Electrochemical sensing

Most of the classical neurotransmitters have been identified via electrochemical methods by the start of the 1970s and further specified with the help of high-pressure liquid chromatogra-phy (HPLC) or combined gas chromatograchromatogra-phy/mass spectrometry (GC/MS) [23], [24], [25], [26], [27]. In 1973, Ralph Adams was the first to apply a traditional voltammetric technique to detect oxidizable biogenic amines in the brain of an anesthetized rat [28]. Nowadays, we have to consider the possibility of interference with ascorbic acid that is also present in the brain and has a similar redox potential to some neurotransmitters [29]. Still, current biological studies would not be possible without the quantitative information about neuro-transmitter concentrations provided by the electrochemical methods [14], [30]. So far, there are two major electrochemical neurotransmitter detection techniques: (1) amperometry and (2) cyclic voltammetry (CV) [31], [32].

Amperometric detection is based on a voltage (potential) applied between two electrodes and its sensitivity to incoming redox-active molecules. Any electroactive analyte will be either oxidized or reduced, and the current measured [33]. In 1990, Wightman et al. could directly monitor time-resolved exocytotic events on the millisecond timescale [34]. To detect vesicle fusion an electrode is held to a positive potential and brought in close proximity to the cell. As soon as the vesicle fuse and release neurotransmitters into the solution, redox active neurotransmitters get oxidized at the electrode tip. Size and sequence of following current spikes provide information about the number of vesicles and the release probability [28], [35].

First experiments involved only single electrodes, thus providing no spatial information.

To improve it, recent electrochemical techniques aim to implement multiple and smaller electrodes. Increasing electrode density can be used to raise both the number of data points for samples with multiple cells or to allow a more accurate imaging of a single cell. Lindau et al. expanded the method to use an electrochemical detector (ECD) array with 4 platinum microelectrodes around the cell (Fig. 4a) [36]. Then chromaffin cells are seeded onto the ECD array and chemically stimulated into catecholamine release. Observed oxidation current reproduces the direction and volume of the neurotransmitter that reach the electrode tip.

Thus, it was possible to achieve electrochemical imaging of fusion pore openings and estimate the most probable exocytosis site on the cell surface. One has to keep in mind possible interference due to introducing an ECD array into the cells and using a patch pipette. Such handling can disturb the normal behavior of the cell and the molecule diffusion pathways, and interfere with an in vivo application.

Figure 4: Electrochemical dopamine detection. (a) Image of a chromaffin cell upon an electrochemical detector (ECD) array with four electrodes (A-D), (b) Currents from the four electrodes (A-D, as black, blue, green, and red line) reveal spatiotemporal differences, (c) Image of a setup with the 16-electrode ensemble over a cluster of PC12 cells and the stimulation pipet. (d) Amperometric traces from different electrodes shown in (c) after cell stimulation. Adapted from [6].

A different microelectrode array (MEA) with a more bio-compatible material was designed by Ewing et al. [37]. Here, 16 electrodes were integrated into a PDMS chamber coated with collagen. PC12 cells were cultured on top of the MEAs device and the single electrodes provided individual readout. Fig. 4f shows traces from different electrodes. Due to this arrangement, it was possible to record exocytotic events and dopamine release down to a spatial resolution of 2 µm. This resolution is useful for collecting information about small cell clusters (e.g. to see how pharmacological treatment could affect dopamine release) but cannot provide any information about the heterogeneity of dopamine release within one cell.

To achieve this resolution the electrodes were minimized even more, until in 2014, Ewing et al. increased the number of electrodes to 36 and achieved subcellular resolution [38].

Cyclic voltammetry (CV) has a slightly different approach. During CV one sweeps the potential of the working electrode at a specific sweep rate (in volts/second). The output results in a curve of measured current vs. time. After the reduction potential of the analyte is reached and no more reducible analyte is available, the reduced analyte starts getting re-oxidized. Thus completing the circle and accounting for the term ofcyclic voltammetry.

Fast cyclic voltammetry (FSCV) is a class of cyclic voltammetry with a high scan rate (up to 1×10⁶ V·s⁻¹ ≈ 10 Hz) which allows for high temporal resolution [39]. FSCV is a frequently used method for detection of neurotransmitters in biological systems [40]. Here, the neurotransmitters are repeatedly oxidized and reduced resulting in a current of electrons on a nanoampere scale. With a known time scale of the voltage oscillation, concentration change of dopamine can be calculated down to 1 nM in real time [41]. The typical size of electrodes for FSCV lies in the order of micrometers, though there are single reports about nanoscale electrodes for dopamine detection [42], [43].

In conclusion: over the last decades, classical electrochemical methods have provided neuro-chemists with important insights into the chemical nature of neurotransmitters, the number of released molecules, and the time resolutions of the processes involved. A clear advantage of electrochemical techniques is their high time resolution (≈100 µs) and the possibility of integration in various designs [44]. As for its drawbacks, there are several:

• Diffusion of released neurotransmitters could be affected by the electrodes implemented into the system, which would require additional calculations to reconstruct the original concentration profiles.

• CV is dependent upon different reduction potentials of analytes. CV can distinguish between molecules with different redox potentials but cannot discriminate compounds with similar potentials, however different their chemical structures might be.

• Another limitation of CV comes from the cross-talk between individual electrodes [42], [43].

• The analytes have to be redox-active which limits the number of neurotransmitters.

Dopamine or serotonin can be detected while other important neurotransmitters, such as L-glutamate orγ-aminobutyric acid (GABA), can not.

• Low spatial resolution. Despite current progress in miniaturization of electrodes, most structures are still too big in comparison to a neurotransmitter release site [44].