Specific Detection Of Pathogenic Bacteria By Fluorescent Biosensor Based On Nucleic Acid Aptamer

Foodborne pathogens bacteria refer to pathogenic microorganisms that invade the human body through food circulation and cause infections and even infectious diseases.In severe cases,they may cause disease or death.According to data from the Centers for Disease Control and Prevention（CDC）,about one-sixth of Americans get sick every year due to contaminated food,at least 3,000 deaths and the economic loss of$15.6billion.For public health safety,it is necessary to check food hygiene and safety regularly and use highly sensitive detection methods to screen for such pathogens.Therefore,rapid and sensitive detection of food-borne pathogenic bacteria is essential for food safety and public health,because these bacteria have a huge impact on the lives of animals and humans.Escherichia coli（E.coli）is one of the most important pathogenic bacteria,usually related to foodborne diseases.In order to better control microbial contamination in food and water samples and reduce foodborne diseases,it is crucial to develop a rapid and highly sensitive detection strategy for E.coli.Currently,conventional methods for detecting E.coli include standard plate count,polymerase chain reaction（PCR）and enzyme linked immunosorbent assay（ELISA）.Colony counting method has high accuracy,but is time-consuming and laborious.In contrast,ELISA and PCR based strategies can quickly detect E.coli.However,in these strategies,sample preprocessing steps（cell lysis and DNA extraction）and expensive instrumentation are often required,and experiments often complicate the test process,resulting in a significant increase in the total test time Therefore,it is of great practical significance to develop a simple,rapid,sensitive,and highly selective detection method to monitor pathogenic bacteria.In this paper,based on the advantages of high sensitivity and good selectivity of quantum dot fluorescent labeling,a series of fluorescent biosensor based on nucleic acid aptamers were established to detect food-borne E.coli.The main contents are as follows:1.This work mainly uses citric acid and polyethyleneimine one-step hydrothermal method to prepare aminated carbon quantum dots（NH₂-CQDs）with excellent fluorescence properties.The synthesis method was simple,the obtained product was uniformly dispersed,and the fluorescence intensity was higher than that of carbon quantum dots prepared by citric acid alone.E.coli O157:H7 was used as the pathogenic bacteria model to be detected,carbon quantum dots（CQDs）modified with specific aptamers were used as energy donors（excitation wavelength at 355 nm）,and graphene oxide（GO）was used as FRET energy acceptor,construct a fluorescent biosensor to realize the quantitative detection of E.coli O157:H7 within 1 h.2.In this work,Fe₃O₄@Au-aptamer magnetic beads（MBs）was used as a carrier,cDNA-coupled N,S-GQDs-MoS₂QDs nanospheres（cDNA-N,S-GQDs-MoS₂QDs）as bioluminescent probes.The nano-biocomplex（Fe₃O₄@Au-aptamer/cDNA-N,S-GQDs-MoS₂ QDs）was prepared by coupling the nano-biological probe and MBs through the hybrid reaction between cDNA and E.coli aptamer.With the increase of the Fe₃O₄@Au-aptamer,the fluorescence intensity decreased accordingly.With the target E.coli O157:H7,the fluorescence intensity was restored due to the specific recognition of E.coli O157:H7 and its aptamer.And the recovery value showed a certain quantitative relationship with the concentration of E.coli O157:H7,so as to specifically detect E.coli O157:H7.3.In this study,Gly-N,S-GQDs was synthesized by glycine coated N,S-GQDs.And then the complex aptamer-Gly-N,S-GQDs as the fluorescence as well as the donor was obtained via the combination of the COOH-aptamer and quantum dots through amide bond.Cobalt oxyhydroxide（Co OOH）acted as the fluorescent acceptor.When Co OOH was introduced,Gly-N,S-GQDs was adsorbed on the surface of Co OOH throughπ-πstacking and hydrophobic interaction,making the fluorescence of Gly-N,S-GQDs was quenched.With the target E.coli O157:H7,the fluorescence resonance energy transfer process was interrupted due to the specific binding of aptamer and E.coli O157:H7,thereby restoring the fluorescence of aptamer-Gly-N,S-GQDs to achieve the detection of E.coli O157:H7.