AND Logic-Gate-Based CRISPR/Cas12a Biosensing Platform for the Sensitive Colorimetric Detection of Dual miRNAs

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) system has been widely explored for the detection of disease-related nucleic acids. Nevertheless, the simultaneous detection of multiple nucleic acids within one assay using the CRISPR-Cas system is still chal...

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Bibliographic Details
Published in:Analytical chemistry (Washington) 2022-11, Vol.94 (45), p.15839-15846
Main Authors: Gong, Shaohua, Wang, Xi, Zhou, Ping, Pan, Wei, Li, Na, Tang, Bo
Format: Article
Language:English
Subjects:
Biomarkers
Biosensing Techniques - methods
Biosensors
Chemistry
Colorimetry
CRISPR
CRISPR-Cas Systems - genetics
DNA, Single-Stranded
Glucose oxidase
Glucose separation
Humans
Logic
Logic circuits
Lung cancer
MicroRNAs - genetics
Nucleic Acid Amplification Techniques - methods
Nucleic acids
Signal generation
Single-stranded DNA
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Summary:Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated) system has been widely explored for the detection of disease-related nucleic acids. Nevertheless, the simultaneous detection of multiple nucleic acids within one assay using the CRISPR-Cas system is still challenging. In this study, we develop an AND logic-gate-based CRISPR-Cas12a biosensing platform to achieve the sensitive colorimetric detection of dual miRNAs. Specifically, the DNA probe was designed to recognize the binary input of miRNAs and to output trigger DNA, which activated the CRISPR-Cas12a system to cut single-stranded DNA (ssDNA). The ssDNA on magnetic beads (MBs) was cleaved by the activated CRISPR-Cas12a, causing the separation of glucose oxidase (GOx) from MB and the subsequent generation of a colorimetric signal. The color change induced by 1 pM of target miRNAs can be directly distinguished by the naked eye and the instrumental limit of detection reaches 36.4 fM. The overexpressed miR-205 and miR-944 in the real human serum can be detected, allowing us to differentiate between lung cancer patients and healthy people. Furthermore, the developed strategy achieves simultaneous detection of dual miRNAs using CRISPR-Cas12a with one kind of crRNA, avoiding sophisticated nucleic acid amplifications and the use of bulky instruments. The current method can broaden the CRISPR-Cas12a-based applications for multiple biomarkers detection and precise disease diagnosis.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.2c03666