A Portable RPA-CRISPR/Cas12a-Based Biosensing Platform for On-Site Detection of the Microcystin Synthetase E Gene in Lake Water

Abstract

Microcystins are potent cyanotoxins produced by toxigenic cyanobacteria during harmful algal blooms (HABs), posing risks to ecosystems and human health. In this study, we developed a portable RPA-CRISPR/Cas12a biosensing platform for the rapid, on-site detection of the microcystin synthetase E (mcyE) gene, a key biomarker for microcystin-producing strains. The developed RPA-CRISPR/Cas12a assays enable detection of the mcyE gene within 50 min, with either fluorescence or lateral flow assay readouts. The fluorescence readouts have an analytical detection limit of 48.4 copies/{micro}L and a dynamic range of 1.2 x 102 to 1.2 x 107 copies/{micro}L. To enable field deployment, a magnetic bead-based DNA extraction method was integrated, achieving extraction within 1 hour without centrifugation. The complete workflow demonstrated a method LOD of 8.4 x 102 cells/mL in spiked lake water. Applicability was validated using non-spiked environmental water samples collected from multiple HAB-affected lakes. Importantly, a systematic matrix effect assessment was conducted for the CRISPR sensing step, evaluating environmental variables such as pH, ions, nutrients, and natural organic matter. This study establishes a practical, sensitive, and selective detection tool for proactive HAB monitoring. The platforms simplicity, portability, and completeness, from sample pretreatment to signal readout, highlight its potential for real-world environmental biosensing applications.