New Methods For High-throughput Protein-chip Analysis And Its Applications In Food Detection

In recent years,food safety incidents had frequently occurred,seriously affecting China's export trade and consumer health.With the increasing supervision of food safety in various departments,higher requirements had been placed on related testing technologies,and traditional instrument testing methods have been difficult to meet market demand.Traditional detection technology had long work time and complicated operation,and it was difficult to adapt to the actual needs of high-throughput rapid detection.Other commonly used rapid detection methods cannot meet the requirements of multi-targets detection.Therefore,the establishment of high-throughput,fast,simple,multi-targets simultaneous detection methods had great significance for food testing.As a highly integrated analytical method and research method,protein chip played an important role in academic research and application for more than ten years with its unique high-throughput,high-sensitivity,high-accuracy and parallel analysis.In recent years,protein chips have been widely used in proteomic,food safety analysis,drug screening,clinical disease diagnosis and other fields.This thesis researched and developed a high-throughput visualization protein chip detection technology based on 96-well plate.One experiment can detect multiple indicators in multiple samples,and at the same time established methods for a variety of foods with multiple harmful substances and multiple nutrients.The main research results are as follows:1.Simultaneous detection of ofloxacin and lomefloxacin in milk by visual protein chipA sensitive,simple,inexpensive,simultaneous analytical method for ofloxacin and lomefloxacin in milk by immunoassay without the need for time-consuming or complex pre-treatment steps was reported.A 96-well microplate was used as solid support,on which ofloxacin antigen and lomefloxacin antigen were immobilized,respectively.After immobilization,a mixture of relevant antibodies and standard solutions containing the analytes or samples were added to the array reaction area,then added silver nanoparticles(AgNPs)labeled secondary antibody.Silver enhancement technique was applied to amplify the detection signals,producing black image on array spots visible with naked eyes.The signals were detected with a microarray scanner;therefore the analyte residues could detect quantitatively.The limits of detection(LOD)(3SD)were estimated to be 0.24 ng/mL(ofloxacin)and 0.35 ng/mL(lomefloxacin).2.Simultaneous detection of quinolones antibiotic residues in milk based on the direct competition methodUsing the principle of direct competition method,the monoclonal antibodies of nine quinolones were immobilized on the bottom of the microplate in the form of microarrays to prepare microplate biochips,which were sequentially labeled with quinolone artificial antigens and quinolones.The drug reacted and finally enhanced coloration with silver to produce an array point visible to the naked eye.The color rendering results were quickly scanned and imaged by a visual chip analyzer,and the images were processed by chip analysis software to extract signals for quantitative detection.The spiked recovery rates of nine quinolones in milk by this method were between 70%and 120%,respectively,and the relative standard deviations were within 15%.The method was based on the direct competition method to achieve the identification and detection of different antibiotics by fixing the position of the antibody,and can be used for simultaneous rapid screening of nine quinolone residues in the milk samples.Avoid cross-reactivity of antibodies with immobilized antigens in indirect competition which cannot simultaneously detect nine quinolone antibiotics.In addition,the method is easy to operate,the detection cost is low,and the detection speed is fast.In the field of food safety testing,this method has great potential.3.Visual protein chip method for simultaneous detection of multiple antibiotic residues in feedSimultaneous detection of multiple antibiotics such as tetracycline,lincomycin,and florfenicol in feed was developed.Tetracycline artificial antigen was synthesized by glutaraldehyde method,and lincomycin as well as florfenicol artificial antigen was prepared by succinic anhydride approach.Artificial antigens were verified by ultraviolet spectrophotometry(UV)and the results showed that the three artificial antigens were successfully synthesized.In addition,the optimizing antigen-antibody concentration,cross-reaction rates were calculated and standard curve were established,and simultaneously evaluate the recoveries and repeatability of tetracycline,lincomycin,and florfenicol in feed.The spike recovery were within 80%-120%,and RSD were less than 12%.At the same time,the established method was applied to the detection of unknown feed samples.The results were consistent with the HPLC-MS method,and the correlation coefficients were all greater than 0.99.4.Smartphone-based visualized microarray detection for multiplexed harmful substances in milkIn this paper,we report a sensitive,simple and inexpensive analytical method for the immunoassay microarray based on a smartphone which various harmful substances in milk could be assayed.Tetracyclines(TCs)and Quinolones(QNs)were selected as the model targets in this study.TCs and QNs antigens were immobilized in the microarray and then samples containing free of antibiotics and corresponding antibodies as well as AgNPs labeled secondary antibodies were added to the microarray.The signal of this competitive format was further amplified by silver enhancement technique based on the development reagents and achieved a visual dots in the array.The resulting microarray could be detected by the smartphone placed in the minicartridge.The limit of detection(LOD)of this novel detection platform was 1.51 ng/mL(TCs)and 1.74 ng/mL(QNs).To achieve one-well quantitative analysis,a series of gradient concentration mouse IgG was immobilized in the same well.As a result,an internal standard curve was plotted by the signal of different concentrations of mouse IgG The results showed that a quantitative detection of TCs and QNs established were consistent with external standard curve.Compared to other methods,this method was superior in terms of detection limit,time saving,and one-well quantitative detected with smartphone which were simple sample-preparation.5.Simultaneous detection of ?-lactoalbumin,?-lactoglobulin and lactoferrin in milk by visual protein chip?-Lactalbumin(a-LA),?-lactoglobulin(?-LG)and lactoferrin(LF)are of high nutritional value which have made ingredients of choice in the formulation of modern foods and beverages.There remains an urgent need to develop novel biosensing methods for quantification featuring reduced cost,improved sensitivity,selectivity and more rapid response,especially for simultaneous detection of multiple whey proteins.A novel visualized microarray method was developed for the determination of a-LA,?-LG and LF in milk samples without the need for complex or time-consuming pre-treatment steps.The measurement principle was based on the competitive immunological reaction and silver enhancement technique.In this case,a visible array dots as the detectable signals were further amplified and developed by the silver enhancement reagents.The microarray could be assayed by the microarray scanner.The detection limits were estimated to be 40 ng/mL(?-LA),50 ng/mL(?-LG),30 ng/mL(LF)(n=6).The method could be used to simultaneously analyze the whey protein contents of various raw milk samples and ultra-high temperature treated(UHT)milk samples including skimmed milk and high calcium milk.The analytical results were in good agreement with that of the high performance liquid chromatography.The presented visualized microarray had showed its advantages such as high-throughput,specificity,sensitivity and cost-effective for analysis of various milk samples.