Direct fluorescence anisotropy approach for aflatoxin B1 detection and affinity binding study by using single tetramethylrhodamine labeled aptamer.

The discovery of aptamers for aflatoxin B1 (AFB1), one of toxic carcinogens, has allowed to develop aptamer-based sensors and assays for aflatoxin. In this work, we reported a direct fluorescence anisotropy (FA) assay for investigation of aptamer-AFB1 binding and detection of AFB1 with the aptamer having single tetramethylrhodamine (TMR) label on a specific site. From a series of labeling sites of a 50-mer aptamer, we screened out the aptamer with TMR labeling at the 26th T, capable of generating good and large FA-decreasing response to AFB1. By using the T26-labeled 50-mer aptamer probe in FA analysis, we determined the affinity and selectivity of aptamer, and identified the crucial region of aptamer and optimum experimental conditions for strong binding. The aptamer could be further truncated to as short as 26 nucleotides in length, and this shorter aptamer possessed a simple stem-loop secondary structure and retained good binding affinity. Nucleotides in the loop region of the aptamer were conserved and important for affinity recognition. We achieved FA detection of AFB1 with a detection limit about 2 nM by using the TMR-labeled aptamer probe. The cross reactivity of aflatoxin B1, aflatoxin B2, aflatoxin M1, aflatoxin M2, aflatoxin G1, and aflatoxin G2 with aptamer were estimated to be 100%, 61%, 23%, 21%, 6.3%, 6.5%, respectively. The aptamer probe presented good selectivity over other mycotoxins and showed potential in complex sample analysis. This study of affinity binding between aptamer and aflatoxins will be helpful for developing other aptamer-based assays and sensors for aflatoxins.