Sawhorse waveform voltammetry for selective detection of adenosine, ATP, and hydrogen peroxide

Abstract

Fast-scan cyclic voltammetry (FSCV) is an electrochemistry technique which allows subsecond detection of neurotransmitters in vivo. Adenosine detection using FSCV has become increasingly popular but can be difficult because of interfering agents which oxidize at or near the same potential as adenosine. Triangle shaped waveforms are traditionally used for FSCV, but modified waveforms have been introduced to maximize analyte sensitivity and provide stability at high scan rates. Here, a modified sawhorse waveform was used to maximize the time for adenosine oxidation and to manipulate the shapes of cyclic voltammograms (CVs) of analytes which oxidize at the switching potential. The optimized waveform consists of scanning at 400 V/s from -0.4 to 1.35 V and holding briefly for 1.0 ms followed by a ramp back down to -0.4 V. This waveform allows the use of a lower switching potential for adenosine detection. Hydrogen peroxide and ATP also oxidize at the switching potential and can interfere with adenosine measurements in vivo; however, their CVs were altered with the sawhorse waveform and they could be distinguished from adenosine. Principal component analysis (PCA) was used to determine that the sawhorse waveform was better than the triangle waveform at discriminating between adenosine, hydrogen peroxide, and ATP. In slices, mechanically evoked adenosine was identified with PCA and changes in the ratio of ATP to adenosine were observed after manipulation of ATP metabolism by POM-1. The sawhorse waveform is useful for adenosine, hydrogen peroxide, and ATP discrimination and will facilitate more confident measurements of these analytes in vivo.

Figure 1

Current versus waveform time plots for the (A) triangle and (B) sawhorse waveform. The triangle waveform is the traditional adenosine waveform for FSCV (−0.4 to 1.45 V and back at 400 V/s). The optimized sawhorse waveform is scanning from −0.4 to 1.35 V, holding for 1.0 ms, and ramping back down to −0.4 V at a rate of 400 V/s. The data were collected at two separate electrodes. The dotted black line shows the shape of the waveform over time and the blue line represents the current vs applied waveform time. Data are plotted as current versus time instead of voltage because of the hold time in the sawhorse waveform; 5 μM adenosine, 10 μM hydrogen peroxide, 5 μM ATP, and 5 μM dopamine were tested.

Figure 2

Background current for both waveforms. (A) Background current for the traditional triangle waveform (−0.4 to 1.45 at 400 V/s) is plotted in red and the black dashed line is the shape of the waveform over time. (B) Background current for the optimized sawhorse waveform (−0.4 to 1.35 V, hold for 1.0 ms at 400 V/s) is plotted in red and the black line denotes the shape of the sawhorse waveform over time. The sawhorse background current shows a drop in capacitive current at the plateau time.

Figure 3

Optimization of the sawhorse waveform switching potential and plateau time. (A) A range of plateau voltage spanning from 1.25 to 1.45 V was tested. The plateau time is constant at 1.0 ms. A noticeable jump in current for 1 μM adenosine is seen at 1.35 V. Overall current was significantly dependent on switching potential (one-way ANOVA, p = 0.0273) and the current with 1.35 V was significantly higher than both 1.25 and 1.30 V (Bonferroni post test, p < 0.01 and p < 0.05, respectively, n = 4). Slightly higher currents were detected at 1.40 and 1.45 V; however, the amount of current was not significantly different than 1.35 V (one-way ANOVA with Bonferroni post test, p > 0.05, n = 4). (B) Three plateau times were tested: 0.5, 1.0, and 1.5 ms for 5 μM adenosine. The plateau voltage was held constant at 1.35 V. Overall, current was significantly dependent on plateau time (one-way ANOVA, p < 0.001). Both 1.0 and 1.5 ms plateau times were significantly higher than 0.5 ms (Bonferroni post-test, p < 0.01 and p < 0.001, respectively, n = 4); however, 1.0 ms was not significantly different than 1.5 ms (p > 0.05).

Figure 4

Comparison of current at both the triangle and sawhorse waveform at various switching potentials. The plot shows average current for each switching potential tested for both the triangle (black) and sawhorse (gray) waveform for 1 μM adenosine. The sawhorse waveform produced significantly more current for adenosine than the triangle waveform at 1.30 and 1.35 V switching potential (unpaired t test p < 0.01 and p < 0.001, respectively, n = 6).The currents for 1.40 and 1.45 V were not significantly different between the sawhorse and triangle waveform (unpaired t test p > 0.05, n = 6).

Figure 5

Mechanically evoked adenosine using the sawhorse waveform. The medial prefrontal cortex of a rat brain slice was mechanically stimulated by a glass pipet lowered approximately 30 μm away from the carbon-fiber microelectrode. After mechanical stimulation, an in slice training set was collected for adenosine, hydrogen peroxide, and ATP via exogenous application near the electrode. (A) An example adenosine training set in a slice. (B) An example of a mechanically evoked adenosine CV in a slice. (C) A comparison of the predicted values using PCA for the sawhorse waveform compared to the actual value if the release was all adenosine (black bar). The actual concentration and predicted concentrations of adenosine were not significantly different from one another (unpaired t test, p > 0.05, n = 8). Negligible amounts of hydrogen peroxide and ATP were predicted.