Development of fouling-resistant physically small carbon electrodes for in vivo dopamine detection

This thesis reports the development of fouling-resistant physically small carbon electrodes to detect the neurotransmitter dopamine in vivo. Dopamine has long been of interest to both chemists and neuroscientists. This is due to its role in modulating many aspects of brain circuitry in a major system of the brain including the extra pyramidal and mesolimbic system, as well as the hypothalamic pituitary axis. In addition, it plays a crucial role in the functioning of the central nervous, cardiovascular, renal and hormonal systems. A loss of dopamine containing neurons or its transmission is also related to a number of illnesses and conditions including Parkinson's disease, schizophrenia, motivational habit, reward mechanisms and the regulation of motor functions and in the function of the central nervous, hormonal and cardiovascular system. In order to detect the neurotransmitter in vivo, high sensitivity, chemical selectivity, and fast temporal resolution are among the desirable characteristics in detecting neurotransmitters. In this respect, electrochemical techniques are well suited for the measurement of transient changes in the concentration of a species. Such techniques are concerned with the interplay between electricity and chemistry, namely the measurement of electrical quantities such as current, potential or charge, and their relationship to chemical parameters. Electroanalytical techniques have been widely developed and, more recently, applied to the investigation of neurochemical systems, leading to a better understanding of neurotransmission. This partly stems from the ease of oxidative detection of many neurotransmitters including dopamine. In addition, the development of structurally small electrodes has made in vivo detection neurotransmitters possible in biological microenvironments. Moreover, the small dimension of such electrodes permits minimal tissue damage upon implantation and, of equal importance, permits very careful selection of the region of tissue where measurements can be performed. Furthermore, the inherent fast response time of structurally small electrodes makes it feasible to follow biochemical events frequently taking place on a millisecond (ms) time scale (e.g. neuronal firing). -- Detection of dopamine in a physiological environment with selectivity and sensitivity has been an important topic of electroanalytical research but one that has also experienced great challenges. Direct voltammetric detection of dopamine at naked electrodes requires high selectivity for the neurotransmitter in the presence of interfering ascorbic acid. In addition, electrode passivation by adsorption of species present in the extra-cellular fluid, known as fouling is another obstacle to electrochemical detection of neurotransmitters. To reduce fouling, electrode surfaces can be modified to deter adsorption. Many of these species are hydrophilic. Therefore, common methods employed to achieve this include film incorporation, conversion to a hydrophobic surface such as that via hydrogenation, application of a diamond layer among others. Therefore, an overall aim of this study is to develop, characterise and apply physically small carbon electrodes that are fouling-resistant in detecting dopamine in vivo. More specifically, this work aimed at (1) fabricating and characterising physically small p-phenylacetate film-coated carbon electrodes; (2) fabricating and characterising physically small hydrogenated carbon electrodes; and (3) assembly of a chemical vapour deposition system for hydrogenating bare carbon electrodes and depositing diamond films. -- The study begins with Chapter 2 describing the fabrication of physically small conical-tip carbon electrodes by pyrolysis of acetylene on pulled quartz capillaries with a micrometer-sized tip (<2 μm electrode radius and ~3 μm axial length). These electrodes were then modified by electrochemically depositing a p-phenylacetate film-layer on the surface. The modified electrodes were subjected to voltammetric characterisation in hexamine ruthenium(III) chloride, dopamine and potassium ferricyanide, respectively, and the results were compared to those at the electrode before modification. Increased limiting currents at the modified electrodes for hexamine ruthenium(III) chloride and dopamine, and decreased currents for potassium ferricyanide were observed, respectively. Characterisation under laboratory conditions showed that the anionic film on the electrode surface was stable over a 40-day period. In detecting dopamine, the modified electrodes demonstrated a limit of detection of 541 pM and a sensitivity of 16 pA/nM, compared to 543 nM and 19 pA/nM, respectively, at bare carbon electrodes. Moreover, the film-coated electrodes also displayed higher sensitivity and lower limit of detection (0.1 nA/nM and 6 nM respectively) for dopamine over ascorbic acid (0.1 nA/μM and 1.4 μM). -- Analytical performance of both film-modified electrodes and bare carbon electrodes was also examined in a synthetic solution consisting of 1.0% (v/v) caproic acid (a lipid), 0.1% (w/v) bovine serum albumin and 0.01% (w/v) cytochrome C (both are protein) and 0.002% (w/v) human fibrinopeptide B (a peptide. The film-coated electrodes displayed a 3-fold increase in sensitivity, while the limit of detection remained about the same. On the other hand, bare carbon electrodes demonstrated evidence of severe surface degradation, with unmeasurable changes to the sensitivity and limit of detection following exposure to the synthetic solution. In detecting dopamine in an anesthetised rat, a greater degradation (62%) of the dopamine oxidation signal after 60 min of monitoring was observed at bare carbon electrodes, compared to 50% at film-coated electrodes. In addition, the film-coated electrodes were observed to offer some resistance to fouling for the first 40 mins of implanting the electrode. This however decreases after 40 min, suggesting possibly more severe fouling. This was attributed to a result of lack of longevity of the p-phenylacetate film on the electrode surface. In comparison, bare carbon electrodes demonstrated a steady decline in the oxidation peak signal. This suggested that the p-phenylacetate film was successful in retarding fouling for the first 40 min after implanting in the rat brain, by which time the carbon electrode surface had degraded to a greater extent.