Ultrasensitive Wash-Free Homogeneous CRISPR Assay Using Spatial Proximity Chemiluminescence Reporter

CRISPR-based diagnostics are promising platforms for point-of-care (POC) testing, but are often hindered by limited sensitivity and complex workflows. Here, we present a spatial proximity chemiluminescence (SPC) reporter that converts CRISPR-Cas12a trans-cleavage activity into a highly sensitive, excitation-free signal. Mechanistically, the intact SPC reporter ensures efficient intramolecular reactive oxygen species (ROS) transfer by spatially confining a catalytic donor and a luminescent acceptor. Upon target-activated Cas12a cleavage, this spatial proximity is disrupted, drastically attenuating ROS transfer and reducing oxidized luminescent acceptors for chemiluminescence. The SPC-CRISPR exhibits an attomolar-level limit of detection with an optimal nearly 50,000-fold sensitivity enhancement, and features an 8-log dynamic range suitable for target quantification. This platform exhibits robust resistance to matrix interference, ensuring high accuracy while requiring only minimal sample preprocessing. We demonstrate SPC-CRISPR is compatible with recombinase polymerase amplification to develop a single-tube reaction with a sensitivity of 1 copy/μL. Extensive clinical validation demonstrated 99.2% overall accuracy for HPV16 identification in 126 cervical swabs, alongside 86.4% accuracy for miR-19a profiling in 22 bladder cancer plasma samples. Furthermore, this wash-free homogeneous workflow is embedded in a portable and sealed microfluidic-based device for sample-to-result diagnostics, showing 100% concordance with qPCR. SPC-CRISPR integrates enhanced sensitivity and simplified operation, holding great potential for POC molecular diagnostics.