CRISPR/Cas12a-powered tri-mode aptasensor for ultrasensitive and multiplexed detection of microcystin-LR

Exploiting CRISPR/Cas12a-powered multiple-mode sensing platforms is urgently needed to meet flexible and multi-scenario analytical requirements while improving detection accuracy for target pollutants. Herein, we developed a novel CRISPR/Cas12a-powered tri-mode aptasensor for ultrasensitive and multiplexed detection of microcystin-LR (MC-LR) in environmental water. First, target-specific recognition and the subsequent release of activator DNA were achieved through an aptamer-based competitive displacement reaction. Then, the trans-cleavage activity of Cas12a was activated in the presence of MC-LR, and a fluorescence-quenched ssDNA (FAM-ssDNA-BHQ1) and a dual -functionalized probe (MBs-ssDNA-AuNPs-4MBA) as signal reporters were employed for the tri-signal readouts. In the fluorescence mode, an ultrasensitive digital droplet fluorescence system was employed. The released activator DNA and Cas12a/crRNA complex were co-encapsulated into monodisperse nanoliter droplets, which not only accelerated the reaction but also amplified local fluorescence signals. This approach enabled rapid detection of MC-LR within 35 min, with a detection limit as low as 1.0 aM. For SERS and colorimetric modes, a novel dual-functionalized reporter was designed to provide both Raman and colorimetric readouts following magnetic separation. This extended the dynamic detection range from 1.0 × 10^-13 M to 1.0 × 10^-6 M. The proposed tri-mode aptasensor, using a single workflow, addresses both trace-level warning and high-concentration detection of MC-LR. It demonstrated good analytical performance in real samples, with recoveries ranging from 81.26% to 113.21% (n = 3). This CRISPR/Cas12a-driven strategy thus provides a versatile tool for reliable MC-LR monitoring across diverse water scenarios.