Influence of Medium Composition and Electrode-Relevant Metal Ions on the Amplex Red Fluorescence Readout During High-Voltage Electric Pulse Treatment

Amplex Red is a widely used fluorogenic probe for hydrogen peroxide (H2O2) detection. In the presence of horseradish peroxidase (HRP), it reacts with H2O2 to form fluorescent resorufin. However, during high-voltage electric pulse treatment, fluorescence readouts may be influenced not only by H2O2 generation but also by the medium composition, pulse conditions, pH-dependent effects, and metal ions released as a result of electrode corrosion. In this study, we examined how medium composition and selected metal ions relevant to electroporation conditions, such as Fe2+, Fe3+, Al3+, and Cr6+, affect the Amplex Red/HRP/H2O2 fluorescence signal. Fluorescence intensity differed markedly among media, with the highest signal in phosphate-buffered saline (PBS, ~34,000 a.u.), followed by HB1 buffer (~19,000 a.u.), cell culture medium (~9000 a.u.), and distilled water (~4100 a.u.). To model ion-mediated interference, metal ions were added directly to the assay mixture. All tested ions reduced fluorescence in a concentration-dependent manner. At 0.4 µM H2O2, Fe2+ reduced fluorescence by up to 92%, Fe3+ by 60%, Al3+ by 35%, and Cr6+ by 40%. At 10 µM H2O2, the quenching effect was reduced but remained evident. In addition, the fluorescence readout showed strong dependence on medium composition and its pH, further demonstrating its vulnerability to the chemical environment. These findings show that the Amplex Red assay is highly sensitive to the assay environment and metal-ion interference. Therefore, it should not be used as a standalone, interference-free measure of H2O2 in electroporation-related experiments without appropriate validation controls.