Issue 18, 2023

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

Abstract

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.

Graphical abstract: A one-tube dual-readout biosensor for detection of nucleic acids and non-nucleic acids using CRISPR-ALP tandem assay

Supplementary files

Article information

Article type
Paper
Submitted
06 มิ.ย. 2566
Accepted
25 ก.ค. 2566
First published
25 ก.ค. 2566

Analyst, 2023,148, 4356-4364

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

X. Ke, Y. Hu, C. Chen and T. Hu, Analyst, 2023, 148, 4356 DOI: 10.1039/D3AN00918A

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