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Sensitive CRISPR-Cas12a-Assisted Immunoassay for Small Molecule Detection in Homogeneous Solution
Fengxi Zhu1,2, Qiang Zhao1,2,3
1State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing, 100085, China.
Analytical Chemistry
|April 20, 2023
Summary
This study introduces a sensitive CRISPR-Cas12a immunoassay for detecting small molecules in solution. The method achieves picomolar detection levels for biotin, digoxin, and folic acid, offering a powerful tool for various applications.
Area of Science:
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Background:
- Sensitive detection of small molecules is vital for biomedical diagnosis, food safety, and environmental monitoring.
- Existing methods may have limitations in sensitivity or require complex sample preparation.
Purpose of the Study:
- To develop a sensitive CRISPR-Cas12a-assisted immunoassay for small molecule detection in homogeneous solution.
- To demonstrate the assay's efficacy for detecting clinically and environmentally relevant small molecules.
Main Methods:
- Utilized an active DNA (acDNA) probe modified with a small molecule as a competitor for antibody binding.
- Leveraged CRISPR-Cas12a's collateral cleavage activity, activated by antibody-free acDNA.
- Employed a steric hindrance mechanism where antibody binding to acDNA inactivates CRISPR-Cas12a activity.
Main Results:
- Achieved sensitive detection of small molecules, including biotin, digoxin, and folic acid, at picomolar levels.
- Demonstrated successful application using streptavidin or antibodies as recognition elements.
- The assay functions in a homogeneous solution, simplifying the detection process.
Conclusions:
- The proposed CRISPR-Cas12a-assisted immunoassay offers a sensitive and versatile platform for small molecule detection.
- This strategy provides a powerful toolbox for expanding applications in diagnostics, food safety, and environmental analysis.
- Further development of DNA-encoded small molecules and antibodies can enhance the assay's capabilities.
