An antifouling, structurally small carbon electrode for detection of the neurotransmitter dopamine

A long-term goal of the present work is to apply structurally small, antifouling carbon electrodes to acquire meaningful results during in vivo detection of the neurotransmitter dopamine. Dopamine is a neurotransmitter in the mammalian brain that plays a crucial role in the central nervous, cardiovascular, renal, and endocrine systems. Under physiological pH (pKa 8.87), dopamine exists as a cation that can be easily oxidised at an electrode. Therefore, electroanalytical techniques have been widely applied to the investigation of neurochemical systems, leading to a better understanding of neurotransmission. Very often, the concentration of dopamine in extracellular fluid is used as a marker for the diagnosis of several neurodegenerative diseases including Parkinson’s, Alzheimer’s, schizophrenia, and depression. However, a major challenge during in vivo measurement of dopamine is electrode fouling, which is caused by the non-specific adsorption of amphiphilic proteins, peptides and lipids present in the extracellular fluid on a hydrophilic carbon surface. This has often yielded compromising results in time dependent in vivo dopamine detection experiments. Notably, application of a carbon electrode with a hydrophobic surface is one of the ways of minimising non-specific adsorption of non-targeted species, which can be easily achieved by hydrogenating the electrode surface using a silane reduction. This hydrogenation mechanism involves the reduction of polycarboxylic acids, ketones, aldehydes, and alcohols to alkanes without any effect on the double bonds and ether. A novelty of this work lies in the application of triethylsilane and phenylsilane hydrogenation to yield a low-oxide surface with sp3 hybridised carbon, leading to a hydrophobic carbon surface. More specifically, this work is aimed at: 1. fabricating and characterising structurally small carbon electrodes hydrogenated by triethylsilane and phenylsilane; 2. evaluating the antifouling characteristics of the hydrogenated carbon electrodes in a laboratory synthetic fouling solution; 3. examining the analytical performance of the hydrogenated carbon electrodes in two real-life biological samples namely the neuroblastoma SH-SY5Y cell line and mouse brain slices. Chapter 1 begins with a general introduction of the topic including literature review on recent research conducted using electrochemical sensors in detecting dopamine in vivo and in real-life biological systems. This Chapter also describes the research conducted to minimise non-specific adsorption by modifying the sensing surfaces modified using a variety of substances including graphene, carbon nanotubes, conducting polymers such as poly(3,4-ethylenedioxythiophene), Nafion, and polyethylene glycol. All the reagents and experimental procedures used in this study to acquire data are described in Chapter 2. Specifically, it includes the detailed description of the fabrication of structurally small carbon electrodes, the protocols and instrumentation used in their microscopic, spectroscopic and electrochemical characterisation experiments. Chapter 3 commences with the fabrication of structurally small carbon electrodes by pyrolysing acetylene in and on pulled quartz capillaries with tip diameters of ~2 μm. Initially, all fabricated carbon electrodes were electrochemically characterised using [Ru(NH3)6]3+ to identify functioning electrodes. These functioning electrodes were then subjected to hydrogenation using either triethylsilane or phenylsilane in the presence of anhydrous dichloromethane and a catalyst, tris(pentafluorophenyl) borane, under ambient conditions. Following hydrogenation, scanning electron microscopy, X-ray photoelectron spectroscopy and Raman spectroscopy were used to determine the elemental composition and the sp3 carbon / sp2 carbon ratio of the electrodes. This Chapter further describes the electrochemical characterisation of hydrogenated electrodes using different redox markers including [Fe(CN)6]3-, dopamine, dihydroxyphenyl acetic acid, 4-methylcatechol and anthraquinone 2,4 disulfonate to determine their responses towards surface sensitive redox analytes. All the results were compared to those of non-hydrogenated carbon electrodes in evaluating the electrochemical behaviour of the hydrogenated carbon surface. In Chapter 4, we initially describe the surface characteristic of hydrogenated carbon electrodes using two-dimensional atomic force microscopy. Next, electroanalytical detection performance of dopamine at the hydrogenated carbon electrodes was assessed. The sensitivity and limit of detection of the triethylsilane and phenylsilane hydrogenated electrodes were then estimated from the analytical detection of dopamine in a pH 7.4 citrate/phosphate buffer to be 23.15 pA μM-1 and 42.89 pA μM-1 (compared to 3.24 pA μM-1 at non-hydrogenated carbon electrodes) and 0.84 μM (compared to 2.31 μM at their non-hydrogenated counterpart), respectively. The selectivity towards dopamine in the presence of ascorbic acid and uric acid is also evaluated at hydrogenated carbon electrodes. Finally, the Chapter concludes with the performance comparison between non-hydrogenated and the hydrogenated carbon electrodes in a laboratory synthetic fouling solution containing 2.0% (w/v) bovine serum albumin (a protein), 0.01% (w/v) cytochrome c (a protein), 0.001% (w/v) human fibrinopeptide (a peptide) and 1.0% (v/v) caproic acid (a lipid). A 30-min incubation of non- hydrogenated carbon electrodes in the fouling solution resulted in a 53% decline in the dopamine oxidation limiting current, while a corresponding loss of 23% and 18% was obtained at triethylsilane and phenylsilane hydrogenated carbon electrodes. This shows the robustness and stability of the hydrogenated carbon electrodes in the presence of proteins, peptides and lipids. As previously discussed in Chapters 3 and 4, one significant challenge during dopamine detection both in vivo and in vitro is electrode fouling, often caused by adsorption of amphiphilic proteins, peptides and lipids present in the biological fluid on hydrophilic carbon electrode surfaces. Chapter 5 examines the performance of triethylsilane and phenylsilane hydrogenated carbon electrodes in the detection of dopamine in two real-life biological samples, the neuroblastoma cell line SHSY-5Y and brain slices. To evaluate fouling in SH-SY5Y cells, a hydrogenated carbon electrode was positioned close (~1 μm) to the cells with the help of an inverted microscope and a micromanipulator. Using amperometry, a constant potential of +0.5 V (estimated from the cyclic voltammogram of dopamine in a pH 7.4 citrate/phosphate buffer) was applied to the carbon working electrode to promote the oxidation of dopamine released from cells that were continuously depolarised using 0.1 M KCl for 1 h. The transient changes in peak height was compared to determine the degree of electrode fouling. To evaluate fouling in a brain slice, a hydrogenated carbon working electrode was positioned in the brain tissue, while fast scan cyclic voltammetry between -0.4 V and +1.2 V (versus a Ag AgCl reference electrode) at a potential scan rate of 400 V s-1, and a repetition rate of 10 Hz, was applied to oxidatively detect dopamine for 1 h. The degree of electrode fouling was evaluated by comparing the dopamine oxidation peak before and after the electrodes were applied to brain tissues. The results show that the non-hydrogenated electrodes are easily fouled by proteins, peptides and lipids, while the hydrogenated carbon electrode electrodes showed resistance towards these biomolecules. We attribute the improved performance of the hydrogenated carbon electrodes to the presence of a hydrophobic surface with low oxygen-containing functional groups that led to weak interaction between the biomolecules and the hydrogenated electrode surface. Chapter 6 concludes with the outcomes of this study including a summary of the performance of the electrodes fabricated in this study. This study demonstrated the antifouling property of the silane hydrogenated carbon electrodes, which was potentially due to low oxygen-containing functional groups with a hydrophobic surface. In addition, one of the features of silane hydrogenation was the formation of siloxane dendrimers on the sensing surface. The use of a bulky siloxane dendrimers has aided in hindering the adsorption of large biomolecules on the electrode surface. Some suggestions for future work are also presented in this Chapter