Cysteine residues reduce the severity of dopamine electrochemical fouling
Introduction
Reliable detection of neurotransmitters is a critical requirement for understanding the mechanisms of the brain, or neurodegenerative diseases. Electrochemistry has many advantages for these applications: it is cheap, in-vivo compatible, it requires only simple equipment and performs real-time measurements. As a consequence, since the seminal work of Adams [1], [2], numerous reports have focused on the electrochemical detection of neurotransmitters to investigate biological issues [3], [4], [5], [6], [7], [8], [9], [10], but several problems still arise.
A significant problem with this type of technique is electrode fouling. Indeed, in biological conditions, the surface of the sensor can be blocked by the biomolecules present in the sample. Two main types of fouling, which arise from different mechanisms, can be described and both have to be addressed. The first type is a passive biological fouling, where background species, such as proteins or lipids, adhere to the electrode surface, whether it is active or not. On the opposite, an active form of electrochemical fouling, where the reaction used for the detection induces the formation of an insulating film on the electrode, can be encountered. This is typical of the oxidation of several neurotransmitters, such as dopamine (DA) [11] and serotonin (5-HT) [12].
To mitigate this matter, special voltammetric waveforms, or electrode coatings have been suggested [13], [14], [15], [16], [17], [18]. However, using an electrode material that is intrinsically resistant to fouling would be beneficial by simplifying the experimental procedures.
New electrode materials have been considered to limit the impact of fouling on measurements. Boron-doped diamond surfaces have shown a substantial resistance to neurotransmitter fouling [12], [19], [20], [21]. Carbon nanotube-based materials were also found to be less sensitive to fouling [22]. Carbon nanotube fibers (CNTF) [23] have recently attracted attention as a potential user friendly and fouling-resistant alternative to the traditional carbon fiber (CF) used in electrode fabrication [24]. We have recently shown the improved resistance to DA fouling of this new type of electrode [11]. We here further emphasize this increased stability by showing that the CNTF resists better to the electrode passivation induced by long oxidation of serotonin and octopamine (OA).
However, the role of the biological matrix has not been considered yet in the fouling mechanisms encountered during electrochemical detection. To investigate this phenomenon, high concentrations (4 wt % corresponding to a concentration of 600 μM) of BSA were used as surrogates for biological fouling. Interestingly, BSA reduced the severity of DA fouling, in a dose-dependent manner. This effect was attributed to the thiol moiety present in the cysteine (Cys) of the BSA molecule undergoing a nucleophilic attack on the oxidized catechol, blocking the fouling cascade. In good agreement with this mechanism (vide infra), glutathione (GSH) was found to reduce the severity of fouling, yet in a lesser extent than BSA. Altogether, our data shows that interactions with the background are to be expected during in situ electrochemical detection. The level of thiols in the matrix has to be taken into account, and BSA could potentially be added to the buffer used for calibrating the sensors before in situ measurements, so that these secondary reactions are accounted for.
Section snippets
Reagents
Analytical grade chemicals were obtained from Sigma-Aldrich (unless stated otherwise) and used as received. All solutions were made using 18 MΩ.cm water from a Millipore purification system, and the solution pH was adjusted to 7.4 with concentrated NaOH if needed.
CNTF synthesis
The CNTF were prepared following previously published methods [23], [24]. Briefly, 0.3 wt% of HipCO single wall CNTs (CNI, Houston, TX, USA) were dispersed in water using 1 wt% sodium dodecyl sulfate as the dispersant. (Safety
Increased resistance of CNTF to neurotransmitters fouling
Previous reports have shown that CNTF electrodes have good electrochemical properties for the detection of neurotransmitters [11], [25], [26]. In a previous article [11], we focused on DA fouling at CNTF electrodes. Here, we also consider two other molecules, 5-HT and OA, to further establish the stability of CNTF surfaces. These molecules induce fouling at the electrode that is more severe than DA, and hence the concentrations chosen for our analysis are lower than for the DA tests [27]. The
Conclusion
This study further underlines the improved resistance of CNTF electrodes over the traditional CF electrodes to fouling during the oxidation of several neurotransmitters. The fiber nature of this composite material facilitates its use as an electrochemical substrate, and makes it highly compatible to the fabrication of microsensors.
More importantly, fouling of the CF and CNTF fibers electrodes in the presence of thiol moieties clearly can affect detection of neurotransmitters. For both types of
Acknowledgements
The work has been supported by the European Research Council (Advanced Grant), the Knut and Alice Wallenberg Foundation, the Swedish Research Council (VR), The Royal Society of Arts and Sciences in Gothenburg and the National Institutes of Health. GS has been supported by a Young Investigator Project Grant from VR (621-2011-4395).
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Present Address: Lohmann & Rauscher GmbH, 2525 Schönau an der Triesting, Austria (WH); Laboratory of Microsystems LMIS2, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland (RT); Division of Biological Physics, Department of Applied Physics, Chalmers University of Technology, 412 96 Gothenburg, Sweden (GS).
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Member of the ISE.