Development Of Label-free Aptasensors For The Rapid Detection Of Tetracycline And Dopamine Residues In Animal Derived Food

At present, veterinary drug residues in animal derived food has become a threat to human health and food safety. Common detection methods are usually expensive, time-consuming and in need of complicated procedures, which limit their applications for onsite and rapid detection. Aptamers can bind with target and often undergo significant conformational changes. With the distinctive properties, including costeffectiveness, high affinity and specificity, aptamers have been widely employed to develop biosensors and used in food analysis field. Aptasensors can be mainly divided into two modes: labeled and label-free aptasensors. The label is usually timeconsuming, labor-intensive and expensive. Moreover, the label to aptamers will affect the binding affinity and selectivity of aptamers. Herein, two kinds of lable-free aptasensors have been developed based on the specific binding between aptamers and targets using dyes as signal probes. The developed methods possess the merits of rapid and convenient detection procedure, cost-effectiveness, high sensitivity and excellent selectivity. Owing to these advantages, the established aptasensors can be applied to detect other harmful substances in food, and have a promosing prosepect in agriculture and environment fields. The research contents are summarized as following:1. A label-free aptasensor based on tetracycline-binding aptamers and thiazole orange(TO) was established for the selective and sensitive detection of tetracycline(TET). TO is essentially nonfluorescent in aqueous solution, while TET-binding aptamers were G-quadruplexes structure. In the absence of TET, TO could intercalate into the G-quadruplex structure, causing a fluorescence emission. In the presence of TET, the specific recognition between the aptamers and TET could undergo significant conformational changes, leading to the separation of TO. Under the optimum conditions, the fluorescence intensity of TO could decrease along with the target TET concentration ranging from 0.05- 1000 μg/m L with the detection limit of 0.022 μg/m L. The developed aptasensor has been successfully applied to detect TET in raw milk, with the recoveries between 89.0 %- 107.9 % and relative standard deviation(RSD) between 0.6 %- 1.4 %. Moreover, the developed aptasensor avoids the procedures of modifying or labeling aptamers, which could detect TET with the advangates of convenient procedure, high sensitivity and excellent selectivity. Based on this strategy, TET detection can be realized in a simple and rapid approach.2. A label-free aptamer-based sensing strategy was developed for dopamine through the fluorescence resonance energy transfer(FRET) between rhodamine B(RB) and gold nanoparticles(Au NPs). The different affinities of unbound and bound aptamers toward Au NPs were employed to modulate the FRET efficiency for signal transfer. Dopamine-binding aptamers could protect Au NPs from salt-induced aggregation, resulting in the fluorescence quenching of RB via FRET. Specific binding of aptamers with dopamine could cause the conformation changes of aptamers, losing the capability to stabilize Au NPs, thus the salt-induced aggregation of Au NPs could lead to the decrease of fluorescence quenching ability. Correspondingly, the fluorescence intensity of RB would recover along with the dopamine concentration ranging from 26 n M to 2.90 × 103 n M with the detection limit of 2 n M. This method was successfully applied for dopamine determination in swine feeds and chicken livers, with the recoveries between 91.4 %- 103.5 % and RSD between 0.9 %- 2.3 %. Owing to its convenient procedure, high sensitivity and excellent selectivity, this strategy will provide a promising alternative for the screening of veterinary drug residues and other harmful small molecues in food.