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CRISPR/Cas12a-Powered Competitive Immunosorbent Assay for Small Molecules.

Fengxi Zhu^1,2, Qiang Zhao^1,2,3

¹State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China.

Analytical Chemistry

|September 18, 2023

Summary

This study introduces a CRISPR/Cas12a-powered assay for sensitive small-molecule detection. The method uses antibodies and DNA probes to quantify targets by monitoring fluorescence changes, enhancing immunoassay performance.

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Area of Science:

Biotechnology
Molecular Diagnostics
Biochemistry

Background:

CRISPR/Cas systems offer precise nucleic acid recognition and trans-cleavage activity for molecular diagnostics.
Existing methods often require converting non-nucleic acid targets into nucleic acid signals for detection.

Purpose of the Study:

To develop a sensitive CRISPR/Cas12a-powered immunosorbent assay for small-molecule detection.
To leverage CRISPR/Cas12a's catalytic activity for quantifying non-nucleic acid targets.

Main Methods:

Utilized antibody-coated microplates to capture small molecule-labeled active DNA (acDNA) probes.
Employed CRISPR/Cas12a to trigger catalytic trans-cleavage of fluorescent DNA reporters, generating a signal.
Small molecule targets displace acDNA probes, reducing Cas12a activation and fluorescence signal for detection.

Main Results:

Successfully detected three model small molecules (biotin, digoxin, folic acid) with low detection limits.
Demonstrated high selectivity and applicability in complex biological media.
Achieved a flexible detection range by adjusting acDNA probe and antibody amounts.

Conclusions:

The CRISPR/Cas12a immunoassay provides a sensitive, rapid, and simple method for small-molecule analysis.
Integration of CRISPR/Cas12a significantly enhances immunoassay analytical performance.
The assay broadens applications for small molecule detection in various fields.