Colorimetric Detection of Extracellular Hydrogen Peroxide Using an Integrated Microfluidic Device

It is well known that hydrogen peroxide (H₂O₂) is a signaling molecule essential for vital physiological reactions in mammalian cells, such as cell survival, intercellular communication, and cancer metabolism. However, to fully understand the function of H₂O₂, it is critical to monitor its intracellular and/or extracellular concentrations. Current techniques implemented to address this need require large sample volumes, expensive instrumentation, and long sample preparation and analysis times, inapplicable to inline or online monitoring. In this paper, a new integrated microfluidic device capable of overcoming these limitations is demonstrated for the colorimetric detection of extracellular hydrogen peroxide H₂O₂. The device contains an optical waveguide to determine absorbance changes and micromixers to enable complete mixing of reagents using a passive approach. This novel H₂O₂-sensing device has allowed the detection of H₂O₂ in the range of 0.5-60 μM with a detection limit of 167 ± 5.8 nM and a sensitivity of 13.5 ± 0.1 AU/mM. Proof of concept of the device was demonstrated by quantifying H₂O₂ release from benign prostatic epithelial (BPH-1) cells upon stimulation with phorbol 12-myristate 13-acetate (PMA). Results show that this integrated device can be potentially utilized to continuously monitor cell-released metabolites autonomously without constant human supervision during the process. Furthermore, this can be achieved without interfering with the cell culture conditions, as only a very small volume of conditioned media (less than 0.4 μL), and not the cells, is required.