5-hydroxytryptamine measurement using paired pulse voltammetry

Purpose: Although paired-pulse voltammetry (PPV) has significantly reduced the effects of confounding factors such as pH changes, its appliance has been limited to triangular waveforms. Here, we extend PPV to N-waveform, known to be effective in differentiating serotonin (5-HT) from other analytes. Methods: Unlike previous PPV that employs a triangular binary waveform with a specified time gap between the comprising pulses, this study experiments PPV with Nwaveform. N-waveform, the most conventional waveform for detecting 5-HT, sweeps from 0.2 V to 1.0 V to -1.0 V and back to 0.2 V at a sweep rate of 1000 V/s, while the electrode is held at a holding potential of +0.2 V between the voltammetric pulses. After experimenting with various gap times (2 ms, 10 ms, 30 ms, and 45 ms), N-shape PPV was optimized to the parameter of 100 ms repetition time (2 Hz) and 2 ms gap time that displayed the highest sensitivity. 5-HT measurement was performed with a carbon fiber microelectrode placed in the flow cell. PPV data was collected with the Wireless Instantaneous Neurochemical Concentration Sensing System. Results: At the optimized parameter, the oxidation peak in secondary pulse of N-waveform PPV recorded about 68% of the peak of the primary pulse. In addition, the fitting of peak currents in primary, secondary, and primary-secondary in Nshape PPV in relation to the concentration level between 0.25 μM to 2 μM displayed high reliability (R-squared values = 0.9823, 0.9895, 0.9914, respectively). When 5-HT 3 μM and 0.1 ΔpH is mixed, the 10 nA artifact created by 0.1 ΔpH in P-S voltammogram was almost completely removed while the oxidative peak by 5-HT was detected. Conclusions: These results demonstrate that N-shape PPV will enable more accurate measures of real-time serotonin changes, especially in complex environment.