Development Of Aptasensors For Rapid Detection Of Tetracycline Residue In Honey

Tetracycline is widely used for animal feeding, because of its low cost and broad spectrum anti-bacterial activity. However, the resulted tetracycline residue in animal-derived food products exposes customers to serious health risks and also impedes development of food industry. It is urgent to develop novel detection methods which have high specificity and sensitivity for preliminary screening and detection of tetracycline.Biosensors combine recognition elements and transduction elements,transducing the specific recognition into quantitative signals, thus can achieve rapid detection. Aptamers are generated in vitro by Systematic Evolution of Ligands by EXponential enrichment (SELEX), and are known as "chemical antibodies" with high affinity and specificity towards their targets. Aptamers can be easily modified to be combined with various transducers, and are good alternatives as recognition elements in biosensors for food safey detection.In this thesis, we focus on the detection of tetracycline in honey and developed four different aptasensors, enzyme-linked aptasenor(ELAA-aptasensor), AuNPs-based aptasensor (AuNPs-aptanseor), surface plasmon resonance-based aptasensor (SPR-aptasensor), and electrochemistry-based aptasensor (EC-aptasensor). All of the four aptasensors can be used for tetracycline detection in real samples after simple pretreatment. Meanwhile, molecule docking package was combined with chemical experiments to explore the intrinsic molecular recognition mechanism between aptamers and tetracycline molecule.Moreover, the "applicability" of aptamers in different systems was deciphered, which can conversely assist the development of aptasensors.The main results are listed as following:1. All the four different biosensors were available to be used to detect tetracycline in honey after simple sample pretreatment, and provided good recoveries at 80?120%, high accuracy, strong anti-interference abi-lity, and high specificity.2. An indirect competitive and a direct competitive ELAA-aptasen-sors were developed simultaneously for high throughput detection of tetracycline. Streptavidin-biotin system was introduced for molecule immobilization and signal amplification. With the optimized conditions,the indirect competitive ELAA-aptasensor provided a linear range of 0.01?100 ng·mL-1 and a detection limit at 0.00966 ng·mL-1. The direct competitive ELAA-aptasensor provided a linear range of 0.1?1000 ng·mL-1 and a detection limit at 0.0978 ng·mL-1. ELISA was then performed for validation. Both the two ELAA-aptasensors and the ELISA obtained comparable revovery rates, and the ELAA-aptasensors showed higher sensitivity than the ELISA.3. Inspired by significantly different performance of two aptamers,Apt76 and Apt40, in the development of ELAA-aptansors, we combined molecular docking package with ITC titration study to explore the intrinsic molecular recognition mechanism. Two scientific points were concluded: (1) Apt76 and Apt40 fold into different conformational structures, contain several binding sites for tetracycline, and bind with tetracycline via "molecular pockets". The complexity of aptamer conformational structures and number of binding sites are closely related to strand length, base composition, and stem-loop structures in secondary structure. Apt76 has more binding sites than Apt40; (2) Different conformational structures and different distribution of binding sites affect the availability of aptamers in detection system, and thus aptamers have"applicability" feature in different systems. Apt76 has advantages in designs which need chemical modification, such as the ELAA, while Apt40 is more preferable to be used in label-free biosensors.4. Based on the molecular recognition study, Apt40 was then used to develop a label-free AuNPs-aptasensor for visual detection. The protective effect of Apt40 on AuNPs was utilized and the interaction among Apt40, AuNPs, and tetracycline was thoroughly studied, in terms of the strand length, base composition, secondary structure, and tertiary structure. Both visual semi-quantitative detection and precise quantitation using a UV-vis spectrometer were performed. The whole analysis could be completed within 30 min. With the optimized conditions, the signal responses present a good linear relationship with the logarithm of tetracycline concentration in the range of 20?320 ng·mL-1, and the detection limit of the AuNPs-aptasensor was calculated to be 12.4 ng·mL-1.5. As Apt76 was more preferable in designs which need chemical modification, Apt76 was labeled with biotin and immobilized on the SPR chip incoprated in Biacore T200 to achieve automatic screening.Streptavidin was immobilized on the surface of CM5 chip via covalent coupling firstly, and the biotin-labeled Apt76 was then immobilized through the specific interaction between strepavidin and biotin.Tetracycline was captured directly when flowing across the surface of CM5 chip and SPR signal was generated at the same time. SPR signals were proportional to the concentration of tetracycline in the range of 0.01?100 ng·mL-1, and the detection limit of the SPR-aptasensor was 0.183 ng·mL-1.6. Similarily, thiol-labeled Apt76 was immobilized on the gold surface of screen printed electrodes to achieve highly sensitive detection with low reagent cost. Single strand Apt76 was firstly used to develop EC-aptasesor; however, the detection signals were not stable due to the entanglement of single strands on the surface of electrode. A DNA tetrahedron structure was then assembled for oriented immobilization of Apt76 to ensure the adaptive folding of Apt76. With the tetrahedron structure, linear detection range of the EC-aptasenor was extended to be 0.01?1000 ng·mL-1 from 0.1?100 ng·mL-1, and detection limit was lowered to be 0.00947 ng-mL-1from 0.0875 ng·mL-1.