Purpose: In this study, we developed a CRET-based biosensor using single strand DNA (aptamer) to detect an OTA in coffee without dilution.
Methods: The CRET was optimized with appropriate concentrations of aptamer toward OTA and hemin, volume of coffee extracts undiluted. To validate the CRET system, OTA-positive coffee samples were prepared by spiking known concentration of OTA at 0.5 to 50000 μg/kg. The coffee samples were extracted with absolute methanol, and the extracts were directly tested by the CRET. Chemiluminescence was immediately measured by Chemi-Doc image (Bio-rad) and analyzed by chemi-doc analyzing program.
Results: As a result, the optimized CRET system could detect 0.005 ng/ml, and cross reaction to other mycotoxins was not observed. Total assay time of the CRET system to detect OTA was in 10 min, more rapid than that of analytical methods previously reported. The optimum concentration of aptamer was 100nM for detecting the chemiluminescence signal in coffee by Chemi-Doc image. Optimal volume of the coffee extracts and aptamer solution was used 4:6 ratio for OTA analysis. Which may be used methanol concentration is important to detect the OTA. Because of extraction method of OTA from real sample had contained methanol and it may used dilution step. In our system removed the dilution step for detection of OTA. As a result, more than 50 ppb of OTA had quenching efficiency of over 20% and less than 0.5 ppb of OTA had had quenching efficiency of over 10%. The over the limit of OTA is 5 ppb and our system can be detect 0.5 ppb in coffee without dilution.
Significance: As a result, we could detected the OTA using quenching or not of chemiluminescence without specific equipment. To detect the chemiluminescence signal just need to the simple image equipment such as CCD camera, CMOS chip and camera of smartphone.