CRISPR/Cas12a-Amplified Aptamer Switch Microplate Assay for Small Molecules

Area of Science:

• Analytical Chemistry
• Biotechnology
• Environmental Science

Background:

• Small molecule contaminants (mycotoxins, heavy metals) pose significant health and economic risks.
• Existing detection methods often lack simplicity, speed, sensitivity, or on-site capabilities.
• There is a critical need for advanced analytical tools for rapid pollutant identification.

Purpose of the Study:

• To develop a sensitive, rapid, and on-site method for detecting small molecule pollutants.
• To leverage aptamer-based signal conversion and CRISPR/Cas12a amplification for enhanced detection.
• To demonstrate the assay's utility for real-world sample analysis.

Main Methods:

• Developed a CRISPR/Cas12a-amplified aptamer switch assay on a microplate.
• Utilized structure-switchable aptamers for target-specific signal generation.
• Employed CRISPR/Cas12a's collateral activity for signal amplification via fluorescent DNA substrate cleavage.
• Immobilized complementary DNA (cDNA) on a microplate to capture aptamer-linked DNA probes (Apt-acDNA).

Main Results:

• Achieved sensitive detection of aflatoxin B1 (AFB1) at 31 pM and cadmium ion (Cd2+) at 3.9 nM.
• Demonstrated good selectivity and high sensitivity for target analytes.
• Validated the assay's performance in actual sample matrices.
• The assay showed a signal reduction in the presence of the target molecule.

Conclusions:

• The developed CRISPR/Cas12a-amplified aptamer switch assay offers a sensitive and rapid platform for small molecule detection.
• The generalizable strategy allows for adaptation to various targets by modifying aptamers and cDNA.
• This method holds significant potential for broad applications in food safety and environmental monitoring.