Antifouling physically small carbon electrodes for dopamine detection

In this work, we have developed physically small carbon electrodes that show antifouling behaviour during detection of the neurotransmitter dopamine in a biological medium to achieve meaningful detection results. In such an experiment, amphiphilic proteins, peptides and lipids present in extracellular fluid can easily adsorb on a hydrophilic electrode surface. This prevents dopamine from making direct contact with the electrode for electron transfer reactions, leading to electrode fouling in which agradually diminishing transient detection signal is observed. In this study, we have developed antifouling conical-tip carbon electrodes by hydrogenating the carbon using an n-butylsilane or a diethylsilane reduction method, followed by a 4-sulfobenzene layer. These modified electrodes were subjected to dopamine detection in the presence of a simulated biological environment to assess their effectiveness in such a detection experiment. In addition, we have also discovered in this work that the n-butylsilane reduction method is capable of activating non-functioning carbon electrodes by removing carbon-oxygen functionalities from an electrode surface prior to terminating the defect sites with carbon-hydrogen. In our laboratory, physically small conical-tip carbon electrodes are routinely fabricated by thermally pyrolysing acetylene in a nitrogen atmosphere to deposit carbon at the tip and on the shank of quartz capillaries already pulled down to a tapered end, typically ~2μm in tip diameter and ~15 μm axial length. These electrodes were characterised by a sigmoidal-shaped cyclic voltammogram of [Ru(NH₃)₆]³⁺ with a small charging current etween the forward and backward scan. Occasionally, a non-sigmoidal shaped cyclic voltammogram with an appreciable charging current was obtained, indicating a nonfunctioning conical-tip carbon electrode. These electrodes were then treated in a onepot reaction involving n-butylsilane reduction capable of reducing carbon–oxygen functionalities present on a carbon surface, e.g. aldehydes, ketones and primary, secondary and tertiary alcohols, polycarboxylic acid group to their corresponding alkyl functionalities, leaving carbon–carbon double bonds and the graphitic structure undisturbed. Phenolic hydroxyl groups, however, were not reduced but were instead silanised to form a dendrimeric butylsiloxane. After such a hydrogenation treatment, these electrodes were found to yield an expected sigmoidal-shaped cyclic voltammogram of Ru(NH₃)₆]³⁺ with negligible charging current, indicating a characteristic functioning microelectrode. In this way, the fabrication success rate of carbon microelectrodes was improved to nearly 100%. X-ray photoelectronspectroscopy was used to systematically characterise the surface of hydrogenatedcarbon electrodes. We also studied the electrochemistry of anthraquinone-2,6-disulfonate (AQDS) at these hydrogenated carbon electrodes to provide evidence for the removal of the carbon-oxygen functionalities from the electrode surface. In addition, after a one-week storage in air and in a pH 7.4 citrate/phosphate buffer, there was a minimal change in the corresponding Ru(NH₃)₆]³⁺ reduction signal of ~1.1%and ~2.8%, respectively, observed at these hydrogenated carbon electrodes. The n-butylsilane hydrogenated carbon electrodes were further characterised using [Fe(CN)₆]³⁻ and dopamine as redox markers. To support the cyclic voltammetric results, electrochemical impedance spectroscopy was also conducted at the hydrogenated carbon electrodes. The results obtained indicated sluggish electron transfer kinetics for both redox markers at the electrodes due to the formation of an sp³ enriched, defect free hydrophobic surface. The electrode surface was also examined using atomic force microscopy. Raman spectroscopic results also confirmed a larger proportion of sp³ hybridised carbon at these hydrogenated carbon electrodes, compared to the corresponding carbon electrodes that were not hydrogenated. Hydrogenated carbon electrodes with such a hydrophobic surface have demonstrated their capability in determining dopamine with minimal interference from ascorbic acid at concentrationas high as 500 μM generally expected in an extracellular fluid. Upon incubating hydrogenated carbon electrodes in a synthetic fouling solution containing 1.0% (v/v) caproic acid (a lipid), 4% (w/v) bovine serum albumin and 0.01% (w/v) cytochrome C (both are proteins), and 0.002% (w/v) human fibrinopeptide B (a peptide) for 30 min, they were used in the cyclic voltammetric detection of dopamine in order to assess their degree of antifouling capability. A ~35% decrease in dopamine oxidation current was observed. However, the magnitude of this oxidation current remained almost unchanged even after incubating the electrodes in the same synthetic fouling solution for one week. The dopamine oxidation product, dopamine-o-quinone, is itself a wellknown fouling reagent. However, there was no observable interference at the hydrogenated carbon electrodes when the dopamine concentration was kept below 1.0μM, which is a 100-fold higher concentration than that generally expected in the central nervous system. These results support minimal fouling at n-butylsilane hydrogenated electrodes during dopamine detection in vitro. Next, a 4-sulfobenzene layer was immobilised on hydrogenated conical-tip carbon electrodes to achieve improved dopamine detection sensitivity and to further enhance the fouling resistance at the electrodes. Cyclic voltammetry and X-ray photoelectron spectroscopy confirmed the grafting of 4-sulfobenzene on n-butylsilane modified electrodes. Similar results in the voltammetric studies of [Fe(CN)₆]³⁻ at the electrodesto n-butylsilane hydrogenated electrodes were obtained. However, a ~4% improved Ru(NH₃)₆]³⁺ detection signal and a ~10% improved dopamine detection signal were estimated at 4-sulfobenzene modified electrodes that were hydrogenated by n-butylsilane reduction, compared to hydrogenated electrodes without any 4-sulfobenzene modification. The former electrodes also displayed a ~13% higher dopamine detection sensitivity over the latter electrodes. We have attributed all these observations to electrostatic attraction between the negatively charged 4-sulfobenzene groups on the electrode surface and positively charged Ru(NH₃)₆]³⁺ and dopamine (a cation under physiological pH). By assessing the antifouling property of the 4-sulfobenzene modified electrodes in the same fouling solution described above, there was only a 15% sensitivity loss, compared to ~38% at hydrogenated electrodes without 4-sulfobenzene modification. In addition, a limit of detection of 52 ± 8 nM was estimated at 4-sulfobenzene modified electrodes, which is approximately a three-fold improvement relative to 138 ± 12 nM estimated at hydrogenated electrodes without any 4-sulfobenzene modification In this work, we have also conducted carbon electrode hydrogenation using a branch-chained alkylsilane, diethylsilane, in place of the linear-chain n-butylsilane, to evaluate their antifouling capability. These diethylsilane hydrogenated carbon electrodes showed a ~36% decrease in the dopamine oxidation signal after a one-week incubation in the fouling solution, which is comparable to 35% obtained at n-butylsilane hydrogenated carbon electrodes. In a similar experiment, a 38% decrease in dopamine signal was observed at a 4-sulfobenzene layer immobilised carbon electrodes that were hydrogenated by diethylsilane reduction. We have also observed faster electron transfer kinetics at both diethylsilane hydrogenated carbon electrodes and those that were subsequently modified by 4-sulfobenzene compared to all other electrodes studied in this work. More significantly, 100% 4-sulfobenzene modified carbon electrodes that were hydrogenated by diethylsilane reduction (compared to 66% of diethylsilane hydrogenated, 33% n-butylsilane hydrogenated, and 33% 4-sulfobenzene modified carbon electrodes that were hydrogenated by n-butylsilane reduction) were found to be stable and demonstrated consistent fouling resistance throughout the experiments after being incubated in the fouling solution for 0, 10, 30 min and 1 week. Finally, all electrodes studied in this work were assessed in their feasibility in the analysis of dopamine in a real-life human serum sample, into which dopamine was spiked and then the recovery was evaluated. In this way, a 97.2 – 129% recovery of dopamine was obtained at all modified electrodes. These results support the potential applications of all the above modified electrodes for determination of dopamine in real lifesamples.