A Dual-mode Nanosensor Based On Aptamer And Aunps For The Detection Of Parvalbumin

In recent years,food allergy has risen as a worldwide public concern as the rapid development of economy and the improvement of living standards.Due to the rich nutrients and savory flavor,fish is recognized as one of the popular food among consumers.However,as one of the Big-eight allergenic food sources,fish has been considered to be related to 13%of food allergy,90%of which is caused by parvalbumin(PV).Moreover,it is difficult to eliminate the allergenicity through common food processing and cooking process due to its resistance to high temperature and enzymatic hydrolysis.For now,the major methods for quantitative detection of PV are ELISA and PCR,which show limits in the complicated operation procedure,high cost,and long detection time.Therefore,this study aims to develop a dual-mode biosensor for rapid detection of PV.The main contents are as follows:(1)The PV in cod was extracted and purified by extraction ammonium sulfate precipitation and DEAE ion exchange chromatography.SDS-PAGE,ELISA and Western blot were used to analyze the properties of the purified protein,and mass spectrometry was used to identify the PV.The results showed that the purity of the extracted PV was over 99%with a molecular weight being 11.5 kDa.The PV was found to possess strong allergenicity.(2)The specific aptamer for PV was screened from random ssDNA library by GO-SELEX.Six candidate sequences were obtained by high-throughput sequencing after 18 rounds of selection.The obtained sequences were subjected to familial analysis,homology analysis and secondary structure analysis by DNAMAN.The candidate sequences were also subject to affinity and specificity analysis by BLI and GO-based fluorescence assay.Finally,a aptamer with good affinity and specificity was selected to be used for further experiment.(3)The thiolated aptamer DNA1 and its complementary short chain DNA2 were used to modify the surface of AuNPs to prepare the AuNPs-DNA1 and AuNPs-DNA2 probes.Then,the third short chain DNA3 that was fluorescently-labeled and complementary to the aptamer,was used to complementary with the AuNPs-DNA1 and AuNPs-DNA2 probes to construct the dual-mode biosensors.When the sample is presented with PV,the assembly of AuNPs would disassemble due to the competitive combination between aptamer modified AuNPs-DNA1probes and the PV.On the one hand,this would bring about the change in the visible light signal of AuNPs solution.On the other hand,it would release the fluorescently-labeled DNA 3 to generate fluorescent signal which was quenched by AuNPs through FRET effect previously.Hence,the dual-model biosensor could realize the qualitative and quantitative analysis of PV by colorimetric method and the fluorescence-based method.The results showed that the biosensor could be used to detect PV qualitatively more than 2.5?g/mL,while detect the PV quantitively with a good linearity in the range of 0~40?g/mL of PV.The final regression equation of standard curve was y=30986x+24750,with good correlation R~2=0.9939.The stability,accuracy and specificity of the biosensor was methodological evaluated,which showed encouraging performance.Therefore,the biosensor could be used to detect PV in the dual mode of visible light and fluorescence light,which is highly promising for the detection of food allergens.