臺灣博碩士論文加值系統

本論文選擇用不同的分子當作感測標的物，包含半胱氨酸(L-cysteine)以及還原態菸鹼醯胺腺嘌呤二核苷酸(NADH)兩種生物分子，並已經過硼氮共摻雜的石墨烯(BN-rGO)與Nafion®修飾的網印碳電極(SPCE)作為工作電極，進行電化學的一系列感測分析。
L-cysteine為人體必要的二十種氨基酸的其中之一，其在人體內的濃度約介於240~360 M之間它涉及人體內抗氧化劑的合成以及許多人體代謝、退化等疾病。NADH為人體許多生化反應的共觸媒，涉及人體能量貨幣ATP的合成、協助進行呼吸作用等重要功能。本文首次利用BN-rGO對於此兩種感測標的物進行電化學感測分析，並探討在不同的製備條件(不同的NH3體積百分比)下，所形成的不同硼氮摻雜比例對於整體感測效果的影響。
第四章主要論述BN-rGOs-Nafion®/SPCE工作電極對於L-cysteine的感測效果。循環伏安法(CV) 實驗顯示，在3% NH3體積百分比下所製成的 BN-rGO (3% NH3)-Nafion®/SPCE對於 在L-cysteine具有最佳的感測效果，此電極相對於未經硼氮摻雜的純石墨烯(Pristine rGO)以及1%, 10% NH3體積百分比下所製成的BN-rGO，具有更低的氧化峰值電位以及更高的氧化催化電流。感測環境的pH值也經過最佳化測試，並選用與人體最接近的pH 7.0作為感測的環境條件。電位掃描速率的分析結果顯示L-cysteine的電極催化反應為一擴散控制的反應機制，利用催化電流與掃描速率的平方根圖所得之迴歸直線斜率，求得L-cysteine之擴散係數為cm2/s。計時電流分析法利用瞬間電極表面電位的改變，得以對感測物進行定量分析，感測結果的線性區間介於10 ~ 1060 M (R2= 0.991)，感測器靈敏度為20.7 μA/cm2-mM，偵測極限則為0.07 μM。本研究亦使用旋轉電極分析BN-rGO (3% NH3)相對於pristine rGO在標準異相電子傳遞速率常數(standard heterogeneous rate constant)以及電活性表面積(electroactive surface area)上的本質差異，研究結果顯示BN-rGO (3% NH3)在兩個參數上都有近約30%的增長，強而有力地證明了經過硼氮共摻雜過後的石墨烯，對於L-cysteine具有更好的電催化效果。干擾物測試方面，本研究探討尿酸(UA)、抗壞血酸(AA)、氯化鉀(KCl)、葡萄糖(glucose)、L-色氨酸(L-tryptophan)、L-酪氨酸(L-tyrosine)對於L-cysteine感測物的干擾影響程度，大多數的干擾物沒有太大程度的干擾，唯有葡萄糖因於人體的相對濃度高於L-cysteine非常多，即使在相同濃度下感測干擾不大，考量到此濃度差距後要應用於實務上仍存在挑戰。
第四章主要論述BN-rGOs-Nafion®/SPCE工作電極對於NADH的感測效果。NADH是一種轉遞質子(氫離子)的輔助因子，出現在細胞很多代謝反應中，用於構建新的細胞生成，抵抗自由基和DNA損傷，並在細胞內發送信號。本研究亦以NADH為標的物探討經過硼氮共摻雜的石墨烯與未經摻雜的石墨烯在 NADH定性與定量分析上的差異。CV結果顯示只有bare SPCE與BN-rGO (3% NH3)-Nafion®/SPCE對NADH具有電催化的效果，BN-rGO (3% NH3)-Nafion®/SPCE成功地降低了NADH的氧化峰值電位約729 mV，此數值在所有文獻中為第二大的降幅，除此之外氧化還原峰值間距(peak separation)、氧化電位以及催化電流上的電化學表現相較於bare SPCE均有較佳的表現。感測環境的pH值也經過最佳化測試，並選用與人體最接近的pH 7.0作為感測的環境條件。電位掃描速率的分析結果顯示NADH的電極催化反應為一擴散控制的反應機制，利用催化電流與掃描速率的平方根圖所得之迴歸直線斜率，求得NADH之擴散係數為cm2/s，藉由此擴散係數數值，我們得以利用Velasco equation計算出標準異相電子傳遞速率常數(standard heterogeneous rate constant)。結果顯示bare SPCE及BN-rGO (3% NH3)-Nafion®/SPCE的異相電子傳遞速率常數分別為 以及，BN-rGO(3% NH3)-Nafion®/SPCE電極表面具有更好的電催化效果。電流分析法對於NADH感測的結果顯示，線性區間介於5~140 M (R2= 0.999)，靈敏度為424 μA/cm2-mM，偵測極限則為0.03 μM，均屬所有可尋文獻中表現傑出者。干擾物測試方面，本研究探討尿酸(UA)、抗壞血酸(AA)、多巴胺(DA)、葡萄糖(glucose)、過氧化氫(H2O2)對NADH的干擾影響程度。此系統在進行NADH感測時，不會受到上述五種常見共存於人體中的干擾物影響，顯示此電極在作為NADH的感測電極時，有很大的潛力能應用於實務上。

In this thesis, boron and nitrogen co-doped reduced graphene oxide with different dopant composition were prepared as the electrochemical sensing platform. L-cysteine (CySH) and the reduced form of nicotinamide adenine dinucleotide (NADH) were selected as the sensing targets. The fabrication of BN-rGOs involved an atmospheric-pressure substitution reaction in the presence of boron oxide (B2O3) vapor and different volume percentage (1%, 3%, 10%) of ammonia gas. The surface morphologies were examined by transmission electron microscope (TEM). The composition and distribution of the dopants on BN-rGOs were verified by X-ray photoelectron spectroscopy (XPS) and energy-filtered transmission electron microscopy (EFTEM) elemental mapping. BN-rGOs were dispersed and drop casted onto the commercial screen printed carbon electrode (SPCE) to make the working electrode of the sensing system. BN-rGO (1% NH3)-Nafion®/SPCE, BN-rGO (3% NH3)-Nafion®/SPCE, BN-rGO (10% NH3)-Nafion®/SPCE and pristine rGO-Nafion®/SPCE were prepared as the working electrodes.
L-cysteine (CySH), a thiol containing amino acid, plays a critical role in regulating the biological activity of certain proteins, peptides and enzymes and involves in a great variety of biological processes. CySH deficiency leads to slow growth in children, hair depigmentation, lethargy, liver damage, loss of muscle and fat, skin lesions, and weakness. From the results, BN-rGO (3% NH3)-Nafion®/SPCE among all the prepared electrodes showed the best electro-catalytic activity toward the electro-oxidation of CySH. It not only showed a decrease in oxidation potential but also an increase in catalytic peak current density of the CySH oxidation reaction with respect to the case of pristine rGO-Nafion®/SPCE. The quantification of CySH by chronoamperometric method could be successfully carried out by using BN-rGO (3% NH3)-Nafion®/SPCE. The sensing parameters included a wide linear range from 0.01 to 1.06 mM, a high sensitivity of 20.7 μA/cm2-mM, and a remarkable low detection limit of 0.07 μM. Rotating disk electrode analysis revealed a 30% increase in electroactive surface area (Ae) and the standard heterogeneous rate constant (k0), which is a powerful evidence accounting for a better electro-catalytic performance of BN-rGO (3% NH3)-Nafion®/SPCE. The system also showed acceptable resistance toward most of the coexisting interference in human serum such as L-tryptophan, L-tyrosine, potassium chloride, glucose, and uric acid.
Reduced form of nicotinamide adenine dinucleotide (NADH) is the most important biological molecule in all living cells acting as a metabolic coenzyme in human body. It has been attributed to the transfer of hydrogen atoms and electrons from one metabolite to another in several intracellular redox reactions, such as glycolysis, the Krebs cycle, and oxidative phosphorylation. BN-rGO (3% NH3)-Nafion®/SPCE showed a unique electro-catalytic ability toward the electro-oxidation of NADH in pH 7.0 PBS, demonstrating a semi-reversible NADH/NAD+ redox peak at around -0.15 V and -0.95 V vs. Ag/AgCl. BN-rGO (3% NH3)-Nafion®/SPCE could also be successfully applied to the quantification of NADH by amperometric method. The sensing parameters include wide linear range from 5 to 140 μM, a high sensitivity of 424 μA/cm2-mM, and a remarkable low detection limit of 0.03 μM. The standard heterogeneous rate constant (k0) of pristine rGO-Nafion®/SPCE, pristine rGO and BN-rGO (3% NH3) were evaluated by Velasco equation, demonstrating a unique electro-catalytic ability and relatively facile electron transfer rate at the surface of BN-rGO (3% NH3)-Nafion®/SPCE. The system resisted most of the coexisting interference in human serum including ascorbic acid, uric acid, dopamine, glucose, and hydrogen peroxide and possessed a good long-term stability.

致謝 I
中文摘要 III
Abstract V
Table of contents VII
List of tables XI
List of figures XII
Nomenclatures XV

Chapter 1 Introduction 1
1.1 Preface 1
1.2 Introduction of sensors 2
1.2.1 Recognition elements 3
1.2.2 Transducers 4
1.2.2.1 Electrochemical transducer 4
1.3 Chemically modified electrodes 5
1.3.1 Carbon materials for electrode modification 6
1.3.2 Heteroatoms doped graphene 8
1.4 Scope of this thesis 10

Chapter 2 Literature Review and Research Motivations 14
2.1 Electrochemical L-cysteine sensor 14
2.1.1 Importance of L-cysteine and its analytical detection methods 14
2.1.2 Preparation of different electrodes for the oxidation reaction of L-cysteine 15
2.1.3 Doping of graphene to enhance their electro-catalytic activities 15
2.2 Electrochemical NADH sensor 18
2.2.1 Importance of NADH and its analytical detection methods 18
2.2.2 Preparation of different electrodes for the oxidation reaction of NADH 18
2.3 Motivation of this research 21

Chapter 3 General experimental descriptions 22
3.1 Materials 22
3.2 Instruments 25
3.3 Solutions 26
3.4 Instrumental analysis 26
3.4.1 Material characterizations 26
3.4.1.1 EFTEM elemental mapping 26
3.4.1.2 X-ray photoelectron spectroscopy 26
3.4.2 Electrochemical analysis 27
3.4.2.1 Three-electrode system 27
3.4.2.2 Rotating disk electrode analysis 27
3.5 Principles of the electrochemical methods 28
3.5.1 Cyclic voltammetry 28
3.5.2 Chronoamperometry 29

Chapter 4 Synthesis of Boron/Nitrogen Co-doped Reduced Graphene Oxide
and Its Enhanced Performance for Electrochemical L-cysteine Biosensor 30
4.1 Overview of Chapter 4 30
4.2 Experimental details of Chapter 4 31
4.2.1 Preparation of BN-rGO 31
4.2.2 Preparation of BN-rGO modified SPCE 32
4.2.3 Chronoamperometric detection of CySH 32
4.3 Results and discussion 33
4.3.1 TEM images and EFTEM elemental mapping of BN-rGOs 33
4.3.2 XPS analysis of BN-rGOs 34
4.3.3 Electrooxidation behavior of CySH at the BN-rGO/SPCE 37
4.3.4 Effect of pH values on CySH oxidation reaction 40
4.3.5 Kinetic study of CySH on the surface of BN-rGO (3% NH3)-Nafion®/SPCE 44
4.3.6 Chronoamperometric (CA) response of CySH 45
4.3.7 Rotating disk electrode analysis 47
4.3.8 Interference studies 50
4.4 Summary of Chapter 4 51

Chapter 5 Synthesis of Boron/Nitrogen Co-doped Reduced Graphene Oxide
and Its Enhanced Performance for Electrochemical NADH Biosensor 53
5.1 Overview of Chapter 5 53
5.2 Experimental details of Chapter 5 54
5.2.1 Preparation of BN-rGO 54
5.2.2 Preparation of BN-rGO modified SPCE 54
5.2.3 Amperometric detection of NADH 54
5.3 Results and discussion 55
5.3.1 TEM images and EFTEM elemental mapping of BN-rGOs 55
5.3.2 XPS analysis of BN-rGOs 55
5.3.3 Cyclic voltammetry analysis of NADH at the BN-rGO (3% NH3)-Nafion®/SPCE 56
5.3.4 Effect of pH values on NADH oxidation reaction 60
5.3.5 Kinetic study of NADH on the surface of BN-rGO (3% NH3)-Nafion®/SPCE 61
5.3.6 Amperometric detection of NADH 65
5.3.7 Interference studies 67
5.4 Summary of Chapter 5 69

Chapter 6 Conclusions and Suggestions 70
6.1 Conclusions 70
6.2 Suggestions 71

References 73

[1]B. R. Eggins, “Chemical Sensors and Biosensors”, John Wiley & Sons, West Sussex, England (2002).
[2]A. JF Griffiths, J. H Miller, D. T. Suzuki, R. C. Lewontin, and W. M. Gelbart, “An Introduction to Genetic Analysis”, 7th ed., W. H. Freeman, New York (2000).
[3]B. Alberts, A. Johnson, J. Lewis, M. Raff, K. Roberts, P. Walter, “Molecular Biology of the Cell”, 4th ed., Garland Science, New York (2002).
[4]http://csrri.iit.edu/~howard/biochem/lectures/cofactors.html (Illinois Institute of Technology, Biological, Chemical, and Physical Science Department. Referred to this website on 2017/01/03)
[5]J. Wang, “Analytical Electrochemistry”, 3rd ed., John Wiley & Sons, Hoboken, New Jersey (2006).
[6]A. J. Bard, L. R. Faulkner, “Electrochemical Methods: Fundamentals and Applications”, 2nd ed.; John Wiley & Sons, New York (2000).
[7]C. E. Banks, R. G. Compton, “Exploring the Electrocatalytic Sites of Carbon Nanotubes for NADH Detection: an Edge Plane Pyrolytic Graphite Electrode Study”, Analyst, 2005, 130, 1232-1239.
[8]C. E. Banks, T. J. Davies, G. G. Wildgoose, R. G. Compton, “Electrocatalysis at Graphite and Carbon Nanotube Modified Electrodes: Edge-Plane Sites and Tube Ends are the Reactive Sites”, Chem. Commun., 2005, 829-841.
[9]E. J. Biddinger, U. S. Ozkan, “Role of Graphitic Edge Plane Exposure in Carbon Nanostructures for Oxygen Reduction Reaction”, J. Phys. Chem. C, 2010, 114, 15306-15314.
[10]E. C. Landis, K. L. Klein, A. Liao, E. Pop, D. K. Hensley, A. V. Melechko, R. J. Hamers, “Covalent Functionalization and Electron-Transfer Properties of Vertically Aligned Carbon Nanofibers: The Importance of Edge-Plane Sites”, Chem. Mater., 2010, 22, 2357-2366.
[11]C. E. Banks, A. Crossley, C. Salter, S. J. Wilkins, R. G. Compton, “Carbon Nanotubes Contain Metal Impurities Which Are Responsible for the “Electrocatalysis” Seen at Some Nanotube-Modified Electrodes”, Angew. Chem. Int. Ed., 2006, 45, 2533-2537.
[12]K. S. Novoselov, A. K. Geim, S. V. Morozov, D. Jiang, Y. Zhang, S. V. Dubonos, I. V. Grigorieva, A. A. Firsov, “Electric Field Effect in Atomically Thin Carbon Films”, Science, 2004, 306, 663-669.
[13]A. A. Balandin, S. Ghosh, W. Bao, I. Calizo, D. Teweldebrhan, F. Miao, C. N. Lau, “Superior Thermal Conductivity of Single-Layer Graphene”, Nano Lett., 2008, 8, 902-907.
[14]C. Lee, X. Wei, J. W. Kysar, J. Hone, “Measurement of the Elastic Properties and Intrinsic Strength of Monolayer Graphene”, Science, 2008, 321, 385-388.
[15]M. D. Stoller, S. Park, Y. Zhu, J. An, R. S. Ruoff, “Graphene-Based Ultracapacitors”, Nano Lett., 2008, 8, 3498-3502.
[16]S. Alwarappan, A. Erdem, C. Liu, C. Z. Li, “Probing the Electrochemical Properties of Graphene Nanosheets for Biosensing Applications”, J. Phys. Chem. C, 2009, 113, 8853-8857.
[17]M. Pumera, A. Ambrosi, A. Bonanni, E. L. K. Chng, H. L. Poh, “Graphene for Electrochemical Sensing and Biosensing”, TrAC-Trends Anal. Chem., 2010, 29, 954-965.
[18]J. Ping, J. Wu, Y. Wang, Y. Ying, “Simultaneous Determination of Ascorbic Acid, Dopamine and Uric Acid Using High-Performance Screen-Printed Graphene Electrode”, Biosens. Bioelectron., 2012, 34, 70-76.
[19]X. C. Dong, H. Xu, X. W. Wang, Y. X. Huang, M. B. Chan-Park, H. Zhang, L. H. Wang, W. Huang, P. Chen, “3D Graphene–Cobalt Oxide Electrode for High-Performance Supercapacitor and Enzymeless Glucose Detection”, ACS Nano, 2012, 6, 3206-3213.
[20]R. Jain, A. Sinha, “A Graphene Based Sensor for Sensitive Voltammetric Quantification of Cabergoline”, J. Electrochem. Soc., 2014, 160, H314-H320.
[21]L. J. Brennan, S. T. Barwich, A. Satti, A. Faure, Y. K. Gunko, “Graphene–ionic liquid electrolytes for dye sensitised solar cells”, J. Mater. Chem. A, 2013, 1, 8379-8384.
[22]K. H. Hung, C. H. Chan, H. W. Wang, “Flexible TCO-free counter electrode for dye-sensitized solar cells using graphene nanosheets from a Ti–Ti(III) acid solution”, Renew. Energy, 2014, 66, 150-158.
[23]G. Ning, Z. Fan, G. Wang, J. Gao, W. Qian, F. Wei, “Gram-Scale Synthesis of Nanomesh Graphene with High Surface Area and Its Application in Supercapacitor Electrodes”, Chem. Commun., 2011, 47, 5976-5978.
[24]Z. Chen, D. Yu, W. Xiong, P. Liu, Y. Liu, L. Dai, “Graphene-Based Nanowire Supercapacitors”, Langmuir, 2014, 30, 3567-3571.
[25]J. B. Raoof, R. Ojani, H. Beitollahi, “L-Cysteine Voltammetry at a Carbon Paste Electrode Bulk-Modified with Ferrocenedicarboxylic Acid”, Electroanalysis, 2007, 19, 1822-1830.
[26]O. Rusin, N. N. St. Luce, R. A. Agbaria, J. O. Escobedo, S. Jiang, I. M. Warner, F. B. Dawan, K. Lian, R. M. Strongin, “Visual Detection of Cysteine and Homocysteine”, J. Am. Chem. Soc., 2004, 126, 438-439.
[27]S. Shahrokhian, “Lead Phthalocyanine as a Selective Carrier for Preparation of a Cysteine-Selective Electrode”, Anal. Chem., 2001, 73, 5972-5978.
[28]D. Rhodes, A.C. Myers, G. Jamieson, “Gas Chromatography-Mass Spectrometry of N- Heptafluorobutyryl Isobutyl Esters of Amino Acids in the Analysis of the Kinetics of [15N]H4+ Assimilation in Lemna minor L”, Plant Physiol., 1981, 68, 1197-1205.
[29]J. Dumonceaux, C. Goujon, V. Joliot, P. Briand, U. Hazan, “Determination of Essential Amino Acids Involved in the CD4-Independent Tropism of the X4 Human Immunodeficiency Virus Type 1 m7NDK Isolate: Role of Potential N Glycosylations in the C2 and V3 Regions of gp120”, J. Virol., 2001, 75, 5425-5428.
[30]P. Schrynemackers-Pitance, S. Schoos-Berbette, “Determination of Aromatic and Neutral Amino Acids by HPLC in Blood Specimens Collected on Filter Paper”, Clin. Chim. Acta, 1987, 166, 91.
[31]Y. Wang, J. Lu, L. H. Tang, H. X. Chang, J. H. Li, “Graphene Oxide Amplified Electrogenerated Chemiluminescence of Quantum Dots and Its Selective Sensing for Glutathione from Thiol-Containing Compounds”, Anal. Chem. 2009, 81, 9710-9715.
[32]M. Zhou, J. Ding, L. P. Guo, Q. K. Shang, “Electrochemical Behavior of l-Cysteine and Its Detection at Ordered Mesoporous Carbon-Modified Glassy Carbon Electrode”, Anal. Chem., 2007, 79, 5328-5335.
[33]L. P. Liu, Z. J. Yin, Z. S. Yang, “A L-cysteine Sensor Based on Pt Nanoparticles/Poly(o-aminophenol) Film on Glassy Carbon Electrode”, Bioelectrochem., 2010, 79, 84-89.
[34]H. Hosseini, H. Ahmar, A. Dehghani, A. Bagheri, A. Tadjarodi and A.R. Fakhari, “A Novel Electrochemical Sensor Based on Metal-Organic Framework for Electro-Catalytic Oxidation of L-cysteine”, Biosens. Bioelectron., 2013, 42, 426-429.
[35]J. A. Reyanud, B. Maltoy, P. Canessan, “Electrochemical Investigations of Amino Acids at Solid Electrodes: Part I. Sulfur Components: Cystine, Cysteine, Methionine”, J. Electroanal. Chem., 1980, 114, 195-211.
[36]D. L. Rabenstein, G. T. Yamashita, “Determination of Homocysteine, Penicillamine, and Their Symmetrical and Mixed Disulfides by Liquid Chromatography with Electrochemical Detection”, Anal. Biochem., 1989, 180, 259-263.
[37]J. L. D’Eramo, A. E. Finkelstein, F. Q. Boccazzi, O. Fridman, “Total Homocysteine Levels in Plasma: High-Performance Liquid Chromatographic Determination with Electrochemical Detection and Glassy Carbon Electrode”, J. Chromatogr. B, 1998, 720, 205-210.
[38]P. J. Vandeberg, D. C. Johnson, “Pulsed Electrochemical Detection of Cysteine, Cystine, Methionine, and Glutathione at Gold Electrodes Following Their Separation by Liquid Chromatography”, Anal. Chem., 1993, 65, 2713-2718.
[39]N. Maleki, A. Safavi, F. Sedaghati, F. Tajabadi, “Efficient Electrocatalysis of L-cysteine Oxidation at Carbon Ionic Liquid Electrode”, Anal. Biochem., 2007, 369, 149-153.
[40]T. R. Ralph, M. L. Hitchman, J. P. Millington, F. C. Walsh, “The Reduction of L-cystine Hydrochloride at Lead Using Static and Rotating Disc Electrodes”, J. Electroanal. Chem., 2005, 583, 260-272.
[41]N. Spãtaru, B. V. Sarada, E. Popa, D. A. Tryk, A. Fujishima, “Voltammetric Determination of L-Cysteine at Conductive Diamond Electrodes”, Anal. Chem., 2001, 73, 514-519.
[42]M. T. Stankovich, A. J. Bard, “The Electrochemistry of Proteins and Related Substances: I. Cystine and Cysteine at the Mercury Electrode”, J. Electroanal. Chem., 1977, 75, 487-505.
[43]F. G. Bãnicã, J. C. Moreira, A. G. Fogg, “Application of Catalytic Stripping Voltammetry for the Determination of Organic Sulfur Compounds at a Hanging Mercury Drop Electrode: Behaviour of Cysteine, Cystine and N-acetylcysteine in the Presence of Nickel Ion”, Analyst, 1994, 119, 309.
[44]T. Inoue, J. R. Kirchhoff, “Electrochemical Detection of Thiols with a Coenzyme Pyrroloquinoline Quinone Modified Electrode”, Anal. Chem., 2000, 72, 5755-5760.
[45]T. Inoue, J.R. Kirchhoff, “Determination of Thiols by Capillary Electrophoresis with Amperometric Detection at a Coenzyme Pyrroloquinoline Quinone Modified Electrode”, Anal. Chem., 2002, 74, 1349-1354.
[46]D. Mimica, F. Bedioui, J. H. Zagal, “Reversibility of the L-cysteine/L-cystine Redox Process at Physiological pH on Graphite Electrodes Modified with Coenzyme B12 and Vitamin B12”, Electrochim. Acta, 2002, 48, 323-329.
[47]A. K. M. Kafi, F. Yin, H. K. Shin, Y.-S. Kwon, “Amperometric Thiol Sensor Based on Prussian Blue-Modified Glassy Carbon Electrode”, Curr. Appl. Phys., 2007, 7, 496-499.
[48]K. P. Gong, X. Z. Zhu, R. Zhao, S. X. Xiong, L. Q. Mao, C. F. Chen, “Rational Attachment of Synthetic Triptycene Orthoquinone onto Carbon Nanotubes for Electrocatalysis and Sensitive Detection of Thiols”, Anal. Chem., 2005, 77, 8158.
[49]N. Sehlothl, T. Nyokong, J. H. Zagal, F. Bedioui, “Electrocatalysis of Oxidation of 2-mercaptoethanol, L-cysteine and Reduced Glutathione by Adsorbed and Electrodeposited Cobalt Tetra Phenoxypyrrole and Tetra Ethoxythiophene Substituted Phthalocyanines”, Electrochim. Acta, 2006, 51, 5125-5130.
[50]C. Y. Deng, J. H. Chen, X. L. Chen, Z. Nie, S. Z. Yao, “Boron-Doped Carbon Nanotubes Modified Electrode for Electroanalysis of NADH”, Electrochem. Commun., 2008, 10, 907-909.
[51]C. Y. Deng, J. H. Chen, X. L. Chen, C. H. Xiao, L. H. Nie, S. Z. Yao, ”Direct Electrochemistry of Glucose Oxidase and Biosensing for Glucose Based on Boron-Doped Carbon Nanotubes Modified Electrode”, Biosens. Bioelectron., 2008, 23, 1272-1277.
[52]N. Q. Jia, L. J. Wang, L. Liu, Q. Zhou, Z. Y. Jiang, “Bamboo-Like CNx Nanotubes for the Immobilization of Hemoglobin and Its Bioelectrochemistry”, Electrochem. Commun., 2005, 7, 349-354.
[53]R. X. Wang, D. J. Zhang, Y. M. Zhang, C. B. Liu, “Boron-Doped Carbon Nanotubes Serving as a Novel Chemical Sensor for Formaldehyde”, J. Phys. Chem. B, 2006, 110, 18267-18271.
[54]L. Wu, X. J. Zhang, H. X. Ju, “Detection of NADH and Ethanol Based on Catalytic Activity of Soluble Carbon Nanofiber with Low Overpotential”, Anal. Chem., 2007, 79, 453-458.
[55]T. J. Davies, R. R. Moore, C. E. Banks, R. G. Compton, “The Cyclic Voltammetric Response of Electrochemically Heterogeneous Surfaces”, J. Electroanal. Chem., 2004, 574, 123.
[56]A. Salimi, C. E. Banks, R. G. Compton, “Abrasive Immobilization of Carbon Nanotubes on a Basal Plane Pyrolytic Graphite Electrode: Application to the Detection of Epinephrine”, Analyst, 2004, 129, 225-228.
[57]M. Zhang, K. P. Gong, H. Z. Zhang, L. Q. Mao, “Layer-By-Layer Assembled Carbon Nanotubes for Selective Determination of Dopamine in the Presence of Ascorbic Acid”, Biosens. Bioelectron., 2005, 20, 1270-1276.
[58]A. Kuznetsova, D. B. Mawhinney, V. Naumenko, J. T. Yates Jr., J. Liu, R. E. Smalley, “Enhancement of Adsorption Inside of Single-Walled Nanotubes: Opening the Entry Ports”, Chem. Phys. Lett., 2000, 321, 292-296.
[59]D. C. Marcano, D. V. Kosynkin, J. M. Berlin, A. Sinitskii, Z. Sun, A. Slesarev, L. B. Alemany, W. Lu, J. M. Tour, “Improved Synthesis of Graphene Oxide”, ACS Nano, 2010, 4, 4806-4814.
[60]S. Park, J. An, J. R. Potts, A. Velamakanni, S. Murali, R. S. Ruoff, “Hydrazine-Reduction of Graphite and Graphene oxide”, Carbon, 2011, 49, 3019-3023.
[61]T. W. Lin, C. Y. Su, X. Q. Zhang, W. Zhang, Y. H. Lee, C.W. Chu, H. Y. Lin, M. T. Chang, F. R. Chen, L. J. Li, “Converting Graphene Oxide Monolayers into Boron Carbonitride Nanosheets by Substitutional Doping”, Small, 2012, 8, 1384-1391.
[62]T. Schiros, D. Nordlund, L. Palova, L. Y. Zhao,M. Levendorf, C. Jaye, D. Reichman, J. Park, M. Hybertsen, A. Pasupathy, “Atomistic Interrogation of B–N Co-dopant Structures and Their Electronic Effects in Graphene”, ACS Nano, 2016, 10, 6574-6584.
[63]R. S. Krsmanović, Ž. Šljivančanin, “Atomic Structure, Electronic Properties, and Reactivity of In-Plane Heterostructures of Graphene and Hexagonal Boron Nitride”, J. Phys. Chem. C, 2014, 118, 16104-16112.
[64]B. Hammer, “Special Sites at Noble and Late Transition Metal Catalysts”, Top. Catal., 2006, 37, 3-16.
[65]T. R. Ralph, M.L. Hitchman, J. P. Millington, F. C. Walsh, “The Electrochemistry of L-cystine and L-cysteine: Part 1: Thermodynamic and Kinetic Studies”, J. Electroanal. Chem., 1994, 375, 1-15.
[66]S. G. Ge, M. Yan, J. J. Lu, M. Zhang, F. Yu, J. H. Yu, X. R. Song, S. L. Yu, “Electrochemical Biosensor Based on Graphene Oxide-Au Nanoclusters Composites for L-cysteine Analysis”, Biosens. Bioelectron., 2012, 31, 49-54.
[67]C. Y. Deng, J. H. Chen, X. L. Chen, M. D. Wang, Z. Nie, S. Z. Yao, “Electrochemical Detection of L-cysteine Using a Boron-Doped Carbon Nanotube-Modified Electrode”, Electrochim. Acta, 2009, 54, 3298-3302.
[68]S. Budavari, M.J. O’Neil, A. Smith, P.E. Heckelman, The Merck Index, 11th ed.; Merck & Co. Inc. Rahway, NJ, 1989.
[69]T. R. Ralph, M. L. Hitchman, J. P. Millington, F. C. Walsh, “The Reduction of L-cystine in Hydrochloric Acid at Mercury Drop Electrodes”, J. Electroanal. Chem., 2006, 587, 31-41.
[70]M. Zhou, J. Ding, L. P. Guo, Q. K. Shang, “Electrochemical Behavior of L-Cysteine and Its Detection at Ordered Mesoporous Carbon-Modified Glassy Carbon Electrode”, Anal. Chem., 2007, 79, 5328-5335.
[71]Y. T. Lai, A. Ganguly, L. C. Chen, K. H. Chen, “Direct Voltammetric Sensing of L-Cysteine at Pristine GaN Nanowires Electrode”, Biosens. Bioelectron., 2010, 26, 1688-1691.
[72]S. Ge, M. Yan, J. Lu, M. Zhang, F. Yu, J. Yu, X. Song, S. Yu, “Electrochemical Biosensor Based on Graphene Oxide-Au Nanoclusters Composites for L-cysteine Analysis”, Biosens. Bioelectron., 2012, 31, 49-54.
[73]S. Fei, J. Chen, S. Yao, G. Deng, D. He, Y. Kuang, “Electrochemical Behavior of L-cysteine and Its Detection at Carbon Nanotube Electrode Modified with Platinum”, Anal. Biochem., 2005, 339, 29-35.
[74]X. Q. Wang, Y. P. Wen, L. M. Lu, J. K. Xu, L. Zhang, Y. Y. Yao, H. H. He, “A Novel L-Cysteine Electrochemical Sensor Using Sulfonated Graphene-poly(3,4-Ethylenedioxythiophene) Composite Film Decorated with Gold Nanoparticles”, Electroanalysis, 2014, 26, 648-655.
[75]Y. Dong, J. B. Zheng, “A Nonenzymatic L-cysteine Sensor Based on SnO2-MWCNTs Nanocomposites”, J. Mol. Liq., 2014, 196, 280-284.
[76]R. X. Shen, X. Z. Li, G. L. Lium, Y. L. Ji, G. F. Wang, B. Fang, “Synthesis and Characterization of Chromium Hexacyanoferrate/Multiwalled Carbon Nanotube Composite and Its Biosensing for L-Cysteine”, Electroanalysis, 2010, 22, 2383-2388.
[77]G. H. Yang, Y. H. Zhou, J. J. Wu, J. T. Cao, L. L. Li, H. Y. Liu, J. J. Zhu, “Microwave-Assisted Synthesis of Nitrogen and Boron Co-doped Graphene and Its Application for Enhanced Electrochemical Detection of Hydrogen Peroxide”, RSC Adv., 2013, 3, 22597-22604.
[78]P. N. Bartlett, Bioelectrochemistry: Fundamentals, Experimental Techniques and Applications. John Wiley & Sons, New York, USA (2008).
[79]F. Ricci, A. Amine, D. Moscone, G. Palleschi, “Surface Chemistry Effects on the Performance of an Electrochemical DNA Sensor”, Biosens. Bioelectron., 2007, 22, 854-862. 
[80]J. Moiroux, P. J. Elving, “Effects of Adsorption, Electrode Material, and Operational Variables on the Oxidation of Dihydronicotinamide Adenine Dinucleotide at Carbon Electrodes”, Anal. Chem., 1978, 50, 1056-1062. 
[81]G. D. Birkmayer, NADH: The Energizing Coenzyme, Basic Health Publications, Inc, United States. (1993) pp. 1-22. 
[82]G. D. Birkmayer, “NADH: The Biological Hydrogen: The Secret of Our Life Energy”, 2009, Basic Health Publications, Inc, United States. 
[83]J. Castro-Marrero, M. D. Cordero, M. J. Segundo, N. Sáez-Francàs, N. Calvo, L. Román-Malo, L. Aliste, T. FernándezdeSevilla, J. Alegre, “Does Oral Coenzyme Q10 Plus NADH Supplementation Improve Fatigue and Biochemical Parameters in Chronic Fatigue Syndrome?”, Redox Signal., 2014, 22, 679-685. 
[84]R. Sahelian, 2014, NADH Supplement Benefit, Side Effects, 5 mg 10 mg and 20 mg Tablets, Fatigue URL: 〈http://www.raysahelian.com/nadh.html〉.
[85]T. S. Blacker, Z. F. Mann, J. E. Gale, M. Ziegler, A. J. Bain, G. Szabadkai, M. R. Duchen, “Separating NADH and NADPH Fluorescence in Live Cells and Tissues Using FLIM”, Nature Commun., 2014, 5, 3936. 
[86]V. B. Ritov, E. V. Menshikova, D. E. Kelley, “High-Performance Liquid Chromatography-Based Methods of Enzymatic Analysis: Electron Transport Chain Activity in Mitochondria from Human Skeletal Muscle”, Anal. Biochem., 2004, 333, 27-38. 
[87]P. Aronov, Y. Kawahara, J. Cox, T. Christison, L. Lopez, 2014, Determinationof NADH and NADPH Ssing ion Chromatography and High Resolution Accurate Mass Spectrometry. In: Thermo Scientific. pp.1-8. 
[88]H. Huck, A. S. Graf, J. Danzer, P. Kirch, H. L. Schmidt, “Bioelectrochemical Detection Systems for Substrates of Dehydrogenases”, Analyst, 1984, 109, 147-150.
[89]S. Mutyala, J. Mathiyarasu, “A Highly Sensitive NADH Biosensor Using Nitrogen Doped Graphene Modified Electrodes”, J. Electroanal. Chem., 2016, 775, 329-336.
[90]J. Balamurugan, T. D. Thanh, N. H. Kim, J. H. Lee, “Facile Fabrication of FeN Nanoparticles/Nitrogen-Doped Graphene Core-Shell Hybrid and Its Use as a Platform for NADH Detection in Human Blood Serum”, Biosens. Bioelectron., 2016, 83, 68-76.
[91]M. Eguílaz, F. Gutierrez, J.M. González-Domínguez, M.T. Martínez, G. Rivas, “Single-Walled Carbon Nanotubes Covalently Functionalized with Polytyrosine: A New Material for the Development of NADH-Based Biosensors”, Biosens. Bioelectron., 2016, 86, 308-314.
[92]P. P. Gai, C. E. Zhao, Y. Wang, E. S. Abdel-Halim, J. R. Zhang, J. J. Zhu, “NADH Dehydrogenase-Like Behavior of Nitrogen-Doped Graphene and Its Application in NAD+-Dependent Dehydrogenase Biosensing”, Biosens. Bioelectron., 2014, 62, 170-176.
[93]C. H. Chen, Y. C. Chen, M. S. Lin, “Urea Based Organic Nanoparticles for Selective Determination of NADH”, Biosens. Bioelectron., 2013, 42, 379-384. 
[94]C. O. Schmakel, K. S. V. Santhanam, P. J. Elving, “Nicotinamide Adenine Dinucleotide (NAD+) and Related Compounds. Electrochemical Redox Pattern and Allied Chemical Behavior”, J. Am. Chem. Soc., 1975, 97, 5083-5092.
[95]A. A. Karyakin, E. E. Karyakina, W. Schuhmann, H. L. Schmidt, “Electropolymerized Azines: Part II. In a Search of the Best Electrocatalyst of NADH Oxidation”, Electroanalysis, 1999, 11, 553-557.
[96]R. D. Braun, K. S. V. Santhanam, P. J. Elving, “Electrochemical Oxidation in Aqueous and Nonaqueous Media of Dihydropyridine Nucleotides NMNH, NADH, and NADPH”, J. Am. Chem. Soc., 1975, 97, 2591-2598.
[97]A. M. Wilson, D. G. Epple, “A Comprehensive Study of the Postnatal Changes in the Concentration of the Lipids of Developing Rat Brain”, Biochemistry, 1966, 5, 3170-3175.
[98]L. Gorton, E. Dominguez, in: A. J. Bard, M. Stratmann (Eds.), NAD(P)+/NAD(P)H in Living Systems, Encyclopaedia of Electrochemistry, Wiley-VCH 2002, p. 77.
[99]L. N. Wu, X. J. Zhang, H. X. Ju, “Detection of NADH and Ethanol Based on Catalytic Activity of Soluble Carbon Nanofiber with Low Overpotential”, Anal. Chem., 2007, 79, 453-458.
[100]T. J. Davies, R. R. Moore, C. E. Banks, R. G. Compton, “The Cyclic Voltammetric Response of Electrochemically Heterogeneous Surfaces”, J. Electroanal. Chem., 2004, 574, 123-152.
[101]M. N. Zhang, K. P. Gong, H. Z. Zhang, L. Q. Mao, “Layer-By-Layer Assembled Carbon Nanotubes for Selective Determination of Dopamine in the Presence of Ascorbic Acid”, Biosens. Bioelectron., 2005, 20, 1270-1276.
[102]J. Moiroux, P.J. Elving, “Mechanistic Aspects of the Electrochemical Oxidation of Dihydronicotinamide Adenine Dinucleotide (NADH)”, J. Am. Chem. Soc., 1980, 102, 6533-6538.
[103]R. R. Moore, C. E. Banks, R. G. Compton, “Basal Plane Pyrolytic Graphite Modified Electrodes:  Comparison of Carbon Nanotubes and Graphite Powder as Electrocatalysts”, Anal. Chem., 2004, 76, 2677-2682.
[104]F. S. Saleh, L. Q. Mao, T. Ohsaka, “Development of a Dehydrogenase-Based Glucose Anode Using a Molecular Assembly Composed of Nile Blue and Functionalized SWCNTs and Its Applications to a Glucose Sensor and Glucose/O2 Biofuel Cell”, Sensor Actuat. B-Chem., 2011, 152, 130-135.
[105]T. Maiyalagan, J. Sundaramurthy, P.S. Kumar, P. Kannan, M. Opallo, S. Ramakrishna, “Nanostructured α-Fe2O3 Platform for the Electrochemical Sensing of Folic Acid”, Analyst, 2013, 138, 1779-1786.
[106]J. G. Velasco, “Determination of Standard Rate Constants for Electrochemical Irreversible Processes From Linear Sweep Voltammograms”, Electroanalysis, 1997, 9, 880-882.
[107]G. P. Keeley, A. O´Neill, M. Holzinger, S. Cosnier, J. N. Coleman, G. S. Duesberg, “DMF-Exfoliated Graphene for Electrochemical NADH Detection”, Phys. Chem. Chem. Phys., 2011, 13, 7747-7750. 
[108]Y. Fan, X. Yang, C. Yang, J. Liu, “Au-TiO2/Graphene Nanocomposite Film for Electrochemical Sensing of Hydrogen Peroxide and NADH”, Electroanalysis, 2012, 6, 1334-1339. 
[109]H. Teymourian, A. Salimi, S. Khezrian, “Fe3O4 Magnetic Nanoparticles/Reduced Graphene Oxide Nanosheets as a Novel Electrochemical and Bioeletrochemical Sensing Platform”, Biosens. Bioelectron., 2013, 49, 1-8.  
[110]G. M. Mota-Ferreira, F. Mota-deOliveira, F. R. Figueiredo-Leite, C. M. Maroneze, L. T. Kubota, F. S. Damos, R. C. Silva-Luz, “DNA and Graphene as a New Efficient Platform for Entrapment of Methylene blue (MB): Studies of the Electrocatalytic Oxidation of β-nicotinamide Adenine Dinucleotide”, Electrochim. Acta, 2013, 111, 543-551. 
[111]V. Serafin, L. Agüí, P. Yañez-Sedeño, J.M. Pingarrón, “A Novel Hybrid Platform for the Preparation of Disposable Enzyme Biosensors Based on Poly(3,4-ethylenedioxythiophene) Electrodeposition in an Ionic Liquid Medium onto Gold Nanoparticles-Modified Screen-Printed Electrodes”, J. Electroanal. Chem., 2011, 656, 152-158.
[112]E. Hua, L. Wang, X. Jing, C. Chen, G. Xie, “One-Step Fabrication of Integrated Disposable Biosensor Based on ADH/NAD+/Meldola''s Blue/Graphitized Mesoporous Carbons/Chitosan Nanobiocomposite for Ethanol Dsetection”, Talanta, 2013, 111,163-169. 
[113]M. Revenga-Parra, C. Gomez-Anquela, T. Garcia-Mendiola, E. Gonzalez, F. Pariente, E. Lorenzo, “Grafted Azure a Modified Electrodes as Disposable β-Nicotinamide Adenine Dinucleotide Sensors”, Anal. Chim. Acta, 2012, 747, 84-91.
[114]http://delloyd.50megs.com/moreinfo/buffers2.html (Delloyd’s Lab Tech, resources reagents and solutions. Referred to this website on 2015/06/18).
[115]McGraw Hill Encyclopedia of Science & Technology, “Graphene and graphene oxide membranes for water treatment”, 11th Edition Keystroked (2016).
[116]L. Zhao, M. Levendorf, S. Goncher, T. Schiros, L. Pálová, A. Zabet-Khosousi, K.T. Rim, C. Gutiérrez, D. Nordlund, C. Jaye, M. Hybertsen, D. Reichman, G.W. Flynn, J. Park, A.N. Pasupathy, “Local Atomic and Electronic Structure of Boron Chemical Doping in Monolayer Graphene”, Nano Lett., 2013, 13, 4659-4665.