One-tube dual-readout biosensor for detection of nucleic acids and non-nucleic acids using CRISPR-ALP tandem assay

Clustered regularly interspaced short palindromic repeats (CRISPR)-based diagnostics have been considered as the next generation molecular diagnosis tool. Single-readout mode has been extensively employed in massive CRISPR/Cas12a-based biosensors. In this work, we propose a one-tube dual-readout biosensor for ultrasensitive nucleic acids and non-nucleic acids detection by harnessing CRISPR-ALP tandem assay for the first time (termed CRISAT). In the presence of target, the Cas12a is activated to randomly cut the single-stranded hyDNA sequence of MB@hyDNA-cALP, thus releasing abundant alkaline phosphatase (ALP) in the supernatant solution. By using 4-aminophenol phosphate as the substrate of ALP, p-aminophenol is produced and then reacts with N-[3-(trimethoxysilyl)propyl]ethylenediamine or diethylenetriamine to generate silicon-containing polymer carbon dots (Si PCDs) or polymer carbon dots (PCDs) in situ, which can be observed by naked eye or detected by a fluorescent device in the same solution. Using this strategy, a fluoroscence and colorimetry dual-readout nanoplatform for CRISPR-based biosensor can be rationally developed. We ascertain the applicability of CRISAT through detecting SARS-CoV-2 pseudovirus, achieving superior sensitivity and specificity. With simple modification of crRNAs, the CRISAT platform can be also employed to detect monkeypox virus (MPXV) and non-nucleic acids of adenosine triphosphate (ATP). This work provides a great potential for nucleic acids and non-nucleic acids detection.