A 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 a 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 (CRISAT) for the first time for the detection of ultrasensitive nucleic acids and non-nucleic acids developed by harnessing CRISPR-ALP tandem assay. In the presence of a target, 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, which 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 the naked eye or detected using a fluorescent device in the same solution. Using this strategy, a fluorescence and colorimetry dual-readout nanoplatform for CRISPR-based biosensors can be rationally developed. We ascertain the applicability of CRISAT by detecting the SARS-CoV-2 pseudovirus, achieving superior sensitivity and specificity. With simple modification of crRNAs, the CRISAT platform can also be employed to detect monkeypox virus (MPXV) and non-nucleic acids of adenosine triphosphate (ATP). This work shows great potential for the detection of nucleic acids and non-nucleic acids.

CRISPR/Cas12a-Enabled Multiplex Biosensing Strategy Via an Affordable and Visual Nylon Membrane Readout.

Most multiplex nucleic acids detection methods require numerous reagents and high-priced instruments. The emerging clustered regularly interspaced short palindromic repeats (CRISPR)/Cas has been regarded as a promising point-of-care (POC) strategy for nucleic acids detection. However, how to achieve CRISPR/Cas multiplex biosensing remains a challenge. Here, an affordable means termed CRISPR-RDB (CRISPR-based reverse dot blot) for multiplex target detection in parallel, which possesses the advantages of high sensitivity and specificity, cost-effectiveness, instrument-free, ease to use, and visualization is reported. CRISPR-RDB integrates the trans-cleavage activity of CRISPR-Cas12a with a commercial RDB technique. It utilizes different Cas12a-crRNA complexes to separately identify multiple targets in one sample and converts targeted information into colorimetric signals on a piece of accessible nylon membrane that attaches corresponding specific-oligonucleotide probes. It has demonstrated that the versatility of CRISPR-RDB by constructing a four-channel system to simultaneously detect influenza A, influenza B, respiratory syncytial virus, and SARS-CoV-2. With a simple modification of crRNAs, the CRISPR-RDB can be modified to detect human papillomavirus, saving two-thirds of the time compared to a commercial PCR-RDB kit. Further, a user-friendly microchip system for convenient use, as well as a smartphone app for signal interpretation, is engineered. CRISPR-RDB represents a desirable option for multiplexed biosensing and on-site diagnosis.