Label-free chemiluminescent CRISPR/Cas12a biosensing strategy for the detection of nucleic acids and non-nucleic acids utilizing DNAzyme Supernova

CRISPR/Cas12a-based biosensors typically output fluorescent, colorimetric, and electrochemical signals, relying on reporters that suffer from low sensitivity, high cost, and low stability. Herein, we introduced a label-free chemiluminescent (CL) reporting system based on a DNAzyme known as "Supernova". Supernova catalyzes the translocation of phosphate from a 1,2-dioxetane substrate (specifically, CDP-Star) to its 5′-OH ends, thereby triggering a CL reaction that produces light. Upon activation by the target, Cas12a cleaves Supernova, rendering it inactive in reacting with CDP-Star. Without pre-amplification, this biosensor can detect synthetic DNA corresponding to the SARS-CoV-2 N gene and monkeypox virus (MPXV) F3L gene with detection limits (LOD) of 1.5 and 2.4 pM, respectively. Integration with pre-amplification further enables the detection of SARS-CoV-2 and MPXV genes down to 1 copy/μL in real samples. Furthermore, the application of this CL system was expanded to the detection of non-nucleic acids with the integration of ATP aptamers, resulting in improved sensitivity in ATP biosensing compared to fluorescent assays. Overall, this work represents the first development of a label-free CL Cas12a reporting system, demonstrating its enhanced performance and broad applicability in synthetic biology-enabled sensing technology.