A competitive thrombin-linked aptamer assay for small molecule: aflatoxin B₁

Area of Science:

• Biochemistry
• Analytical Chemistry
• Molecular Biology

Background:

• Aflatoxin B1 (AFB1) is a potent mycotoxin with significant health implications.
• Accurate and sensitive detection methods for AFB1 are crucial for food safety and public health.
• Existing detection methods may have limitations in sensitivity, selectivity, or ease of use.

Purpose of the Study:

• To develop a novel competitive thrombin-linked aptamer assay (TLAA) for the sensitive detection of small molecules.
• To utilize the enzymatic activity of thrombin and aptamer affinity binding for signal generation.
• To establish a versatile platform for detecting mycotoxins like AFB1 in complex matrices.

Main Methods:

• Design of a dual-functional DNA probe incorporating aptamers for both thrombin and AFB1.
• Immobilization of thrombin-labeled DNA probes onto a microplate via AFB1-bovine serum albumin (BSA-AFB1) conjugate.
• Enzymatic cleavage of a peptide substrate by immobilized thrombin to generate a detectable signal.
• Competitive binding assay where free AFB1 competes with immobilized BSA-AFB1 for the DNA probe.

Main Results:

• The TLAA successfully detected AFB1 in a concentration range of 0.5 nM to 1 μM using a fluorogenic substrate and fluorescence analysis.
• A chromogenic substrate allowed AFB1 detection from 0.5 to 125 nM via simple absorbance measurements.
• The assay demonstrated good selectivity for AFB1 and was effective in detecting AFB1 spiked in diluted beer and corn flour samples.

Conclusions:

• The developed thrombin-linked aptamer assay provides a sensitive and selective method for small molecule detection.
• This TLAA strategy offers a novel application for thrombin and aptamers in analytical chemistry.
• The assay's adaptability to different detection substrates and matrices highlights its potential for real-world applications in food safety and diagnostics.