Rigidity-Responsive Fluorescence Polarization Detection of Aflatoxin B₁ via Programmable RCA-Coupled CRISPR/Cas12a...

Area of Science:

• Analytical Chemistry
• Biosensing Technology
• Food Safety

Background:

• Aflatoxin B1 (AFB1) is a highly toxic and carcinogenic mycotoxin.
• Accurate detection of AFB1 in food is challenging due to complex matrices and limitations of current methods like chromatography and fluorescence-based CRISPR biosensors.

Purpose of the Study:

• To develop a robust and matrix-tolerant biosensing strategy for trace-level determination of AFB1.
• To overcome the limitations of existing AFB1 detection methods, particularly background interference and signal instability.

Main Methods:

• Integration of aptamer recognition, rolling circle amplification (RCA), and CRISPR/Cas12a trans-cleavage.
• Utilized a rigidity-responsive fluorescence polarization (FP) readout with a conformation-restricted depolarization reporter (CRD-Reporter).
• Cas12a activation leads to cleavage of the CRD-Reporter, causing a decrease in FP signal.

Main Results:

• The proposed FP-based CRISPR assay demonstrated high sensitivity with a detection limit of 0.00113 ng/mL.
• Achieved a wide linear range (0.003–300 ng/mL) and high specificity.
• Showed excellent accuracy and reliability in analyzing real food samples like grains, peanuts, and tea.

Conclusions:

• Established a robust FP-based CRISPR sensing paradigm for reliable mycotoxin monitoring.
• The developed method offers a promising alternative for sensitive and accurate AFB1 detection in complex food systems.
• This approach effectively suppresses background interference and signal fluctuations, enhancing reliability.