Development Of Colorimetric And Photothermal Dual-mode Aptasensors For Shrimp Tropomyosin

Food allergy has attracted more attention due to the large number of people involved and the faster onset of symptoms.At present,there is no effective solutions for the treatment of allergic reaction in the world.If allergic population want to get rid of the potential threats,they can only avoid ingesting allergens.Therefore,it is of practical significance to establish more sensitive,rapid and portable allergen detection methods.Shrimp tropomyosin(TM)is a kind of crustacean allergen.It is popular because of its rich nutrition and fresh taste.At the same time,its structure is relatively stable,and its allergenicity is not easy to eliminate during processing.Therefore,shrimp was selected as the target to be tested in this study.Compared with directly extracting tropomyosin from prawns,this study used in vitro recombination and affinity chromatography to express and purify shrimp tropomyosin,which has the advantages of simple operation,high expression level,and no impurity pollution.Currently,methods for detecting shrimp tropomyosin mainly include ELISA,mass spectrometry,and PCR,etc.,which are complex in operation,high in cost and partly dependent on largescale instruments.Based on the above problems,in this study,aptamers with low synthesis cost and strong specificity were selected as target recognition element,combined with the principle that ss DNA can be adsorbed to the surface of gold nanoparticles(Au NPs)to enhance its peroxidase-like activity,using the oxidation product ox TMB,which both has color rendering and photothermal properties,and the introduction of colorimetric and temperature two signals that are more convenient to read out is used to construct the sensor,which realizes the high-sensitivity and rapid detection of shrimp tropomyosin,and the photothermal sensor also has the potential to develop into Point Of Care Testing.The main content and results are as follows:(1)The pET28a-TM plasmid was constructed and introduced into E.coli BL21 for successful expression;the target protein TM was designed to be fused with His tag,and purified by nickel column affinity chromatography;the results of SDS-PAGE and Western blot showed that the obtained TM protein is correct and the purity is high,which can be used in subsequent experiments.(2)Taking the colorimetric characteristics of ox TMB as an entry point,a colorimetric aptasensor with absorbance as the response signal was constructed for the quantitative detection of TM.The key parameters of the detection system were optimized by single factor experiments.Under the optimal conditions,the linear detection range of the constructed colorimetric aptasensor was 0.856 ～ 21.128 n M,the limit of detection was as low as 0.805 n M,and the recoveries in beef paste and potato chips were 95.00～116.2%,and the specificity of TM is high,the effect of fast,simple and high sensitivity detection of TM is achieved.(3)Taking the photothermal properties of ox TMB as an entry point,a photothermal aptasensor with temperature change as the response signal was constructed for the quantitative detection of TM.Continue to use the best parameters related to the solution detection system in(2),and further optimize the parameters related to near-infrared radiation.Under the optimal conditions,the limit of detection for the constructed photothermal aptasensor is 1.515 n M,and it has a good linear range(1.599～12.093 n M),specificity and recovery(94.13-117.80% in mutton).Rapid,portable and highly sensitive detection of TM is achieved.Molecular docking experiments were carried out between the aptamer A5 and the target TM using MOE software to simulate the binding and provide theoretical verification for the specific binding of A5 and TM.In summary,this study successfully expressed eukaryotic shrimp tropomyosin with a simple structure in the prokaryotic system E.coli BL21 by using in vitro recombination technology..The protein band has the correct size,relatively good purity and sensitization ability,and is used as the detection target for subsequent experiments.Photothermal two-mode biosensor used in shrimp tropomyosin This study provides a good research basis for the rapid,sensitive and convenient detection of Oita allergen protein,and has good application potential.Using aptamer as recognition element,a colorimetric and photothermal biosensor was successfully constructed for the rapid detection of shrimp tropomyosin.This study provides a good research basis for the rapid,sensitive and portable detection of macromolecular allergen proteins,and has good application potential.