(657d) Novel Subsecond Voltammetric Separation between Neurotransmitter Molecules in the Presence of Ascorbate

Electrochemical methods of analysis offer excellent and tuneable spatial resolution, low cost and extreme miniaturisation and can therefore be used to considerable advantage in applications as diverse as neurochemistry, biosensors, scanning probe microscopy and environmental analysis [1-3]. For instance amperometry, where the electrode voltage is kept constant, and voltammetry, where the electrode voltage is perturbed according to a predefined waveform, are the methods of choice for a wide range of measurements in neuroscience [4-6]. While amperometric methods offer good temporal resolution they cannot discriminate between different molecules undergoing an electrochemical reaction step at the electrode. Voltammetry offers improved selectivity as well as improved discrimination against non-faradaic contributions [1]. More recently, micro-meter carbon-fibre electrodes, because of their excellent biocompatibility, have been combined with voltammetry to monitor in vitro and in vivo biological processes related to various physiologica and pathological states [2, 4-6].

In general, the major difficulty of voltammetric methods lies in the interpretation of the electrochemical sensor's current response. Electrochemical signals are intrinsically nonstationary and nonlinear. Thus, for a long time voltammetruc measurements were confined in small-amplitude perturbations to avoid nonlinear effects of electron transfer. Lately, ac voltammetry which a 'slow' ramp superimposed on a 'faster' large amplitude harmonic as voltage excitation has drawn considerable attention due to its enhanced voltammetric detail as well as its ability to investigate phenomena on different timescales. Such large-amplitude/high-frequency excitation waveforms though cause capacitance interference to the current to become substantial [1]. Hitheto, there have been few alternatives to the fast Fourier transform technique for frequency analysis although fundamental assumptions of the theory, namely stationarity and linearity, are not satisfied [7]. As a result, ac voltammetric data could not be fully interpreted [8, 9].

Neurotransmitters, which are related to various biological processes ranging from motor control and sensory processing to central nervous system developemnt, learning and memory, attention, control of mood, stress and anxiety, are easily oxidised at carbon electrodes; therefore electrochemical methods provide a convenient, sensitive and localised means of their measurement. Despite this, hitherto it has been very difficult to separate between neurotransmitters such as dopamine, serotonin or noreadrenaline because of their similar electrochemical signature [6].

In this work, we present how ac voltammetry can be used to monitor simultaneously and from the same electrode a number of neurotransmitter molecules both in vitro as well as in vivo. We will show that the application of novel signal processing techniques - the windowed Hilbert transform - allows the efficient separation of neurotransmitter-molecules as similar as dopamine and serotonin offering unprecedented detail in biological measurements [10]. This is only achieved because the herein introduced methodology (i) considerably suppresses the impact of capacitance and (ii) allows monitoring changes of the capacitance baseline in the duration of the experiment [11, 12]. Additionally, we will illustrate that ascorbate, the main electrochemical interference in most biological systems which is present in 100- to 1000-fold higher concentrations than the neurotransmitters, can be judiciously monitored, if wanted, using different voltage excitation parameters. Our in vitro findings we will apply in in vivo measurements of dopamine and serotonin in the biological model Lymnea stagnalis (i.e. the pond snail). Measurements from different regions of the CNS will illustrate the fact that the ability to simultaneously monitor more than one analytes significantly enhances our ability to study/understand the biological information behind neurotransmitter (co-)release dynamics. At the end, we will discuss important biosensing issues such as electrode fouling and sensing optimisation strategies.

References

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[10] Anastassiou CA, Patel BA, Arundell M, Yeoman MS, Parker KH, O'Hare D. Anal. Chem. (submitted)

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[12] Anastassiou CA, Ducros N, Parker KH, O'Hare D. Characterisation of AC voltammetric Reaction-Diffusion Dynamics: From Patterns to Physical Parameters. Anal. Chem. (in press) DOI: 10.1021/ac060122v.