An integrated smartphone-based lateral flow assay utilizing enzyme-loaded nanoprobes for rapid and ultrasensitive on-site detection of microcystin-LR

초록

The increasing prevalence of harmful cyanobacterial blooms has intensified concerns over microcystin-LR (MC-LR) contamination in freshwater, creating an urgent demand for rapid and decentralized water quality monitoring. Although liquid chromatography-tandem mass spectrometry (LC-MS/MS) and enzyme-linked immunosorbent assay (ELISA) provide laboratory-grade sensitivity, these methods require complex instrumentation and trained personnel, which limits their applicability in field settings. Conventional lateral flow assays (LFAs) offer portability and ease of use but generally lack the sensitivity required for reliable detection of MC-LR at sub-regulatory concentrations. In this study, we present a user-friendly semiautomated paper-based lateral flow immunosensor engineered as a point-of-care testing (POCT) platform for on-site MC-LR screening. To address intrinsic sensitivity limitations, enzyme-loaded signal-amplifying nanoprobes (ELNPs) were engineered to enable catalytic signal amplification through an on-chip TMB reaction. The multilayer paper-based microfluidic device supported capillary-driven fluid flow and incorporated dedicated washing and substrate inlets, which allowed controlled enzymatic reactions while minimizing background interference. A custom 3D-printed smartphone reader was also constructed to enable standardized imaging and RGB-based quantitative analysis of the device output. The resulting platform detected MC-LR across a range of 85 to 400 pg/mL with a limit of detection of 85 pg/mL within 15 min, which is significantly lower than the guideline value established by the World Health Organization (WHO) for drinking water. Moreover, the system demonstrated high selectivity and strong agreement with LC-MS/MS when applied to real freshwater samples. Overall, this integrated platform provides a practical and portable POCT solution for rapid on-site water quality monitoring.

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