The Study On The Detection Methods For Foodborne Pathogens Based On Aptamer-molecular Motor Sensor

Foodborne diseases in developed countries and developing countries are a common and serious public health problem and they are serious threats to human safety.Microbial pathogens is the main cause,and bacteria accounted for the main part of that.Traditional testing methods of detection procedure was complicated time-consuming which not only increase the workload but also be harmful to quality control in the food industry production and government departments.So the reliability of detection results is not good.Traditional testing methods also have limitations in the detecting speed,sensitivity and specificity.So developing the more fast,more convenient,higher sensitivity and stronger specificity of detection methods for food safety is of great significance.Molecular motor is the biological macromolecules which can convert chemical energy to mechanical energy.They widely exist in cells and have obtained the widespread attention at home and abroad in recent years.The biological rotated molecular motor F₀F₁-ATPase was repeatedly used in the detection of foodborne pathogenic bacteria.Using F₀F₁-ATPase as a biosensor for foodborne detection has high sensitivity and strong specificity.This study first uses the aptamer as specific recognition probe to conjugate with F₀F₁-ATPase and constitute the aptamer-based molecular motor.The aptamer biosensor is successfully applied to detecting the S.typhimurium and V.parahaemolyticus.The main work of this study is as follows:Firstly,a novel biosensor was developed by conjugating aptamer with the‘‘rotator''?-subunitofF₀F₁-ATPasewithinchromatophoreswithananti-?-subunit antibody-biotin-avidin-biotin-aptamer linker to capture S.typhimurium.The bacterial load decreased the rotation rate of F₀F₁-ATPase,which led to the decrease in ATP synthesis.The detection of S.typhimurium was based on proton flux change driven by the F₀F₁-ATPase-mediated ATP-synthesis,which was indicated by F-DHPE,monitored by a fluorescence spectrometer.The correlation between the fluorescence signal and the concentration of S.typhimurium was observed to be linear within the range of 10¹ to 10⁴cfu/m L?y=1085.13x+363.98,R²=0.9944?.The limit detection for the developed method was10 cfu/m L for S.typhimurium.The method has been successfully applied in actual milk sample testing.Secondly,a novel aptasensor with quantum dots as fluorescent indicator was constructed.The p H sensitive quantum dots were tab to the outside of the chromatophore membrane like the previous studies.Conjugate anti-?-subunit antibody-biotin-avidin-biotin-aptamer with F₀F₁-ATPase within chromatophores to capture V.parahaemolyticus.When the bacteria exists,the rotation of the ATP synthase fall which causes the H⁺concentration outside the membrane decreases,then the p H value increases and the fluorescence of quantum dots increased at last.The correlation between the fluorescence signal and the concentration of Vibrio parahaemolyticus was observed to be linear within the range of 50 to 10⁶ cfu/m L?y=138.3x+13.05,R²=0.9962?.The limit detection for the developed method was 5 cfu/m L for V.parahaemolyticus.The proposed method has been successfully applied in actual salmon samples.