Fluorescence-Enabled Portable Droplet Microfluidic Paper-based Analytical Device for Rapid and Sensitive Biotoxicity Assay

There is an increasing campaign for advancing analytical strategies that require the least sample and reagents while maintaining enhanced sensitivity and selectivity. Paper-based microfluidic analytical devices (µPADs) have revolutionized the assays in various fields such as environment, toxicology, drug testing, and point-of-care infection diagnosis. This study proposes a simple strategy for integrating fluorescence assays into µPADs to take advantage of the unique benefits of fluorescence spectroscopy. The µPADs were designed to entrap sample droplets within the reaction zones coated with viability probe compound, resazurin (RZ), using impressed hydrophobic boundaries. The change of fluorescence induced by the metabolic reduction of RZ to resorufin (RS) by Enterococcus faecalis (E. faecalis) was detected using a microplate reader and a new handcrafted black box equipped with fluorescence light method (SBFL). The innovative µPAD-fluorescence techniques afford swift analysis of different densities of E. faecalis, as well as the biotoxicity of chlorophenols (pentachlorophenol, 2,4-dichlorophenol, and 4-chlorophenol) and heavy metal ions (Pb^2 +, Cu²⁺, and Zn²⁺). The assays could be completed in a few minutes, providing a significant time advantage over the traditional methods. The reliability of the data was statistically confirmed. The findings reveal that hyphenating RZ-coated µPADs with fluorescence techniques advances sensitivity towards bacteria and bio-toxicant assays. © 2025 Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.