| In recent years,there is a trend that the domestic food safety incidents emerged one after another,among which the contamination of food-borne pathogenic bacteria is the most common problem.The microbial contamination in food,at home and abroad,is the most important factor affecting food security.The microbiological testing of food is the last pass to prevent tainted food entering into the market.However,there exists many problems in the existing national industry testing standards of food-born pathogen in China,such as the defects of tech hysteresis,inconvenient operability,complex testing process,expensive cost and etc.,which are unable to adapt to the existing large-scale food production and consumption demand.Protein chipis to fix protein(such as antigen,antibody),polypeptide,receptor and ligand,etc.onto a carrier(e.g.membranes,gels,slides,beads and micro-pore plate)in order,analyzing the ingredients capable of having the specific interaction with samples.Nowadays,protein chips have been used in many research fields such as proteomics,food testing,disease diagnosis,drug screening,agriculture,forestry,animal husbandry,forensic and other fields.Moreover,some of the mature protein chip products have come into the market.However,there is no easy solution to solve the problem of quality control of spots in protein chips.The first problem to solve in this research was the quality control of spots in protein chips.On the basis of basic single detection protein chip for detection of Escherichia coli O157:H7,by adding Ponceau stain in spotting buffer,allowing the dyed antibody after the spotting,there was a possibility to have a forecast of degree of spot roundness and uniformity,which could achieve the purpose of spot quality control.At the same time,optimizing the fixed conditions of antibody and the relative humidity of spotting by the spotting buffer,and then the optimal group was selected.The results showed that,adding Ponceau in spotting buffer had the effect of staining antibody spot.In addition,Ponceau added in spotting buffer could be eluted into colorless after blocking,which did not affect subsequent chromogenic reaction.Confirmed by experiments,the optimum dosage of Ponceau in spotting buffer is 1%(w/v).Adding glycerol in spotting buffer could eliminate the "coffee ring" phenomenon of spots,making the antibody spot diffusion on NC membrane more uniform.Glycerol could keep the moisture of antibody spot,prolonged valid period of chips.Confirmed by experiments,the optimum dosage of glycerin was 10%(v/v).The optimized spotting buffer was used to dilute the antibody and to make protein chip.Optimizing the relative humidity for spotting,the relative humidity was 55%(ambient temperature: 20±1℃).In the meanwhile,the best fixed conditions of antibodies was 37 ℃ 2h through experiments.Then,this experiment evaluated the influence to the performance of protein chip by adding Ponceau.Whether with or without Ponceau stain in spotting buffer,the results showed that,there was no effect to sensitivity,multi-examination and specificity.Adding Ponceau in spotting buffer as method of spot quality control of protein chip had no effect in the detection performance.On this basis,selecting Escherichia coli O157:H7,Shigella boydii,and Enterobacter sakazakii three food-borne pathogens as detection object,using nitrocellulose membrane to make protein chip for detection,and using "HRP(horseradish peroxidase)catalyzing DAB(diaminobenzidine)" technology made the detection results visible to the naked eye.These procedures got rid of conventional dependence on large equipment,and were convenient for general laboratory use.By optimizing the conditions of spotting polyclonal antibody of three food-borne pathogenic bacteria having no cross reactivity on a nitrocellulose membrane to produce a visual detection protein chip for rapid multi-inspection of three food-borne pathogens through only one sample,could reduce the workload of detection at present,and improve the detection efficiency.Finally it realized the complete detection of three food-borne bacteria within 2.5 hours.Results showed that the detection sensitivity of the protein chip of Escherichia coli O157:H7 was 7.1×10~5CFU/mL,Shigella boydii was 2.2×10~5CFU/mL,Enterobacter sakazakii was 2.6×106CFU/mL.Moreover,this protein chip could detect three kinds of pathogens at the same time,and without the detection of the other 33 kinds of pathogenic bacteria.Finally,this research established an antigen chip for the detection of Escherichia coli O157:H7,which adopted the "principle of competition" immune detection method.Compared with "sandwich" immunoassay,"competition" immune detection method has its advantage,which only needs one antibody in the experiment,and greatly reduces the cost of chip production.In the experiment,the working concentration of detecting antibody(Escherichia coli O157:H7 HRP labelled monoclonal antibody)was optimized.The results showed that the optimal concentration of the chip required for detection antibody was only 1:20000,while the optimal concentration of the "sandwich" immunoassay was 1:1000,which obviously reduced the detection antibody dosage,and saved the experimental cost.Meanwhile,the detection results of the sensitivity and specificity of the antigen chip showed that the sensitivity of the chip is 7.2 ×10~5CFU/mL,and only could be detected in Escherichia coli O157:H7,which was basically identical with the "sandwich" immunoassay.Finally,the results of detection of liquid milk(sterile)purchased on the market showed that the detection sensitivity was 7.2×10~5CFU/mL,exactly identical with that of pure bacteria solution.At last,this antigen chip could realize the normal detection of Escherichia coli O157:H7,and the chip only needed one kind of antibody,making this chip be widely used in the market at a lower price. |
