A Gold Nanoparticle Trimer-Based Biosensor For Ultrasensitive Detection Of Tropomyosin In Shellfish Foodstuffs

In recent years,food allergy increasingly represents an important issue in the field of food safety in industrialized countries with the improvement of consumer living standards.Due to the rich nutrients and savory flavor,shellfish has become popular worldwide,however,as one of the "big eight" categories of food allergens,shellfish contain various sensitizing ingredients.Tropomyosin(TROP),a major allergen of shellfish,is suggested to be a cross-reacting allergen.It is difficult to reduce the allergenicity of TROP through food processing and cooking processes because of its stable structure.Currently,the only effective management for food allergy is the complete avoidance of the allergen-containing food.Therefore,Therefore,it is urgently required to develop reliable,highly sensitive and feasible analytical methods for detecting allergenic ingredients in food products.So far,two kinds of analytical methods are widely applied for quantitative detection of shellfish TROP,such as enzyme-linked immunosorbent assay(ELISA)and polymerase chain reaction(PCR).However,the disadvantages of these two methods are complicated pre-processing,high cost,complicated operation,low precision and longer reaction time.Therefore,based on the above problems,this study aims to develop a rapid and convenient quantitative method for TROP detection.The main contents are as follows:1.The recombinant plasmid pGEX4T-MBP-TROP-lO×His was constructed and introduced into BL21 E.coil by thermal stimulation method to express recombinant Metapenaeus ensis TROP.The recombinant protein was modified by TEV protease and purified using a Histrap HP affinity chromatography column to obtain high purity recombinant TROP.Finally,the protein was identified as Metapenaeus ensis TROP(Met el)by liquid chromatography-mass spectrometry(LC-MS),suggesting its molecular weight is 32.8 kDa,and its concentration is 1.8460 mg·mL-1 obtained by BCA method.The sensitization of recombinant tropomyosin and the natural Metapenaeus ensis TROP obtained by acetone powder-salting out method were compared by ELISA.The results indicate that the sensitization ability of the two TROP was close,while the optimum coating antigen concentration was 0.5 ?g ·mL-1,and the optimal dilution times of serum was 1000 times.2.Gold nanoparticles(AuNPs)were prepared by sodium citrate reduction tetrachloroauric acid and characterized by transmission electron microscope(TEM),UV-Vis spectroscopy(UV-vis)and dynamic light scattering(DLS).Monoclonal antibody against TROP(mAb)was modified on surface of 33.82 nm gold nanoparticle,while recombinant tropomyosin was modified on surface of 21.04 nm gold nanoparticle.AuNPs-mAb and AuNPs-TROP were mixed at a ratio of 4:1 by volume.After incubation,with assembly rate of more than 90%,the AuNPs trimer was obtained,and the optical properties of AuNPs trimer were characterized by UV-visible spectrum and circular dichroism.The AuNPs trimer showed no significant change in absorption of UV-vis,but it has a significant chiral absorption signal at a wavelength of 525 nm.3.In this work,an ultrasensitive and simple biosensor using a chiral assembly trimer of gold nanoparticles was developed for the quantitative determination of TROP in shellfish.The AuNPs trimer-based biosensor allowed the selective determination of TROP in the range of 0.1-15 ng·mL-1,the calibration curve for TROP detection corresponds to ACD of each working solution was:y=-4.607 In(x)+17.251 with good correlation(R2=0.9927),And the limit of detection(LOD)at 21 pg·mL-1(S/N=3),and the limit of quantitation(LOQ)at 70 pg·mL-1(S/N= 10).Compared with the traditional ELISA quantitative method(LOD=90 pg·mL-1),AuNPs trimer biosensor has higher sensitivity,and more convenient pre-processing operations.4.Whole protein extracting solutions were extracted from Penaeus chinensis,Metapenaeus ensis,Litopenaeus Vannamei,Pandalus borealis,Penaeus monodon,Oratosquilla oratoria and Procambarus clarkia.The TROP concentrations in these in whole protein extracting solutions were analyzed by AuNPs trimer biosensor and indirect ELISA.The results obtained by the two analytical methods are consistent,demonstrating the good accuracy of the AuNPs trimer biosensor.