Glycine/PVP-Enabled One-Pot CRISPR/Cas13a Detection of Vibrio parahaemolyticus by Tetrahedron-Mediated Electrochemistry

Vibrio parahaemolyticus is a major foodborne pathogen widely distributed in aquatic environments and seafood supply chains, necessitating rapid and ultrasensitive detection strategies adaptable to diverse testing scenarios. Here, we present a glycine/ PVP- and tetrahedron-integrated one-pot CRISPR sensing platform (termed GPT-CRISPR) for robust and ultrasensitive nucleic acid detection. The platform introduces chemical regulation into a multienzyme one-pot RAA−CRISPR/Cas13a network, where glycine may help reduce nonspecific Cas13a background activity, possibly through weak competitive interactions, while polyvinylpyrrolidone (PVP) may enhance reaction compatibility through macromolecular crowding and spatial shielding. This coordinated microenvironment enables stable amplification and CRISPR activation within a single closed vessel while minimizing background interference. Upon target recognition, activated Cas13a cleaves uracil-containing, surface-immobilized DNA tetrahedra, translating molecular recognition into amplified electrochemical signals. This transduction strategy enables quantitative detection with a linear dynamic range of 1.5 to 3 × 103 copies μL−1 and a limit of detection of 0.38 copies μL−1. The same chemically regulated one-pot CRISPR framework remains compatible with fluorescence and lateral flow readouts. The assay operates under isothermal conditions and delivers results within 30 min without complex sample preparation. Validation across real-world samples demonstrates robustness in complex matrices. Collectively, GPT-CRISPR integrates chemical stabilization of a one-pot CRISPR framework with electrochemical transduction, defining a robust sensing architecture with adaptable readout capability.