Electrochemical study of some selected neurotransmitters at metal oxide doped phthalocyanine supported on carbon nanotubes sensor platform

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ELECTROCHEMICAL STUDY OF SOME SELECTED

NEUROTRANSMITTERS AT METAL OXIDE DOPED PHTHALOCYANINE

SUPPORTED ON CARBON NANOTUBES SENSOR PLATFORM

NTSOAKI G MPHUTHI

BSc (NWU), BSc (Hons) (NWU)

Thesis submitted in partial fulfillment of the requirements for the

degree Master of Science in the

Department of Chemistry

Faculty of Agriculture, Science and Technology

North-West University (Mafikeng Campus)

Supervisor:

Prof Eno. E. Ebenso

Co-supervisor:

Dr Abolanle S. Adekunle

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DECLARATION

I hereby declare that the dissertation entitled “Electrochemical study of some selected Neurotransmitters at metal oxide doped phthalocyanine supported on carbon nanotubes sensor platform” submitted to the Department of Chemistry, North West University, Mafikeng Campus in the fulfilment of the requirements for the degree Master of Science in Chemistry is the original Research work conducted by myself under the supervision of Prof. Eno Ebenso and Dr. AS. Adekunle. None of this Research work has been submitted by any student, at the North West University or any other University.

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ACKNOWLEDGEMENTS

I would like to thank Jesus Christ as my Lord and Saviour for granting me with new mercies and steadfast love every day. I wouldn’t be here if it wasn’t for His Grace.

Thank you to my supervisor Prof Ebenso for believing and seeing the potential in me. For giving me the opportunity to go places i never thought i would go.

Thank you to Dr Adekunle for his guidance and support throughout my research work and for always encouraging me to do my best.

I would like to thank the Department of Education, Free State province for financing my undergraduate studies. Thank you to Sasol-Inzalo, NRF and North West university postgraduate bursary for funding my postgraduate studies.

Special thanks to my colleagues, Fayemi Esther, Lukman Olasunkanmi, Dr Sasikumar Yesudass, Diseko Boikanyo, Dr Mwadham Kabanda, Mashuga Motsie, Palesa Tsele and Lorato Gwala, I really appreciate their kindness and help.

Thank you to MaSIM research group and chemistry department, Miss Maggy Medupi for her support and help. Special appreciation to Kagiso Mokalane, Sizwe Loyilani, Peter Mahlangu and Dr Damian Onwudiwe for their technical support.

Thanks to my high school teacher Mr Nthako for his support, guidance and for helping to get me admitted to the university. And I would like to appreciate the HOD of Free State Department Mr Malope for his support and encouragement.

Thank you Miss Fayemi Esther and Elijah Mashuga for spiritual support and guidance. God Bless you exceedingly and abundantly above all.

Thank you to my special mom; Modiehi Motaung, brothers; Simon Mphuthi, Dallas Mphuthi, Tankiso Mphuthi and Hlomla Mphuthi, sisters; Makgala Mphuthi and Refiloe Pilane. Acknowledgements to my friends, for their moral support, I love them so much, God bless them.

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ABSTRACT

The electrocatalytic properties of metal oxides (MO = Fe3O4, ZnO) nanoparticles doped

phthalocyanine (Pc) and functionalized multi-walled carbon nanotubes (MWCNTs), decorated on glassy carbon electrode (GCE) was investigated. Successful synthesis of the metal oxide nanoparticles and the MO/Pc/MWCNT composite were confirmed using Ultraviolet-visible spectrophotometry (UV-Vis), Energy-dispersive X-ray spectroscopy (EDX), X-ray powder diffraction (XRD) and Transmission electron microscopy (TEM) techniques. Successful modification of GCE with the MO and their composite was also confirmed using cyclic voltammetry (CV) technique. The potential of the developed sensor towards dopamine (DA) oxidation was explored using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopic (EIS). Results indicate that GCE-MWCNT/Fe3O4/2,3-Nc, GCE-MWCNT/Fe3O4/29H,31H-Pc, GCE-MWCNT/ZnO/2,3-Nc and

GCE-MWCNT/ZnO/29H,31H-Pc modified electrodes demonstrated fast electron transport or lower charge transfer resistance, and enhanced DA oxidation current compared with other electrodes studied. Electroanalysis results indicated that the four GCE-MWCNT/MO/Pc modified electrodes were stable towards DA oxidation with minimum current drop (5-10%) after 20 scans. GCE-MWCNT/ZnO/29H,31H-Pc was the best electrode towards DA detection with very low detection limit (0.75 µM) that compared with literature, good sensitivity (1.45 µA/ µM), resistance to electrode fouling, and excellent ability to detect DA without interference from ascorbic acid (AA) signal. Electrocatalytic oxidation of DA on GCE-MWCNT/ZnO/29H,31H-Pc electrode was diffusion controlled but characterized with some adsorption of electro-oxidation reaction intermediates products. The fabricated sensors are easy to prepare, cost effective and can be applied for real sample analysis of dopamine in biological samples.

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Furthermore, glassy carbon electrode (GCE) was also modified with metal oxides (MO = Fe3O4, ZnO) nanoparticles doped phthalocyanine (Pc) and functionalized MWCNTs, and the

electrocatalytic properties was studied. Successful synthesis of the metal oxide nanoparticles and the MO/Pc/MWCNT composite were confirmed using Fourier transform infrared spectroscopy (FTIR), Raman and Scanning electron microscopy (SEM) techniques. The electrodes were characterised using cyclic voltammetry (CV) technique. The electrocatalytic behaviour of the electrodes towards epinephrine (EP) and norepinephrine (NE) oxidation was investigated using cyclic voltammetry (CV), differential pulse voltammetry (DPV) and electrochemical impedance spectroscopic (EIS). Result showed that GCE-MWCNT/Fe3O4/2,3-Nc, GCE-MWCNT/Fe3O4/29H,31H-Pc, GCE-MWCNT/ZnO/2,3-Nc and

GCE-MWCNT/ZnO/29H,31H-Pc electrodes gave enhanced EP and NE current response. Stability study indicated that the four GCE-MWCNT/MO/Pc modified electrodes were stable against electrode fouling effect with the percentage NE current drop of 5.56%, 5.88%, 5.56% and 5.56% for MWCNT/Fe3O4/2,3-Nc, MWCNT/Fe3O4/29H,31H-Pc, MWCNT/ZnO/2,3-Nc and

MWCNT/ZnO/29H,31H-Pc modified electrodes respectively after 20 scans. GCE-MWCNT/ Fe3O4/29H,31H-Pc gave the lowest limit of detection (4.6 µM) towards EP while

MWCNT/ZnO/29H,31H-Pc gave the lowest limit of detection (1.7 µM) towards NE. The limit of detection and sensitivity of the electrodes compared well with literature. Electrocatalytic oxidation of EP and NE on GCE-MWCNT/MO/Pc electrodes was diffusion controlled with some adsorption of electro-oxidation reaction intermediates products. The electrodes were found to be electrochemically stable, reusable and can be used for the analysis of EP and NE in real life samples.

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LIST OF ABBREVIATIONS

Ag/AgCl Silver/silver chloride reference electrode

CNT Carbon nanotubes

CV Cyclic voltammetry

CV Cyclic voltammogram

DMF Dimethylformamide

EIS Electrochemical impedance spectroscopy GCE Glassy carbon electrode

LoD Limit of detection

LSV Linear sweep voltammetry

MO Metal oxides

MWCNTs Multi-walled carbon nanotubes

NPs Nanoparticles

PBS Phosphate buffer solution R.E. Reference electrode

SCE Standard calomel electrode SWCNTs Single-walled carbon nanotubes. SWV Square wave voltammetry W.E. Working electrode

Pc Phthalocyanine

Nc Naphthalocyanine

DPV Differential pulse voltammetry

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EP Epinephrine

NE Norepinephrine

AA Ascorbic acid

UA Uric acid

TEM Transmission electron microscopy SEM Scanning Electron Microscopy XRD X-ray powder diffraction

EDX Energy-dispersive X-ray spectroscopy FTIR Fourier transform infrared spectroscopy VI-Vis Ultraviolet-visible spectrophotometry

ST Serotonin

MPc Metallophthalocyanine MNc Metal naphthalocyanine

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LIST OF SYMBOLS

Г Surface coverage or concentration

π Pi bonding

c Molar concentration of analyte

Cdl Double-layer capacitance CPE Constant phase electrode

D Diffusion coefficient

Epa Anodic peak potential Epc Cathodic peak potential

E Potential

Eº Standard potential

E1/2 Half-wave potential

F Faraday constant

Hz Hertz

ipa Anodic peak current

ipc Cathodic peak current

K Kelvin

n Number of electron

R Universal gas constant

Rct Charge transfer resistance

Rs Resistance of electrolyte

υ Scan rate

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LIST OF FIGURES

Figure 1: Sequence of enzymatic reactions generating other catecholamines

from L-DOPA ... 10

Figure 2: Oxidation mechanism of DA ... 12

Figure 3: Oxidation mechanism of EP ... 13

Figure 4: Oxidation mechanism of NE ... 15

Figure 5: Oxidation mechanism of (a) AA and (b) UA ... 16

Figure 6: Structure of (a) SWCNT and (b) MWCNTs ... 19

Figure 7: Nitric acid oxidation method of pristine CNT ... 21

Figure 8: Molecular structures of Pc and MPc ... 27

Figure 9: Molecular structure of Ncs ... 28

Figure 10: A 3-electrode electrochemical cell ... 31

Figure 11: A typical chronoamperogram obtained from modified Pt electrode ... 40

Figure 12: Randles’ equivalent circuit for an electrode in contact with an electrolyte ... 43

Figure 13: UV-Vis spectra of (a) Fe3O4, Fe3O4/2,3-Nc, Fe3O4/29H,31H-Pc and (b) ZnO, ZnO/2,3-Nc, ZnO/29H,31H-Pc in DMF solution ... 56

Figure 14: FTIR spectra of (a) Fe3O4, (b) ZnO and (c) MWCNT-COOH ... 57

Figure 15: Raman spectra of pristine and functionalized MWCNTs ... 59

Figure 16: EDX spectra of (a) MWCNT/ZnO/Pc (b) MWCNT/Fe3O4/Pc ... 60

Figure 17: X-ray diffraction patterns of (a) Fe3O4 (b) ZnO (c) functionalized and pristine MWCNTs ... 62

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Figure 18: SEM images of (a) Fe3O4, (b) ZnO, (c) 2,3-Nc, (d) 29H,31H-Pc, (e)

MWCNTs ... 64 Figure 19: SEM images of (a) MWCNT/ZnO/2,3-Nc, (b) MWCNT/Fe3O4/2,3-Nc

(c) MWCNT ZnO/29H,31H-Pc and (d) MWCNT/Fe3O4/29H,31H-Pc ... 65 Figure 20: TEM images of (a) Fe3O4 NPs, (b) ZnO NPs, (c) 2,3-Nc, (d) 29H,31H-Pc,

(e) MWCNT ... 67 Figure 21: TEM images of (a) MWCNT/ZnO/2,3-Nc, (b) MWCNT/Fe3O4/2,3-Nc

(c) MWCNT ZnO/29H,31H-Pc and (d) MWCNT/Fe3O4/29H,31H-Pc.... ... 68

Figure 22: Cyclic voltammetry evolutions of modified electrodes in 5mM

[Fe(CN)6]4- /[Fe(CN)6]3- (b) and (c) and in pH 7.2 PBS (b) and (d). ... 70

Figure 23: Cyclic voltammetry evolutions of modified electrodes in 5mM

[Fe(CN)6]4- /[Fe(CN)6]3- (b) and (c) and in pH 7.2 PBS (b) and (d). ... 71

Figure 24: Typical Nyquist plots obtained for the bare and modified electrodes in [Fe(CN)6]4- /[Fe(CN)6]3- redox probe at fixed potential 0.22V (vs

Ag/AgCl, Sat’d KCl). (e) Represents the circuit used in the fitting of

the EIS data in (a) – (d). ... 73 Figure 25: Cyclic voltammograms of bare and modified electrodes in pH 7.2 PBS

containing 0.085 mM of DA at scan rate of 25mVs-1_{. (After}

background current subtraction) Figure 25 (a) show cyclic voltammograms of the bare and Fe3O4 modified electrodes before

background current subtraction... 76 Figure 26: Current response (20 scans) of (a) MWCNT/Fe3O4/2,3-Nc, (b)

MWCNT/Fe3O4/29H,31H-Pc, (c) MWCNT/ZnO/2,3-Nc and (d)

MWCNT/ZnO/29H,31H-Pc in pH 7.2 PBS containing 0.085 mM of DA

at scan rate of 25mVs-1_._{... 80}

Figure 27: Cyclic voltammograms of (a) MWCNT/Fe3O4/2,3-Nc and (c)

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25 – 1000 mVs-1_{in pH 7.2 PBS containing 0.085 mM of DA. (b) And}

(d) are peak current vs. square root of scan rate plots of

MWCNT/Fe3O4/2,3-Nc and MWCNT/Fe3O4/29H,31H-Pc respectively ... 82

Figure 28: Cyclic voltammograms of (a) MWCNT/ZnO/2,3-Nc and (c) MWCNT/ZnO/29H,31H-Pc modified electrodes at scan rate range 25 – 300 mVs-1_{in pH 7.2 PBS containing 0.085 mM of DA. (b) And (d) are}

peak current vs. square root of scan rate plots of

MWCNT/ZnO/2,3-Nc and MWCNT/ZnO/29H,31H-Pc respectively ... 83 Figure 29: Plots of peak potential (Ep) versus log υ of (a) MWCNT/Fe3O4/2,3-Nc,

(b) MWCNT/Fe3O4/29H,31H-Pc (c) MWCNT/ZnO/2,3-Nc and

(d)MWCNT/ZnO/29H,31H-Pc in pH 7.2 PBS containing 0.085 mM of

DA. ... 86 Figure 30: Differential pulse voltammograms of (a) MWCNT/Fe3O4/2,3-Nc and

(c) MWCNT/ Fe3O4/29H,31H-Pc modified electrodes at scan rate

range 25mVs-1_{in pH 7.2 PBS containing different concentrations of}

DA (3.27, 6.3, 9.11, 11.7, 14.2, 16.5, 18.6, 20.6, 22.5 and 24.3 µM). (b) And (d) are peak current vs. concentration plots of MWCNT/

Fe3O4/2,3-Nc and MWCNT/ Fe3O4/29H,31H-Pc respectively ... 89

Figure 31: Differential pulse voltammograms of (a) MWCNT/ZnO/2,3-Nc and (c) MWCNT/ ZnO /29H,31H-Pc modified electrodes at scan rate range 25mVs-1_{in pH 7.2 PBS containing different concentrations of DA}

(3.27, 6.3, 9.11, 11.7, 14.2, 16.5, 18.6, 20.6, 22.5 and 24.3 µM). (b) And (d) are peak current vs. concentration plots of MWCNT/ZnO

/2,3-Nc and MWCNT/ZnO/29H,31H-Pc respectively ... 91 Figure 32: Cyclic voltammograms of binary mixture of DA and AA at (a)

MWCNT/Fe3O4/2,3-Nc, (b) MWCNT/Fe3O4/29H,31H-Pc, (c)

MWCNT/ZnO/2,3-Nc and (d) MWCNT/ZnO/29H,31H-Pc with constant concentration of DA (0.085 mM) and increasing

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concentration of AA (0.031, 0.058, 0.081, 0.1, 0.12, 0.13, 0.14, 0.16,

0.17, 0.18 mM) ... 94 Figure 33: Cyclic voltammograms of bare and modified electrodes in pH 7.2 PBS

containing 0.14 mM of EP at scan rate of 25mVs-1_{. (After background}

current subtraction) Figure 33 (a) show cyclic voltammograms of the bare and Fe3O4 modified electrodes before background current

subtraction ... 96 Figure 34: Current response (20 scans) of (a) MWCNT/Fe3O4/2,3-Nc, (b)

MWCNT/Fe3O4/29H,31H-Pc, (c) MWCNT/ZnO/2,3-Nc and (d)

MWCNT/ZnO/29H,31H-Pc in pH 7.2 PBS containing 0.14 mM of EP at

scan rate of 25mVs-1_._{... 99}

MWCNT/Fe3O4/29H,31H-Pc modified electrodes at scan rate (range

25 – 300 mVs-1_{) and (range 25 – 1000 mVs}-1_{) respectively in pH 7.2}

PBS containing 0.14 mM of EP. (b) And (d) are peak current vs. square root of scan rate plots of MWCNT/Fe3O4/2,3-Nc and

MWCNT/Fe3O4/29H,31H-Pc respectively. ... 101

Figure 36: Cyclic voltammograms of (a) MWCNT/ZnO/2,3-Nc and (c) MWCNT/ZnO/29H,31H-Pc modified electrodes at scan rate range 25 – 1000 mVs-1_{in pH 7.2 PBS containing 0.14 mM of EP. (b) And (d) are}

peak current vs. square root of scan rate plots of

MWCNT/ZnO/2,3-Nc and MWCNT/ZnO/29H,31H-Pc respectively. ... 102 Figure 37: Plots of peak potential (Ep) versus log υ of (a) MWCNT/Fe3O4/2,3-Nc,

EP.... ... 104 Figure 38: Differential pulse voltammograms of (a) MWCNT/Fe3O4/2,3-Nc and

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EP (7.5, 16.5, 23.3, 29.5, 35, 40, 44.5, 48.7, 52.5 and 56 µM). (b) And (d) are peak current vs. concentration plots of MWCNT/Fe3O4/2,3-Nc

and MWCNT/Fe3O4/29H,31H-Pc respectively... 106

Figure 39: Differential pulse voltammograms of (a) MWCNT/ZnO/2,3-Nc and (c) MWCNT/ZnO/29H,31H-Pc modified electrodes at scan rate range 25mVs-1_{in pH 7.2 PBS containing different concentrations of EP (7.5,}

16.5, 23.3, 29.5, 35, 40, 44.5, 48.7, 52.5 and 56 µM). (b) And (d) are peak current vs. concentration plots of MWCNT/ZnO/2,3-Nc and

MWCNT/ZnO/29H,31H-Pc respectively. ... 107 Figure 40: Cyclic voltammograms of binary mixture of EP and AA at (a)

MWCNT/ZnO/2,3-Nc and (d) MWCNT/ZnO/29H,31H-Pc with constant concentration of EP (0.14 mM) and increasing concentration of AA (0.32, 0.58, 0.81, 1.0, 1.2, 1.3, 1.4, 1.6, 1.7, 1.8

mM) ... 109 Figure 41: Cyclic voltammograms of bare and modified electrodes in pH 7.2 PBS

containing 0.12 mM of NE at scan rate of 25mVs-1_{. (After background}

current subtraction) Figure 41 (a) show cyclic voltammograms of the bare and Fe3O4 modified electrodes before background current

subtraction. ... 112 Figure 42: Current response (20 scans) of MWCNT/Fe3O4/2,3-Nc,

MWCNT/Fe3O4/29H,31H-Pc, MWCNT/ZnO/2,3-Nc and

MWCNT/ZnO/29H,31H-Pc in pH 7.2 PBS containing 0.12 mM of NE at

scan rate of 25mVs-1_.._{... 114}

MWCNT/Fe3O4/29H,31H-Pc modified electrodes at scan rate (range

25 – 300 mVs-1_{) and (range 25 – 1000 mVs}-1_{) respectively in pH 7.2}

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square root of scan rate plots of MWCNT/Fe3O4/2,3-Nc and

MWCNT/Fe3O4/29H,31H-Pc respectively... 116

Figure 44: Cyclic voltammograms of (a) MWCNT/ZnO/2,3-Nc and (c) MWCNT/ZnO/29H,31H-Pc modified electrodes at scan rate (range 25 – 1000 mVs-1_{) and (range 25 – 300 mVs}-1_{) respectively in pH 7.2}

PBS containing 0.12 mM of NE. (b) And (d) are peak current vs. square root of scan rate plots of MWCNT/Fe3O4/2,3-Nc and

MWCNT/Fe3O4/29H,31H-Pc respectively. ... 117

Figure 45: Plots of peak potential (Ep) versus log υ of (a) MWCNT/Fe3O4/2,3-Nc,

NE. ... 119 Figure 46: Differential pulse voltammograms of (a) MWCNT/Fe3O4/2,3-Nc and

(c) MWCNT/Fe3O4/29H,31H-Pc modified electrodes at scan rate

NE (7.5, 14.1, 20, 25.3, 30, 34.3, 38.2, 41.7, 45 and 48 µM). (b) And (d) are peak current vs. concentration plots of MWCNT/ Fe3O4

/2,3-Nc and MWCNT/ Fe3O4/29H,31H-Pc respectively ... 121

Figure 47: Differential pulse voltammograms of (a) MWCNT/ZnO/2,3-Nc and (c) MWCNT/ZnO/29H,31H-Pc modified electrodes at scan rate range 25mVs-1_{in pH 7.2 PBS containing different concentrations of NE (7.5,}

14.1, 20, 25.3, 30, 34.3, 38.2, 41.7, 45 and 48 µM). (b) And (d) are peak current vs. concentration plots of MWCNT/ZnO/2,3-Nc and

MWCNT/ZnO/29H,31H-Pc respectively. ... 122 Figure 48: Cyclic voltammograms of binary mixture of NE and AA at (a)

MWCNT/ZnO/2,3-Nc and (d) MWCNT/ZnO/29H,31H-Pc with constant concentration of NE (0.12 mM) and increasing

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concentration of AA (0.32, 0.58, 0.81, 1.0, 1.2, 1.3, 1.4, 1.6, 1.7, 1.8

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LIST OF TABLES

Table 1: Impedance data obtained for bare and and modified electrodes in [Fe(CN)6]4- /[Fe(CN)6]3- redox probe at fixed potential 0.22V (vs

Ag/AgCl, Sat’d KCl). The values in brackets represent the percentage

errors of the data fitting. ... 74 Table 2: Cyclic voltammetric parameters obtained for bare and modified

electrodes in pH 7.2 PBS containing 0.085 mM of DA at scan rate 25

mVs-1_{. ... 78}

Table 3: Cyclic voltammetric parameters of binary mixture of DA and AA obtained for modified electrodes with constant concentration of DA (0.085 mM) and increasing concentration of AA (0.031, 0.058, 0.081,

0.1, 0.12, 0.13, 0.14, 0.16, 0.17, 0.18 mM). ... 93 Table 4: Cyclic voltammetric parameters obtained for bare and modified

electrodes in pH 7.2 PBS containing 0.14 mM of EP at scan rate 25

mVs-1_{. ... 97}

Table 5: Cyclic voltammetric parameters of binary mixture of EP and AA obtained for modified electrodes with constant concentration of EP (0.14 mM) and increasing concentration of AA (0.32, 0.58, 0.81, 1.0,

1.2, 1.3, 1.4, 1.6, 1.7, 1.8 mM)... 110 Table 6: Cyclic voltammetric parameters obtained for bare and modified

electrodes in pH 7.2 PBS containing 0.12 mM of NE at scan rate 25

mVs-1_{. ... 113}

Table 7: Cyclic voltammetric parameters of binary mixture of NE and AA obtained for modified electrodes with constant concentration of NE (0.12 mM) and increasing concentration of AA (0.32, 0.58, 0.81, 1.0,

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CHAPTER ONE

INTRODUCTION

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2 1.1 Introduction

Neurotransmitters are the brain chemicals that communicate information throughout our brain and body. They are synthesized, stored, and released from specialized cells called neurons, where they relay, amplify and modulate signals between nerve cells [1, 2]. They are present in many body fluids, including serum, cerebral spinal fluid, saliva, and urine [2]. Their metabolism, receptor formation and catabolism are controlled by genes [3].The interest in studying neurotransmitters started in 1902 when Ernest Starling and William Bayliss discovered the first hormone called secretin, and that introduced the world to the existence of an internal communication system [4]. After this discovery, scientists have identified numerous other chemical messengers like Dopamine (DA), Serotonin (5-HT), Epinephrine (EP), Norepinephrine (NE) and Acetylcholine (Ach) to name the few. Studies that were carried out in the past decades improved the understanding of the relationship between chemistry in the central nervous system and the behavioural, cognitive, and emotional state of an organism [5]. Recently, there has been considerable interest in developing electrochemical techniques for determination of neurotransmitters [6]. Clinicians and researchers are interested in the function and measurement of neurotransmitters as they have the potential to serve as clinically relevant biomarkers for specific disease states or to monitor treatment efficacy [1].

Many diseases are directly related to alter patterns of neurotransmitters. According to the World Health Organization, approximately 1 in every 100 of the world’s inhabitants suffers from some form of neurological disorder [7]. Abnormal neurotransmitter concentrations can reveal the state of diseases such as epilepsy, Parkinson’s disease, or Alzheimer's disease. Modern methods of treatment of those diseases focus primarily on pharmaceutical and electroceutical neuromodulation techniques to suppress the effects of the disease or to

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restore healthy signalling [7]. The development of electrochemical sensors has been widely researched as an inexpensive method to sensitively detect a variety of neurotransmitters.

Sensors and sensor arrays for the detection of chemical and biological substances have attracted much attention in recent years [8]. Sensors are devices that consist of a signal transducer, which is an active sensing material, that transmit the signal in form of electrical, thermal or optical output without any amplification from selective compound [9, 10].In the past few decades there have been a great progress in development of electrochemical sensors and their application in environmental protection studies, biotechnology, food safety, medical diagnosis, drug screening and security [10]. Construction of electrochemical sensors has proven to be a simple analytical method that provides opportunity to perform biomedical, environmental and industrial analyses away from a centralized laboratory; hence an ample amount of time has been devoted in fabrication and application of these sensors [11]. Sensors can be broadly classified in to two categories as chemical sensors and biosensors, and the combination of the two has given rise to electrochemical biosensor [12]. Chemical sensors are devices that provide a certain type of response or continuous information directly related to the quantity of a specific chemical species [13]. While biosensor can be classified in terms of sensing aspects, where an active sensing material or a catalyst on the surface of an electrode should catalyse the reaction of the biomolecule compound to attain a signal output [12].

Electrochemical sensing has been proven as a simple analytical method that can determine the presence of a wide range of substances at relevant concentration levels with sufficient selectivity, sensitivity, reproducibility, and ease of miniaturization [8, 14]. A range of analytical techniques such as chromatographic methods, mass spectroscopy,

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spectrophotometry, fluorimetry, capillary electrophoresis, chemiluminescence [7, 8], fast-scan cyclic voltammetry, amperometry and differential pulse voltammetry [14], are reported in the literature for detection of neurotransmitters. However, these methods are relatively expensive, require long time analysis, multi-step sample preparation procedures and in some cases have low sensitivity and selectivity [15]. Electrochemical sensors are small, relatively simple to fabricate, easily implantable, can provide real-time measurements and fast response [6]. The recent development in the nanotechnology has paved the way for large number of new materials and devices of desirable properties which have useful functions for numerous electrochemical sensor and biosensor applications [14, 16].

The potential of nanomaterials for industrial and medical use was first recognised by Feynman [17] and they have attracted universal attention since 1990s, due to their specific and outstanding features that differ from bulk materials [18]. Their unique characteristics have given rise to a great economic and technological growth and development in a large number of industrial sectors. With current advances in nanotechnology, there has been a considerable progress in application of nanomaterial in sensors, medicine, material science, chemistry, biology and physics because attractive electronic, optical, magnetic, thermal and catalytic properties [19]. As a result nanomaterials have received attention in fabrication of electrochemical sensors and biosensors, which led to the production of selective and sensitive sensors. Nanomaterials such as nanotubes, nanoparticles, nanosheets and nanofibers have been investigated for their ability as electrode surface modifiers, due to high surface-to-volume ratios and electrical conductance [19, 20]. Although the use these nanomaterials improve the sensing capacity of modified electrodes [21], the controlled growth and synthesis with minimal aggregation and functionalization with molecular recognition molecules is a major challenge to the use of nanomaterials [20]. However their

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ability to have high capacity of charge transfer makes them suitable to improve reversibility, give lower detection limits, real life-time measurements and longer operation life time of electrodes [20, 21].

Phthalocyanines (Pc) and their derivatives are well-known organic semiconductor materials for their electrocatalytic activity for many electron transfer reactions and have been identified as promising candidates for neurotransmitters sensors [22, 23]. As an excellent sensing material, Pc has been extensively studied based on its high sensitivities, excellent thermal and chemical stability, film-processing ability and structure adjustability [24]. Furthermore, metal phthalocyanine (MPc) derivatives possess high sensitivity, fast response, ease of processability, as well as wide scope of operation at room temperature; they have therefore been studied extensively as thin films for chemical and gas detection [25].

1.2 Problem statement

It is suggested that everything known about human behaviour is entirely regulated by human brain; therefore, neurotransmitters have been studied in relation to psychology and human behaviour. Neurotransmitters are chemical messengers that carry, boost and modulate signals between neurons and other cells in the body, hence they play an important role in everyday life functioning. There are two kinds of neurotransmitters, viz inhibitory and excitatory. Excitatory neurotransmitters stimulate the brain and the inhibitory neurotransmitters calm the brain and help create balance of moods and feelings. Since they are freely floating in our body, they are easily modified by either internal or external forces. For example, drugs can decrease the rate of synthesis of neurotransmitters by affecting the synthetic enzymes for neurotransmitters. Some drugs block or stimulate the release of specific neurotransmitters, resulting in a dysfunction of neurotransmitter activity.

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When neurotransmitters are affected by drugs or certain diseases in our bodies, they cause adverse effects such as Alzheimer's disease and Parkinson's disease etc. Therefore, it is important to detect the level of neurotransmitters at all times for diagnosis and pharmaceutical purposes. It is proposed that this problem can be resolved by fabrication of electrochemical sensors.

1.3 Research aim & objectives

1.3.1 Aim

The aim of the research is to explore the sensing properties of phthalocyanine doped with metal oxides (ZnO, Fe3O4) nanoparticles and supported on multi-walled carbon nanotubes

platform towards the catalysis and analysis of Dopamine, Epinephrine and Norepinephrine in biological systems.

1.3.2 Objectives

 Quantitatively synthesise metal oxide (ZnO, Fe3O4) nanoparticles from commonly

available laboratory salts using a synthetic route that is cost effective, requires few steps, and produces particles with better electron transport and catalysis than the bulk.

 Confirm successful synthesis of the metal oxide (ZnO, Fe3O4) nanoparticle using

different characterization techniques such as Scanning Electron Microscopy (SEM), Transmission Electron microscopy (TEM), Fourier Transform Infrared spectroscopy (FTIR), Raman spectroscopy, Ultraviolet/Visible (UV-vis) spectroscopy, X-ray Diffraction (XRD), Electron Dispersive X-ray Spectroscopy (EDX), and Electrochemical Technique.

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 Explore the potential of the synthesised nanomaterials for sensor application towards the detection and quantification of some selected neurotransmitters such as dopamine, norepinephrine, and epinephrine.

 Study the synergistic effect of the composite formed between MWCNT-Phthalocyanine-MO composite on the sensing properties of the developed sensor.

1.4 Research justification

The use of electrochemical sensors is found to be important in various applications such as medicine, environmental monitoring, bioprocessing, pharmaceutical and food industries. For instance in medicine, application of biosensors for neurotransmitters is of high necessity. Timelines in both the detection and monitoring of levels of neurotransmitters can be critical in the diagnosis and later the treatment of the diseases caused by the dysfunctions of neurological activities. Simultaneous detection of neurotransmitters in the presence of ascorbic acid (AA) and uric acid (UA) is a critical challenge in the field of biomedical chemistry and neurochemistry. A range of analytical techniques such as chromatographic methods, mass spectroscopy, fluorimetry, capillary electrophoresis, chemiluminescence, fast-scan cyclic voltammetry, amperometry and differential pulse voltammetry have been developed in the past. However, these methods require several derivatization procedures which are time consuming and very expensive. Therefore, there is need for the fabrication of highly sensitive and selective chemical sensor or biosensors that would discriminately detect neurotransmitters in the presence of other interfering species such as ascorbic and uric acids. Electrochemical methods provide a simple, cost effective and quick way of analysing biologically and environmentally important molecules. ZnO and Fe3O4 are used as catalysts for this study since there is dearth of information on their use as

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electrocatalysts doped with phthalocyanine for neurotransmittance detection and quantification. Secondly, these metal oxide nanoparticles can readily be made from cheap, common and readily available laboratory salts of their metals as compared with other expensive electrode materials that have been reported in literature.

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CHAPTER TWO

LITERATURE REVIEW

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10 2.1 Neurotransmitters

Neurotransmitters are the primary chemical messengers released from neuron. The most important monoamine neurotransmitters are serotonin (ST) and the catecholamines dopamine (DA), norepinephrine (NE), and epinephrine (EP). DA is the most abundant of the four monoamine neurotransmitters [26]. Catecholamines are a class of important monoamines that serves as markers for the mammalian central nervous system activities [27]. Concentration measurements of catecholamines in body fluids are important for diagnosis of several neurological disorders in the central and peripheral adrenergic functions [27, 28]. Catecholamine drugs are widely used in the treatment of bronchial asthma, hypertension, heart failure associated with organic heart disease and cardiac surgery [29]. Figure 1 shows formation of DA by decarboxylation of L-DOPA and is a precursor of epinephrine and norepinephrine.

Figure 1: Sequence of enzymatic reactions generating other catecholamines from L-DOPA [30].

2.1.1 Dopamine

Dopamine (DA) is a neurotransmitter in mammalian brain tissues that belongs to the family of inhibitory/catecholamine neurotransmitters; it plays an important physiological role in the functioning of central nervous, renal, hormonal and cardiovascular systems as an extra

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cellular chemical messenger, as well as in drug addiction; its function is to regulate neural interactions by reducing the permeability of gap junctions between adjacent neurons of the same type [7]. Abnormal DA level may relate to many diseases, such as Parkinson’s disease, Huntington’s disease, Alzheimer’s diseases and tardive dyskinesia [23], where the dopaminergic activity is lower than in healthy individuals. The opposite is true in schizophrenia where the activity of the dopaminergic neurons is increased due to abnormalities in their regulation. Furthermore the development of anorexia nervosa and bulimia nervosa has also been associated with altered dopaminergic activities [16, 22, 23]. Therefore, it is very important to rapidly and accurately measure DA concentrations in biological fluids for clinical diagnosis.

Although DA is an electrochemically active compound on some electrode surfaces, it is a major problem to determine DA in biological fluids by electrochemical methods at a bare electrode because of interfering species such as ascorbic acid (AA) and uric acid (UA) [31]. As a result the accuracy of its detection it’s very low in real sample analysis. The basal concentration of DA is 0.01 – 1 µM, while the concentration of AA and UA is 100 - 1000 times higher than that of DA, hence it is crucial to develop sensitive and selective methods for determination of DA [22]. A great contribution to disease diagnosis would be a development of electrochemical sensor that would measure DA at low levels of characteristic of living system (26 – 40 nmolL-1_{) [30, 32]. Figure 2 shows the oxidation}

mechanism of DA, where DA is oxidized to dopaminequinone and then dopaminequinone is transformed into leucodopaminechrome and then leucodopaminechrome is oxidized to dopaminechrome [33].

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Dopamine dopaminequinone

Dopaminequinone leucodopaminechrome

Leucodopaminechrome dopaminechrome

Figure 2: Oxidation mechanism of DA [33].

2.1.2 Epinephrine

Epinephrine (EP) also known as adrenaline exists as an organic cation [34], which is about nmolL-1 in human serum [35]. It belongs to the family of inhibitory/catecholamine neurotransmitters in mammalian central nervous systems. It was first discovered by Takamine and Aldrich in 1901, and synthesized by Stolz and Dalkin in 1904 [36]. EP acts as a cellular chemical messenger [37], and many diseases are related to the change of its concentration. It is a hormone synthesized by the adrenal medulla of the adrenal glands [38-40], it stimulates a series of actions of the sympathetic nervous system called “fight or flight” response [41]. EP controls the performance of the nervous system, and its abnormal levels affect the regulation of the blood pressure heart rate, and glycogen metabolism [34, 42]. Determination of the level of EP is important for diagnosis of Parkinson’s disease, among other mental disorders [8]. It has been used as a common healthcare medicine, for

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instance EP drugs are used to treat anaphylactic shock, bronchial asthma and organic heart disease [43]. Therefore, a quantitative determination of EP in physiological pH in our body fluids has become increasingly significant. The molecular structure of epinephrine is made of a benzene ring with two hydroxyl groups and an alkylamine chain [44]. The major problem related to EP is that it is easily oxidized by air, hence antioxidants formulations such as sodium metabisulfite helps to minimize the analyte oxidation [38]. Figure 3 shows the processes involved in the oxidation mechanism of EP.

Figure 3: Oxidation mechanism of EP [38, 45]. 2.1.3 Norepinephrine

Norepinephrine (NE) is an important catecholamine neurotransmitter in the mammalian central nervous system. It is secreted and released by the adrenal glands, the noradrenergic

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neurons during synaptic transmission and relinquished as a metabotropic neurotransmitter from nerve endings in the sympathetic nervous system and some areas of the cerebral cortex [11, 46]. The human adrenal medulla releases about 20% NE, while the adrenergic neurons are responsible for the major NE production [20]. NE is responsible for increased heart rate, blood pressure, dilation of pupil, dilation of air passage in lungs and narrowing of blood vessels. NE is important for attention and focus, learning, memory, and the sleep– wake cycle; hence it is also used as performance enhancing drug in competitive games by athletes; therefore prohibited by the World Anti-Doping Agency [47, 48]. It promotes the conversion of glycogen to glucose in the liver and helps in converting the fats into fatty acids, resulting in an increment in energy production [47]. Extreme abnormalities of NE concentration levels caused by its metabolic dysfunction may lead to occurrence of many pathological conditions such as thyroid hormone deficiency, congestive heart failure, arrhythmias and idiopathic postural hypotension [49]. Recent studies of NE in animal models is a substantial step in the process of discovery and evaluation of new drugs in many disease areas such as diabetes, heart disease, pain, anxiety, and other neurological disorders [46]. Furthermore, quantification of NE concentrations in plasma and urine is considered clinically important for diagnosis and for evaluating the hemodynamic function of pheochromocytoma, paraganglioma, ganglia neuroblastoma and Parkinson’ disease [11]. It is therefore, very essential to develop fast, accurate and sensitive methods for direct determination of NE. The oxidation mechanism process of NE is shown in figure 4, where NE is oxidized to norepinephrinequinone, and then norepinephrinequinone is oxidized to leuconorephinechrome [50].

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Norepinephrine norepinephrinequinone

Norepinephrinequinone leuconorephinechrome

Figure 4: Oxidation mechanism of NE [50].

2.1.4 Ascorbic acid (AA) and uric acid (UA)

Ascorbic acid, also known as vitamin C, is one of the most important vitamins popularly known for its antioxidant properties [23]. It is found in various plants, food and animals, but it cannot be synthesized by human body due to the lack of L-gulono-lactone oxidase [51-53]. It plays a role in some biological and enzymatic reactions such as fee radical scavenging in different metabolic processes that involve redox mechanism, cancer preventing and improving immunity [51, 54]. AA is also used for prevention and treatment of infertility, cancer, common colds, some mental illness and in some clinical manifestations of HIV infections [23, 54, 55]. AA is vital in human diet, daily intake of AA recommended is about 70-90 mg [51] and insufficient dietary intake of AA will result in the symptoms of scurvy and gingival bleeding [51, 55]. In addition the level of AA in the body decrease with both age and smoking, and this is due to chronic diseases such as rheumatoid arthritis and cancer which result to an increase in oxidative stress [53]. However this can be resolve by multivitamin preparations, which are commonly used to supplement inadequate AA dietary intake.

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Whereas high intake of AA have nocuous effects such as urinary stone, metabolic acidosis, oxaluria, renal tubular disease, gastrointestinal disturbances, cholesterol disturbances, vitamin B12 destruction, fatigue and sterility [53, 56]. AA coexists with other biomolecules such as uric acid and catecholamines in biological fluids such as blood and urine. Numerous reports have shown their coexistence in biological systems and how they influence each other in their respective activities. UA (2,6,8-trihydroxypurine) is an important principal end product of purine metabolism in the human body [57-59]. It is generated by the xanthine oxidase catalyzed conversion of xanthine and hypoxanthine [60]. In healthy individuals the concentration of UA is 240-520 µM in human blood, whereas in urine is 1.4-4.4 mM [57]. Abnormal concentration in body fluids leads to several diseases and pathological disorders such as gout, arthritis, kidney disease, cardiovascular disease, neurological diseases [60] hyperuricemia Lesch-Nyan disease and leukaemia [23, 58, 61, 62]. Figure 5 show the oxidation process of (a) AA [58, 63] and (b) UA [64, 65].

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Investigation of neurological behaviour and simultaneous determination of neurotransmitters is of great importance for the illustration of their precise physiological functions [7]. However, the challenge involved in working with multiple neurotransmitters simultaneously is that the electrochemical detection suffers interference from other biological compounds, such as uric acid (UA) and ascorbic acid (AA), since they have similar oxidation potentials [16, 22]. The determination of ST is often complicated by the presence of DA, since the oxidation potential of ST (0.38 V) is close to that of DA (0.22 V) [25]. Furthermore, EP and NE are electrochemically difficult to distinguish because of their similar structures. In the past years, many chemically modified electrodes have been developed to carry out selective detection of various neurotransmitters in the presence of high concentrations of UA and AA [16]. Various materials that have been employed to modify electrodes include nanoparticles, carbon nanotubes, carbon nanofibers, graphene, polymer films, assembled monolayers, metal oxides [23], ionic liquids, magnesium ferric nanoparticles [16], pyrolytic graphite and dye doped sol–gel [43].

2.2 Nanomaterials

Since their discovery, nanomaterials are changing many basic concepts of science [66] and they have received undivided attention because of their fundamental unique physical and chemical properties [67]. Nanomaterials are defined as materials with at least one dimension of approximately 1 – 100 nm range [68-72]. These materials include natural and manmade materials; natural nanomaterials are present in forest fires and volcanoes, viral particles, biogenic magnetite, and protein molecules such as ferritin [68]. There is an interest in preparation and application of these materials because they possess good magnetic, electrical, chemical and optical characteristics, which differs or could not be

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directly achieved by their bulk counterparts chemical and compounds [71, 73]. These properties make them important; hence they can be exploited for commercial and medical applications such as acting as catalyst in chemical reactions, drug delivery devices, as imaging agents in medicine, and in consumer products such as paints, suntan lotions, cosmetics and food [68, 70, 71]. Nanomaterials possess a diverse array of chemical and structural composition, produced as nanotubes, nanowire, nanofibers, nanosheets, nanorods and nanoparticles [66].

2.2.1 Carbon nanotubes (CNTs)

Since the first report by Iijima in 1991 [74], carbon nanotubes (CNTs) have been studied as novel form of carbon materials and they have received tremendous attention in the past decades because of their extraordinary properties. A wide range of studies have been conducted based on their structure, properties and potential applications. CNTs can be synthesised using the three main methods namely chemical vapour deposition, electric arc-discharge methods and laser evaporation [75, 76]. CNTs are hexagonal networks of carbon atoms forming nanotubes approximately 1 nm in diameter and 1–100 nm in length [5], they consist of one or several concentric graphene tubules or cylinders each with a helically wound hexagonal honeycomb lattice [77, 78]. They could either be single walled carbon nanotubes (SWCNTs) or multi-walled carbon nanotubes (MWCNTs) depending on the arrangement [5]. SWCNTs consist of sp2_{hybridized carbon in a hexagonal honeycomb}

structure that is rolled into hollow tube; it comprises of one single layer of graphene sheet which is favourable for the effective functionalization leading to high specificity to analytes [48]. Their diameters range from 0.4 to 2 – 3 nm, and their length is usually of the micrometer order [78]. Although SWCNTs usually aggregate, forming bundles that consist of

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numerous nanotubes in parallel attached to each other [79], they are known to have excellent mechanical and thermal properties and can be either metals or semiconductors depending on their geometry [75, 79]. The distinction between semiconducting and metallic activity is important for their use in different sensors although the physical separation of allotropes has proven to be one of the difficult challenges to overcome [48]. MWCNTs are similar to a collection of SWCNTs and they were the first to be discovered [76, 80]. They are made of several layers of graphene cylinders that are concentrically placed around a common central hollow and from a nest like rings of a tree trunk [76, 80]. The length, diameter and characteristics or properties of MWCNTs differs from those of SWCNTs [79]. MWCNTs are regarded as metallic conductors [81], the spacing between the concentric nanotubes is 0.34 nm and the diameter range of 2 – 100 nm [80]. The way graphene sheets are wrapped around also play a role in properties of CNTs. Figure 6 shows the structure of (a) SWCNT and (b) MWCNTs.

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CNTs are an important class of materials due to their unique electronic, mechanical, and structural characteristics. The physical and catalytic properties make CNTs ideal for use as chemical sensors and for electrochemical detection [43]. However their electrochemical properties are sensitive when chemically doped with various molecules [75], furthermore Liu [83] reported that the electron localization and barriers to electron transport through CNTs is mechanically caused by structural distortion that is due to atomic force microscopy tip of CNTs [83]. It has also been reported that the application of pristine CNTs is limited due to their poor solubility in both organic and inorganic solvents [84] and also the CNTs walls are not reactive [75]; therefore it is of best interest to attach functional groups such as – COOH, -OH or –C=O to their surface to optimize their use and increase their solubility [85]. Functionalization of non-covalent sidewalls of CNTs can be based on weak attraction forces such as hydrogen bonding, π-π stacking, van der Waals and electrostatic forces which can be used to attach these small active molecules or wrap polymer chains onto the sidewalls of CNTs [75]. These attachments can be achieved by using simple treatments involving the use of oxidative methods with nitric acid and sulphuric acid, as a result the caps of both ends of CNTs are removed and functional groups such –COOH on the surface walls and at the ends are revealed [79, 84]. Figure 7 shows the attachment of -COOH on pristine CNTs by nitric acid oxidation method.

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Figure 7: Nitric acid oxidation method of pristine CNTs [84, 86].

The mechanically strong, small size and chemically stable nature of CNTs is very appealing for sensing applications [6]. Furthermore, CNT based platforms were cited as biocompatible sensors because of the similarity in size with analytes such as cells, proteins and even DNA; they have shown a significant development because of their promising applications in nanoelectronics as well as in highly sensitive biosensors [43]. There are many different types of CNT-based electronic sensors including ionization sensors, capacitors, resistors and transistors [5]. The first electrochemical application of CNTs by Britto [77], where MWCNTs where used to study the electrochemical oxidation of DA. The results obtained from the study showed a two electron transfer redox reversible process and the oxidation of DA showed a low potential with a faster rate than that observed for graphite electrodes. These remarkable results suggest that MWCNTs possess properties such as high electrical conductivity, larger surface-active groups to-volume ratio, chemical stability and significant mechanical strength, as a consequence, MWCNTs can serve as excellent substrates for the development of biosensor devices [23]. This study demonstrated good electrocatalytic

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features of CNT-modified electrodes, meaning that CNTs are capable of mediating electron transfer reactions with electroactive species in solution when used as the electrode modifying material [77, 87]. From 1996, the application of CNTs in electrochemistry increased drastically towards detection of biological analytes and gases using sensors or biosensors. Thomas et al [88] studied the oxidation detection of EP in the presence of UA and AA using MWCNTs modified carbon paste electrode. The modified electrode showed good electrocatalytic effects and the current sensitivity of EP was enhanced to about five times as compared to bare electrode. A complete resolved peak of EP from that of UA and AA was observed with detection limit of 2.9 x 10-8_{M. Wang et al [89] studied}

electrocatalytic effect of MWCNTs based gas sensor towards detection NH3 at room

temperature. It was reported that MWCNTs based sensor was sensitive to NH3 gas and that

suggested the MWCNTs were good candidate materials for NH3 detection at room

temperature. CNTs sensors exhibit low limit of detection (LOD) and fast response due to the signal enhancement provided by high surface area, low overvoltage, rapid electrode kinetics and high thermal conductivity [6].

2.2.2 Metal oxide nanoparticles

Metal and metal oxide nanoparticles (NPs) are known for their excellent physical and catalytic properties; therefore they are the most widely applied nanomaterials. The application of these nanomaterials on medicine, information technology, energy storage, catalysts, electronics and sensors has driven research in developing synthetic pathways to such nanostructures [90]. It has been discovered that reduction of materials to a very small size result into a profound effects to their chemical and physical properties. Factors such as crystal structure, size, shape, morphology and surface chemistry play significant roles in

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metal oxide properties [90]. There is currently an interest in the use of metal oxide NPs, metal-doped metal oxides, metal oxide-CNTs nanocomposites, and metal oxide-polymer composites [93] in electrochemistry to improve the performance of electrochemical detection of biological and environmental analytes [11]. Analytical devices based on nanostructured metal oxides are cost-effective, highly sensitive due to the large surface-to-volume ratio of the nanostructure and additionally show excellent selectivity [93]. Metal and metal oxide NPs have been used to modify electrodes for use as electrocatalysts and biosensors; hence they play an important role in diagnostic devices [94]. Metal-oxide NPs are prepared using chemical methods/techniques, such as hydrolysis, sol-gel, co-precipitation, impregnation or chemical-vapour synthesis or hydrothermal techniques, physical methods, including sputtering, evaporation (thermal and electron beam), pulse-laser deposition and ion implantation [95].

It is well known that metal oxide NPs have excellent conductivity properties, which make them ideal to increase the electron transfer between redox reactions in electrode surfaces and also act as catalysts to enhanced electrochemical reactions. It has been reported that metal oxide NPs have shown the ability to decrease overpotentials of many analytical electrochemical reactions, and also convey reversibility of some redox reactions, which are irreversible at common unmodified or bulkmetal electrodes [92]. The basic functions played by nanoparticles to electrochemical sensors or biosensors can be classified as, (1) immobilization of biomolecules, (2) catalysis of electrochemical reactions, (3) enhancement of electron transfer, (4) labelling biomolecules and (5) acting as reactant [96].

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24 2.2.2.1 Iron oxide nanoparticles

Iron oxides nanoparticles play distinct roles in environmental, electrical, chemical, medical, and industrial fields. There are different types of iron oxides namely, FeO, α-Fe2O3,

(hematite) β-Fe2O3, γ-Fe2O3 (maghemite), ε-Fe2O3 and Fe3O4 (magnetite) [97]. Fe3O4 NPs are

one of the most important nanomaterials due to their excellent strong super paramagnetic properties, biocompatibility, low toxicity high coercivity, low Curie temperature, high magnetic susceptibility and high adsorption ability [98, 99]. On this basis Fe3O4 NPs have

been widely investigated in catalysis, detection of biological molecules (DNAs, enzymes, proteins, neurotransmitters etc.), clinic diagnosis, and therapy [99], and also as electrode modifiers in electrochemical sensors and biosensors. However the encountered challenge with these NPs its size, shape and stability control. Because they have large surface-to-volume ratio and strong magnetic dipole-dipole attractions between the particles [100], they are prone to aggregation and they easily undergo oxidation due to their high chemical activities thus resulting in poor magnetism and dispersibility [101, 102]. Therefore for many applications, providing surface coating is very crucial to chemically stabilize and protect them against degradation during or after the synthesis [101].

It is suggested that among a wide variety of metal oxide NPs, Fe3O4 NPs are particularly

attractive due to presence of iron cations in two valence states, Fe2+_{and Fe}3+ _{[103], higher}

bioactivity and better contact between biocatalyst and its substrate [104]. Shan et al [105] investigated Fe3O4/chitosan modified electrode as a feasible sensor for detection of H2O2.

He found that there was limited interference, prompt response, good reproducibility and the electrode showed long term stability, therefore he suggested that these might be beneficial for future environmental and biological applications towards detection of H2O2. In

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addition Adekunle et al [106] conducted a study on the voltammetric detection of DA using easily prepared nano-scaled iron oxide (Fe2O3) catalyst supported on MWCNTs modified

pyrolytic graphite electrode. Furthermore Fang et al [31] also studied the voltammetric sensing of DA using Fe3O4 NPs modified gold electrode. The modified electrode gave

promoting effect and high stability toward the electrochemical oxidation of DA, which showed reversible redox peaks, great sensitivity and selectivity in interference studies with AA. The limit of detection obtained was 3.0 x 10-8_{M, this suggests that Fe}

3O4 NPs have good

electrocatalytic properties. Another vital property of iron oxide NPs as electrochemical biosensors is the ability to provide a favourable microenvironment in which biomolecules (such as proteins) may exchange electrons directly with an Fe3O4 modified electrode [107].

2.2.2.2 Zinc oxide nanoparticles/rods

Among the wide variety of semiconductor nanomaterials, ZnO NPs have showed excellent and distinguished performance in electronics, optics and photonics, they have received wide applications as sensors, transducers and catalysts [108]. ZnO has wide band-gap (3.37 eV), high exciton binding energy of 60 meV at room temperature and high mechanical and thermal stabilities [109, 110], hence it is suitable for short wavelength optoelectronic applications operating in the visible and near ultraviolet spectral regions [111]. Strong piezoelectric and pyroelectric characteristics of ZnO are due to the lack of a centre of symmetry in wurtzite, combined with large electromechanical coupling [108]. Therefore Chen et al [110] suggested that combining good conductivity of ZnO NPs and remarkable properties of CNTs; it could result in potential applications of semiconducting CNTs as novel photocatalysts. ZnO NPs have a wide range of use and applications in industrial purposes, solar cells UV light-emitting devices, gas sensors, photocatalysts, pharmaceutical and

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cosmetic [111]. Aravind et al [112] mentioned that because of the great surface area, high electrocatalytic activity, excellent electron transfer rate of CNTs, strong adsorption ability and fast electron transfer kinetics of ZnO, a biosensor was fabricated using ZnO NP-decorated MWCNTs and tested for the selective detection of DA. ZnO/MWCNT nanocomposites gave best sensitivity, stability and excellent anti-interference ability, with detection limit of 1 mM towards DA, linearity up to 300 mM and sensitivity of 785 nA/mM. Studies have shown the use of other metal oxide NPs in electrochemical sensors. In simultaneous detection of DA, UA and AA using nanoSnO2/MWCNTs/carbon paste

electrode, Sun et al [16] used SnO2 NPs due to their unique properties such as high

conductivity, catalytic activity, and chemical sensitivity. From the results obtained, nanoSnO2/MWCNTs/carbon paste electrode showed excellent separation peaks of AA, UA

and DA at optimal conditions, with good sensitivity and detection limits of 0.03, 1, and 50 μM for DA, UA, and AA, respectively.

2.3 Phthalocyanines and their metalloderivatives

In 1907 Braun and Tcherniac were the first to discover phthalocyanines (Pcs) as by-product during o-cyanobenzamide preparation from phthalamide and acetic anhydride at high temperature and their structures were later studies by Linstead in 1934 [113]. Pcs are porphyrin synthetic analogues made up of four isoindole groups that are linked together through nitrogen atoms [114]. Pcs and their derivatives are well-known blue-green organic semiconductor materials that belong to the family of aromatic heterocyclic conjugated molecules, with alternating single-double bond structures [115]. They are made of delocalised π-electrons system [116], where π-electron delocalisation and interactions with central mental atom on metal phthalocyanines MPcs determine the redox properties [117].

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Because of their optical and electrical characteristics [118], Pcs and (MPcs) have received great attention in technological applications. Pcs complexes are attractively used as photoconducting agents in photocoping machines and widely as pigments and dyes [113] because of the ligand-based π-π transitions near 655 and 330 nm, respectively, labelled as Q and B [118]. Pcs are special compound because they have the ability having more than 70 different metallic and non-metallic ions in the ring cavity, furthermore they can consolidate array of different peripheral substituents around the Pc core and also replace some of the four isoindole units by other heterocyclic moieties, producing different Pc analogues [119]. Figure 8 shows the molecular structure of Pc and MPc (dashed line, M = metal).

Figure 8: Molecular structures of Pc [120] and MPc [114,119]

2.3.1 Naphthalocyanines

Naphthalocyanines (Ncs) and metal naphthalocyanines (MNcs) were first reported in 1936 [121], not much work has been carried out on naphthalocyanines systems as compared to phthalocyanines. Ncs have extension of π-electron systems as compared to Pcs analogues; hence they have more pronounced nonlinear optical and electrical properties such as redox potentials, electrical conductivity, photoconductivity and catalytic activity [121, 122]. Their

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superior electrical properties are due to the shift in Q-bands near IR at about 800 nm [122] and the rendering of benzofusion on the macrocyclic core, resulting in strong absorption near-IR region [123]. Nc can be prepared by using 1,2- and 2,3-dicyanonaphthalenes as starting materials, and the final product is four isomers, however isomerism can be avoided by utilising the starting materials [121]. 2,3-Naphthalocyanines have recently received attention because of their intriguing physical properties and potential use as semiconducting materials, electrochromic displayers, in optical storage media, in nonlinear optics and in photodynamic therapy [124, 125]. 2,3-Naphthalocyanine have more extended π-electron-delocalized system and contains Pc core onto which four linearly annelated benzene rings are attached. It absorbs strongly in the near-IR region, thus exhibiting high electrical conductivity as compared with Pcs [125]. 2,3-Naphthalocyanines can be prepared by heating a mixture of 2,3-dicyanonaphthalene with the metal or the corresponding metal salt in an inert solvent [124]. Figure 9 shows the molecular structure of metal free Ncs.

Electrochemical study of some selected neurotransmitters at metal oxide doped phthalocyanine supported on carbon nanotubes sensor platform