Label-free Fluorescent Aptasensor Based on Exonuclease-assisted Target Recycling Strategy for Sensitive Detection of Oxytetracycline

A label-free fluorescence detection method was developed for quantitative determination of oxytetracycline (OTC) based on aptamer recognition capability and exonuclease I (Exo I) -assisted target recycling. In the absence of OTC , SYBR green I (SGI) molecules could insert the double-stranded DNA ( dsDNA ) of OTC aptamer and its complementary DNA , resulting in strong fluorescence emission of SGI with excitation wavelength of 495 nm. In the presence of OTC , due to the strong affinity of the aptamer to its target, aptamer preferentially bound with OTC and unwound the dsDNA. Thus, SGI molecules released from the hydrogen bonds of dsDNA, accompanied with the obvious decrease of SGI fluorescence. To amplify the fluorescence quenching effect of SGI caused by the addition of OTC , Exo I-assisted target recycling was involved in. The results of acrylamide gel electrophoresis confirmed that Exo I could digest single stranded DNA selectively and the aptamer bound with OTC. The released OTC participated in the next cycle, continuously destroyed the dsDNA structure, releasled the free SGI, and gradually weakened the fluorescence. Based on this Exo I-catalyzed target recycling strategy, the sensitivity of this aptamer-based fluorescent strategy was significantly improved. The linear range of OTC was 0. 01-10 mu g/mL with a detection limit (3 sigma) of 6. 77 ng/mL. The method realized the detection of OTC in milk and honey sample ssuccessfully. For milk samples, the recoveries were 93. 0% -105. 1% with relative standard deviations (RSD) of 0. 5% -6. 2% , and for honey samples, the recoveries were 94. 0% -95. 8% with RSD of 0. 3% -7. 7% , respectively. The fluorescence sensor had many advantages such as low cost, high sensitivity and good specificity, showing great potential in the rapid detection of residue of harmful substances in food.