Nitric Oxide Sensing [1 ed.] 9814877670, 9789814877671

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Table of contents :
Cover
Half Title
Title
Copyright
Contents
Preface
1. Introduction
2. Small-Molecule and Metal Complexes as Nitric Oxide Sensors
2.1 Introduction
2.2 Nitric Oxide Homeostasis
2.3 Fluorescent Detection Kit for NO Sensors
2.3.1 Different Strategies
2.3.2 Fluorophore Displacement without Metal Reduction
2.3.3 Metal Reduction without Fluorophore Displacement
2.3.4 Metal Reduction with Fluorophore Displacement
2.4 Metal Complexes for NO Sensing
2.4.1 Cobalt Systems
2.4.2 Iron Systems
2.4.3 Reversible NO Sensing by Rhodium Complexes
2.4.4 NO Detection with Ruthenium Complexes
2.4.5 Copper Complex as a NO Sensing Probe
2.4.6 Copper (II) Conjugate Polymer
2.4.7 Copper (II) Anthracyl Cyclam Complex
2.5 Polymer-Based Sensors
2.6 Biomedical Applications
2.7 Conclusion
3. Nitric Oxide Sensing with Carbon Nanomaterials
3.1 Carbon Nanomaterials
3.2 Nitric Oxide Sensing with Carbon Dots
3.3 Nitric Oxide Sensing with Carbon Nanotubes
3.4 Nitric Oxide Sensing with Graphene
3.5 Conclusion
4. Electrochemical Nitric Oxide Detection
4.1 Introduction
4.2 Scope of Electrodes for NO Detection
4.3 NO Detection on Noble Metals and Pt Electrodes
4.4 Biosensors with Modified Electrodes
4.5 Metallocycle-Modified Electrodes
4.6 Nanocomposite Electrodes
4.7 Conclusion
5. Nitric Oxide–Sensing Devices: A Practical Application
5.1 Introduction
5.2 Devices for Exhaled NO Detection
5.2.1 Chemiluminiscence Devices
5.2.2 Electrochemical Devices
5.2.3 Laser-Based Device
5.3 In Vivo NO Measurement Devices
5.4 Conclusions
Index
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Nitric Oxide Sensing

Nitric Oxide Sensing

Sagarika Bhattacharya Subhra Samanta Biswarup Chakraborty

Published by Jenny Stanford Publishing Pte. Ltd. Level 34, Centennial Tower 3 Temasek Avenue Singapore 039190

Email: [email protected] Web: www.jennystanford.com British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library.

Nitric Oxide Sensing Copyright © 2022 by Jenny Stanford Publishing Pte. Ltd. All rights reserved. This book, or parts thereof, may not be reproduced in any form or by any means, electronic or mechanical, including photocopying, recording or any information storage and retrieval system now known or to be invented, without written permission from the publisher.

For photocopying of material in this volume, please pay a copying fee through the Copyright Clearance Center, Inc., 222 Rosewood Drive, Danvers, MA 01923, USA. In this case permission to photocopy is not required from the publisher. ISBN 978-981-4877-67-1 (Hardcover) ISBN 978-1-003-14218-8 (eBook)

Contents

Preface 1. Introduction 2. Small-Molecule and Metal Complexes as Nitric Oxide Sensors 2.1 Introduction 2.2 Nitric Oxide Homeostasis 2.3 Fluorescent Detection Kit for NO Sensors 2.3.1 Different Strategies 2.3.2 Fluorophore Displacement without Metal Reduction 2.3.3 Metal Reduction without Fluorophore Displacement 2.3.4 Metal Reduction with Fluorophore Displacement 2.4 Metal Complexes for NO Sensing 2.4.1 Cobalt Systems 2.4.2 Iron Systems 2.4.3 Reversible NO Sensing by Rhodium Complexes 2.4.4 NO Detection with Ruthenium Complexes 2.4.5 Copper Complex as a NO Sensing Probe 2.4.6 Copper (II) Conjugate Polymer 2.4.7 Copper (II) Anthracyl Cyclam Complex 2.5 Polymer-Based Sensors 2.6 Biomedical Applications 2.7 Conclusion

3. Nitric Oxide Sensing with Carbon Nanomaterials 3.1 Carbon Nanomaterials 3.2 Nitric Oxide Sensing with Carbon Dots 3.3 Nitric Oxide Sensing with Carbon Nanotubes

vii 1

7 7 10 12 12 13 13 13 13 13 18 19 20 23 25 26 27 32 37

43 43 44 51

vi

Contents

3.4 3.5

Nitric Oxide Sensing with Graphene Conclusion

4. Electrochemical Nitric Oxide Detection 4.1 Introduction 4.2 Scope of Electrodes for NO Detection 4.3 NO Detection on Noble Metals and Pt Electrodes 4.4 Biosensors with Modified Electrodes 4.5 Metallocycle-Modified Electrodes 4.6 Nanocomposite Electrodes 4.7 Conclusion

5. Nitric Oxide–Sensing Devices: A Practical Application 5.1 Introduction 5.2 Devices for Exhaled NO Detection 5.2.1 Chemiluminiscence Devices 5.2.2 Electrochemical Devices 5.2.3 Laser-Based Device 5.3 In Vivo NO Measurement Devices 5.4 Conclusions

Index

58 72

79 79 82

87 91 97 102 107

117 117 118 118 121 124 130 132 137

Preface

Preface

Nitric oxide, an important bio-interesting signaling molecule in living organisms, is associated with cardiovascular, neuronal, and immunological cell regulatory functions. Beside these, it plays a major role in vasodilation and signal transduction. The irregular NO homeostasis causes several diseases like hypertension, cardiovascular diseases, stroke, neuro-degeneration, and gastrointestinal distress. However, nitric oxide is typically biosynthesized by a group of enzymes called nitric oxide synthase (NOS) during the conversion of arginine to citrulline at the intravascular/extravascular interface having a life time of 2–6 sec at a physiological pH that depends on oxygen concentration. Consequently, the selective detection and quantification of NO concentration in bio-samples is particularly important for signaling point of view by various sensors, whether they are molecular complexes, nanomaterials, electrochemical methods, or devices. This book summarizes the recent developments in NO detection by small molecules, metal-organic probes, carbon nanomaterials, metal nanoparticles, and even modern devices developed or commercialized. The methodologies adopted for NO identification are based mainly on fluorescence quenching, electrochemical, and colorimetric detection. This book comprises five chapters containing sensing capability of NO as gas and in aqueous solution. A brief introduction of NO, including electronic structure and its influence on the reactivity is discussed in Chapter 1. Chapter 2 describes the development of small organic molecules, transition metal complexes, and their polymerembedded structure for NO detection. Recently developed carbon nanomaterials (especially carbon dots, nanotubes, and graphenes) for NO sensing via fluorescence quenching mechanism pathway have been discussed in Chapter 3. In addition, electrochemical recognition of NO by single- or multi-walled carbon nanotubes and functionalized graphene have also been summarized in Chapter 3. Details of electrochemical NO sensing by metal complexes [Pt(II), Ni (II)], metal(0) nanoparticles (Au, Pt, Ag), different nanocomposites, and nano-biocomposites are discussed in Chapter 4. The last chapter

vii

viii

Preface

is dedicated to the modern devices that have been fabricated or commercialized for practical NO detection in nasal breath samples for inflammation study in humans. This book is an ideal guideline for researchers working in the NO-sensing area. It will also enrich the knowledge of master’s students and is recommended as a reference book for universities and research institutes. We express our sincere gratitude to Prof. Sasankasekhar Mohanta (University of Calcutta, India), Prof. Abhisek Dey (IACS Kolkata, India), Prof. Tapan Kanti Paine (IACS Kolkata, India), Prof. Ashok K. Ganguli (IIT Delhi, India), and Prof. Nicolai Lehnert (University of Michigan, USA) for their invaluable reviews for this book. Sagarika Bhattacharya Subhra Samanta Biswarup Chakraborty April 2021

Chapter 1

Introduction

Investigation on air by the British polymath Joseph Priestley (Experiments and Observations on Different Kinds of Air) led to the discovery of most of the gases present in air. At the same time, Priestley first identified nitrous air, a colorless gas now known as nitric oxide (NO), as another component of air present in insignificant amounts. Additionally, he developed the nitrous air test to monitor the quality of air, where he used NO as a probe, and it is believed to be the major pollutant of air [1]. Presently, a significant amount of NO present in air is produced by different chemical plants [2, 3]. An extensive study on the chemical behavior of NO establishes its radical character, short life time, and fast reaction kinetics with paramagnetic and diamagnetic molecules [4]. Molecular orbital (MO) analysis of this heterodiatomic molecule reveals that it has an unpaired electron in the p*2p MO (Fig. 1.1) with an overall bond order of 2.5 that commonly represents as conventional two solid double bonds and one dotted bond between N and O atoms (Fig. 1.2). The N–O bond length is 1.15 Å, which is shorter than the double bond, indicating the removal of the last electron in the antibonding MO [5]. Due to the unpaired electron in the antibonding p orbital, the NO molecule is highly reactive, although gaseous NO shows no sign of dimerization, whereas partial dimerization is observed in liquid NO. This highly reactive freeradical molecule can react with oxygen and can convert into NOx, Nitric Oxide Sensing Sagarika Bhattacharya, Subhra Samanta, and Biswarup Chakraborty Copyright © 2022 Jenny Stanford Publishing Pte. Ltd. ISBN 978-981-4877-67-1 (Hardcover), 978-1-003-14218-8 (eBook) www.jennystanford.com

2

Introduction

which, in turn, can cause acid rain [6] and also the greenhouse effect [7]. Besides these harmful effects on the environment, gaseous NO radicals can cause acute respiratory illness in humans.

Figure 1.1 Molecular orbital (MO) energy level diagram of NO. Mixing of atomic orbitals (2s–2p) has been considered to form four sets of hybrid atomic orbitals (φhy) of nitrogen and oxygen, which then form MOs.

N

O

N

O

N

O

Figure 1.2 Electron dot structure of NO, following the octet rule and MO diagram.

NO, generated from inducible and constitutive nitric oxide synthase (iNOS and cNOS), plays an important role in neuronal, cardiovascular, and immunological processes associated with macrophage and neutrophil activation [8–11]. In 1980, it was discovered that NO is an endothelial-derived relaxing factor, which intrigued extensive research [12, 13]. Eighteen years later (1998),

References

Furchgott, Ignarro, and Murad received the Nobel Prize in Medicine for recognizing NO as a signaling molecule for many physiological processes, which inaugurated a new avenue for threranostic study [14]. Later, the role of NO in mitochondrial regulatory function was established, and it is also assumed that NO can inhibit radical induce damage and oxidative stress [15]. However, irregular NO homeostasis is an indication of various diseases and disorders like hypertension [16], atherosclerosis [16], diabetes [17], many neurodegenerative diseases [18], parthenogenesis of Parkinson’s disease, and tumor angiogenesis [19]. The diverse biological function of NO is concentration and location dependent. At low concentration, NO regulates long-term potentiation (LTP) of the brain and vasodilation, while high concentration leads to the formation of reactive nitrogen species (RNS), which, in turn, causes carcinogenesis and neurodegenerative disorders. NO, being a free-radical species, diffuses spontaneously through cells and tissues and reacts with most biological targets [20–22]; hence real-time monitoring of NO is a potential challenge, and realtime detection of NO released from living cells is a new avenue of biological research and drug discovery. Previously, several spectroscopic, chromatographic, electrochemical, and other methods have been reported for the detection of NO, although mass spectrometric and gas chromatographic methods are not sensitive [23–25]. Common spectroscopic techniques such as UV-visible (UVvis), electron spin resonance (ESR), and fluorescence spectroscopy have also been used for the detection of NO [23, 24, 26]. The focus of this book is mainly to cover the detection and sensing of NO with small molecules, metal-organic probes, carbonbased and other nanomaterials, and also modern devices developed and commercialized so far. Herein, we have attempted to provide an up-to-date review of NO sensing with devices and their potential applications.

References

1. Fontijn, A., Sabadell, A. J., Ronco, R. J., Homogeneous chemiluminescent measurement of nitric oxide with ozone. Implications for continuous selective monitoring of gaseous air pollutants. Anal. Chem., 1970, 42(6), 575–579.

3

4

Introduction

2. Sun, J., Yan, Y., Sun, M., Yu, H., Zhang, K., Huang, D., Wang, S., Fluorescence turn-on detection of gaseous nitric oxide using ferric dithiocarbamate complex functionalized quantum dots. Anal. Chem., 2014, 86(12), 5628–5632. 3. Beychok, M. R., NOX emission from fuel combustion controlled. Oil Gas J., 1973, 71, 53–56. 4. DeRosa, F., Keefer, L. K., Hrabie, J. A., Nitric oxide reacts with methoxide. J. Org. Chem., 2008, 73(3), 1139–1142.

5. Greenwood, N. N., Earnshaw, A., (eds.) Nitrogen. In Chemistry of the Elements (2nd Ed.). Butterworth-Heinemann, Oxford, 1997, pp. 406– 472.

6. Koenig, J. Q., Jansen, K., Mar, T. F., Lumley, T., Kaufman, J., Trenga, C. A., Sullivan, J., Liu, L. J. S., Shapiro, G. G., Larson, T. V., Measurement of offline exhaled nitric oxide in a study of community exposure to air pollution. Environ. Health Perspect., 2003, 111(13), 1625–1629.

7. Lammel, G., Graßl, H., Greenhouse effect of NOX. Environ. Sci. Pollut. Res., 1995, 2(1), 40–45. 8. Brown, G. C., Nitric oxide and mitochondrial respiration. Biochimi. Biophysic. Acta -Bioenergetics, 1999, 1411(2), 351–369.

9. Cooke, J. P., Mont-Reynaud, R., Tsao, P. S., Maxwell, A. J., Nitric Oxide Biology and Pathobiology. Academic, San Diego, 2000.

10. Roszer, T., The Biology of Subcellular Nitric Oxide. Springer, Netherlands, 2012.

11. Kerwin, J. F., Lancaster, J. R., Feldman, P. L., Nitric oxide: a new paradigm for second messengers. J. Med. Chem., 1995, 38(22), 4343–4362.

12. Ignarro, L. J., Buga, G. M., Wood, K. S., Byrns, R. E., Chaudhuri, G., Endothelium-derived relaxing factor produced and released from artery and vein is nitric oxide. Proc. Natl. Acad. Sci., 1987, 84(24), 9265.

13. Viktor Bauer, R. S., Nitric oxide-the endothelium-derived relaxing factor and its role in endothelial functions. Gen. Physiol. Biophys., 2010, 29, 319–340. 14. Murad, F., Discovery of some of the biological effects of nitric oxide and its role in cell signaling (nobel lecture). Angew. Chem. Int. Ed., 1999, 38(13–14), 1856–1868. 15. Darley-Usmar, V. M., Patel, R. P., O’Donnell, V. B., Freeman, B. A., Nitric Oxide Biology and Pathobiology. Academic, San Diego, 2000, pp. 265– 276.

References

16. Heitmeyer, M. R., Corbett, J. A., Nitric Oxide Biology and Pathobiology. Academic, San Diego, 2000, pp. 785–810.

17. Gonzales-Zulueta, M., Dawson, V. L., Dawson, T. M., Nitric Oxide Biology and Pathobiology. Academic, San Diego, 2000, pp. 695–710. 18. Giulivi, C., Poderoso, J. J., Boveris, A., Production of nitric oxide by mitochondria. J. Biol. Chem., 1998, 273(18), 11038–11043.

19. Kavya, R., Saluja, R., Singh, S., Dikshit, M., Nitric oxide synthase regulation and diversity: implications in Parkinson’s disease. Nitric Oxide-Biol. Chem., 2006, 15(4), 280–294.

20. Lancaster, J. R., A tutorial on the diffusibility and reactivity of free nitric oxide. Nitric Oxide, 1997, 1(1), 18–30.

21. Figueroa, X. F., Lillo, M. A., Gaete, P. S., Riquelme, M. A., Sáez, J. C., Diffusion of nitric oxide across cell membranes of the vascular wall requires specific connexin-based channels. Neuropharmacology, 2013, 75, 471–478.

22. Heinrich, T. A., da Silva, R. S., Miranda, K. M., Switzer, C. H., Wink, D. A., Fukuto, J. M., Biological nitric oxide signalling: chemistry and terminology. Br. J. Pharmacol., 2013, 169(7), 1417–1429.

23. Yoshimura, T., Yokoyama, H., Fujii, S., Takayama, F., Oikawa, K., Kamada, H., In vivo EPR detection and imaging of endogenous nitric oxide in lipopolysaccharide-treated mice. Nat. Biotechnol., 1996, 14(8), 992– 994. 24. Kojima, H., Nakatsubo, N., Kikuchi, K., Kawahara, S., Kirino, Y., Nagoshi, H., Hirata, Y., Nagano, T., Detection and imaging of nitric oxide with novel fluorescent indicators: diaminofluoresceins. Anal. Chem., 1998, 70(13), 2446–2453.

25. Li, C. M., Zang, J., Zhan, D., Chen, W., Sun, C. Q., Teo, A. L., Chua, Y. T., Lee, V. S., Moochhala, S. M., Electrochemical detection of nitric oxide on a SWCNT/RTIL composite gel microelectrode. Electroanalysis, 2006, 18(7), 713–718. 26. Taha, Z. H., Nitric oxide measurements in biological samples. Talanta, 2003, 61, 3–10.

5

Chapter 2

Small-Molecule and Metal Complexes as Nitric Oxide Sensors

2.1

Introduction

Joseph Priestly first discovered nitric oxide (NO), a simple diatomic molecule having a N–O bond length of 1.15 Å in 1772; then it was used as an anesthesia agent for dentistry [1, 2]. From the molecular orbital (MO) diagram, it is seen that the unpaired electron present in the lowest unoccupied molecular orbital (LUMO) (p*) plays an important role in the greater binding affinity with biological electron-rich molecular species as well as some transition metal ions having the dp orbital, mainly iron (first and second transition series) [3]. The electronic structure revealed from the MO diagram confirms the serenity of oxidation to nitrosonium (NO+) and reduction to nitroxide (NO–), and being labile toward an oxygen attack leads to oxide formation. A very close relationship toward isoelctronicity with regard to dioxygen monocation (O2+) and NO+ with CO and CN– as well as NO– with regard to dioxygen (O2) having a triplet ground state accounts engrossment in studying various metal-nitrosyl complexes as probes for sensing of biological networks [4]. The NO+ species has been formed as various stable salts leading to oxidizing agents that function as transport agents which reflected from there ionization potential of 9.26 eV and the electron affinity of 0.024 eV. Nitric Oxide Sensing Sagarika Bhattacharya, Subhra Samanta, and Biswarup Chakraborty Copyright © 2022 Jenny Stanford Publishing Pte. Ltd. ISBN 978-981-4877-67-1 (Hardcover), 978-1-003-14218-8 (eBook) www.jennystanford.com

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Small-Molecule and Metal Complexes as Nitric Oxide Sensors

The reduced part (NO–) has tethered with the metal ions in the biological system for a structure–function relationship. Being a redox-active molecule, the redox potential ENO+/NO is 1.2 V versus the normal hydrogen electrode (NHE), while the potential depends on the solvent and pH. Notably, under a basic condition, the reduction potential ENO+/NO is –0.46 V versus the NHE [4, 5]. The reduction of NO to triplet NO– and singlet NO– is (–0.8±0.2) V3 for triplet NO– and (–1.7±0.2) V1 for singlet NO– [5], altering the inaccessible NO– formation at physiological pH. From the MO diagram, it is obvious that the bond order of NO is 2.5. Oxidation of the NO molecule to NO+ depicts the contraction of the bond length to 1.06 Å and a bond order of 3, whereas reduction to NO– leads to electron population in p* to finalize a bond order of 2, resulting in an increment of the bond length to 1.26 å [6, 7]. Nitrogen (N2) plays a crucial role in living organisms, which fulfill nitrogen uptake in the form of NH4+ and also in the form of various nitrogen oxides (NO3–, NO2–) (Fig. 2.1) [8]. NO is one of the major environment pollutants, mainly generated from the burning of fossils fuels [9] and some pathogenic bacteria [10]. It can cause depletion of the ozone layer in its further reduced state during lightning [11, 12]. Besides these environmental issues caused by NO and/or its congeners, NO plays a vital role in some biological processes, and currently sensing of nano- and subnanomolar concentrations of NO in biological samples have gained immense interest. A nanomolar concentration of NO acts as a signaling agent for the cardiovascular and nervous systems, which controls blood vessels in mammals, whereas a micromolar concentration of NO leads to stress, nerve damage, organ failure, and other severe health-related issues, such as vasodilation [13–15]. In 1980 Robert Furchgott first observed the effect of this simple gaseous molecule NO in vasodilation [16]. It was found that the endothelial cells in blood vessels are relaxed under the action of acetylcholine. Generally, endothelial cells are situated as an array of cells above some special smooth muscle cells inside blood vessels. Vasodilation is responsible for the relaxation and contraction of such muscle cells; without the endothelial cells, this physiological phenomenon would not be possible. Some factors are required for the excitation of endothelial cells to be relaxed or contracted; one factor is the endothelial-derived relaxing factor (EDRF) [16, 17].

Introduction

Figure 2.1 Main species involved and interconversion in the nitrogen cycle. N2 is reduced to NH3 through biological or industrial N2 fixation (red arrows), providing N-containing fertilizers for plants (green arrows). However, excess NH3 is processed by microorganisms in the soil by nitrification (pale-blue arrows) and denitrification (blue arrows), which transform the N-containing fertilizer into environmental pollutants. Adapted by permission from Ref. [8], Copyright 2018, Springer Nature.

NO is biosynthesized by nitric oxide synthase (NOS) and endogenously released by endothelial cells at a rate of approximately 0.5–4.0 × 10–10 mol cm–2 min–1. The rapid NO diffusion is explained by the aquaporin channel-1 [18]. Several biochemical phenomena were regulated by NO as a potent stimulant, such as soluble guanyl cyclase (sGC) to cyclic guanyl monophosphate (cGMP) [19, 20]. NO has been found to be bound to several metalloproteins and has been associated with several biological and physiological processes; namely Cyt.P450nor is catalyzed for NO reduction, leading to further denitrification, which is one step before molecular dinitrogen [8]. Basically, irregular NO homeostasis is regulated by mettaloproteines, which keeps our body system free from this NO pollutant [21, 22]. Several groups are trying to get a suitable platform to sense this NO quickly to regulate the biological process [3, 23–25]. On the basis of the MO diagram and its affinity toward transition metals, several groups have developed potential molecular probes as NO sensors that operate at extremely low concentrations of NO [26].

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Figure 2.2 Biological effects on grafts that release NO and pathological events on graft surface in the absence of NO. (A) Small-diameter vascular grafts following implantation tend to fail mainly because of thrombosis and intimal hyperplasia. Biologically and mechanically unoptimized vascular grafts do not promote endothelialization, but they do facilitate the adhesion and proliferation of smooth muscle cells from the site of anastomosis as a response to injury and lead to overall thrombus formation with the adhesion of red blood cells and platelets on the graft lumen, leading to graft occlusion. Highlighted are the molecular events leading to platelet adhesion, aggregation, and thrombus formation. (B) The presence of NO prevents platelet adhesion and aggregation, smooth muscle cell proliferation, and migration, as well as promoting endothelialization, thus maintaining a smooth blood flow through vascular grafts. Highlighted are the NO-mediated mechanisms that modulate platelet activities and are discussed further in this section [28]. Adapted with permission from Ref. [3], Copyright 2011, American Chemical Society.

2.2

Nitric Oxide Homeostasis

Nature has given the power to all living systems to regulate the internal physical and chemical environment and keep it free from toxins that can be generated by the system. One of the important parts is NO homeostatis. NO is generated in the body system by endothelium nitric oxide synthase (eNOS) from l-arginine in the

Nitric Oxide Homeostasis

presence of plasma O2 [21, 22]. This then passes through the endothelium layer to the smooth muscle cells below and causes dilation of the blood vessels, regulates the blood pressure, and acts as signal transduction (Fig. 2.2) [3, 27]. l-arginine + O2 + eNOS

NO + l-citrulline

Figure 2.3 Red blood cell interactions with NO include (1) NO binding to the deoxygenated heme in oxygenated red blood cells, forming iron-nitrosyl hemoglobin (FeII_NO); (2) oxyhemoglobin scavenging NO and transferring it to the β-globin Cys-93 residue to form SNOHb (NO transport); (3) hemoglobin deoxygenation and structural transitions from R (oxy) to T (deoxy), facilitating the release of NO; and (4) T-state Hb reacting with NO species and undergoing hemoglobin nitrosylation. Note: In the R state, Cysβ93 is enclosed in a hydrophobic pocket, and the heme pocket is more accessible. In the T state, Cysβ93 is exposed to reactions, and the heme pocket is less accessible [3, 29, 30]. Reprinted with permission from Ref. [3], Copyright 2011, American Chemical Society.

Second, NO generated in the living system also diffuses to the blood, reacts with free oxygen forming nitrite, and reacts with oxyhemoglobin to form methomoglobin and hemenitosyls with

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Small-Molecule and Metal Complexes as Nitric Oxide Sensors

deoxyhemoglobin (Fig. 2.3). The first pathway releases oxidized products, nitrates, which are regulated by nitrate reductase; the hemenitrosyls are further reduced by several metalloenzymes like cytochrome P450nor, multicopperoxidase, etc., to limit the free NO production in the local production area [3]. NO + O2 + H2O

Oxyheme (Fe2+) + NO

Deoxyheme (Fe2+) + NO

2.3

NO2– + H+

Oxyheme (Fe3+) + NO3–

Deoxyheme (Fe3+) – NO

Fluorescent Detection Kit for NO Sensors

Being a reactive free radical, NO can diffuse into the cell membrane so fast that it cannot be easily detected by any instrument. Bioimaging is one possibility to trap NO by combining with other microscopic and spectroscopic instruments, such as EPR, chemiluminescence [31], amperometry, etc. There are two commercially available organic molecule–based sensors for bioimaging of NO by making a triazole ring [32]. Depending on the fluorescent response, it can’t be real-time measurement. For efficient fluorescent sensors, the probe should be nontoxic, reversible, fast, and selective. It is obvious that a fluorophore should be excited and emit energy between the visible and near-infrared regions so that it can avoid interference or cellular damage in UV light. Therefore, there should be some strategy for detecting NO by metal complexes [23].

2.3.1

Different Strategies

Several groups are attempting to solve the problems in NO bioimaging. For the fluorescent effect, intensity enhancement is greater over the quench for the monitoring substance. They are developing metal-based fluorophore complexes, reacting them with NO, and studying the fluorescence that shows a diminished effect on NO binding to the metal center. The first example was an ironbased cyclam complex, which showed decreased emission intensity on NO binding. Similarly, the iron-dithiocarbamate complex linked with an acridine-TEMPO ligand also generated same spectroscopic

Metal Complexes for NO Sensing

signature. Therefore some strategies were taken into consideration [33].

2.3.2

Fluorophore Displacement without Metal Reduction

This strategy is mainly due to metal-NO adduct formation. NO has a strong trans directing ability so that it can release the axial fluorophore ligand when coming into contact with a metalfluorophore complex. Initially the complex was quenched, but now the released fluorophore might give more intensity, which we call switch-on. This effect is observed for iron cyclam, ruthenium porphyrins, and dirhodium carboxylate complexes [34]. This will be further discussed throughout the chapter [33, 35].

2.3.3

Metal Reduction without Fluorophore Displacement

NO is a strong self-reducing agent. Some metal ions, when bound to the fluorophore on its high valent paramagnetic states, show quench intensity, but introduction of NO makes the metal in its reduced states transform the system to a more stable and labile one, resulting in retention of fluorescent intensity. This approach is observed for some copper complexes [26, 36, 37].

2.3.4

Metal Reduction with Fluorophore Displacement

This case arises for redox-active metal ions where the nitrosated ligand must be displaced under exposure of NO because of producing a stable fluorophore NO adduct. This is observed for certain Co– dansyl-aminotroponimine (DATI) species [26, 38, 39].

2.4

2.4.1

Metal Complexes for NO Sensing Cobalt Systems

These systems are important from the viewpoint of fluorescence displacement strategies. Lippard and coworkers studied various

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Small-Molecule and Metal Complexes as Nitric Oxide Sensors

cobalt complexes for enhanced fluorescence emission for NO detection. They synthesized a series of cobalt complexes, [Co(iprDATI)2] (1), [Co(tBuDATI)2] (2), [Co(BzDATI)2] (3), and [Co(DATI)2] (4), with pseudotetrahedral arrangement around the metal center having dihedral angles 76.1°, 81.4°, 73.8°, and 62.2° respectively (Fig. 2.4) [38, 40]. The metal ions formed a chelating ring with the nitrogen atoms. So the dihedral angle should be the angle containing the metal ions. The differences are due to various ligands such as tBu, Bz, ipr, and simple hydrogen. Depending on the angles, it was clear that steric hindrance can contribute toward the absorption for fluorescence. The last two complexes (3 and 4) has the dansyl group in parallel fashion, and the distance between two dansyl groups was 3.5–3.7 Å, resulting in p–p stacking interaction, which leads to fluorescence quench of the dansyl group. The fluorescence intensity of the complexes decreased by 5%–6% due to the hypothesis of interaction between the cobalt d orbital and the fluorophore’s exited state by direct electron or energy transfer (Fig. 2.5). On exposure to NO, there was an approximately steady eightfold increase in fluorescence for complex 1, which was far lower than complex 4, as the tetramethylene chain might distort the Co(II) ion to the Co(I)-dansyl adduct. This caused a differential NO reactivity toward the complexes. This phenomenon was well characterized by infrared (IR) and 1H NMR spectroscopy. They found two NO stretching vibrations at 1838 cm–1 and 1760 cm–1, indicating a dinitrosyl complex after several hours of NO addition. Similarly, two sets of resonance appeared in 1H NMR for the diamagnetic cobalt and free ligands that lead to reductive nitrosylation mechanism toward turn on emission from fluorophore displacement upon NO addition [23]. Air-stable dicobalt(II)dansylpiperzine(Ds-Pip) tetracarboxylate species [39, 41] [Co2(m-O2CArTol)2(O2CArTol)2(Ds-Pip)2] (5) and [Co2(m-O2CArTol)4(Ds-Pip)2] (6), where –O2CArTol = 2,6-di(ptolyl) benzoate and (Ds-Pip) = dansylpiperazine, have also shown enhanced fluorescence intensity upon NO addition via the N-nitrosated pathway. When excess amount of NO is added to the dicobalt species (6), there is an approximately tenfold increment in fluorescence emission and a shift in the absorption maxima from

Metal Complexes for NO Sensing

Figure 2.4 Chemical structures of cobalt complexes showed 50% thermal ellipsoids with HRDATI and H2DATI-4. Adapted with permission from Ref. [38], Copyright 2000, American Chemical Society.

503 nm to 513 nm, resulting in the appearance of two stretching bands on Fourier transform infrared (FTIR) spectroscopy at 1864 cm–1 and 1783 cm–1, indicating dinitrosyl Co(I) species. A band around 1610 cm–1 before NO addition for C=O stretching shifted to 1745 cm–1 and represents free carboxylic acid. This phenomenon

15

16

Small-Molecule and Metal Complexes as Nitric Oxide Sensors

revealed that there should be conformational changes in the metal sites, which is also confirmed by X-ray analysis (Fig. 2.6).

Figure 2.5 Fluorescence response of 1 compared to HiPrDATI (right) and of 1 upon addition of excess NO (left), excited at 350 nm. Adapted with permission from Ref. [38], Copyright 2000, American Chemical Society.

Thus reductive nitrosylation via the N-nitrosated pathway at the metal center is responsible for fluorescence enhancement by fluorophore dissociation. Irrespective of the dansyl species, the authors synthesized nonfluorescence-based [Co(Ds-AMP)2] (7) and [Co(Ds-AQ)2] (8) complexes (Fig. 2.7) [42], which also exhibited around twofold enhancement of emission by DS-Amp and DS-AQ species; conjugate bases of dansylaminopyridine (DS-Amp) and dansylaminoquinoline (DS-AQ) respectively, upon addition of excess NO in acetonitrile and methanol, forming [Co(NO)2] species characterized by FTIR (1763 cm–1, 1693 cm–1) and 1H NMR (two sets of diamagnetic peak) spectroscopy. Thus, turn on fluorescence is also observed by ejecting one of the fluorophores via reductive nitrosylation. Other fluorescein-based Co(II) complexes [Co(iPrFATI-3)] (9) and [Co(iPrFATI-4)] (10) [42] have been developed due to low excitation energy, which is very important for NO bioimaging. Complex 9 was found to be mononuclear, which also exhibits an intense NO stretching vibration at 1630 cm–1 and dinitrogen at 2114 cm–1 after NO exposure. A band at 1759 cm–1 shows that the fluorescein carboxylate may overlap [Co(NO)2]. On the contrary, complex 10 has also been shown to have a dinitrogen generation with multiple products formation have been speculated.

Metal Complexes for NO Sensing

Figure 2.6 ORTEP diagrams of windmill 5 and paddlewheel 6 showing 50% probability thermal ellipsoids. The phenyl rings of ArTolCO2– ligands have been omitted for clarity. Reprinted with permission from Ref. [34], Copyright 2004, American Chemical Society.

Figure 2.7

Chemical structures of 9 (n = 3 for CH2) and 10 (n = 4 for CH2).

17

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Small-Molecule and Metal Complexes as Nitric Oxide Sensors

2.4.2

Iron Systems

Not only carefully designed cobalt complexes but also iron complexes synthesized on purpose can detect NO in its nanomolar level. An oxygen-sensitive methoxycoumarin tethered cyclam-based (Mmccyclam) iron (II) complex [43] containing a fluorescamine-PROXYL (11) moiety exhibits NO-sensing ability through the fluorophore displacement pathway. When the complex is excited at 360 nm, fluorescence resonance energy transfer occurs from the Mmccyclam to fluorescamine-PROXYL. The small fluorescence intensity changes have been observed upon NO addition to the metal center, followed by fluorescamine-PROXYL displacement (Fig. 2.8) [43].

Figure 2.8 NO detection by an iron-cyclam complex 11. Reprinted with permission from Ref. [23], Copyright 2007, American Chemical Society.

Very similar to dicobalt species, metal interchanges with iron lead to the same type of complex [Fe2(m-O2CArTol)4(Ds-Pip)2] (12) (Fig. 2.9) [44], where –O2CArTol represents 2,6-di(ptolyl) benzoate and Ds-Pip indicates dansylpiperazine also show better NO detection than the cobalt complex via the ligand dissociation pathway. Upon NO addition, fluorescence increases approximately fourfold within 5 min, resulting in two new FTIR bands at 1797 cm–1 and 1726 cm–1, indicating the generation of diirontetranitrosyl species via two bridging carboxylate ligands, as the authors didn’t observe the 1605 cm–1 band that is responsible for the carboxylate ligand before [44]. Thus ligand reorganization can take place throughout the reaction. The complex is unsuitable for biological NO imaging due to its oxygen sensitivity.

Metal Complexes for NO Sensing

Figure 2.9 ORTEP diagram of di-iron carboxylate bridged complex 12 showing 50% probability thermal ellipsoids. The phenyl rings of ArTolCO2– ligands are omitted for clarity. Reprinted with permission from Ref. [34], Copyright 2004, American Chemical Society.

2.4.3

Reversible NO Sensing by Rhodium Complexes

Rhodium also detects NO in a reversible manner. Robinson and coworkers have shown that NO can bind to a dirhodiumtetra bridge core complex [45] and NO can also be displaced upon heating at 120oC, revealing a novel idea that the complex might show reversible NO sensing capacity by modifying with a turn-on fluorophore. They synthesized [Rh2(m-O2CMe)4(Ds-Pip)] (13) and [Rh2(mO2CMe)4(Ds-Im)] (14), where (Ds-Im) is dansyl imidazole. Exposure of the complexes to excess NO (Fig. 2.10) increased the fluorescence intensity by approximately 26-fold, resulting in a dinitrosyl species with a NO stretching band (FTIR) at 1729 cm–1 and 1698 cm–1. The resulting complexes were characterized by X-ray crystallography. The dirhodium core [34] also shows turn-on fluorescence by displacing the dansyl fluorophore in a reversible pathway. Due to its reversible sensing nature, the complex has been utilized for real-time NO imaging in biological imaging applications. The only problem was the water, as it displaced the fluorophore from the rhodium core. The problem could be overcome by introducing a silastic membrane, which permits the NO gas but not the water. By this technique, when NO was added to the dirhodium core, fluorescence turn-on was observed (Fig. 2.11). This potential approach toward NO sensing in a biological system was further fabricated by making optical fiber– or film-based strategy.

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Small-Molecule and Metal Complexes as Nitric Oxide Sensors

Figure 2.10 ORTEP diagram of [Rh2(μ-O2CMe)4(NO)2] showing 50% probability thermal ellipsoids. Adapted with permission from Ref. [34], Copyright 2004, American Chemical Society.

Figure 2.11 (a) Fluorescence response of a CH2Cl2 solution of 13 protected by a silastic membrane against water in the outer vial (right) and upon introduction of 1.9 mM aqueous NO (aq) into the outer vial (left). (b) Fluorescence response of silastic membrane–embedded [Rh2(m-O2CPr)4(Ds-pip)] in water (left) and after exposure to a saturated NO aqueous solution (right). Reprinted with permission from Ref. [34], Copyright 2004, American Chemical Society.

2.4.4

NO Detection with Ruthenium Complexes

Ruthenium has a strong tendency to bind p acid ligands like CO and NO in their low valent states. Therefore Lorkovic and coworkers [26] synthesized fluorophore-modified Ru(II) porphyrin [46], [Ru(TPP)(CO)(Dan-Im)] (15), and [Ru(TPP)(CO)(Dan-Tm)] (16)

Metal Complexes for NO Sensing

(Fig. 2.12), where the two axial positions were occupied by CO and dansyl-based fluorophore, namely dansyl imidazole (Dan-Im) or dan-thiomorpholine (Dan-Tm). Upon NO addition, an immediate increase of approximately 19-fold fluorescence in 20 min resulted in displacement of the dansyl group as well as CO, generating [Ru(II) por(NO)(ONO)] species, which was confirmed by FTIR spectroscopy [47].

Figure 2.12 ORTEP diagrams of ruthenium complexes 15 and 16 showing 50% probability thermal ellipsoids. Reprinted with permission from Ref. [34], Copyright 2004, American Chemical Society.

Recently it was observed that the bipyridine-based ruthenium complex bis(2,2¢-bipyridine)(4-(3,4-diaminophenoxy)2,2¢-bipyridine)ruthenium(II) hexafluorophosphate ([(Ru(bpy)2(dabpy)][PF6]2) [47] is a potent sensor for inhibiting the NO-binding stage. The sensor’s ability toward NO in a concentration-dependent manner in vivo has been demonstrated spectrophotometrically. The endothelial cells were externally cultured by activating agents like peroxide to induce the production of endogenous NO. The luminescence intensity was found to be pH independent, confirming the strength to be a probe. The response was selective toward NO upon exposure. Furthermore, the complex was easily injectable into living cells, although the complex was impermeable to the cell membrane. The strong color change indicates a metal-to-ligand charge transfer having a strong absorption peak at 455 nm and emission peaks at 610 and 616 nm (Fig. 2.13) [48]. It was indicated that the luminescence response was increased by

21

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Small-Molecule and Metal Complexes as Nitric Oxide Sensors

Figure 2.13 Reaction of [Ru(bpy)2(dabpy)]2+ with NO under aerobic conditions. The photos show the luminescence colors of solutions of the two complexes under a 365 nm lamp. Adapted from Ref. [48], Copyright 2010, with permission from John Willey and Sons.

Figure 2.14 Luminescence intensities of the products of [Ru(bpy)2(dabpy)]2+ (10 mM) treated with various reactive oxygen species (ROS) and reactive nitrogen species (RNS) in 0.1 M borate buffer at pH 7.4. NO, 40 mM; H2O2, 100 mm; .OH, 100 mM H2O2 + 100 mM ferrous ammonium sulfate; OCl–, 100 mM NaOCl; 1O2, 100 mM H2O2 + 100 mM NaOCl; NO2–, 100 mM NaNO2; NO3–, 100 mM NaNO3; ONOO–, 100 mM NaONOO; O2–, 100 mM KO2. Adapted from Ref. [48], Copyright 2010, with permission from John Willey and Sons.

approximately 16.9-fold on exposure to NO gas, confirming that the 3,4-diaminophenyl moiety represents an effective probe as a

Metal Complexes for NO Sensing

chemically irreversible off-on switch for NO via the N=N-NH ring formation reaction. Again, the selectivity has been checked by the presence of various ions at pH 7.4 in 0.1 M borate buffer with 10 μ M concentration of the complex, resulting in a several-fold increase in luminescence intensity with NO in just 2 min of addition (Fig. 2.14). The initial reaction rate with a slope of 2.7 × 10–1 with a constantly increasing total rate constant (ktot) of (Ru-(bpy)2(dabpy)]2+/NO was found to be 2.7 × 1010 M–1 s–1 in air-saturated borate buffer (Fig. 2.15).

Figure 2.15 Excitation and emission spectra of [Ru(bpy)2(dabpy)]2+ treated with various amounts of saturated NO solution at room temperature in 0.1 M borate buffer at pH 7.4. The spectra were measured after the addition of NO aqueous solution (2.2 mm; 0–60 mL) to a solution of [Ru(bpy)2(dabpy)]2+ (10 mm, 3.0 mL) for 30 min. Reproduced from Ref. [48], Copyright 2010, with permission from John Willey and Sons.

2.4.5

Copper Complex as a NO Sensing Probe

Unlike other transition metals, a copper complex has also shown the NO-induced fluorescent phenomenon. Lippard and coworkers synthesized two complexes, namely [Cu(II)(Ds-En)2] (17) and [Cu(II)(Ds-AMP)2] (18) (Fig. 2.16), where Ds-En and Ds-AMP are the conjugate bases of dansyl-ethelenediammine

23

24

Small-Molecule and Metal Complexes as Nitric Oxide Sensors

Figure 2.16 ORTEP diagrams of 17 and 18 showing 50% probability thermal ellipsoids. The figures are adapted from Ref. [42], Copyright 2004, American Chemical Society.

Metal Complexes for NO Sensing

and dansylaminopyridine, respectively [36]. These complexes exhibit fluorescence by NO-induced metal reduction followed by NOfluorophore switch-on. In an organic solution of 17 and 18 exposed to NO gas, the emission intensity promptly increased by around 6-fold and 9-fold, respectively, whereas emission intensity was around 2 to 2.3 fold for the buffer solution irrespective of the physiological pH. These complexes could sense NO with a detection limit greater than 10 nM in an organic solvent again in a purely aqueous solution. Due to the advantage of the autoreduction capacity of NO, Cu(II) complexes are reduced by NO to form Cu(I) species with subsequent dissociation of sulfonamide functionality by protonation. Cu(I) has been well characterized by EPR (decreased intensity tends to EPR silent species) spectroscopy. The protonation was characterized by IR spectroscopy with a characteristic stretching band around 3083 cm–1 (–N=N-H).

2.4.6

Copper (II) Conjugate Polymer

A bipyridyl-based conjugated polymer (poly-p-phenylenevinylene) with Cu(II), Cu(II)-CP1a (19) (Fig. 2.17) also showed NO detection [37]. The fluorescence intensity of the conjugated polymer fluorophore significantly increased after addition of NO to the organic solution (DCM:methanol) by around threefold. The intensity enhancement occurs due to the same phenomenon, that is, metal reduction by NO in its low oxidation states. The intrinsic studies on nitrosothiol have revealed that fluorescence increases due to nitroxyl species generated in the reaction mechanism. Thus the complex shows the fluorescent probe for both NO and HNO with a lower limit of 6 nM.

Figure 2.17

Chemical structure of CP1a.

25

26

Small-Molecule and Metal Complexes as Nitric Oxide Sensors

2.4.7

Copper (II) Anthracyl Cyclam Complex

A cyclam-based pendent anthrcenyl derivative of Cu(II) complex[Cu(DAC)2+ (20), where DAC = bis(9-anthracylmethyl) cyclam] [49] (Fig. 2.18), has also shown very slow fluorescent NO detection. On exposure to excess NO, the fluorescence intensity increases slowly over 45 min due to the release of the N-nitrosated DAC ligand from Cu(II) to Cu(I). The result was confirmed by the optical spectroscopy of Cu(II); the disappearance of the d-d band and the formation of n-nitrosated DAC was confirmed by electrospray ionization mass spectrometry (ESI-MS) at m/z = 610 (DAC + NO)+. The N-nitrosoamine formed during the reaction with NO triggers the tetrahedral arrangement and favors the mechanism due to less basisity [49].

Figure 2.18 Chemical structure of copper cyclam complex with anthracene in backbone 20.

For a better view of the roles of HNO and NO toward physiological and pathological insight, Lippard and coworkers [25, 50] developed some copper-based small molecules that selectively respond to the fluorescent process toward NO detection in both in vitro and in vivo conditions. The intensity can be tuned by modulating the oxidation states of the copper ion.

Polymer-Based Sensors

Figure 2.19 NO sensing by copper complex 21.

The authors developed a copper-bound benzoresorufin fluorophore [Cu(II)FL1] (21) and [Cu(II)BOT1] (22) (Fig. 2.19) [24], which contained a secondary amine that might interact with the NO exposure. The fluorophore thus absorbs at a high energy region which was shifted to 30 nm Stokes shift resulted a low signal-tonoise (S/N) ratio. Therefore, a low-energy ratio can penetrate more into the tissue. Exposure of Cu complexes to NO leads to fluorescent quenching by reduction of the paramagnetic Cu ion, followed by the triggered on emission responses. The methyl group on the naphthalene site gives the Cu ion the binding affinity toward the ligand. Benzoresorufin was chosen as it shows absorption in the red region, leading to penetration of the tissue more effectively.

2.5

Polymer-Based Sensors

Unlike metal complexes, polymer-based metal complexes are used to detect NO by using thin-film-based methods. The organic thin-film transistor (OTFT) has attracted significant interest as a gas sensor due to its low power consumption and also due to its reversible responses on sensing target gases. Torsi and coworkers from Italy developed a NO-selective organic field effect transistor (FET). A spin-

27

28

Small-Molecule and Metal Complexes as Nitric Oxide Sensors

coated film of 9,10-bis[(10-decylanthracen-9-yl)ethynyl]anthracene oligomer was utilized as an OTFT NO sensor (Fig. 2.20). A facile charge transport was observed as three neighboring anthracene units were connected via two ethynylene bonds, forming a planar π framework. This device was operated at room temperature, displaying very low sensitivity toward NO and NO2 with a 250 ppb detection limit with interference from carbon monoxide and hydrogen sulfide. Figure 2.20a illustrates the chemical structure of the oligomer serving as the OTFT NO sensor, and Fig. 2.20b represents the schematics of the device [51].

(a)

(b)

Figure 2.20 (a) Molecular structure of p-channel D3A organic active and sensing layers. (b) Schematic view of the architecture of the bottom contact– organic field effect transistor (organic thin-film transistor [OTFT]) used in the study. Reprinted from Ref. [51], Copyright 2009, with permission from Elsevier.

Polymer-Based Sensors

Figure 2.21 Responses of PEDOT:PSS films with different thicknesses, controlled by dropping different volumes of the PEDOT:PSS solution to 10 ppm NO gas at room temperature. Reprinted from Ref. [51], Copyright 2009, with permission from Elsevier.

Recently, conducting polymers, for example, polythiophene, polyaniline, and polypyrrole, were implemented as gas sensors due to their porous morphology and satisfactory sensitivity. Ho and his group from National Taiwan University implemented a conducting polymer, poly(3,4-ethylenedioxythiophene) (PEDOT), as a NO gas sensor. The device was fabricated by combining PEDOT with poly(styrene-sulfonate) (PSS) onto an Al2O3 substrate with a screen-printed interdigitated gold electrode. A larger response of the NO sensor was obtained with increasing thickness of the film, as illustrated in Fig. 2.21. An excellent response time of 527 s and a recovery time of 1780 s were visualized for the PEDOT:PSS-modified electrode sensor with a limit of detection (LOD) (S/N = 3) of 350 ppb. The device can retain its efficiency up to 74.5% with respect to the initial response even after 1 month of operation [52]. A flexible polymer semiconductor thin film was utilized at room temperature as a NOx gas sensor by Kong and coworkers from the Korea Research Institute of Chemical Technology, Republic of Korea. High sensitivity and selectivity of the gas-sensing polymer

29

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Small-Molecule and Metal Complexes as Nitric Oxide Sensors

semiconductor was achieved by the breath-figure molding technique, increasing the nanoporosity of the polymer film. Poly(2,5{2-octyldodecyl}-3,6-diketopyrrolopyrrole-alt-5,5-{2,5-di[thien2-yl]thieno[3,2-b]thiophene}) (DPP-DTT) was used as a polymer for developing a NO-sensing organic FET. The average pore size of the film was 750 nm, displaying a 104% maximum response with sensitivity more than 774%/ppm and the lower detection limit was 110 ppb against NO. The device can differentiate between oxidizing and reducing analytes, depending on their polarity and extent of sensitivity [53]. A real-time NO sensor was constructed using a conducting polymer ligated with a coordinatively unsaturated paramagnetic transition metal complex. Swager and Shioya from the Massachusetts Institute of Technology, Cambridge, synthesized a cobalt-salen polymer (shown in Fig. 2.22) for detecting NO. The redox potential of Co2+/3+ closely matched with the polymeric organic matrix, facilitating charge transportation throughout the material. Cyclic voltammogram experiments were performed to understand the interaction of NO with Co(II/III). Two closely spaced peaks at –0.1 and 0.05 V were assigned to the Co2+/3+ couple and the organic part of the polymer. On exposure to NO the cobalt peak shifted toward the more positive side, indicating oxidation of the Co(II) center in the presence of NO, as portrayed in Fig. 2.22b. The electrochemical sensor exhibits reversibility, and the conductivity returns to normal after five cycles at 100 mV s–1 in the NO-free electrolyte [54].

Figure 2.22 Cobalt salen–based polymer 23 used for the detection of NO [54].

Similarly, another conducting metallopolymer was developed by the same group only by modifying the amine residue from ethylenediamine to 2,2-dimethyl-1,3-propanediamine (Fig. 2.23).

Figure 2.23 Schematic illustration of the fabrication of conducting metallopolymer/electrode devices: (i) electropolymerization of 24 across interdigitated microelectrodes (IMEs) and (ii) chemoresistive response to NO gas exposure. Adapted with permission from Ref. [55], Copyright 2006, American Chemical Society.

Polymer-Based Sensors 31

32

Small-Molecule and Metal Complexes as Nitric Oxide Sensors

The lower LOD was less than 1 ppm for the solid-state sensor. The alteration in the ligand backbone introduces flexibility to the ligand, generating a conformational change in the metal center. The metallopolymer films were studied by cyclic voltammetry (CV), in situ conductivity, profilometry, and several other spectroscopic methods like X-ray photoelectron spectroscopy (XPS), ultravioletvisible (UV-Vis), and FTIR spectroscopic techniques. For gas-phase testing, commercially available interdigitated microelectrodes (IMEs) were used from the metallopolymer. The surface area of the film was increased with high porosity having the ability to absorb more gaseous analytes [55]. Sensitive and fast real-time detection of NO was observed by Prakash and coworkers from the Industrial Toxicology Research Centre, India, utilizing differential pulse voltammetry in aqueous solution. A platinum electrode was modified with a polycarbazoleconducting polymer for NO detection in phosphate-buffered saline (PBS) buffer. The geometrical surface area of the electrode was 0.0078 cm2 in deoxygenated 0.1 M PBS buffer at pH 7.4 at room temperature. The detection limit of the electrode was 50 nM, with negligible interference from ascorbic acid and dopamine, essential for biological samples. The plot of NO concentration versus anodic peak current gave a linear fit on standard addition in a concentration range from 10 nM to 0.1 mM [56].

2.6

Biomedical Applications

Due to high precision in NO detection (in micro-level), metal complexes are employed as important component in biomedical NO sensor design. It was observed that NO has excellent therapeutic properties against bacteria and prevents thrombosis. Endogenously produced NO has a radical nature with a very short life, which is beneficial for thrombosis. This property has a crucial effect on catheter-related blood stream applications [57]. Nowadays, catheters play an essential role in blood-related delivery of drugs or nutrients to patients [58]. Complications arise due to microbial infection or thrombosis and/or clotting. These catheter-based problems can be overcome by heparin infusion. Moreover, heparin is an anticoagulant that also may have side effects. NO can be used for solving this problem [59]. Mayerhoff and coworkers developed

Biomedical Applications

a copper-based model complex Cu(II)-tri(2-pyridylmethyl)amine [Cu(II)TPMA)] (25) to catalyze the electrochemical reduction of nitrite to NO in a buffer solution (pH = 7.2) [57, 60]. The generation of NO can be measured by faradic efficiency, which is a measurement of the current produced due to reduction. Figures 2.24 and 2.25 present the schemes of electrode configuration and response.

Figure 2.24 Schematics of (a) single- and (b) dual-lumen electrochemically modulated NO-releasing catheter configurations examined in this work. Reprinted with permission from Ref. [57], Copyright 2014, American Chemical Society.

Figure 2.25 Modulation of NO flux from a single-lumen catheter with 0.0798 cm2 Pt wire. The solution contains 4 mM Cu(II)TPMA, 0.4 M NaNO2, 0.2 M NaCl, and 0.5 M MOPS (pH 7.2). Flux calculated on the basis of the 3 cm silicone surface area near the Pt wire. Adapted with permission from Ref. [57], Copyright 2014, American Chemical Society.

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Small-Molecule and Metal Complexes as Nitric Oxide Sensors

Figure 2.26 Antithrombotic effect of e-chem NO-releasing catheters in veins of six rabbits for 7 h. Representative pictures of (a) single- and (b) dual-lumen catheters after removal from the vein; (c) thrombosis coverage percentage on the catheters (single lumen, SL 1−3; dual lumen, DL 1−3). Adapted with permission from Ref. [57], Copyright 2014, American Chemical Society.

Catheters have been designed with single and dual lumens for testing NO efficacy in both in vivo and in vitro arrangements. NO flux was measured in rabbit jugular veins for 7 days, ending up with a flux of approximately 2.0 × 10−10 mol min−1 cm−2, which is

Biomedical Applications

in the physiological range (Fig. 2.26). The authors already tested the antimicrobial effect on Escherichia coli biofilms grown over the catheters. It was observed that the bacterial strength decreased up to 1000-fold [57]. On further development, they designed a series of copper complexes (Fig. 2.27) that all showed electrochemical nitrite reduction to generate NO [57].

Figure 2.27 Structural representations of six BMPA- and BEPA-carboxylate Cu complexes investigated in this work. X = acetate (OAc−), OH2, Cl−, or nitrite, depending on the conditions [60].

It was found that [Cu(BEPA-Bu)](OAc) (28) is the best complex for NO detection by faradic efficiency data over a large concentration of nitrite under an oxygen atmosphere. The Cu(II) species is first reduced electrochemically (working electrode) to Cu(I), which subsequently reduces nitrite to generate NO (Eqs. 2.1 and 2.2). Cu(II)(L) + e− Æ Cu(I)(L),

Cu(I)(L) + NO2– + 2H+ Æ Cu(II)(L) + NO + H2O,

(2.1)

(2.2)

where L is the ligand bound to Cu(II), namely BMPA or BEPA. Depending on the faradic efficiency (Table 2.1), it was clear to measure the NO level (Fig. 2.28) [57, 60, 61].

35

11

9

7









24.75

5

3

54.5

1

TPMA

14.44

23.45

31.8

52.8

85.3

93.34

BMPA-Pr

87.97

89.22

93.55

92.04

97.53

95.46

BEPA-Pr

Faradic efficiency (%) BEPA-Bu









1.98

1.52

TPMA

6.9

6.89

6.75

6.25

5.16

2.41

BMPA-Pr

25.94

22.08

17.46

12.49

7.37

2.39

BEPA-Pr

BEPA-Bu

NO flux/mol∙min–1∙cm–2∙1010

Faradaic efficiencies and NO fluxes obtained for examined Cu(II) complexes at the applied current

Applied current (μA)

Table 2.1

36 Small-Molecule and Metal Complexes as Nitric Oxide Sensors

Conclusion

Figure 2.28 Comparison of Faradaic efficiencies of the six Cu catalysts with standard errors. Shown for 1 mM catalyst solutions in the presence of 50 mM NaNO2, measured at the complex’s reduction potential (ER). Adapted with permission from Ref. [61], Copyright 2018, American Chemical Society.

2.7

Conclusion

The past few decades’ research has found the pathway for bioimaging and sensing the environmental pollutant NO not by simple organic ligands but by a variety of metal complexes using fluorescence probes. In living system, NO binds primarily to the soluble guanylyl cyclase (sGC) to form the cyclic guanylate cyclase (cGC), leading to a secondary messenger that modulates cardiovascular-protective roles. The advantage of this pathway toward the efficiency of physiological pH range draws our attention to the biomedical application. Copper-based NO sensors are most important as they give have broad in vivo and in vitro applications with a larger period in sheep and rabbits.

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Small-Molecule and Metal Complexes as Nitric Oxide Sensors

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Chapter 3

Nitric Oxide Sensing with Carbon Nanomaterials

3.1

Carbon Nanomaterials

Carbon nanomaterials, a class of low-dimensional materials, have emerged as a very useful vehicle for scientific research and technological processes [1–5]. Interesting properties are observed in carbon nanomaterials because of their low dimensionality, which is absent in bulk materials, such as graphite and diamond; hence carbon nanomaterials have been widely used in the fields of electronics and catalysis, as well as in biological and medical sciences [3, 5–10]. Carbon-based nanomaterials include 0D fullerene [11– 14], 1D carbon nanotubes (CNTs) [15–18], 2D graphene (graphene oxide [GO], reduced graphene oxide [rGO]) [19–22], and emerged sp2-core-hybridized carbon dots (C-dots) [23–26]. Recently, carbon nanomaterials have been implemented as sensors due to the ease of surface functionalization regulating water solubility and biocompatibility. The large π-conjugated surface area of the carbon nanomaterial enables supramolecular binding of the hydrophobic molecule on the surface via weak interaction; thus a selective sensor can be constructed. Carbon nanomaterials have a huge impact on nitric oxide (NO) sensing and detection. Three distinct sections Nitric Oxide Sensing Sagarika Bhattacharya, Subhra Samanta, and Biswarup Chakraborty Copyright © 2022 Jenny Stanford Publishing Pte. Ltd. ISBN 978-981-4877-67-1 (Hardcover), 978-1-003-14218-8 (eBook) www.jennystanford.com

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Nitric Oxide Sensing with Carbon Nanomaterials

of this chapter will focus on specifically NO-sensing properties of carbon nanomaterials. Figure 3.1 presents the types of carbon nanomaterials employed for this purpose. (a)

(b)

(c)

(d)

Figure 3.1 Scheme of various carbon nanomaterials employed for NO-sensing applications: (a) single-walled carbon nanotube (SWCNT), (b) multiwalled carbon nanotube (MWCNT), (c) graphene, and (d) carbon dots.

3.2

Nitric Oxide Sensing with Carbon Dots

C-dots are quasi-spherical fluorescent carbon nanoparticles (0.01 0.30

+1.0 V Ag/AgCl

5.6x10-4

~0.5

0.44

0.003

Sensitivity (mA/mM)

+0.75 V Ag/AgCl

+0.8 V Ag/AgCl

+0.8 V SCE

+0.824 V SCE

cytochrome c. (3-mercaptopropyl)-trimethoxysilane.

b MPTS,

a Cyt-c,

ITO/MPTSb/AuNPs

ITO/Au film (