A Polydopamine-based Fluorescent Aptasensor And Its Application In Arginine Kinase Detection

In recent years,more and more people are allergic to allergens in aquatic products and their products.Due to the lack of effective treatments,the main way to prevent food allergies is nothing but avoiding intake of allergen food.Herein,rapid and efficient detection and identification methods of potential allergens in food are highly required.Arginine kinase(AK)is one of the major allergens in crustaceans.For now,PCR method and mass spectrometry analysis are the main detection methods for AK.However,these two methods are complicated to operate,expensive,and prone to give out false positive results.Give the above issues,this study aims to develop a simple,accurate,and environmentally-friendly method for the detection of AK.The main contents are as follows:1.In this paper,the protein in Penaeus vannamei was preliminarily extracted by ammonium sulfate precipitation method and the anion exchange column.The protein was identified as AK in Penaeus vannamei by LC-MS with the molecular weight being 39,675 Da according to MALDI-TOF analysis.The most abundant amino acids were Aspartic acid,Glutamate acid and Lysine in AK.According to UV-Vis spectra,AK has two absorption peaks at 210 nm and 280 nm.The secondary structure studied by circular dichroism spectroscopy showed there are 64% ?-helix,11% ?-sheet and 25% other structures in AK.The AK extracted by this method was proved to possess allergenicity by ELISA.The structure and allergenicity of AK be determined by various experiments,which can be used to screen aptamer and construct biosensors.2.In this paper,the aptamer of AK was screened 15 rounds by Bead-SELEX method with three screening modes.Each screening was optimized protocol and PCR amplification protocol.The nucleic acid sequences of aptamer were obtained by high-throughput sequencing,and their familiality was analyzed to select representative aptamer.The secondary structure of these aptamers was predicted to contain stem loops and hairpin structures.Affinity experiments and specific experiments showed the equilibrium dissociation constant of aptamer II was 40.41 nmol/L,and its specificity was good.The aptamer II was selected as the optimal aptamer,sequence of which was 5?-GGCGAACAGCAGCGCGATTCGGGTTGCGGATAGTGACAT A-3?.3.In this paper,a fluorescent biosensor for AK was developed by the fluorescence resonance energy transfer(FRET)phenomenon between polydopamine and fluorescent aptamers.The polydopamine was subjected to surface modification of polyethylene glycol to reduce background interference and achieve rapid,efficient detection of AK.The results showed that the fluorescence biosensor had good stability and could resist nonspecific interference of background substances.The methodological study showed that the standard curve equation of the method was y=1897x+82.66 and the correlation coefficient was 0.9905.The linear range was 0-2.5 ?g/mL.The limit of detection(LOD)was 0.437 ?g/mL(S/ N=3)and the limit of quantitation(LOQ)was 1.455 ?g/mL(S/N=10),relative standard deviation was 9.59%.This proposed methodology was rapid,sensitive and precise.Finally,the quantitative analysis of AK in nine shrimp and five shrimp processed products was carried out by the proposed biosensor,which proved that the biosensor can be applied to analyze the real-world samples.