Construction Of Three Multifunctional Nanoprobes And Improvement Of Rapid Detection Methods For Two Food Pathogenic Factors

Firstly,we designed an enzyme-linked immunochromatography assay combined with personal glucose meter;then,a colorimetric immunoassay combined with nano-probes of pseudo enzyme to realize the quantitative detection of Escherichia coli 0157:H7;finally,a quantitative immunochromatographic method based on fluorescence quenching technique was designed for the determination of chloramphenicol residues in raw milk.A series of rapid detection methods with simple operation,low detection cost,accurate detection results and certain innovative are obtained.The specific work is as follows:1.Portable and quantitative point-of-care monitoring of Escherichia coli 0157:H7 using a personal glucose meter based on immunochromatographic assayWith the improvement of detection demand and the innovation of detection technology,quantitative detection has become one of the most important research and development directions.Immunochromatographic technique has been widely used because of its many advantages,but the existing quantitative immunochromatographic assay requires expensive instruments.Personal glucose meter is a very successful quantitative detection device and bifunctional enzyme-linked magnetic nanoparticles(MNPs)can significantly improve the specificity and sensitivity of detection.Based on the above advantages,a novel quantitative immunochromatographic assay combining personal glucose meter was developed for the rapid detection of Escherichia coli 0157:H7.Firstly,the carboxylated MNPs were synthesized by one-step method.The activated MNPs were combined with invertase and E.coli 0157:H7 monoclonal antibody to form bifunctional MNPs.Before laminating onto the baking card,the absorbent pad was soaked in sucrose solution and desiccated.MNPs produced brown band at the detection zone of the ICA when acting as direct labels.As they were also coupled with invertase,the invertase catalyzed the hydrolysis of sucrose on the absorbent pad into glucose,which was detected by the PGM.The innovative aspect of this approach lies in the visualization and quantification of E.coli 0157:H7 through bifunctional MNPs and the detection of glucose concentration using PGM.Although the feasibility is demonstrated using E.coli 0157:H7 as a model analysis,this approach can be easily developed to be a universal analysis system and applied to detection of a wide variety of foodborne pathogens and protein biomarkers.This study proposed a qualitative and quantitative analysis device for the food safety analysis and clinic diagnostics.2.Highly sensitive colorimetric immunoassay for Escherichia coli 0157:H7 based on probe of pseudo enzyme and signal dual amplificationA highly sensitive colorimetric immunoassay was designed for the detection of Escherichia coli 0157:H7.Carboxylated magnetic nanoparticles labeled with E.coli 0157:H7 antiserum were used as the capture probes and the Au@Pt nanoparticles with peroxidase-like activity were bound to the neutral red modified reduced graphene oxide(rGO-NR)to form the probe.In order to improve the sensitivity,horseradish peroxidase(HRP)was used instead of BSA as a blocking agent to form a double amplification system with Au@Pt nanoparticles.The capture probe formed sandwich structure with target bacteria and chromogenic probe,and catalytic oxidation of 3,3',5,5'-tetramethylbenzidine(TMB).The absorbance of the product at 450 nm was linearly correlated with the concentration of bacteria in a certain range.This study provides a simple and quantitative method for food safety analysis and clinical diagnosis.3.Quantitative detection of chloramphenicol in milk based on background fluorescence quenching nanoprobes immunochromatographyA background fluorescence quenching nanoprobes immunochromatography method was developed for the quantitative detection of chloramphenicol(CAP).Colloidal gold nanoparticles with a diameter of about 30 nm were synthesized and combined with CAP monoclonal antibodies to prepare quenching probes,a special fluorescent baking card was used to prepare immunochromatographic strip.When the detected substance is present,the target and quenching probe competitive antigen coated on the strip,showing red detection line(T line)and control line(C line),and making the background fluorescence quenching.Reading the background fluorescence value(T0)and fluorescence value on T line(T1)through the instrument,then calculate T0/T1 to quantitative detection of CAP.This study successfully designed a new quantitative immunochromatographic method for the detection of CAP.The linear range was 0.1-2.0 ng/mL.The detection rate of standard samples was 100%and the recovery rate was 95.8%?109.6%.The method was easy to be popularized and applied.In summary,this paper established three rapid detection methods to achieve accurate quantitative detection of targets.All the three methods have good sensitivity,specificity and stability,and can be widely applied to different target substances by replacing antigen and antibody,but these methods also have their own shortcomings and need further research and improvement.