Biphasic spatiotemporal regulation of Cas12a substrate cleavage enables one-pot autocatalytic CRISPR biosensing of non-nucleic-acid targets.
One-pot autocatalytic CRISPR biosensing offers a promising route for signal amplification without nucleic acid pre-amplification, but its efficiency is limited by an intrinsic readout-amplification conflict: the ssDNA reporter required for signal output competes with the autocatalytic mediator required for feedback amplification. Here, we report a biphasic spatiotemporal regulation strategy (BS-Cas12a system) to resolve this substrate competition for one-pot autocatalytic CRISPR biosensing of non-nucleic-acid targets. Mechanistic studies revealed that Cas12a preferentially cleaved the ssDNA reporter over the autocatalytic circular mediator, leading to insufficient mediator linearization and impaired autocatalytic amplification. A glycerol/water biphasic system was therefore constructed to spatially delay reporter access to activated Cas12a, allowing preferential cleavage of the autocatalytic mediator and subsequent generation of additional Cas12a activators. This biphasic system achieved a 3-fold increase in the autocatalytic amplification efficiency. By integrating an aptamer-mediated target-to-activator conversion module, non-nucleic-acid recognition was programmably converted into Cas12a activation. Using sulfadimethoxine as a model target, the platform achieved a detection limit of 65 pM and showed good recoveries in fish samples. The system was further extended to Cd2+ and thrombin detection with limits of detection of 0.65 nM and 11.63 pM, respectively. Moreover, by replacing the fluorescent reporter with a FAM/biotin-labeled reporter, a lateral-flow readout was achieved. This work provides a kinetic-regulation strategy for modular one-pot Cir DNA autocatalytic biosensing with an independent and exchangeable reporter and expands CRISPR-based detection toward diverse non-nucleic-acid targets.