal epithelial mucosa secretions for testing, this material is based on small and flexible rods with 15 cm length and a tip of 0.35–0.40 cm diameter. They are inserted into the nose until reaching the back of the nasopharyngeal cavity, and rotated, keeping the biomaterial adhered for further analysis [25, 30]. With the increase in the number of infected people, a highly increased demand for NP swabs was faced during the ongoing pandemic, making this material urgently required equipment [31]. The capacity of the 3D printing technology to produce objects on large scale, besides its simplicity and versatility in prototyping, makes this technique an ideal tool in the obtention of NPs and has been explored for this aim. A great advantage of using 3D printing for the obtention of NPs is the decentralization of the manufacturing process, which drastically simplifies the

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transportation, since a simple digital file can be sent by the internet, reaching instantly the user that can print the object with low cost [30]. Studies regarding the 3D printing of NP swabs can easily be found in the literature, providing different ways for the obtention of this equipment in an optimized way. In this aspect, Arjunan and coauthors [30] proposed the obtention of an innovative framework of NP swabs via SLA 3D printing technique using a biocompatible resin FLSGAM01. For this, many tip structures (Fig. 6) were obtained and evaluated regarding tensile and resistance tests. The obtained swabs were able to shrink under axial resistance, allowing a sample collection with less stress in the nasal cavity tissue, being a great option for nasopharyngeal tests.

Fig. 6 Different designs evaluated for the obtention of sample swab heads using SLM 3D printing, reprinted from Ref. [30], with the permission of Elsevier

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Fig. 7 Image of SLA 3D printed NP swabs (left) and alternative NP swab designs for 3D printing (right), reprinted from Ref. [32], with permission of BMC

Given the race for containing the pandemic, and help to gather important equipment, many groups have been pooling their efforts. As reported by Ford et al. [32], the teams from USF Health Radiology and Northwell Health System looked for an alternative to production through 3D printing. The authors aimed to provide impact for 3D printing labs in hospitals to leverage efficient NP swab designs during the pandemic by giving a complete and descriptive workflow study, including guide information for 3D printing labs. For this, they prototyped over 20 different swab tip designs for printing utilizing the SLA technique. Figure 7 presents the scalable obtention of the NP swabs, as well as five different swab tip models, designed to maximize surface area, sample retention, and comfort. Biocompatible and affordable materials were employed, providing cost-efficient and fast production of NP swabs for COVID-19 testing kits. Together, they were capable to print millions of NP swabs, demonstrating the ability of 3D printing to address the shortages of critically needed equipment. Besides SLA, the FDM 3D printing technique can also be employed for the obtention of NP swabs. Cox and co-authors [33] in turn, sought to assess the feasibility of producing a 3D-printed NP swab using the FDM technique, due to the cost of the printer and the material used. Thus, the NP swab model was proposed and applied, in comparison to a commercial swab to evaluate the viability. According to the authors, the results obtained have shown that there was no significant variation between the proposed 3D printed or commercial NP swab, demonstrating that the application of the 3D swab is viable and reliable. Furthermore, it is also reported that 5500 swabs were prepared and delivered within 20 days, already incorporated into the analysis kit, with a cost equal to the commercial one. Besides SLA and FDM, other 3D printing techniques can be employed for the obtention of NP swabs, including SLS and DLP [14]. In addition, the literature also presents studies regarding the validation of different proposed swabs obtained by 3D printing, as presented by Callahan et al. [34]. The authors evaluated different 3D printed NP swabs, obtained from medical centers,

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individuals, and academic laboratories, and provided feedback on the proposed prototypes, aiming to aid in the obtention of safe clinical swabs rapidly, after a trial of several prototyped swabs, supporting the scientific literature.

3.3 RT-PCR The analysis of viral RNA of SARS-CoV-2 has a gold-standard method the RTPCR. The PCR is a very powerful and sensitive method for DNA amplification, which presents a wide range of applications, in molecular biology, diagnostics, and forensic analysis [35]. The RT-PCR method is responsible to convert the virus RNA into complementary DNA (cDNA), for this, a probe is linked to a fluorophore on one side, and a quencher on the other. After successive rounds of PCR, the probe is cleaved, generating an increase in the fluorescence until obtention of a quantifiable fluorescence signal. Since this method is highly reliable, sensitive, robust, sensitive, and specific, it is considered a gold-standard method for the detection of viruses in general [36]. RT-PCR analysis involves some steps, initiating with sample collection, in this case, the use of NP swabs is highly employed, and they can be 3D printed as presented in the previous section. After collection, the storage and transportation proceed and finally, the analysis is performed. The RT-PCR analysis is performed using a relatively bulky instrument, thus, this analysis needs to be performed in a centralized laboratory, and requires skilled personnel [8]. Due to the cost, complexity in maintenance, and use of instruments for performing PCR for quick diagnoses in the field, the search for alternatives that aim to solve these problems has intensified in the last decade. Independently from the pandemic, 3D printing has been previously employed for the construction of accessible PCR devices, which could be employed nowadays. Mulberry and co-authors [37] proposed, in 2017, the use of 3D printing for the manufacture of a low-cost portable PCR device to increase the accessibility of these devices. The structure of the system consists of custom-designed parts, such as a heating element, circuit board, and fixtures for the light path and casing. For this, besides the 3D printer, the authors also employed a 3D milling machine and a hand drill. The external structure, the holder for the heating element, the cooling system, including the fan, and the structure for the light path were all 3D printed employing the FDM technique. The device was shaped to use a typical PCR tube, with various key features and completely sealed. The mill was employed in the obtention of the electronic controls in the circuit board. A complete view of the involved parts and assembly scheme is presented in Fig. 8. The obtained equipment is battery controlled, has a low-cost and is portable, being able to perform diagnoses of diverse infectious diseases, such as malaria and HIV, using 20 µL of samples. Though this system had been proposed years before the emergence of SARSCoV-2, the proposed system could be well employed nowadays, as an alternative to commonly employed instrumentation. In addition, besides the presented work, the literature reports the use of 3D printing for the obtention of thermocyclers [38], and

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Fig. 8 3D printed PCR device parts, structure, and assembly reprinted from Ref. [37], with permission of PLOS ONE

microfluidic devices for PCR analysis [39], showing that this technique has potential in combat COVID-19.

3.4 ELISA The enzyme-linked immunosorbent assay (ELISA) is an immunological assay, widely used in the detection of viruses or viral antigens. This assay is based on the identification of antibodies and/or antigens, which are labeled with an enzyme. The antibody reacts with the antigen immobilized in a microplate and is detected by a secondary antibody labeled by the enzyme. This reaction causes a color change with a chromogen, indicating the presence of the antigen [40, 41]. The ELISA assay is highly sensitive and precise, and the antibody detection is typically performed on serum samples of the patient, which requires trained personnel to perform the

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collection procedure [42]. This technique is performed in 8 steps: 1—activation of the plate, 2—washing, 3—immobilization of antibodies, 4—washing, 5—incubation, 6—washing, 7—adding chromogenic substrate, and 8—reading optics of the wells [43]. One example that 3D printing could be used to contribute to ELISA assays is in the production of optimized microwells, improving the performance of the microplates. The microplates employed for ELISA analysis are varying in capacity and size, which directly influences the incubation time. The obtention of 3D printed microplates could significantly improve the performance of ELISA tests, by optimizing incubation time [44]. In this aspect, Singh and co-authors [45] have fabricated a 3D printed platform for application in ELISA tests. The platform consisted of 96 wells, initially designed to have a larger surface area than the conventional platform wells [46]. Figure 9a presents a schematic diagram of the 3D well (top view). Also, Fig. 9b shows a 3D well prototype showing the top layer composed of 8-half oval shapes (left) and a side view showing 5 layers of Part A interspersed by 4 layers of Part B (circular shape) (right), with the placement of 3D well in the 96-well plate. According to the authors, the development of the 3D platform provided rapid validation through the diagnostic performance of ELISA in infectious diseases. Furthermore, the 3D well showed up to a 2.25-fold improvement in sensitivity compared to the conventional 96-well ELISA [45]. In addition, the literature reports the FDM 3D printed obtention of an automated colorimetric ELISA assay for the detection of malaria, which consists of a disposable cartridge, and a non-disposable frame [47], and an accessible way to perform ELISA essay, by employing a 3D printed pipette tip, employing SLA technique [48]. This shows that 3D printing can be a powerful tool for the performance of ELISA assays, being an interesting combination that could be employed in the combat of COVID-19.

4 Electrochemical 3D Printed Biosensors Besides the construction of accessories and apparatus, 3D printing can also be explored in the obtention of electrochemical sensors [49–51]. With the advent of conductive filaments, 3D printing has brought to light a new generation of electrochemical sensors, called “3D printed sensors”. The fabrication of conductive filaments for 3D printing enabled the creation of electrochemical sensors via FDM 3D printing, which is the most employed method for its low cost and accessibility. 3D electrochemical sensors have rapidly expanded their development, from simpler analysis, such as drugs and metals, to more complex analysis, involving the anchorage of biomaterials for the detection of viruses, for example. This great versatility is due to the intrinsic qualities of 3D printing, allowing the prototyping of different sensor designs that can be adapted to different environments and can be used in portable systems for field and point-of-care analysis. The electrochemical biosensors, as discussed in Chap. 2 of this book, are analytical tools for biorecognition, which can be classified regarding the anchored biomolecule

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Fig. 9 a Schematic diagram of the 3D-well; b 3D well prototype individual, and placed in the plate, reprinted from Ref. [45], with permission of MDPI

and the recognition method. The methods which employ an antibody or antigen as a biorecognition element, reporting immunoreactions are classified as immunosensors, the sensors produced using DNA/RNA probes as recognition elements are classified as genosensors, and, the use of sensors composed of several single-stranded artificial nucleotides are reported as aptasensors [4]. In this section, we reported works from the literature that employ 3D printed electrochemical platforms for the construction of different biosensors for the detection of SARS-CoV-2.

4.1 Immunosensors Immunosensors are analytical devices which employ antibodies, antibody fragments or antigens in the monitoring of immunochemical reactions, generating a response signal related to the specific antigen–antibody binding. In an immunosensor, either

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the antibody or the antigen can be used as biorecognition element. Once immobilized in a transducer surface, an analytical response proportional to the concentration of the analyte is obtained after the imunnoreaction [4, 52]. The first work involving the use of 3D printed electrodes for the development of an immunosensor for virus detection was recently presented by Martins et al. [53]. The authors showed the possibility of using a carbon black/PLA filament for the construction of an immunosensor based on the immobilization of the specific antibody by using EDC/NHS directly at the electrode surface. As a proof-of-concept, the biosensor was tested in the detection of Hantavirus Araucaria nucleoprotein (Np). This work showed the suitability of 3D printed electrodes as platforms for the obtention of biosensors. To our knowledge, until the moment only 4 works are reported in the literature employing a 3D printed electrode as a platform for the construction of immunosensors for the detection of COVID-19. Muñoz and Pumera [54] proposed an antigenbased 3D printed electronic device based on a 3D printed graphene/PLA electrode, obtained from a commercial filament, modified with gold nanoparticles, and later a cys-glu-biomarker crosslink for the detection of the S1 spike protein of SARS-CoV2. Figure 10 shows the immunosensor production steps. The immunosensor proposed was able to detect the SARS-CoV-2 virus with a LOD of 0.5 µg mL−1 . According to the authors, the analysis of human serum samples showed satisfactory responses within a period of 20 min per analysis. Finally, the 3D-printed immunosensor successfully demonstrated the potential of using 3D printing to produce analytical devices at a low cost, fastly and without the need for a specialized operator, showing the feasibility of employing electrochemical immunosensors for the detection of the virus, in comparison to RT-PCR and ELISA assays for example, that are time spending and costly. Another option for the construction of 3D printed electrochemical sensors when involving FDM printing is the manufacturing of lab-made conductive filaments. This option provides the advantage to obtain electrochemical sensors with desired characteristics and materials, which can provide improved platforms for the construction of biosensors, since the commercial filaments present a low amount of conductive material, making necessary surface activation or pre-treatment steps. In this sense, Stefano and co-authors [55] proposed a filament with a high load of graphite (40% w/w) for the construction of ready-to-use electrochemical (bio)sensors that eliminate the need of surface pre-treatment steps. The proposed sensors were excellent platforms for the construction of a SARS-CoV-2 immunosensor, by the direct bonding of EDC/NHS at the electrode surface, with no need to metal modification. Figure 11 presents the filament preparation method, as well as the biosensor production and monitoring mechanism. For the construction of the biosensor, fewer steps than usual (metallic particle—ligands—antibody/biomarker) are required, involving only one ligand responsible for the crosslink between the base sensor and the specific antibody for the SARS-CoV-2 virus. Apart from FDM 3D printing, another 3D printing technique explored for the construction of electrochemical immunosensors is material jetting. Ali and coworkers

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Fig. 10 a Illustration of the 3D-printed electrochemical COVID-19 immunosensor fabrication steps. b Indirect competitive assay carried out for detecting the COVID-19 recombinant protein (antigen), the one against the SARS-CoV-2 virus. Reprinted from Ref. [54], with permission of Elsevier

[56] fabricated a 3D-printed biosensor using the aerosol jet technique for the obtention of an array of nanomaterials at the electrode surface and coupled the obtained biosensor to a smartphone for portable analysis (Fig. 12). The produced sensor microarray was coated with reduced graphene oxide (rGO), and subsequently, the immobilization of specific viral antigens to determine the SARS-CoV-2 virus was performed. The proposed 3D printed biosensor was integrated into a microfluidic device and used in a standard electrochemical cell. Figure 12 presents the electrochemical system developed, surface images of the 3D printed biosensor, and the modification procedure and mechanism of action for virus detection. Furthermore, according to the authors, the proposed platform could also be useful to detect biomarkers for other infectious agents, such as Ebola, HIV, and Zika. Following the same approach, Ali and co-authors [57] also developed a 3D-printed biosensor for SARS-CoV-2 virus detection, by monitoring a biomarker (N nucleocapsid). The 3D printed biosensor is similar to the one shown in Fig. 12. According to the authors, this new system for monitoring the nucleocapsid N has excellent sensitivity and fast response (only in a few seconds).

4.2 Genosensor Genossensors are DNA/RNA compact analytical devices, based on the immobilization of DNA/RNA probes as recognition element. This type of biosensors relies

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Fig. 11 a Representative scheme of the obtention of the conductive filaments and 3D printed electrochemical system; b Immunosensor fabrication steps and spike protein detection, reprinted from Ref. [55], with permission of Elsevier

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Fig. 12 A Electrochemical system and assembling, and electrodes characterization; B Functionalization of the 3D printed electrode and sensor operation, reprinted from Ref. [56], with permission of Wiley Online Library

on DNA-DNA or DNA-RNA hybridization reactions by molecular recognition providing highly specific analysis. With a genosensor, the detection of pathogens such as viruses or bacterias can be performed through their genetic material [4]. The use of 3D printing for the obtention of COVID-19 genosensor is poorly explored. The literature reports only one study of the development of genosensor using 3D printed electrodes as a platform, reinforcing the need to explore better this technological tool for the obtention of electrochemical biosensors. The production of electrochemical sensors from a 3D printing pen has attracted attention in recent years, mainly due to its easy handling, reduced cost, and the

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Fig. 13 a Digital photograph of the lab on a chip system containing the 3D printed electrodes placed in the reservoir and schematic representation; b Scheme representative of the SARS-CoV-2 genosensor, reprinted from [58], with permission of Wiley Online Library

possibility of being used in any environment quickly. The genosensor created by Crevillen and co-authors [58] explores this tool to develop a lab-on-chip system, consisting of a microfluidic channel integrated into an electrochemical cell fully printed with a 3D printing pen for SARS-CoV-2 detection. Figure 13 features the 3D printing pen, full lab-on-chip system, and proposed genosensor detection engine. The genosensor proposed is modified with an ssDNA probe capable of interacting with the N gene sequence of the SARS-CoV-2 virus. The detection mechanism depends on the electro-oxidation of adenines present in ssDNA when in contact with SARS-CoV-2 RNA. According to the authors, the 3D printed genosensor developed presented high sensitivity and rapid response in the analysis of SARS-CoV-2.

4.3 Aptasensor Aptamers are artificial single-stranded nucleic acid ligands (DNA or RNA) capable of binding to diverse targets, from molecules to whole cells. The interaction between the aptamer immobilized and the specific analyte generates a measurable response [4, 59]. Regarding the obtention of aptasensors from 3D printed electrodes for the detection of SARS-CoV-2, no works are reported in the literature to date. However, the development of aptasensors has been proposed using electrochemical platforms, for the detection of the SARS-CoV-2 virus as mentioned before. This indicates that electrodes with potential in electroanalysis, such as 3D printed electrodes, could be successfully applied in the manufacture of aptasensors. An aptasensor platform, as proposed by Rahmati et al. [60], which employed a screen-printed carbon electrode to develop an aptasensor capable of detecting the SARS-CoV-2 virus, could be successfully developed on the surface of a 3D printed electrode. The aptasensor

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development protocol was based on the modification of screen-printed carbon electrodes with copper hydroxide nanorods and later immobilizing 5' -NH2 -aptamer on the surface, which is capable of binding to the SARS-CoV-2 virus. In this aspect, 3D printed electrodes can be an alternative to commercial disposable sensors for the production of aptasensors following well-established modification protocols, as described previously. Thus, as can be seen, the production of 3D printed electrochemical sensors would facilitate the decentralized manufacture of electrochemical platforms for the construction of biosensors, which can assist in the troubleshooting of unexpected problems, such as the emergence of a pandemic, fighting diseases, or even protecting people against them. Given the astonishing characteristics of this technology, the exploration of 3D printing in the development of biosensors is highly worthy, and there is still a lot to be explored.

5 Conclusion and Future Perspectives 3D printing has proven to be a versatile technology, which can be employed successfully in the combat of pandemic situations, either by providing diagnosis tools, such as platforms and devices, by the 3D printing of electrochemical sensors or in the construction of PCR and ELISA devices, or by providing apparatus for personal protective equipment. In addition, the ease of production coupled with low cost, versatility, fast response, and no need for specialized operators make from 3D printing technology an accessible and viable technology to be implemented in the fight against COVID-19 pandemic, allowing decentralized analysis and the supply of specific apparatus according to the need. As can be seen, the diagnosis of COVID-19 can be carried out with the use of 3D printed electrochemical devices, and its simple manufacture, gathered with the low costs, low amount samples required, fast analysis, and mainly due to the ease of portability and miniaturization shows the great potential in the obtention of in situ analysis tools. Though these astonishing characteristics are present, there are still many possibilities to be explored with the use of 3D printing technology. A great diversity of materials can be employed, as well as the development of adaptable designs, which can be developed for the construction of equipment pieces, apparatus for individual protection, or even in the creation of diagnosis tools for point-of-care analysis, with a view in wearable sensors. This exploitation can be very useful in the combat of COVID-19 pandemic, or even preparing caretakers to deal with other viral diseases.

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Post-COVID-19 Metabolomics: Pursuing the Sequels of a Pandemic Leonardo Santos Alexandre and Emanuel Carrilho

1 Introduction COVID-19 is an infectious disease caused by SARS-CoV-2, a new coronavirus discovered in December 2019 in Wuhan, China, in the middle of a pneumonia outbreak. This disease became pandemic in March 2020, launching the world into a social distancing and isolation era. In December 2020, the first COVID-19 vaccine was applied. The year 2021 was marked by mass vaccination, SARS-CoV-2 variants, relaxation of distancing methods, and extensive political discussions about science and vaccine safety [1]. Vaccination efficiently contained the disease spread, while non-vaccinated people presented significant issues. The new variant, discovered in November 2021 in Africa, B.1.1.529 named omicron, became responsible for a new outbreak of cases [1, 2]. This outbreak is due to the mutations that boosted its transmissibility in exchange for being a milder disease [3]. To this date, in mid-2022, the total cases sum more than 430 million, with more than 5.9 million deaths and 10 billion vaccine doses administrated around the globe [4]. SARS-CoV-2 belongs to the Betacoronavirus genus in the Coronaviridae family, having many genomic and phylogenetic similarities with SARS-CoV. The latest virus was responsible for an outbreak of severe acute respiratory syndrome (SARS) in 2003 [5]. The viruses in this family have a genomic RNA of ~30 kb and are responsible for various respiratory infectious diseases in humans and animals [6]. COVID-19 primarily affects the lungs, causing shortness of breath, sore throat, and dry cough. The disease can be mild or evolve into severe cases, with the need for intensive care for the patient, commonly with the need for mechanical ventilation [7]. Disease variability is not well understood, with viral load and pre-existing health conditions impacting disease severity [8, 9]. L. S. Alexandre · E. Carrilho (B) Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP 13566-590, Brazil e-mail: [email protected] © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 F. N. Crespilho (ed.), Covid-19 Metabolomics and Diagnosis, https://doi.org/10.1007/978-3-031-27922-5_6

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The disease, when in mild to moderate cases, lasts approximately two weeks. In some cases, the symptoms persist for more than two weeks, even without the virus in the body. This disease causes a decrease in the overall health quality of the world population once even asymptomatic cases report this condition. It is called long COVID-19 or post COVID-19 syndrome [10, 11]. Metabolites are small molecules at the end of biological information flow. As such, they represent what was encoded by the genome and modified by diet, environmental factors, and the gut microbiome. Profiling these small molecules and correlating them with the biological mechanism involved in their expression gives an idea of the metabolic state of an individual at that determined moment. Based on this concept, metabolomics uses advanced analytical methods (mass spectrometry and nuclear magnetic resonance) to profile a significant number of these small molecules and compare the metabolic state of individuals in different conditions. These conditions can be versatile, such as healthy versus sick, sedentary versus athletic, and different diets. This opportunity provides knowledge of biology and biological mechanisms under these differences [12–15]. In this chapter, COVID-19 and post-COVID-19 syndrome will be discussed in light of metabolic changes. For that, we show how the disease promotes changes in the body, starting from cell infection and spreading through the organism. The known mechanism of its progression, post-COVID-19 syndrome characterization, and the metabolic changes throughout the disease are also discussed.

2 Biochemistry of COVID-19 Infection SARS-CoV-2 invades the body via the upper respiratory tract. It disrupts the immune system and entails complications throughout the whole body. The ability of the virus to infect different types of tissues resides in its cell entry mechanism. That mainly works around the acceptor angiotensin-converting enzyme 2 (ACE2) in host cells. This protein work on blood pressure regulation and can be found in many organ systems (guts, kidney, heart) [7, 16]. After entering the cells, the virus starts replicating and spreading through tissues. The mechanism of how the virus enters cells, its replication, and the progression of the disease will be discussed in further detail.

2.1 Viral Entry and Replication COVID-19 virus is composed of four structural proteins. They are nucleocapsid (N), membrane (M), envelope (E), and spike (S) proteins. The cell entry is mediated by the S glycoprotein, relying on ACE2 present on the host cell surface [17]. The S protein is a homotrimer with many copies spread in the virus membrane surface, giving its characteristic crown (corona) shape. The S protein consists of 2

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Fig. 1 Two simplified viral entry mechanisms of the SARS-CoV-2. On the left, endocytosis and cathepsin mediated membrane fusion. On the right, TMRSS2 mediated membrane fusion. Adapted from: https://commons.wikimedia.org/wiki/File:Entrada_SARS-CoV-2.svg

subunits, S1 and S2. The interaction of the receptor-binding domain (RBD) in S1 with ACE2 anchors the virus on the cell surface and exposes the S2 site of cleavage [17]. From this point forward, the viral RNA has two ways to enter the cytoplasm, as depicted in Fig. 1. The first is by transmembrane protease, serine 2 (TMPRSS2), on the cell surface, cleaving the S2 site, which causes S1 to shed and provoke intense conformational changes in S2. These changes release and propel a fusion peptide from S2 to the target cell membrane. The fusion peptide starts the cell fusion, unifying the cell and virus membrane, and opening a pore, whereas the viral RNA enters the cytoplasm. If the cell does not express sufficient TMPRSS2, or the virus cannot find one near where it is anchored, it suffers endocytosis forming an endolysosome. Inside the cell, the S2 site will be cleaved by cathepsins, mainly cathepsin L, promoting cell fusion with the endosome membrane and releasing the viral RNA [17]. To properly induce cell fusion, the boundary S1-S2 must be cleaved. This fusion happens by furin or furin-like proteases during virus maturation in the host cell. This mechanism is crucial for viral entry and is one of the particularities that differs SARS-CoV-2 from SARS-CoV [17]. Inside the cell cytoplasm, the genomic RNA (gRNA) makes use of host ribosomes serving as mRNA, translating two large open reading frames (ORFs), ORF1a and ORF1b, to polyproteins pp1a and pp1b. These ORFs compose three-quarters of total gRNA. The polyproteins are cleaved 15 times by virus-encoded papain-like protease, releasing sixteen non-structural proteins (nsp), nsp1 to nsp16. The nsp1 mediates the shutdown of translation of host mRNAs, while the other nsps form Replication-Transcription Complexes (RTCs) [18]. The replication and transcription are primarily made by enzymes from nsp12 to nsp16, while the RNA-dependent RNA polymerase (RdRp) domain performs the catalysis of RNA synthesis. This domain is present in nsp12, and with aid of nsp7

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Fig. 2 Summary of SARS-CoV-2 replication. Adapted from: https://commons.wikimedia.org/ wiki/File:The_life_cycle_of_SARS-CoV.svg

and nsp8 they form the holo-RdRp, a central component of RTC. The other RTC subunits act as support modulating the host’s innate immune response or remodeling cell membranes to form a double-membrane replication organelle, where all the RNA synthesis occurs [18]. The RTC starts RNA synthesis by generating a complementary strand of gRNA that will serve as a template to generate new gRNA. It also produces subgenomic RNAs downstream from ORF1a and ORF1b in the gRNA. The sgRNA directs the synthesis of subgenomic mRNAs. It is responsible for producing the structural proteins, N, M, E, and S, all essential for virus assembly. Once complete, the new virion leaves the cell by lysosomal trafficking [18]. Based on the mechanisms illustrated in Fig. 2, the virus can enter de cells and replicate continuously, starting the infection in the body. However, some of the specific functions of the nsps are not entirely elucidated as the complete action of RTCs. Understanding these mechanisms can progressively give ways to disturb this process and stop the viral spread.

2.2 Physiopathology and Disease Progression COVID-19 is known for causing pneumonia-like symptoms and disturbing the immune system, causing it to overreact and sending a storm of cytokines. The mechanism of how the virus promotes these damages and induces the body to overreact includes four loops: the viral loop, hyperinflammatory loop, non-canonical reninangiotensin system (RAS) axis loop, and the hypercoagulation loop. Through these loops, the virus causes direct viral toxicity, endothelial damage, microvascular injury,

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immune system dysregulation and stimulation of hyper-inflammatory state, hypercoagulability with resultant thrombosis and macrothrombosis, and maladaptation of the ACE2 pathway [7, 8, 19]. The viral feedback loop, observed in SARS-CoV infection, evades the host’s innate response, becoming accessible for viral replication and turning on a hyperactive immune response. The virus promotes the production of double-membrane vesicles to evade the innate immune system. Then the virus can replicate in these vesicles without being detected. The nsps generated from ORFs translation and cleavage, provides further support to this undetected state, as they act inhibiting interferon responses, a defense molecule responsible for disturbing viral replication. With innate response controlled, the virus can freely replicate. This free viral replication induces pattern recognition receptors to cause an aberrant inflammatory response. SARS-CoV-2 can replicate 3.20 folds higher than SARS-CoV without inducing interferons significantly [19]. The aberrant inflammatory response constitutes the hyperinflammatory loop. The uncontrolled viral replication induces apoptosis, causing the production of elevated levels of cytokines and chemokines. In turn, they start to infiltrate the lungs massively. Also, in response to the virus, white blood cells (monocyte-macrophages and neutrophils) start accumulating in the lungs, which releases more cytokines and chemokines. This accumulation can cause cell death in endothelial tissues, affecting the lung microcirculation and resulting in vascular leakage and alveolar edema [19]. Virus-specific T cells must stop and control this chaos to eliminate the virus and soften the hyperactive inflammatory response. However, the TNF-α-mediated cell apoptosis and upregulation of interleukin-6 (IL-6) caused by the virus can decrease its action, leaving the inflammatory responses uncontrolled. This response is observed as lymphopenia present in COVID-19 severe cases [19]. The RAS consists of a canonical ACE/Ang II/AT1R and a non-canonical ACE2/Ang (1–7)/Mas1R pathways. Putting aside the intricate biochemical pathways and their connections, the canonical side is responsible for increasing tension on the sympathetic nervous system, causing vasoconstriction, increasing blood pressure, and promoting inflammation. Ang II in the canonical route activates several cellular functions and signaling pathways related to tissue injury, inflammation, and fibrogenesis. Conversely, the counterregulatory non-canonical path negatively modulates leukocyte migration, cytokine expression and release, and fibrogenesis pathways. The deficiency in ACE2 in the non-canonical route increases vascular damage by increasing gene expression of vascular adhesion molecules, cytokines, chemokines, and matrix metalloproteases [19]. The virus binding in ACE2 and further degrading it causes ACE2 to be scarce. The loss of ACE2 results in a higher increase in Ang II-induced expression of inflammatory factors, enhanced vascular permeability, increased lung edema, and neutrophil accumulation. This strategy facilitates the permeability of cytokines, leaving the lung helpless against its storm [19]. The hypercoagulation loop happens as a result of inflammation and coagulation interactions. Neutrophil Extracellular Traps (NET) activate endothelial cells,

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platelets, and proteases that inactivate endogenous anticoagulants. Platelets activate the coagulation system and stimulate the release of NET, which activate more platelets, creating a feedback loop. These interactions are triggered during inflammation [19]. Additionally, the ACE2/Ang (1–7)/Mas1R path acts, activating Mas1R in platelets and protecting endothelial dysfunction. While this pathway is disturbed by SARSCoV-2, the presence of a hyperinflammatory state, endothelial injury, and activation of coagulation lead to a full expression of a hypercoagulable state. This state is seen as pulmonary thrombosis, venomous thromboembolism, or other thrombotic events during the disease [19]. These insights in the body’s biochemistry give an idea of how the disease initiates and progresses. The virus, deceiving the innate immune system, causes an overreaction of defense cells worsening inflammation, which can be further increased by downregulation of ACE2 and a hypercoagulation state. The latter two processes are linked to the disrupted ACE2/Ang (1–7)/Mas1R pathway [19]. Although these mechanisms seem present during the disease, not all people suffer from severe disease. This phenomenon can be related to metabolism, environmental factors, and the virus and its variants. Viral load also seems to correlate with the severity of the disease [19].

2.3 COVID-19 Infection Outside of Lungs The expression of ACE2 is not exclusive to respiratory system cells, thus allowing the virus to infect other cells throughout the body. In this way, the virus infects tissues like the guts, heart, and kidney, causing more damage besides the previously discussed lung damage and thrombo-inflammatory responses. The abnormal conditions promoted include myocardial dysfunction and arrhythmia, acute coronary syndromes, acute kidney injury (AKI), gastrointestinal symptoms, hyperglycemia and ketosis, neurologic illness, and dermatologic complications [8]. Figure 3 highlights the affected organ and symptoms. The multi-organ damage is mainly caused due to direct viral damage that promotes apoptosis of infected cells. The most infected cells are those with high ACE2 expression, including cardiac myocytes, fibroblasts, endothelial cells, smooth muscle cells in the heart, kidney endothelial cells, intestinal glandular cells, and cholangiocytes in biliary ducts, and, but inconsistently, in the endocrine pancreas. The virus can reach the central nervous system (CNS) via nasal mucosa, olfactory bulb, or retrograde axonal transport. Cytokine storms also can worse extrapulmonary damage [8]. The cardiovascular damage includes myocarditis, myocardial ischemia, arrhythmia, and cardiomyopathy. The primary clinical complication in the kidney is AKI, with acute tubular injury, diffuse erythrocyte aggregation, and endothelial damage. The gastrointestinal system disturbs present diarrhea, abdominal pain, nausea, and vomiting, which can be worsened by intestinal microbiota alteration. Endocrinologic effects are hyperglycemia and ketosis or the worsening of these

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Fig. 3 Affected organs and symptoms of COVID-1 through the body. The most common symptoms are in bold

conditions. It is possible that damage to the pancreas can be responsible for insulin deficiency. Diabetic complications can also be linked to reduced renal function, coagulation state, and cardiac dysfunction. When the CNS is affected, the observed symptoms are headache, dizziness, and fatigue. Erythematous rash, urticaria, and chickenpox-like vesicles are common dermatologic manifestations [8]. The SARS-CoV-2 biochemical mechanism is not entirely understood, but the more is found and known, the better it is possible to treat people and stop the virus rampage through the body. The virus can act in many body tissues and have several sequelae. So, also understanding the after-disease condition is crucial for completely recovering patients.

3 Post COVID-19 Syndrome The beginning of SARS-CoV-2 infection can be very soft, with no symptoms, with the possibility that some people are asymptomatic. Others develop a mild disease and some progress to a severe stage. In general, the disease lasts one to two weeks after symptoms onset. However, some people have reported symptoms for more than six months after recovering from the disease. This condition is nominated as a postCOVID-19 syndrome, post-acute COVID-19 syndrome, or simple long COVID-19 [7, 10, 11]. Several studies have characterized the post-COVID-19 syndrome. The syndrome can be divided post severe COVID-19 and post-mild COVID-19 [10, 11]. The post-severe syndrome has a considerable asset of sequelae across several biological systems. Not all symptoms are directly linked to the infection but are related

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to neurological symptoms, such as anxiety from a near-death experience and body stress from health support equipment, like mechanical ventilation applied during the severe stages of the disease [11]. Otherwise, the mild syndrome has fewer and more spread reported symptoms across patients [10].

3.1 Post Severe COVID-19 Syndrome The post-severe COVID-19 syndrome has persistent symptoms and sequelae in pulmonary, hematologic, cardiovascular, neuropsychiatric, renal, endocrine, gastrointestinal, and dermatologic systems [11]. This wide range of symptoms is according to multi-organ infection by SARS-CoV-2. The pulmonary system presented prolonged symptoms of dyspnea, hypoxia, and decreased exercise capacity. Also, reduced diffusion capacity and restrictive pulmonary physiology were observed, and imaging exams showed ground-glass opacity and fibrotic changes in the lungs [11]. Thromboembolic events are observed in the hematological system, with persistent cardiovascular symptoms, like palpitations, dyspnea, and chest pain. The sequelae observed include increased cardiometabolic demand, myocardial fibrosis, arrhythmias, tachycardia, and autonomic dysfunction [11]. The disturbance in the neuropsychiatric system is frequent and can directly affect the quality of life and the functional capabilities of patients [20]. It can include fatigue, myalgia, headache, dysautonomia, and cognitive impairment (brain fog). Survivor patients commonly report anxiety, depression, sleep disturbance, and Post-Traumatic Stress Disorder. Such symptoms are also seen in survivors of other coronavirus diseases [11]. The fatigue and cognitive impairment are confirmed significantly and can persist for over 12 weeks. However, the precise mechanism is unclear [21]. The causes of those symptoms can arise from different pathophysiology mechanisms such as immune dysregulation, inflammation, and microvascular thrombosis or be driven by the effects of medicaments and psychosocial effects of the disease [11]. AKI during COVID-19 can cause a prolonged impairment in renal function. In the endocrine system, sequelae are observed to worsen control of diabetes, subacute thyroiditis, and bone demineralization. Residual COVID-19 particles in the guts can be involved with alterations in the microbiome, opening space for opportunistic microorganisms. Furthermore, hair loss after COVID-19 is a recurrent reported [11]. The complete range of post-severe COVID-19 is not entirely understood. Characterizing the symptoms and their causes, such as direct viral toxicity, side effects from the health support protocols, or psychosocial conditions, will help treat and foster recovery of COVID-19 patients.

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3.2 Post-Mild COVID-19 Syndrome In mild cases, it is possible to observe the evolution of the symptoms. In COVID-19 symptom onset, the most reported ones are cough, ageusia, anosmia, body aches, headache, and fever. After 4–7 months, the reported symptoms were anosmia, ageusia, fatigue, and shortness of breath. Less common symptoms were observed in the 7-month range with headache, alopecia, and diarrhea. Some of the patients who presented the syndrome were asymptomatic. The prolonged symptoms ranged from 12.8 to 27.8%, with a duration of up to seven months, with at least one symptom [10]. The prolonged fatigue can be seen in other post-infectious diseases, such as Epstein-Barr virus (glandular fever), Coxiella burnetti (Q fever), and Ross River virus (epidemic polyarthritis), with a duration of 6 months [22]. Post COVID-19 syndrome, in general, and these infectious diseases can be cross-linked with chronic fatigue syndrome [22, 23]. Anosmia is a ubiquitous reported symptom and can occur with two mechanisms. The direct viral damage in the olfactory epithelium causes deprivation of olfactory inputs or allows virus invasion of the central nervous system. The prolonged anosmia seems to be related to CNS damage, as a neurological sequela that alters cognition and impairs memory [24]. In post-mild COVID-19, diarrhea symptoms can relate to the persistence of COVID-19 particles in the intestine, as observed in severe cases, too [10, 11]. Most of this data is from the patient descriptions, not having actual clinical data to confirm them and their gravity. Because of this, it is not possible to complete link all symptoms observed to post COVID-19 symptoms. In this manner, the post-mild COVID-19 syndrome is likely to present ageusia, anosmia, fatigue, and shortness of breath as the main symptoms once they cannot be related to any other diagnosis or pre-COVID-19 conditions [10].

3.3 Occurrence and Distribution of Symptoms Across Age, Race, and Sex Once characterized, it is essential to know how the syndrome affects different groups of patients. Generally, at least one symptom post-COVID-19 syndrome varies largely across age, race, and sex. The symptoms are pronounced in people with poor baseline health or those who suffer from severe diseases [25]. Most sequelae have a higher incidence in old adults. However, hyperlipidemia, chest pain, sleep disorders, cough, and anosmia happen to be more common in people younger than 60 years old [25]. Slight differences were observed in burden across races. Black people seem to suffer more frequently from kidney injury, diabetes mellitus, chest pain, cough, substance abuse, thromboembolism, headache, and tachycardia. In comparison,

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white people have gastroesophageal reflux disease and tachycardia as more common symptoms [25]. It is known that African descendants have a higher risk of kidney injury because of the higher presence of polymorphisms in APOL1 genes. This elevated risk of kidney injury is also seen during COVID-19 [26]. Shortness of breath, cough, chest pain, arrhythmia, headache, anosmia, and hair loss were more commonly seen in female patients. Moreover, the more the symptoms pose a burden, the more the necessary health support [25]. Still, not all symptoms can be directly linked to SARS-CoV-2 infection. Pre-existing health conditions and health support equipment worsen the syndrome [11, 25]. COVID-19 spreads more and more each day, meaning that more and more people are susceptible to suffering from the syndrome. Its characterization helps to recognize it faster and be aware of what patients are more likely to develop a severe case. Knowing its occurrence frequency and its potential risk of decreasing the quality of life of the general population heightens the need to find ways to treat the patients efficiently. Happily, evidence of vaccines offering protection and lowering the incidence of post-COVID-19 symptoms has been reported recently [27].

4 Metabolic Changes During and Post COVID-19 Infection 4.1 Plasma Metabolome Changes During COVID-19 Science has long been evaluating the metabolic effects of viral infections. The main characteristic of a virus is to undergo a free multiplication inside the host cells; it uses the cell machinery by reprogramming its functions to replicate the viral structures and release new viral particles in the host organism. Then the host cell metabolism is subdued by massive changes to support the viral spread. In response, the body starts the adaptative immune system to eliminate the invader organism, causing the metabolism to undergo more transformations [28–30]. The metabolic change mechanisms are not fully understood, and all these modifications at a subcellular level can lead to the disease symptoms. Looking into the metabolome, mainly the plasma metabolome is possible to observe these biochemistry changes circulating through the whole body by comparing patients in different disease stages. With this approach, it is possible to have a deeper understanding of how COVID-19 develops [15, 28, 31, 32]. Many studies have found metabolic changes through the progression of the disease, showing that COVID-19 affects the whole-body metabolism. The findings suggest that fatal cases suffer from a metabolic disturbance with an increase or decrease in levels of several metabolites [33–35]. Other studies show correlations between metabolic disturbance with cytokines release [31] and inflammation [29, 35, 36] with a general worsening of the disease [28]. Moreover, the metabolic disturbance further draws an escalated correlation from mild to severe in a cohort of patients [31].

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This disturbance shows how reprogramming the cellular metabolism is critical for viral replication [29]. Many studies reported the tryptophan metabolism as being altered during the stages of COVID-19 infection [28, 34–38]. Its pathway merge with kynurenine and nicotinamide pathways [39] and is presented in Fig. 4. The diminished tryptophan levels correlated with an active consumption caused by the disease in the kynurenine pathway. This pathway produces anthranilate with marked immunosuppressive effects linked to IL-6 levels [34, 37]. Fraser et al. suggested that the arginine/kynurenine ratio could discriminate between healthy and COVID-19 patients and the creatinine/arginine ratio to predict disease mortality [35]. Arginine is positively correlated to proinflammatory cytokines. It is related to T cell activation and could be involved in an immune response loop during COVID-19 [31]. The 3-indole acetic acid and nicotinamide were found in the univariate and multivariate models to predict COVID-19 diagnosis based on plasma metabolome. These two metabolites are central to the tryptophan/nicotinamide pathway and may be linked with inflammatory signals of tryptophan/kynurenine metabolism [38]. Another study found ceramide metabolism, tryptophan degradation, and disturbing metabolic reactions of nicotinamide adenine nucleotide to be associated with respiratory severity and correlated with inflammation [36]. These two studies expand the effects of the tryptophan/kynurenine pathway to the tryptophan/nicotinamide pathway. This pathway also influences mTOR activation, which is involved in the expression of antimicrobial peptides in the intestine and opens the discussion on the role of microbiota during the infection [38]. The purine metabolism was also recurrent in different studies. It was found that the inhibition of purine metabolism potentializes the inflammatory response, but inhibition of pyrimidines suppressed the inflammatory cytokine release. This duality shows that the balance between purine/pyrimidine is relevant to viral replication and immune response [31]. Valdés et al. also report that purine metabolism metabolites (urea and xanthine) increased with the severity stage [28]. The malic acid and aspartic acid were decreased in the plasma metabolome of COVID-19 patients. They participate in purine metabolism and may be caused to decrease because of the virus replication, which consumes the nucleic acids and cause the metabolism to prioritize nucleotides biosynthesis. This preference indicates how the disease progression affects nucleotide metabolism [33]. Additionally, alterations observed in guanosine monophosphate could imply an immunomodulatory enzyme imbalance. Carbamoyl phosphate changes suggest liver damage. These findings can reinforce the effects of the disease on immune disfunction [33]. There is a report on the relevance of cytosine, which is a coordinator agent in the cell metabolism of SARS-CoV-2. The decrease of cytosine in the virus genome may be associated with its increase in host biological fluids and could be used as a marker to predict disease evolution [38]. The lipidome also showed exciting changes. Wu et al. observed up- and downregulation of lipids and suggested the occurrence of dyslipidemia in COVID-19

Fig. 4 Tryptophan/Kynurenine/Nicotinamide connected pathway. NaMN: nicotinic acid mononucleotide; NaAD: nicotinic acid adenine dinucleotide; NMN: nicotinamide mononucleotide; MNA: N-methylnicotinamide; 2-Py: N-methyl-2-pyridone-5-carboxamide; 4-Py: N-methyl-4-pridone-3-carboxamide

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patients [33]. Furthermore, increased levels of unsaturated fatty acids correlate with the worsening of COVID-19 infection. This increase may be due to polyunsaturated fatty acids disturbing the virus-host cell interaction, and the change in lipid homeostasis is considered a strategy to make the organism friendly to replication [28]. The lysophosphatidylcholine (LPC) and phosphatidylcholines (PC) levels were found as a predominant change in serum metabolome [28, 29, 35]. These changes are related to disease progression to a severe state and correlate with inflammatory markers IL-6 and CRP [28, 29]. Additionally, were reported significant metabolic changes through a vast array of metabolite classes. The citric acid metabolites (citrate, isocitrate, oxalosuccinate, malate) were reduced, indicating a lower energy production during the infection. A decrease in amino acids, glutamine, citrulline, and urea and an increase in succinate levels were reported [31]. Furthermore, changes in sarcosine levels [35] and anthranilic acid increase with disease severity [34]. Acylcarnitines were observed to increase in COVID-19-positive patients. It participates in the formation of acyl-CoA, which takes part in beta-oxidation. It can be related to the observed increase in ketone bodies, sub-products of betaoxidation. Ketone bodies work as energy supply and have roles in the modulation of inflammation, oxidative stress, and immune cell function [28]. The bile acids were found an increase in levels prior to severe disease. They can, in different cases, inhibit or potentialize viral replication, which is unclear for COVID-19 [28]. The way COVID-19 differentiates from mild to severe is bound to the metabolism! In the future, an even better dissection of how the metabolism in plasma and different tissues respond to viral infection is expected. As shown by many studies, the impact of COVID-19 on the body metabolism is not fully understood, yet many metabolites and affected pathways were solidly identified. Understanding how the disease evolves in the biochemistry pathway is fundamental to elaborate efficient forms of treatment. Biomarkers and possible drug targets are the main focus of the studies, but this knowledge can be spread to different virus infections or other respiratory diseases.

4.2 Persistent Metabolic Changes in Post COVID-19 Syndrome The metabolism is expected to return to normal after the disease, but as discussed, many sequelae are present in COVID-19 recovered patients. On the metabolism disorder side of sequelae, several metabolites turned to normal levels at hospital discharge in mild patients. These metabolites involve acylcarnitines, unsaturated fatty acids, some bile acids, and most of the purine metabolism molecules [28]. On the other hand, many recovered patients that tested negative for the virus were still

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found to have metabolic changes, meaning no total recovery or persistent alteration in the metabolism [33]. Holmes et al. analyzed the blood of post-COVID-19 patients three months after severe disease and with symptoms that carried until six months, indicating the presence of residual COVID-19 biomarker signatures. They found that the plasma apolipoprotein B100/A1 ratio returned to normality, but the values were significantly lower, meaning a partial recovery of the phenotype [32]. The disbalance in mitochondrial functions by the virus can change energy metabolism and be associated with muscle degeneration, fatigue, and metabolic transformations [23]. Observation of elevated taurine and reduced glutamine/glutamate ratio are signals of possible liver and muscle damage and higher energy demand [32]. Also, imbalances in energy metabolism are connected to inflammation signaling [23]. A glycometabolic control, insulin resistance, and beta-cell function were reported in COVID-19 patients without any history or diagnosis of diabetes [40]. Also, it was reported glycemic abnormalities in patients two months after recovery. Al-Aly et al. found persistent lipid metabolism alteration and loss in controlling diabetes and obesity. The prolonged effects observed seem to be mediated by abnormal secretome, while the metabolic alteration is due to viral infection [40, 41]. Plasma neopterin was found to normalize after the disease, indicating the reduced adaptative immune activity. Systemic inflammatory biomarkers, GlycA and tryptophan, and kynurenine/tryptophan ratio remained elevated in some but not all patients [32]. This elevation indicates how we do not fully understand how each person’s metabolism adapts and recovers after a severe disease. The understanding of the metabolism changes is beneficial for the post-COVID-19 syndrome. Knowing what happened helps reverse it and ease the burden on recovered patients. Fortunately, there is evidence that the vaccine protects from the worsening of the disease and prolonged symptoms of COVID-19 [27]. There are no metabolic data around it, but it is expected that the metabolism stays unchanged with no symptoms. With the global spread of vaccination, we expect long COVID-19 cases to be decreased and a significant issue for non-vaccinated people or pre-vaccination cases.

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Metabolic Behavior of Covid-19 Infection Severity Vinícius G. Ferreira, Mariana B. Almeida, and Emanuel Carrilho

1 Introduction As hypothesized by several scientists over history, such as the microbiologists David White and Macfarlane Burnet, the emergence and outbreaks of viral infections are not less than natural and are expected to occur over our timeline [1, 2]. A proof of this hypothesis is the several epidemics and pandemics that humanity already had faced over time, like influenza, H1N1, H3N2, SARS-CoV, MERS-CoV, Dengue fever, Zika virus, Ebola, Smallpox, and the most recent, SARS-CoV-2. As a matter of fact, as more globalized and interlinked the human society is, the larger the probability of an outbreak from an isolated viral infection occurring in a small community and eventually leading to a global impact. The spread of SARS-CoV-2, the virus responsible for the Coronavirus Disease—19 (Covid-19), precisely followed this process, starting in a relatively small province in China, Wuhan. After less than three months, the World Health Organization (WHO) declared SARS-CoV-2 infection as a pandemic, i.e., worldwide spread, eventuating in the disease of approximately 3% of the global population after two years [3–5]. After the recognition of the severity of the disease, the world testified an unprecedented global scientific effort, resulting in approximately 300 thousand papers about the Covid-19 outbreak, leading to several impacting discoveries, which, in its turn, resulted in the development of several diagnostics tests, adaptative treatment protocols, ten different approved vaccines [6] and countless lives saved [7, 8]. It is unclear how these efforts will influence the future. However, the world witnessed the importance of open science, the value of faster peer-reviewing systems, the relevance of the culture of a scientific background for the general population, and the importance V. G. Ferreira · M. B. Almeida · E. Carrilho (B) Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, SP 13566-590, Brazil e-mail: [email protected] Instituto Nacional de Ciência e Tecnologia de Bioanalítica—INCTBio, Campinas, SP 13083-970, Brazil © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 F. N. Crespilho (ed.), Covid-19 Metabolomics and Diagnosis, https://doi.org/10.1007/978-3-031-27922-5_7

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of well-established economic and sanitary protocols for controlling the impact of a pandemic. From the scientific perspective, efforts against Covid-19 will undoubtedly result in significant advances, such as the mRNA vaccines, the most effective vaccine against the infection so far, and it was firstly applied for SARS-CoV-2 infection [9–12]. Virus sequencing was never as common and essential as nowadays due to constant surveillance of the virus mutations and the surge of new variants [13, 14]. Analytical chemistry applied to health had never developed as quick as in the past years, resulting in several platforms that could be rapidly adapted for different diseases according to the needs [15–17]. Another massive impact was the biochemical understanding of severe infections in human organisms. Several important proteomics and metabolomics studies revealed the biological, environmental, and behavioral relevancies for the infection progress [9, 18–20]. Although all the lessons from the Covid-19 pandemic are of great utility, this chapter highlights the discoveries related to the importance of understanding the patient’s metabolism for the disease outcome. Comparisons between patients’ metabolism before, throughout, and after the infection can shed some light on the prognosis. Because of that, understanding how the metabolism can affect the infection outcome are of an enormous application once the discoveries can eventually be used as a diagnostic and prognostic tool in the clinics. The following pages will discuss the metabolism under Covid-19 infection, the Covid-19 high-risk conditions and their metabolic signatures, the state-of-the-art Covid-19 metabolic markers, and how analytical chemistry may help bring the discoveries to the bedside.

2 SARS-CoV-2 Infection: What You Need to Know Before further discussing how metabolism can influence the clinical evolution of Covid-19 infection, it is essential to overview a few of SARS-CoV-2 characteristics and how the infection process occurs and affects the human body. In this section, we will focus on summarizing the most relevant aspects, while for further and deeper information, the reader may reach the many qualified reviews available nowadays [21–24]. SARS-CoV-2 belongs to the coronavirus family, i.e., Coronaviridae, which implicates in a few characteristics, such as the presence of the structural proteins: spike glycoprotein (S), the envelope protein (E), membrane protein (M), and nucleocapsid protein (N), Fig. 1 [25]. Amongst the proteins, the spike glycoprotein is the main responsible for the viral entry in the human cells [22, 26, 27], once its surface’s receptor-binding domains (RBD) binds to the angiotensin-converting enzyme 2 (ACE2), Fig. 2, which is expressed in different tissues, including intestine, colon, and lung [22, 26–28]. Along with ACE2, the transmembrane serine protease 2 (TMPRSS2) and cathepsin L are also essential proteins to enhance the infectious capacity of the SARS-CoV-2 virus, acting as priming agents for the virus entry into the host cell [29].

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Fig. 1 SARS-CoV-2 virus morphology. The virus particle is formed by four proteins, Spike (S), responsible for the virus entry into the host cell; Nucleocapsid (N), the only protein that binds with the viral RNA inside the particle, and besides keeping the RNA structure, it is also relevant for viral assembly and budding; Membrane (M), responsible for maintaining the viral structure; Envelope (E), necessary for viral assembly and budding. Besides the proteins, the virus is built of a lipidic membrane and an RNA single tape. The S protein is divided into two subunits, S1, containing the binding site for ACE2, and S2, mainly responsible for the viral and host cell membrane fusion. Created with BioRender.com

Specifically, during the virus entry, the S protein breaks down into two subunits, S1, responsible for ACE2 binding, and S2, responsible for anchorage in the host cell membrane [30, 31]. After S1-ACE binding, the viral entry into the cell may occur in two different manners, (i) through endocytosis of the viral particle, in which the particle is surrounded by the host cell membrane, or (ii) through direct fusion in the cell membrane, Fig. 2 [30, 32–34]. For both entry processes, the S2 subunit is exposed after S1-ACE binding. It undergoes a cleavage—by Cathepsin L for endocytosis entry or by TMPRSS2 for direct membrane fusion—exposing the called S2’ site, which includes a fusion peptide, which is the main responsible for the viral fusion into the host cell membrane. [30, 31, 33] Subsequently, as the fusion process advances, a pore starts to form, leading to the viral genome being released in the host cell’s cytoplasm, Fig. 2 [23, 30–35]. Once the viral RNA is inside the host cell, it starts to be translated by the host cell ribosomes in the cytoplasm, allowing the synthesis of viral proteins and viral RNA replication, Fig. 3 [36]. Essentially, the viral RNA makes use of the host cellular machinery for replicating its own proteins, originating new viral particles [23, 31, 37, 38]. The N proteins bind to the replicated RNA strand as the proteins are translated. In contrast, the remaining proteins are incorporated in the endoplasmatic reticulumGolgi intermediate (ER-Golgi), where the viral membrane is produced, Fig. 3 [31, 37, 38]. To the same extent the S protein is vital for viral entry in the host cell, the M protein is essential for new viral particle formation, once it helps the S protein to be incorporated in ER-Golgi; it allows the curvature of the membrane, starting the construction of a new viral particle; and in the last step of viral particle formation, the M protein binds to N protein to stabilize the nucleocapsid-RNA complex inside

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⏴Fig. 2 Mechanisms of SARS-CoV-2 entry in the host cell. The entry process after the viral binding to ACE2 protein in the host cell membrane has two potential pathways, (i) through a direct merging of the viral membrane, or (ii) by endocytosis. Each pathway uses different priming proteases, TMPRSS2 for direct fusion and Cathepsin L for endocytosis. Adapted from “Mechanism of SARSCoV-2 Viral entry” by BioRender.com (2022). Retrieved from https://app.biorender.com/biorendertemplates

the newly formed particle [31, 38]. The E protein is essential for viral assembly and release, including controlling the ion channel activity. Additionally, recombinant coronavirus without E protein presented malfunction and was less likely to correctly replicate, supporting that E protein is fundamental for a successful viral cycle [31, 39, 40]. After the viral particle is completely replicated inside the host cell, it is released from the cell through exocytosis, completing the viral lifecycle, Fig. 3. Besides the seizure of energy and supplies from the host cell, viral replication is not responsible for the Covid-19 symptoms. The main complication caused by the virus is the excessive immune response that the virus promotes in the human body, which starts immediately during the process of viral entry into the host cells. As the

Fig. 3 The SARS-CoV-2 lifecycle. In short, after the cell entry, its genome starts to be translated, replicated, and transcripted. The structural proteins S, M, and E are integrated into the ER-Golgi intermediate, while the N protein binds to the viral RNA strand. The M protein promotes the membrane curvature and stabilizes the N-RNA complex inside the new viral particle, which is ultimately released from the host cell through exocytosis. Reprinted from “Coronavirus Replication Cycle” by BioRender.com (2022). Retrieved from https://app.biorender.com/biorender-templates

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reader may know, the universe around us is full of different viruses and bacteria; however, most of them cannot cause illness in humans, an accomplishment that must be thanked our innate immune system [41–45]. The innate immune system is the first defense line, creating a harmful environment around infected cells, recognizing the antigens, recruiting effector cells, such as the natural killer (NK) cells, and triggering the adaptative immune system cells, such as the B cells—responsible for antibody production—and the T cells—responsible for mediating the cell death in the host infected cells through inducing phagocytosis of cellular lysis [44, 46–50]. Some viruses, however, can evade the innate immune system [44], as in the case of SARS-CoV-2, a particularly competent virus at obstructing the signaling pathway for type-I interferon (IFN) release [41, 50]. Considering that IFNs are responsible for interfering in the viral replication, and for quickly triggering the adaptative immune system, the viruses able to prevent its release slows down the immune response, which will lead to an uncontrolled viral replication [41, 50]. As INF1 release is restricted, the adaptative immune system cells are not quickly summoned, and the innate immune system cells—such as neutrophils and macrophages—try to overcome the absence of T and B cells through the release of higher levels of different cytokines and chemokines. On its turn, this release is harmful not only for infected cells but also for the host healthy cells, provoking extensive tissue damage including acute lung injury and pneumonia. The cytokine and chemokine massive release is called “cytokine storm”, and its occurrence is highly related to COVID-19 increase severity [41, 44, 51]. The reader can recognize that the SARS-CoV-2 infection is a complex process, and differences in any stage can significantly affect the disease outcome. Heterogeneity in the immune system, genetic conditions, pre-acquired diseases, and dietary habits are just a few topics that can influence the Covid-19 prognosis for better or worsening scenarios. Age, sex, metabolism, obesity, exercises are other factors that dictated the severity of the disease. The following section will further discuss the risk factors for Covid-19 and considered why the conditions affect the disease outcome.

3 Covid-19 Severity-Related Factors Several aspects can contribute to our immune system properly managing an infection; for example, undernourishment leads to a higher mortality rate since it is more difficult for the organism to supply the necessary energy and molecules in a malnourished state. In that sense, even socioeconomic status may be a differential for the mortality of a viral infection, as can be exampled by the seasonal influenza excess deaths, which shows higher mortality in lower-income countries, such as in Sub-Saharan African territories [52]. For the Covid-19 pandemic, a similar correlation was reported in several nationwide studies, proving that higher mortality is associated with poverty due to limited hospital access, poor testing policies, and scarce living conditions [53–57].

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Socioeconomic factors aside, the most impacting demographic factor for Covid19 mortality and severity regards the patient’s age, with the risk increasing along with aging [44, 58–63]. The most reported cause for such behavior is a weaker immune response in elderly patients. However, older patients often suffer from other comorbidities, increasing the mortality risk [62, 64, 65]. In a study performed by Ho et al. [64], patients aged ≥ 75 years showed a 13-fold higher mortality risk in comparison with ≤ 65 years old patients, and considering only patients with no comorbidities, ≥ 75 years old patients had 12-fold higher risk of death due to Covid-19 compared to ≤ 65 years elderly with no comorbidities. The elderly immune system is known for immunosenescence, a condition in which the innate and adaptative immune systems are impaired. Briefly, during immunosenescence, the production of naïve B and T cells—i.e., B and T cells before activation and differentiation to effector cells— decreases substantially, leading to a malfunction of the innate immune response and a late progression to adaptative immune response [65–70]. This information, accompanied by the fact that the elderly present lower IFN1 production, as explained in the previous section, leads to higher cytokines and chemokines release, provoking extensive tissue damage and the worsening of the Covid-19 symptoms [44, 71]. Several studies have also evaluated genetic predisposition, motivated by the fact that there were young and healthy patients between the casualties, indicating possible genetic causes [41, 72, 73]. At this point, the reader may think of a few possible genetic modifications that can weigh on the balance of Covid-19 prognosis, for example, the higher expression of ACE2 or TMPRSS2 in the host cells or even differences in the genes that encode immune system proteins, such as antibodies. Indeed, changes in such genes were identified in COVID-19 patients, starting with modifications on the Major Histocompatibility Complex (MHC), which is responsible for the immune system cells’ recognition of antigens, resulting in difficulties in T cells activation, and, therefore, late immunological response [72–75]. A list of MHC alterations related to covid-19 severity can be found in Fricke-Galindo and Falfán-Valencia’s review, including the world population in which such alterations are most common [73]. Several ACE2 and TMPRSS2 protein variations were linked to susceptibility to SARS-CoV-2 infection [74, 76–79]. TMEM189-UBE2V1 genes, part of the Interleukine-1 (IL-1) pathway, were related to Covid-19 severity [75]. Therefore, genetic alterations influence the symptoms undergoing, and in a personalized medicine perspective, such alterations could be used to enhance the patient medical care. Nowadays, it is also proved that different comorbidities, or preexisting diseases, enhance the probability of severe Covid-19 symptoms, such as diabetes, obesity, hypertension, and cardiovascular disease [59, 60, 80–83]. Each disease presents a specific role in SARS-CoV-2 infection and/or in the priming of the immune system. Diabetes, a condition defined as high glucose levels in the bloodstream, affects the patient’s innate immunity, mainly through malfunctioning some types of leukocytes, leading to lower phagocytic capacity, linked to viral neutralization [84–88]. Additionally, drugs prescribed for diabetes can elevate the ACE2 expression on the cells, thus, resulting in a more accessible entry for the SARS-CoV-2 and leading to a higher viral load [81, 86].

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Higher expression of ACE2 protein can also explain the higher risk of severe Covid-19 in obese patients, which shows two times higher risk of hospitalization and severity than non-obese patients [60, 81, 89–91]. ACE2 is highly expressed in adipose tissue cells, and as obese individuals present a great extent of adipose tissue, the viral entry and replication are eased, leading to a higher viral load and poorer outcomes [81, 89–91]. Additionally, in obese patients, complex metabolic interactions occur, such as leptin signaling, which is responsible for satiety control and crucial for the innate immune system [89, 92, 93]. Secreted at higher levels in obese patients, leptin regulates the innate immune response by inducing monocytes and T cell proliferation and influencing cytokines’ release [94]. Due to high leptin levels, obese patients often present a chronic inflammatory state. Also, they suffer from leptin resistance, a state in which the cells no longer respond appropriately to leptin stimuli, upsetting the proper leptin signaling and leading to inadequate immune response, contributing to the high severity of Covid-19 infection in obese patients [94, 95]. Cardiovascular diseases also increase the risk of severe Covid-19 in patients with a four times higher chance of death [96, 97]. Heart cells express ACE2 receptors on their surface; thus, they are susceptible to SARS-CoV-2 infection, which results in heart tissue damage [98]. ACE2 also plays a key role in Renin-angiotensin system (RAS) activation, which is responsible for maintaining blood pressure through angiotensin II (Ang II), a protein in charge of vasoconstriction, but also linked to cytokines release and innate immune cells assignment [99–101]. ACE2 is a negative regulator of RAS, and with SARS-CoV-2 binding to ACE2, the protein is downregulated, generating an increase in angiotensin II levels in the metabolism [81, 99]. With high levels of Ang II, the blood pressure is elevated, and more cytokines are released; therefore, Covid-19 infection also presents an increased risk for patients with hypertension [81, 102]. The knowledge of such factors related to covid-19 severity shows the importance and relevance of preliminary tests and diagnostics to assess the patient’s overall risk of evolving into a better or worse clinical condition. Therefore, many of the relevant risk factors are chronic conditions presented in the patient history; however, such conditions occur at different levels. Additionally, some patients may suffer from comorbidities and have never been properly diagnosed. Therefore, it would be of great clinical interest the discovery of biomarkers for Covid-19 prognosis, together with analytical tests and protocols to apply in the population. The following section will discuss the state-of-the-art biomarker discovery for Covid-19 infection and the application of analytical tests for its measurement.

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4 “Omic” Signature: An Analytical Perspective of Severity Markers The COVID-19 epidemic brought to the scientific field at the beginning of 2020 a race for the development of diagnostics tests, vaccines, and treatments. Knowing the pathogenic effects on the host organism became crucial to finding treatment strategies and providing a prognosis. During the last decades, the omics sciences displayed great evolution. Thanks to that, deeper discussion about virus-induced reprogramming of cells is now widely described and available in the literature and complete metabolic pathways. Previous studies of viruses, mainly of respiratory diseases, had helped researchers with a starting point to answer the many questions that have emerged with the COVID-19, although a deeper individualized study for SARS-CoV-2 infection is still indispensable [103, 104]. As previously mentioned in Sect. 2, SARS-CoV-2 infection causes an unregulated immune response. The elevated production of pro-inflammatory cytokines, as in the case of IL-1 and IL-6, suggests its use as a stratification marker for mild to severe cases [105, 106]. However, small molecules present in saliva, plasma/serum, urine, cells, tissues, and other fluids can play an essential role in this prognosis [104]. The discovery of a significant biomarker for the clinical question, preferably, should follow a workflow, as suggested in Fig. 4. The biological question needs to be aligned with the samples available. Following the workflow, untarget analysis and identification of potential biomarkers must precede the quantitative analysis that, in the last instance, should be replicated with different groups to evaluate the biomarker specificity [107].

Fig. 4 Analytical chemistry perspective of a successful biomarker discovery by omic analysis. Reprinted with permission Ref. [107]. Quality assurance (QA) and Quality control (QC)

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Among the instrumental techniques available to perform this analysis, the spectroscopic methods, such as nuclear magnetic resonance (NMR) and mass spectrometry (MS), are highlighted by identifying and quantifying the signatures left on the host after an infection by the SARS-CoV-2 [108–110]. Although the dataset is challenging, discerning and advanced statistical analysis can be considered the best friends of researchers to answer two key questions: (1) Is it important or unimportant? (2) Was it caused by the pathogenicity of the virus or by the host’s defense mechanism [111, 112]? The first attempt to understand a physio-pathological state under infection is throughout the blood plasma since it carries high levels of small molecules, mostly metabolites. A second approach is to understand the role of lipids, crucial components for cellular functions [112, 113]. In this case, it is primordial to remember that prior to any analysis, the storage of the samples and the choice of processing may affect the results, and each type of biofluid and technique of analysis has its proper sample preparation [113, 114], and currently still exist a lack of standard operating procedure for omic analysis, which difficult the implementation of this kind of analysis in the clinical routine [107, 115]. While the genomic data might infer the proteome, the metabolites of a living organism, on the other hand, are highly diverse, reflecting the complexity of the analysis, and parallel detection methods are needed for full coverage [107]. Among the biosamples available, the blood, represented by plasma or serum, covers as many metabolites as possible since it flows continuously through the body. Urine is less common but still valuable for caring for the waste metabolites and by-products. While saliva is a non-invasive way to collect a sample [115]. A single sample, for example, if properly prepared, covers all the biochemical classes under investigation, such as lipids, hydrophilic and hydrophobic metabolites, proteins, and macromolecules [116–118]. Indeed, this approach is quite interesting, especially during a pandemic situation, where the evolution of the disease can be deeply assessed and concurrently with the changes caused by the viral replication, with a small sample amount. In the temporal evaluation of the disease, it is useful to direct markers to be monitored and used as the risk of stratification. Thus, based on the host–pathogen interaction and time of infection, the specific mechanisms of action of new or redirected drugs can be assessed [119, 120]. Interestingly, the omics approach is that distinct researchers’ groups might reach similar results even with different cohorts of patients, as in the case of three independent studies of COVID-19 severity showing ~71% of data overlap [116, 121, 122]. The numerous analytes, also called targets or markers, have different physical–chemical properties and abundance levels [123]. Direct data comparison of quantitative presence versus absence is not always possible since scientists worldwide have different equipment and technologies available, although gene symbols and proteins or molecules ids are easier to compare [121]. In the case of compatible identifications, the lipidome/metabolome/proteome can also be compared between different diseases, intending to recognize patterns of an

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effective immune response, for example [111]. One of the most discriminating signatures ratios might be given by the citrulline/ornithine (liver), valerylcarnitine (mitochondrial), glutaminolysis, and kynurenine/tryptophan (immune response), being the last one vastly reported [105, 106, 111, 124]. Researchers have also reported changes in ceramides, arginine, and disturbances in the TCA cycle, among several other compounds [125]. However, there are strong indicators that the kynurenine and tryptophan deserve a closer follow-up [126, 127]. Potential biomarkers are currently being discovered and validated, and this process can reach the values of precision medicine if correctly considering the individual variations (https://www.fda.gov/medical-devices/in-vitro-diagnostics/precis ion-medicine). So far, a consensus on the best severity marker for COVID-19 disease was not reached yet. However, an example of the advantages of using personalized medicine and databases can be checked by the unprecedented project CoVSeq realized in Andalusia, Spain (http://clinbioinfosspa.es/projects/covseq/indexEng.html) [128].

5 Conclusion Although the countless losses are not recoverable, the COVID-19 pandemic has brought significant technological and scientific advances. The possibility of obtaining data about the pathogen-host interaction practically in real-time and worldwide demonstrates that clinical studies allied to the research of severity biomarkers bring new perspectives. The standardization of bioanalytical methods and the sharing of information in databases is not a typical attitude in the field. However, they could help physicians promote quick and assertive prognoses, more effective treatments, and more tranquility in times of uncertainty.

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“Pandemics-on-a-Chip”: Organ-on-a-Chip Models for Studying Viral Infections Amanda Maciel Lima, Jéssica Freire Feitor, Vinícius Guimarães Ferreira, Mariana Bortholazzi Almeida, Laís Canniatti Brazaca, Daniel Rodrigues Cardoso, and Emanuel Carrilho

1 Introduction The organ-on-a-chip (OoC) platform presents as a low-cost alternative for preclinical drug screenings and vaccine development, possibly replacing conventional experiments in animal models, which are time-consuming and have many failures in clinical trials by not being so faithful to the human organism. In addition, research has shown that since the 1950s, there has been a considerable reduction in the number of drugs approved by the Food and Drug Administration (FDA) per billions of dollars spent [1, 2]. In recent years, the global crisis caused by COVID-19 has highlighted the urgent need to develop platforms that are faithful models of the human organism to develop drugs and vaccines that fight viral diseases and reduce the risk of a pandemic. The OoC aims to mimic an organ of the human body, seeking to simulate aspects of the physiological system that favors the formation of morphology, gene expression, and cell signaling that more reliably approximates a tissue of an organ of the human body [1, 3]. Thus, this chapter will briefly introduce OoC in the pandemic scenario A. M. Lima · J. F. Feitor · V. G. Ferreira · M. B. Almeida · L. C. Brazaca · D. R. Cardoso · E. Carrilho (B) Instituto de Química de São Carlos (IQSC), Universidade de São Paulo (USP), São Carlos, SP 13566-590, Brasil e-mail: [email protected] A. M. Lima · V. G. Ferreira · M. B. Almeida · L. C. Brazaca · E. Carrilho Instituto Nacional de Ciência e Tecnologia de Bioanalítica—INCTBio, Campinas, SP 13083-970, Brazil L. C. Brazaca Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA E. Carrilho Bioanalysis, Microfluidics and Separations Group, São Carlos Institute of Chemistry, University of São Paulo, 400, Trabalhador São-Carlense Av, São Carlos, SP 13566-590, Brazil © The Author(s), under exclusive license to Springer Nature Switzerland AG 2023 F. N. Crespilho (ed.), Covid-19 Metabolomics and Diagnosis, https://doi.org/10.1007/978-3-031-27922-5_8

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and discuss the challenges of studying viral diseases, such as COVID-19. The cell studies must be carried out in Biosafety Level 3 (BSL-3) facilities, and there is a need to design a safe chip model with the choice of biocompatible materials that can be suitable for manufacturing OoC in these studies. This chapter will also outline other topics, such as presenting the main OoC prototypes described in the literature and highlighting successful studies using this technology. In addition, the use of OoC to learn about infection kinetics, virus-host interactions, drug responses, and vaccine development is described. Also, here we will discuss coupling techniques to OoC for online and offline biomolecule detection that can be implemented for viral diseases. Moreover, the chapter addresses the importance of the OoC for a pandemic scenario (pandemic-on-a-chip).

2 Organ-on-Chip in the Pandemic Scenario With the COVID-19 pandemic caused by the new coronavirus—Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), the last few years have been marked by an unprecedented political, economic, and public health crisis in world history. Although several vaccines have been developed to combat SARS-CoV-2, the virus mutated rapidly, and variants with reduced vaccine sensitivity to the original strain emerged [4]. Patients affected by COVID-19 can be classified as asymptomatic or have symptoms related to respiratory problems and the gastrointestinal tract, for example, and can evolve to an inflammatory condition that favors lesions in several organs [5]. Therefore, there is an immediate need to improve the compression of the inflammatory action of this pathogen in the human body and search for drugs with antiviral properties to develop an effective therapy [6, 7]. Generally, virology studies are performed in animal models and 2D cell cultures to understand the effects caused by a viral infection, develop vaccines, and investigate therapeutic targets [8]. Studies conducted with SARS-CoV-2 demonstrate that the Spike (S) protein favors the invasion of the virus into the target cell by binding to protein receptors on human cells, such as Angiotensin-Converting Enzyme 2 (ACE-2) and transmembrane serine proteinase 2 (TMPRSS2) [9]. To study the mechanism of viral invasion, some 2D cell cultures that present these receptors were used for a drug investigation, isolation, and replication of SARS-CoV2, such as Vero E6 [10], Calu-3 [11], and CaCo-2 [12]. Monolayer cell cultures are often used in vitro assays by culturing plates and/or flasks under highly controlled static cultivation conditions and well-established protocols for cell-specific culture media, pH, temperature, and CO2 level. Although this traditional culture technique has played a crucial role in drug discovery science through high-throughput screening and vaccine development, the results are generally not as efficient due to the lack of similarity to the organ’s physiological environment [13–15]. Some animal models usually used in clinical trials, such as rodents, are not natural hosts of the virus [5]. Thus, some genetic modification studies were carried out with rodents, seeking to favor the formation of transgenic animals that can express

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ACE2 [16] that confer pathogenicity in these animals. There are reports in the literature showing that other animal models can be used for viral investigation, such as primates (Rhesus macaques and Cynomolgus monkeys) [17, 18] and ferrets (Mustelidae family) [19]. Bear in mind that these models do not reflect substantially similar responses to SARS-CoV-2 infection due to differences in the animal and human systems [5]. In vitro alternatives using organ-on-a-chip biomimetic devices have great potential to replace animal and plate models in preclinical stages for drug toxicological testing [20]. Thus, studies indicate that this device can precisely mimic an area of an organ considered a “histological section.” OoC mimics some aspects of the complexity of cellular organisms, such as (i) cell– cell interaction of vascular interfaces with epithelia, as in the lungs, (ii) mechanical and electrical stimuli, as simulating heartbeat in the heart, and nerve impulses in the brain, respectively, (iii) application of a dynamic microflow environment (10–9 a 10–18 L min–1 ) that promotes fluid, gas, shear stress, and surface chemistry over the cells, mimicking, for example, the current of a blood flow (Fig. 1). So far, numerous microdevices have been developed, including the heart, brain, kidney, veins, intestine, liver, and muscle [13–15, 21, 22]. Thus, for the respiratory disease model, in 2010, Huh presented the first lung model on a chip, being considered a microdevice that simulates lung characteristics, creating a cell–cell barrier through the co-culture of epithelial and endothelial cells, lung and vascular, as well as recapitulating the movement of breathing [21]. In this pioneering work, a polydimethylsiloxane (PDMS) device with three layers was created, two layers containing micropatterned channels where epithelial and endothelial cells were cut, with a porous membrane between them. In order to mimic physiological aspects of the alveolar composition, an airflow was applied to the apical chamber, and a microflow of culture medium was maintained in the vascular chamber. In addition, a parallel chamber with a cyclic vacuum application (10% cyclic tension at 0.2 Hz) was placed to mimic lung breathing movement. The effect of these mechanical stimulations was investigated from an immunological point of view through pulmonary edema, attracting inflammatory cytokines (Tumor Necrosis Factor-α, or TNF-α) into cells and by measuring the expression of InterCellular Adhesion Molecule-1 (ICAM-1), significant immunological differences were observed in the response of chips submitted to mechanical movements of breathing. Furthermore, these also revealed the identification of potential new therapies concerning IL2 toxicity, such as Angiopoietin-1 (Ang-1) and a new Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel inhibitor (GSK2193874) [20, 21]. Currently, in the context of a pandemic, studies carried out with lung-on-a-chip have already demonstrated that this platform can mimic the inflammatory responses of SARS-CoV-2 in lung cells [23] and contribute to the mechanisms of drug discovery be used as prophylactic and therapeutic measures to combat COVID-19. Drugs can be considered a low-cost alternative as preventive measures in countries with no financial resources to obtain vaccines [13, 24]. While OoC has been quite promising, it still lacks some elements to make it a standard protocol capable of performing preclinical tests. For example, the dimension or

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Fig. 1 a Advantages and disadvantages of different preclinical models that can be used in virology studies and drugs toxicology assays, being animal models and 2D cell culture the most used ones, while the organ-on-a-chip platform has emerged as a promising alternative to mimic physiological system features. b Illustration of an OoC and the critical aspects of human physiology it aims to recreate. Created with BioRender.com

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thickness of the membrane, aiming to recapitulate epithelial and vascular interfaces, does not accurately simulate the thickness of blood vessel membranes (300–400 nm). Furthermore, most OoC models use immortalized cells lacking primary features of human cells, such as cell structure, morphology, and metabolism [20]. Thus, the scientific community and funding bodies such as the US Food and Drug Administration (FDA) and the National Agency for Institutes of Health (NIH) are not measuring efforts to improve the OoC platform [1, 2, 14].

2.1 Microbiological Challenges for Viral Disease Research in OoC Although OoC platforms are advancing rapidly, several challenges still exist to replace traditional in vitro and in vivo experiments, mainly when researching diseases of pathogenic organisms such as viruses. The study of viral and microbial pathogens requires biosafety norms, which correspond to standard operating procedures to carry out safe research, and any failure in one of these protocols can be a risk to the health of researchers and the population. Four main controls are followed in biosafety laboratories: laboratory engineering control, in which the facility must conform to a specific design; personal safety equipment and a biosafety cabinet; an administrative control that provides training; and laboratory access control. Within this context, the biosafety level (BSL) can be determined by the nature of the infectious agent or biological risk. As a result, in the United States, laboratories follow safety levels designated with BSL-1, BSL-2, BSL-3, and BSL-4, which provide standards for safe work. The study of pathogens can vary from levels BSL-2, BSL-3, and BSL-4, depending on their risk [13, 22, 25–27]. Because the OoC is not yet considered a standard protocol for biological assays, there are no regulations that can be consulted to direct its biomanufacturing for virology studies. However, we raise some observations to consider in manufacturing and handling OoCs for viral studies. Some suggestions that can guarantee the analyst’s safety when using the OoC with the pathogen are: the manufacture of a biodevice that must be highly sealed so that it does not present risks of leakage between the layers of the chip; as well as guaranteeing the second layer of protection on the chip, which can be wrapped in protective support, such as the biosafety box, to minimize leakage risks; not present risks of leaks in the microfluidic inlet and outlet channels; fluid disposal should be performed by suitable and readily available decontamination methods (Table 1). The first obstacles to be observed by the researchers were mainly related to the infrastructure of the OOC research laboratories. Considering that conventional research is carried out in BSL-2 laboratories, to develop the study of infectious viral diseases is necessary to have a BSL-3 laboratory (Table 1). To circumvent the biological risks and simulate the SARS-CoV-2 and lung-on-chip (Airway-Chips) interaction, the researchers decided to reduce the risk of infection and work in the

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Table 1 List of key biosafety elements that should be employed in the lung OoC platform with pseudotyped SARS-CoV-2 and native SARS-CoV-2 [13, 22, 25–28] Lung OoC

Airway-chips

Human alveolar epithelium

Virus

Pseudotyped viral particles (SARS-CoV-2pp)

SARS-COV-2

Biosafety elements for OoC BLS-2 Lab engineering • Entrances must have self-closing doors • A sink and eyewash station must be readily available Safety equipment • PPE including lab coats and gloves, eye protection, and face masks • Procedures performed in the biological safety cabinet • Available decontamination methods (e.g., Autoclave) Laboratory policies • Access to the laboratory is restricted to the researcher OoC • Biodevice without risk of leakage in microfluidic inlet and outlet channels • Fluid disposal carried out by decontamination method

BLS-3 Lab engineering • Entrance through two interlocked and self-closing doors • A hands-free sink and eyewash station are available near the entrance doors • Exhaust air cannot be recirculated, and the laboratory must have a one-way air flow Safety equipment • Appropriate PPE must be worn, and respirators may be required • Procedures performed in the biological safety cabinet Laboratory policies • The researcher should receive immunizations from the infectious agents or toxins they work with, if available • Access to the laboratory is permanently restricted and controlled OoC • Biodevice risk-free of leakage in microfluidic inlet and outlet channels • Biodevice poured with protective support, such as a biosafety box, to minimize biological hazards • Fluid disposal carried out by decontamination method

BLS-2: BioSafety Level 2; BLS-3: BioSafety Level 3; PPE: Personal Protective Equipment; SARSCoV-2pp: pseudotyped viral particle of SARS-CoV-2

BSL-2 laboratories. Based on the genomic sequence of SARS-CoV-2 deposited in GenBank, a pseudo-type viral particle (SARS-CoV-2pp) that expresses the Spike (S) protein was used by Si et al. [13]. The S protein is the main gateway to some human cells by using angiotensin-converting enzyme 2 (ACE2), essential in studying COVID-19 [13]. Then, another study promoted the first successful attempt to cause infection of human alveolar epithelium by SARS-COV-2 in OoC. With this, it was

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possible to verify the immune response of lung cells by increasing the inflammation by releasing cytokines and cell detachment, suggesting an intense inflammation in the alveolar barrier observed in the clinical picture of COVID-19 [28].

2.2 Biofabrication of Organs-on-a-Chip for Viral Disease The OoC models are mainly developed by joining microengineering and cell biology concepts [29]. The structure of these 3D microfabrics features multilayers of polymeric biomaterials or glass, which contain hollow microchannels (in scale from nanometers to micrometers), and aim to grow cells to mimic the specific microarchitecture of an organ in vitro [22]. To design an OoC, the physiological characteristics of the organ and the experimental goals on the platform must be considered in advance [14]. Thus, to design a robust and reproducible chip for a viral disease, one must consider some factors, such as chip design, techniques used in biofabrication, and microfluidics (Fig. 2).

Fig. 2 Fabrication of organ-on-chip for viral infections steps. (1) Design of OoC, (2) the microfluidics submitted the channels through a syringe pump that can evaluate the shear stress caused by the flow of the medium culture. (3) The biomanufacturing of OoC can be carried out by material cutting (laser cutter), 3D printing, and soft lithography. Created with BioRender.com

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Organ-on-a-Chip Design and Biofabrication

Biodevices can be designed in specialized software (AutoCad™ and CorelDraw™), in which all the layers that will compose the microchip are drawn, containing upper (apical) and lower (basal) microchannels created to grow cells. Cells grown in these channels can interact through a barrier, usually porous (pores of 10–0.4 μm), between both cell channels [22, 29]. When creating the architecture of the OoC for virus study, it is essential to consider the dimensions of the biodevice, the width and the height of the channels, and the size of the membrane pores [2]. The dimensions of the channels, corners, and entry and exit ports of the microchip must be carefully studied. These characteristics influence the resulting forces on the biological tissue, such as shear forces and increased pressure within the microchip, which may provide dangerous biological leaks [30]. The dimensions of the channels can also be adjusted for the organ of interest. For example, the channels of an intestine chip have greater width and height than those in a lung chip, seeking to favor the simulation of intrinsic characteristics of the intestine as a peristalsis-like movement instead stronger shear stresses experienced in veins or arteries [22]. Entrance channels maintain biological tissue sterility while allowing virus entry and culture medium exchange. Furthermore, in the design stage, importance should be given to the number of cells that will grow in the microchannels [22, 30, 31], bearing in mind that the number of cells must meet the minimum analytical requirements for the detection and analysis of products secreted by them. Recent studies of viral infection with SARS-CoV-2 on an alveolar-pulmonary barrier chip have shown that lesions are caused by the virus in cells, projecting cell deaths and, therefore, reducing the number of viable cells for further studies [15].

2.2.2

Microfluidics

Through the different microchannels of the OoC, nutrient concentration gradients can be constantly favored for the cells. A culture medium is flown in a controllable manner using syringe pumps, peristaltic pumps, or pressure-control systems [17, 18]. Pathogens, drugs, toxins, and cytokines can be administered to perform experimental assays on both cell types or mimic transmembrane transport. Furthermore, shear stress can be simulated in the tissues, exploring the chip’s actual characteristics related to the shear stress in the blood vessels [32]. According to fluid mechanics, shear stress is generated when a tangential force in a fluid generates stress on a surface. Therefore, the shear stress in the living organism is the friction force of a biological fluid on a cell or tissue surface. Therefore, in OoC microfluidics, it is crucial to evaluate the shear stress caused by the flow of the culture medium in regions of the body that features flow, such as veins, arteries, and even renal cells. Studies in the literature have shown that hemodynamic forces caused by fluid friction confer morphological changes on endothelial cells, such as elongation of cells in the flow direction. Furthermore, shear stress can alter gene expression,

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cell signaling, actin distribution, reorganize cytoskeleton, and align microtubules [32, 33].

2.2.3

Potential Materials and Fabrication Techniques for OoC Devices

The material used in the fabrication of the viral OoC must be transparent and biocompatible and have the properties to seal the 2D layers to form the 3D device firmly. Materials that have been widely used in microfluidics are biocompatible polymers such as polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polycarbonate (PC), polystyrene (PS), polyvinyl chloride (PVC), polyimide (PI), and olefin polymers cycles [29, 34]. PDMS is the most used among these materials, allowing the soft-lithography technique, which relies on the deposition of soft organic or polymeric matter using molds containing the desired features. Soft lithography allows rapid prototyping, enabling the formation of desired micro or nanostructures in the chip layers, and is usually followed by a gluing step, which can be performed using plasma treatment in the case of PDMS and glass, for example [29, 34]. A disadvantage of PDMS is its high absorption rate of small hydrophobic molecules, which is a problem for drug studies [2]. Another quick and easy biofabrication method that can be applied for the OoC of viruses is 3D printing, in which a computer design is used to deposit the materials layer by layer. A photocurable resin solidified when UV exposure can be applied in this case [35]. In addition, rapid, facile, economical fabrication techniques use a laser cutter and assemble the layers of the device. This method used thermoplastic material such as PMMA sheets and 3 M® tape to be a viable alternative to minimize steps in the fabrication process and have reliable sealing and robust chips that avoid leaks to chips that can be applied to study viral diseases [36].

3 The Study of Viral Infectious Diseases Using Organ-on-a-Chip Originally, OoCs were manufactured to meet the pharmaceutical industry needs. The most common applications of OoCs are for drug pharmacokinetics and disease awareness, even though this technique does not yet replace the in-use methods such as conventional 2D and 3D culture and animal testing. Undoubtedly, it is more of a comparative assay [37, 38]. As previously mentioned, the current methods used for drug screening and disease evaluation present large failure rates in predicting clinical trial results. OoCs are being extensively explored to decrease these failure rates to become a more precise and comparative method to the human system [39, 40]. It is possible to find several types of OoC in the literature, each one with a specific objective. Depending on the

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aims, the device may present one or more types of cell culture, and different designs are available for academic and industrial use (Fig. 3) [1]. This topic collects the recent applications of OoCs for infectious diseases focusing on organs with ACE2 as a receptor for SARS-CoV-2. Also, it covers what is known about the host-vector relationship, the kinetics of contamination, the evolution of viral diseases, and the responsiveness of drugs and vaccines, especially for COVID-19 target organs.

Fig. 3 The purposes of organ-on-chip technology for infectious viral diseases. On top of the cut view of each organ-on-chip, including a intestine, b lung, c kidney, and d liver. At the bottom are the potential applications, including the study of viral mechanisms, the development of vaccines, and the progress and study of new drugs. Source Created by FEITOR, J.F. with BioRender.com

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3.1 Gut-on-a-Chip The gastrointestinal (GI) tract is a challenging system to develop due to its dynamicity and the complexity of its structure. Animal testing has proven to be inefficient for evaluating infectious diseases compared to humans, as this methodology often shows a lack of similarity in physiological factors. Conventional in vitro testing also cannot represent the complexity involved in the GI tract, lacking key factors such as the constant fluid flow [24, 41]. Several models of intestine-on-chip and gut-on-chip [29, 42, 43] were developed and reported in the literature as capable of simulating the human GI tract for specific physiological and pathological behaviors [44]. Typically, a gut-on-chip is composed of a hollow microchannel containing a porous membrane that interfaces an apical and a basal side. This architecture is an essential feature because it recreates a similar environment to the intestine. Generally, two types of cells are cultured inside these chips: intestinal epithelial cells on one side and vascular endothelial tissue on the other. Other important characteristics are the mechanical pulse (to mimic the peristaltic movements) and the constant fluid flow (to mimic blood and nutrients flow) present in these models with the assistance of peristaltic and syringe pumps. Figure 3a shows a cut view of a general intestine-on-chip for viral studies. The most advanced models (Gut Chips) can co-culture even immune and microbial cells with the characteristics as mentioned earlier [44]. With all these factors, it is possible to precisely study parameters such as pharmacokinetics, drug absorption, disease development, and infection dynamics, among others, in an out of equilibrium system. In the past decade, with the development of more evolved models of gut chips, the possibility of evaluating infectious diseases became a reality. It was proven that these prototypes could recapitulate human responses at the transcriptional, metabolic, and immunological levels [45]. A recent study [46] has shown that an intestine-on-chip prototype evidenced an intestinal infection by two types of coronaviruses, SARSCoV-2 and NL63. This study detailed essential receptor interactions and infection mechanisms, and some meaningful insights about drug responses were revealed. The most important feature found in this study is that the organ-on-a-chip system was comparable with the results observed in humans, while conventional 2D and 3D culture methodologies failed to give similar responses to the human setup. Another relevant study [42] with coxsackievirus B1 (CVB1), an enterovirus, showed that a guton-chip faithfully reproduced cytopathic effects of the gut infection. It was possible to compare the cytokines (immunomodulating agents) released via different infection routes, which is very important since animal and conventional culture models cannot provide precise responses. Both mentioned studies show how powerful OoC can be as a tool for studying intestinal diseases and defining their potential cure.

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3.2 Lung-on-a-Chip A lung is a remarkably complex organ to simulate because of all the many tissues making it up. Animal models and traditional cell culture are well developed to study respiratory viral diseases like influenza. However, those methods often provide false adaptive mutations on behalf of nonhuman systems [24, 41]. With the increasing necessity of having a more loyal technique, the Lung Airway Chip was developed for influenza evaluation purposes [47, 48]. Like any current Lung-on-Chip prototype, the Lung Airway Chip comprises two hollow channels separated by a porous membrane that defines an air–liquid interface. Epithelial lung cells are established in the air interface, while endothelial microvascular lung cells are grown in the liquid (culture medium) interface. This whole system allows the establishment of a microenvironment to recapitulate critical behaviors such as barrier function and mucus secretion, which are essential for the study of infectious diseases [48, 49]. Figure 3b represents the Lung Arway Chip for viral infection. A few works have reported using lung-on-chip devices to study viral infections to show tissue disruption, malfunctioning, significant specific molecule production, and virulence [47, 50–52]. Two influenza studies demonstrated relevant factors for the propagation of this virus within the body, such as the production of serine proteases by the infected cells [47, 48]. For these studies, it was essential to simulate an airway in contact with a vascular way since respiratory viruses commonly infect the airway to reach the blood flow. This behavior is observed in the lung chip, in which influenza enters the airway affecting both epithelial and endothelial tissues, causing issues that lead to barrier function despair and production of immunomodulating agents. Lungson-chip is also helpful in studying host-parasite interactions, promoting insights on mechanisms of the infection and the host organism response. Different types of strains can be analyzed (H1N1, H3N2, and H5N1), and it is possible to define the virulence of each strain and the interactions among them under a co-infection situation. Considering this scenario, it is viable to determine drug efficacy and its ability to generate mutations. In addition, two or more microchips can be connected to simulate a populational virus transmission, defining even more critical parameters such as molecules and organ-organ interactions [47, 48]. The study of two promising drugs, amodiaquine, and toremifene, for COVID-19 treatment, revealed their potential as inhibitors to the virus in the airway, with one of them being a potential inhibitor and the other one not [47, 51, 52]. Moreover, essential aspects such as replication mechanisms, evolutionary factors, infection rate, and the host response performance are also being investigated for SARS-CoV-2 [51]. Finally, other respiratory diseases are also being studied in the lung chip, such as asthma caused by viruses [52]. Scientists can now establish parameters for lung infections that may lead to a better understanding of how these organisms act, evolve, and persist in the human body. With the same tool, it is now feasible to elucidate drug efficacy and pharmacokinetic parameters for a more efficient and ethical launch of these products.

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3.3 Kidney-on-a-Chip The complexity of organs is widely discussed, and the kidney is a very sophisticated system containing multiple types of cells. It is essential to study this organ’s structure and mechanisms mainly because of its potential in the metabolism of drugs and nutrients. As already mentioned, 2D traditional cell culture is very limited in the sense of not being able to support more than one cell culture type and not having a dynamic flow. Besides this, conventional in vitro analysis is associated with the lack of key proteins, transporters, and metabolites produced in vivo [53]. The most recent development of a model for virus infection on the kidney is a distal tubule-on-a-chip (DTC). This chip is fabricated in the typical three-layer way, in which a porous membrane supports Madin Darby Canine Kidney (MDCK) cells while the basal side of the chip keeps a static culture media pool. The apical side of the chip allows the constant perfusion of culture media, simulating the shear stress caused by the flowing pro-urine on top of the cells [54]. Figure 3c shows a cut view representation of the distal tubule-on-chip. Under pseudorabies virus (PRV) infection, this microchip was capable of simulating responses that are normally observed in human subjects. This virus disrupted the epithelial barrier, leading to a series of electrolytes disbalance and finally generating signaling and transport pathways issues. Ions such as Na+ , Cl– , and K+ could be identified in this system as part of the misregulation caused by the virus. This chip can be a tool for evaluating important mechanisms involved in the host-parasite relationship and a safe way to test drugs and their metabolites that could be harmful to the kidney. Furthermore, this chip proved to be effective for this specific virus infection behavior, although improvements in this system are required to mimic the whole complexity of this organ and predict relevant outcomes better [55].

3.4 Liver-on-a-Chip The liver is an intense object of study since it is an organ with frequent disease development and an essential organ for drug metabolism. Overall, hepatic diseases lead to inadequate performance and sometimes exhaustion of liver functionality, making elucidating these disorder mechanisms and potential drug candidates fundamental [56]. It has been reported in the literature [57–59] a series of studies involving in vitro evaluation of common liver disorders with 3D primary liver cell models. Although these studies were essential to understanding examples of fatty liver, metabolic dysregulation, and viral hepatitis, it was observed that the in vitro approach had made cells lose essential functions in a short time. These examples and a few more from studies involving 2D culture of primary liver cells had proven to be ineffective due to their limitations around mimicking both the liver morphology and the interactions between host and pathogens [56, 60]. Animal testing has also proven to work only as an alternative technique since viral mechanisms are different for distinct

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organisms. Furthermore, the most common viral liver disease, hepatitis, does not affect animals as it does in humans [60]. With the urge for a method that closely resembles the human liver, a few liver microdevices were developed to study mainly liver functionality, metabolism, and biological processes of infections. Improvements in the expression of critical signaling agents and transporters, and the structural conformation were observed, obtaining results more similar to those observed in the human liver [61]. An important virus that affects the liver is the hepatitis B virus (HBV), which might cause issues leading to cirrhosis and liver cancer under chronic infection [62]. To investigate such impacts caused by the virus, important advances were achieved using liver chips cultured with animal pulmonary cells. A combination of rat hepatocytes and bovine aortic endothelial cells was established in this chip, obtaining consistent differentiation, morphology, and function and finally leading to the progression of a human cell model [60]. It was possible to investigate the infection process using the human cell model, the viral gene behavior, and the host immune system performance. Figure 3d shows a representative liver-on-chip. More advanced models of liver chips were created later. Ortega-Prieto et al. [59] reproduced a liver chip with a co-culture of macrophages and primary human cells, which brought more fidelity to this system compared to the real one. Important routines were observed in this device facing viral contamination, such as the secretion of specific enzymes, proteins, and immune factors [59]. The gradual advance in these chip models can contribute enormously to understanding the disorders and the development of drugs and treatments in more effective and prompt means.

4 Analytical Techniques Applied in Organ-on-a-Chip Models Ever since it was featured as one of the top ten emerging technologies by the World Economic Forum in 2016 [14, 63], the OoC devices had many additional features developed to enhance it, including forms to perform cell count, pH analysis, and newer analytical measurements of biomarkers [64, 65]. As the reader reviewed in the previous sections of this chapter, OoC is an excellent platform for biomedical and drug discovery applications; however, paradoxically, the low cell count in the OoC limits the available material for analytical measurements and consequently limits the concentration of the biomolecules, limiting not only the number of analyses that can be performed per device but more importantly, increasing the difficulty in achieving precise quantifications, due to the necessity of very low limits of quantification and detection [66, 67]. Accordingly, in the past years, several analytical chemistry scientists have been spending resources and efforts to develop new platforms and protocols to enable direct measurements in the OoC or even hyphenate the devices with standard analytical techniques such as electrochemical monitoring, enzyme-linked immuno sorbent

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assay (ELISA), liquid chromatography coupled with mass spectrometry (LC–MS), and lab-on-a-chip devices [64, 68–70], permitting fast, cheap, and in some cases, real-time analysis. In this section, the reader will be introduced to analytical technologies integrated with OoC, and the latest breakthroughs in analytics applied to virus studies will also be pointed out.

4.1 Detection Methods Adapted to OoC Recently, the OoC fabrication process can be considered highly similar to modern low-cost biosensors, developed with paper or PMMA, enabling easier integration between the devices [67, 72]. Biosensors are devices that use a bioreceptor (antibody, DNA, aptamers, among others) to recognize specific targets in biological samples or fluids, and its transducer transforms the signal into analytical measurements [73]; hence, the integration of biosensors with OoC permits the analytical assessment of compounds or physiological aspects directly in the device. Amongst biosensors, electrochemical and optical instruments are the most commonly integrated with OoC for compound analysis, while electrical biosensors are frequently used to access cell properties [65, 67, 74]. Cellular mechanical properties such as the tight junction formation in co-culture OoC are currently performed by impedance sensors, which measure the electrical resistance between the apical and basal channels [75–79]. An increase in impedance indicates the formation of a tight junction [80], an intercellular intersection responsible for bar passage of fluids and molecules in between the cells; therefore, its formation composes a barrier that increases the resistance of the current passage. Such sensors are especially interesting for evaluating the absorption of particles, drugs, and nutrients in specific organs, such as the lung, liver, and intestine. The tight junction is essential to ensure the integrity of the cellular monolayer and properly simulate the organ tissue. Currently, its formation is measured by specific voltohmmeters or dyes, such as ZO-1 immunostain [81–83]. In pursuit of optimizing OoC devices, several researchers have been using the measurement of tight junctions, or transepithelial electrical resistance (TEER), to different types of organs. TEER can also be adapted to an already established labon-a-chip system, Intelligent MObile LAb for In Vitro Diagnostics (IMOLA-IVD), to the OoC [74]. Table 2 summarizes a few examples of different organ models on-achip and their assessment methods, like TEER, impedance, capacitance, or LC–MS, highlighting the in-situ measurements. Such devices can be considered as proof of principle and are fascinating if adopted in commercial OoC. Electrochemical biosensors (ECB) have also been adapted for OoC devices, focusing on detecting biomarkers [65, 88]. From the authors’ perspective, although different analytical techniques enable an enormity of different projects and objectives, we believe that ECBs are potentially the most suitable type of sensors for direct integration with OoC, mainly due to their characteristic high sensitivity and, in some cases, their capability of real-time continuous analyte monitoring, which could be a

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Table 2 Summary of organ-on-a-chip models and target detection modes Organ on-a-chip

Target/goal

Detection method/measurement

In-situ References

Skin

Skin integrity, toxicity

TEER

Yes

[74]

Blood–brain barrier

Drug screening

TEER

Yes

[84]

Gut

Tissue differentiation

Impedance spectroscopy, TEER

Yes

[85, 86]

Liver

Drugs toxicity

Impedance, TEER, colorimetric, ELISA, pH, albumin

–

[77]

Amperometry

Muscle

Cytokine levels

Yes

[87]

Multiple

Multiorgan interaction, Cyclic voltammetry and Yes drug screening electrochemical impedance spectroscopy (EIS)

[88]

Placenta

Transport of molecules Offline LC–MS through the placental barrier - fetus

[89]

Intracellular

Drugs detection

No

Online MS

Yes

[90]

Human–Multiorgans Tolcapone effect

Offline LC–MS

No

[91]

Blood–brain barrier

Offline LC–MS

No

[92]

Inflammation metabolic pathway

Renal barrier

Mass transfer

UV–Vis

Yes

[93]

Kidney

Nephrotoxicity

Fluorescence microscope

Yes

[94]

TEER: Transepithelial or transendothelial electrical resistance measurements; LC–MS: Liquid chromatography coupled to mass spectrometry

game-changing characteristic in biochemical process research. Between ECBs, we can highlight the Electrochemical Impedance Spectroscopy (EIS) and Amperometric based biosensors. Each works by measuring a specific physical or electrochemical phenomenon occurring on the biosensor surface. EIS works by measuring the change in one impedance element, i.e., resistance or capacitance, on the electrode surface as function of the analyte concentration [95]. EIS biosensors are commonly label-free, which means they use a single bioreceptor for analyte detection, and with the analytes binding to the bioreceptor, the charge transfer resistance is altered, originating a signal [86, 96]. Amperometric biosensors, in its turn, measure the electrical activity from oxidation or reduction reactions occurring in the system. Some molecules enable direct analysis due to a redox reaction on the applied potential [96]; on the other hand, for molecules that do not react in the desired potential, a secondary bioreceptor is commonly employed in amperometric biosensors, besides the first bioreceptor linked to the electrode surface [96]. The second bioreceptor contains a catalyst site for a specific redox reaction, and it binds to the counterpart of the analyte bound to the

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first bioreceptor. Therefore, a higher concentration of the analyte leads to a higher concentration of the secondary bioreceptor, leading to higher reaction rates and higher electrons release [96]. Although the system does not enable constant monitoring, it is fully integrated with the OoC, facilitating the time-point analysis. However, OoC can be constructed as a multi-modular system integrated with multiple real-time sensors, including electrochemical biosensors, allowing the in-situ monitoring of different biological activities [87]. The optical-based analysis is the most widespread and versatile analytical measurement in OoC devices, being applied to a wide range of analytes, from phenol red analysis for pH measurement to fluorescent stains and labels to perform protein quantification, imaging of protein expression, and cell count [67, 97, 98]. A special advantage of optical analysis is the easy device integration with the analytical platform, simply hyphenating the OoC output to the optical equipment, such as a UV–vis spectrometer, or the insertion of the OoC in a fluorescence microscope [67, 99, 100]. Metabolite analysis and biomarkers discovery research are other exciting applications for OoC; however, considering that OoC is an inherently miniaturized platform, the amount of analytes present in the devices can be regarded as low, requiring extremely sensitive equipment to reach the necessary low limits of detection and quantification [66]. Some biochip models were developed to allow real-time UV– Vis measurements in a continuous flow for a few small molecule studies, such as the experiments to understand the mass transfer on the tight junctions of tissues. However, a spectrophotometer does not present an excellent detection limit, and biomolecules are usually present in the organism in low concentrations. Thus, to mimic the system and reach the expected sensitivity, the online monitoring of molecules must be performed by robust detectors, such as mass spectrometers (MS) [93]. Currently, the most applied analytical instruments in biomarker research consist of MS integrated with liquid chromatography (LC–MS), nano liquid chromatography (nLC–MS), capillary electrophoresis (CE–MS), or paper spray (paper spray-MS), which enables analysis of target analytes or even a full scan of several metabolites or proteins present in the samples [69, 101–104]. A great example of this approach is measuring trace levels of molecules that might be considered inoffensive for adults, but when trespassing the placenta barrier is harmful to the fetus, as in the case of caffeine [89] (Table 2). The integration of OoC and mass spectrometry can be performed in two main approaches, online and offline. The online analysis consists of the direct application of the OoC fluids in an MS-based system (regardless of separation techniques), whereas offline approaches compromise the sample collection from the OoC followed by a sample preparation step prior to MS analysis [105]. Following the advance of paper-based devices, the OoC models found an excellent opportunity to control 3D cultures of cells on the paper substrate, allowing physical sections without the need for optical or histological sectioning [106]. We must remember that paper-spray ionization devices for direct analysis by mass spectrometer are already known for their great advantages like low cost and minimal sample handling [107, 108]. Thus, these two techniques combined created a new scenario for simultaneously tissue engineering and analytical measurements, although there exists the single-use limitation [90].

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4.2 Methods Applied to Viruses Studies on OoC As previously discussed in Sect. 3, OoCs have enabled a more realistic assessment of viral effects and viral entry in the host cells. Among all the techniques available, fluorescence is the most applied detection method for cellular processes investigation, such as the visualization of tight junctions [47, 54], barrier function [13, 32, 34, 57], cell type differentiation [34], viral particles [13, 57, 58], and live/dead cells [13, 23, 42, 45, 46, 52, 54, 71]. The variety of commercial fluorescent-labeled dyes and its facilitated application make this technique highly viable and easy for OoC employment. In contrast, a few papers also applied offline analytical technologies, such as ELISA tests for cytokines and chemokines evaluation [13, 57], and western blot analysis for specific protein analysis [13, 23, 42, 54, 57], and even LC–MS based proteomics [13]. The offline protocols enable state-of-the-art analytical technologies; however, it also increases the difficulty and time–cost of the analysis. Although host–pathogen studies on OoC have been performed, virology applications are more recent in OoC devices, representing a new area for drugs and vaccine development and is a platform for careful studies on how the virus acts in the host cells [28]. Like everything new, the methodological aspects of previous OoC models can be used as inspiration, and we anticipate the application of biosensors directly integrated into the OoC for future virus-related projects, enabling specific measurements directly on the devices. Notwithstanding, the analytical protocol adopted in OoC devices is dependent on the project goals, and the use of online or offline analytical protocols depend on the project needs. With the ongoing efforts for miniaturized analytical techniques and minimal sample manipulation, the online assays on OoC are promising and still have room to grow and overcome the limitations of studies, particularly for studies that mimic multi-organs interactions.

5 Conclusions and Future Perspectives Although relatively recent, OoC technology has already demonstrated its potential to revolutionize the study of the behavior of biological systems. Due to its unique microfluidic designs and characteristics such as malleability and selective porosity, it is possible to closely mimic the microenvironment of different tissues and/or barriers present in vivo, achieving more reliable results, especially in the study of diseases and in drug and vaccine development. Among the many existing microdevices, lung-, gut-, liver- and kidney-on-a-chip have been the most used in the study of SARSCoV-2 due to the importance of such organs in disease development. The use of these devices was able to bring important information that is not obtainable by static 2D or 3D cell cultures, including the integrity of interfacial layers, a better-modeled inflammation response, and drug efficacy studies that promoted results more closely related to the human body. Furthermore, the integration of analytical techniques into

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OoCs allows unprecedented opportunities to study specific biomarkers and understand disease development, an essential tool for finding effective treatments. With such statements, it is clear that OoCs are an essential tool for combating pandemics, allowing the simultaneous testing of different drugs in human-like models without any harm to living beings, making vaccine developments quicker and more efficient and unraveling the diseases’ unique mechanisms. Soon, we expect OoCs to become even more robust tools, with standardized and commercial protocols for producing devices to closely mimic specific organ structure and access a variety of biomarkers. Furthermore, we expect these devices to be increasingly used in personalized assessments for costumes medicine. Evaluating the effects of a drug on a specific type of individual (elderly, newborns, or diabetics, for example) is a good example of predicting specific harmful health effects.

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