Simultaneous measurement and quantitation of 4-hydroxyphenylacetic acid and dopamine with fast-scan cyclic voltammetry

Abstract

Caged compounds have been used extensively to investigate neuronal function in a variety of preparations, including cell culture, ex vivo tissue samples, and in vivo. As a first step toward electrochemically measuring the extent of caged compound photoactivation while also measuring the release of the catecholamine neurotransmitter, dopamine, fast-scan cyclic voltammetry at carbon-fiber microelectrodes (FSCV) was used to electrochemically characterize 4-hydroxyphenylacetic acid (4HPAA) in the absence and presence of dopamine. 4HPAA is a by-product formed during the process of photoactivation of p-hydroxyphenylacyl-based caged compounds, such as p-hydroxyphenylglutamate (pHP-Glu). Our data suggest that the oxidation of 4HPAA occurs through the formation of a conjugated species. Moreover, we found that a triangular waveform of −0.4 V to +1.3 V to −0.4 V at 600 V/s, repeated every 100 ms, provided an oxidation current of 4HPAA that was enhanced with a limit of detection of 100 nM, while also allowing the detection and quantitation of dopamine within the same scan. Along with quantifying 4HPAA in biological preparations, the results from this work will allow the electrochemical measurement of photoactivation reactions that generate 4HPAA as a by-product as well as provide a framework for measuring the photorelease of electroactive by-products from caged compounds that incorporate other chromophores.

In Situ Electrochemical Monitoring of Caged Compound Photochemistry: An Internal Actinometer for Substrate Release

“…Figure 3A shows a CV of 4HPAA (0.1 mM) obtained by flow injection analysis and FSCV. As described by Shin et al, 30 we observed two well-resolved peaks, suggesting that two distinct electrochemical processes occur. The first peak occurred at +1.23 V as the potential swept toward the most positive value, while the second peak occurred at +1.12 V as the potential swept toward the most negative value.…”

“…We have previously shown that 4HPAA can readily be measured by FSCV. 30 Thus, we sought here to demonstrate that the liberation of 4HPAA from p-hydroxyphenacyl-based caged compounds, such as p-hydroxyphenacyl glutamate, could be used to quantify the formation of the resulting bioactive molecule. Our approach quantifies glutamate photorelease by measuring differences between currents produced by the liberation of 4HPAA after light application and currents present prior to light application.…”

“…The method described here uses a microliter photoreaction vessel to generate a calibration curve that relates the electrochemical oxidation signal of photoreleased 4HPAA measured by FSCV to the concentration of 4HPAA obtained by HPLC with ultraviolet–visible detection. We have previously demonstrated the simultaneous, electrochemical quantitation of 4HPAA vs dopamine by FSCV . Thus, the method proposed here extends our capability to include the direct measurement of caged compound degradation in conjunction with dopamine release.…”

“…We have previously demonstrated the simultaneous, electrochemical quantitation of 4HPAA vs dopamine by FSCV. 30 Thus, the method proposed here extends our capability to include the direct measurement of caged compound degradation in conjunction with dopamine release. Solutions used for generating the calibration curves were prepared by serial dilution of the stock solution.…”

“…For the detection of 4HPAA, a triangular waveform was used in which potential (vs Ag/AgCl electrode) was linearly scanned from −0.4 V to +1.3 V to −0.4 V. The potential between scans was held at −0.4 V. The scan rate was 600 V/s and the update rate was 10 Hz. 30 Electrochemical Measurement of pHP-Glutamate Photoactivation. A microliter reaction vessel (Figure 2) was fabricated with 1 mm plastic tubing (Cole Parmer, Vernon Hills, Illinois) that allowed a 3 μL volume of aqueous pHPglutamate solution to be held in place by surface tension.…”