Study On Novel Electrochemical Biosensor For Rapid Detection Of Escherichia Coli O157:H7 And Salmonella

Diseases caused by foodborne pathogens can cause not only human casualties but also serious economic losses,especially in limited resources developing countries or regions.Therefore,it is important to establish a rapid,inexpensive,sensitive and specific point of care test（POCT）method for immediate on-site detection of foodborne pathogens.In recent years,electrochemistry has attracted much attention in the field of POCT due to its relatively low cost,rapid response,miniaturization,and flexible construction.Meanwhile,novel developed technologies such as organic-inorganic nanoflower materials,CRISPR/Cas,and primer exchange reaction（PER）have been applied to the biorecognition elements and signal amplification strategies of electrochemical biosensors,resulting in a leap forward in the performance of electrochemical biosensors in terms of sensitivity and specificity.Based on this,this thesis designs and establishes novel electrochemical biosensor detection methods for the pathogenic bacteria of Escherichia coli O157:H7 and Salmonella typhimurium,which mainly includes the following three section.1.An electrochemical biosensor based on methylene-blue-loaded nanocomposite as signal-amplifying tags to detect pathogenic bacteria.A sandwich-type electrochemical biosensor was successfully constructed for the sensitive detection of pathogenic bacteria.In this biosensor platform,methylene blue（MB）organic-inorganic nanocomposites（MB@MI）were synthesized from magainin I(MI,antimicrobial peptides specific to Escherichia coli O157:H7,Cu₃（PO₄）₂and MB via a one-pot method,and were explored as a novel electrochemical signal label of biosensors generating amplified electrochemical signals by differential pulse voltammetry（DPV）.E.coli O157:H7 specifically sandwich bound to the aptamers on the electrode surface and MB@MI nanocomposites,and the changes in current signal generated on the electrode surface were used for quantitative determination of E.coli O157:H7.Under optimum conditions,the proposed biosensor showed excellent performance with a wide linear range of 10²～10⁷ CFU/m L,with a low detection limit of 32 CFU/m L,featuring favorable selectivity,repeatability and stability.According to the experiments conducted on real samples,the proposed approach is capable of detecting pathogenic bacteria in clinical diagnostics.2.Ferrocene-functionalized nanocomposites as signal amplification probes for electrochemical immunoassay of Salmonella typhimurium.Herein,an electrochemical immunosensor based on ferrocene（Fc）-functionalized nanocomposites as an efficient electroactive signal probe to amplify electrochemical signals was fabricated for Salmonella typhimurium detection.The electrochemical signal amplification probe was constructed by encapsulating ferrocene into S.typhimurium specific antimicrobial peptides Magainin I（MI）-Cu₃（PO₄）₂ organic-inorganic nanocomposites（Fc@MI）through a one-step process.Dynabeads（DBs）coupled with antibody were used as capture ingredient for target magnetic separation,and Fc@MI nanoparticles were used as signal labels in immunoassays.The sandwich of DBs-target-Fc@MI assay was performed using a screen-printed carbon electrode as transducer surface.The immunosensor platform presented a lower limit of detection（LOD）of 3CFU/m L and linear range from 10 to 10⁷ CFU/m L,with good specificity and precision,and was successfully applied for S.typhimurium detection in milk.3.Ultrasensitive detection of pathogenic bacteria by CRISPR/Cas12a coupling with a primer exchange reaction.The detection of pathogenic bacteria is extremely important to maintain food safety.In the present study,we constructed an electrochemical biosensor for point-of-care testing（POCT）of pathogenic bacteria through the cascade signal amplification of CRISPR/Cas12a and primer exchange reaction（PER）without nucleic acid extraction.Functional DNA aptamers locked the hairpin of PER,thereby preventing primer extension in the absence of target pathogenic bacteria.The presence of target pathogenic bacteria triggered the unlocking of the hairpin of PER and extended the primer into a long single-stranded DNA（ss DNA）,which then activated the cleavage of ss DNA modified on an Au electrode by Cas12a,resulting in the decrease of the electrochemical signal detectable by an electrochemical workstation.The sensor could quantify Escherichia coli O157:H7 concentration from 10 to 10⁶ CFU/m L,with a detection limit of 19 CFU/m L.Target bacteria in milk samples can be successfully detected by this method.On the basis of these characteristics,the proposed electrochemical biosensor based on CRISPR/Cas12a and PER could serve as a prospective tool for new point-of-care applications in food safety.In summary,based on organic-inorganic nanoflower materials,nucleic acid isothermal amplification technology and CRSIPR/Cas12a gene editing system,three sensitive and rapid electrochemical biosensors for foodborne pathogens were developed in this paper,providing a new scientific reference for clinical research in the field of food safety.