Paper-based Detection Methods Of Food-borne Pathogenic Bacteria Using The Photo-thermal Effect Of Nanomaterials

Food-borne pathogens are the main factors that endanger food safety and human health.Food safety problems caused by food-borne pathogens have been widely concerned by public.There are many bacteria of food-borne diseases.Common Gram-negative bacteria are Salmonella,Shigella and Escherichia coli.Common Gram-positive bacteria are Bacillus,Streptococcus and Listeria Genus.Most of intestinal bacteria are Gram-negative bacteria.Salmonella are the main food-borne pathogens that caused human intestinal diseases.It is necessary to establish portable,rapid and effective detection methods for effectively monitoring food safety.Traditional Salmonella detection methods are time-consuming,low efficiency,and require complex equipments.As a new type of paper analysis technology,test strip technology has attracted much attention and has been applied in many fields,because of its high efficiency,portability,simple manufacture,convenient operation and low consumption.In general,the sensitivity of the test strip is not satisfied and is susceptible by external environmental factors.Therefore,this thesis used the photo-thermal effect of nanomaterials to improve the test strip.Thequantitativedeterminationofbacteriawasachievedbythelinearelationshipbetweenthenumbero fbacteriaandtemperaturecontrast.In this thesis,three new methods were established.They were paper-based detection method of Escherichia coli using the photo-thermal effect of colloidal gold(Au),paper-based detection method of Salmonella using the photo-thermal effect of graphene oxide(GO)and paper-based detection method of Salmonella using the photo-thermal effect of microbeads(MBs).These three methods achieved the rapid detection of food-borne pathogenic bacteria.It is simple,fast,less consumption of samples and no large instruments.The thesis consists of five parts as follows:In the first chapter,types and hazards of food-borne pathogens,principles of test strip and classification of food-borne pathogens were briefly introduced.Application in the detection of food-borne pathogenic bacteria and application of nanomaterials based on photo-thermal effect were mainly introduced.In the second chapter,paper-based detection method of Escherichia coli using thephoto-thermal effect of Au was established.Antibody-Escherichia coli complex was formed on the surface of the nitrocellulose membrane(NC),other nonspecific sites were blocked,Au-antibody-bacteria complex were added to the NC.The temperature contrast caused by Au was greater with increased number of Escherichia coli.Coating buffer,blocking buffer,washing buffer and laser irradiation time were optimized.Specificity and sensitivity of the test strip methods were tested.The results showed that the optimum conditions as follows: 6% methanol was chosen as coating buffer,3% BSA + 0.6% Tween-20 was chosen as blocking buffer,0.4% Tween-20 was chosen as washing buffer.The optimal laser irradiation time was 1 min.The standard curve equation: y = 3.49x-8.662,R2 = 0.972.The limit of detection of this strip method was 410CFU/m L.In the third chapter,paper-based detection method of Salmonella using the photo-thermal effect of GO was established.Antibody-Salmonella was formed on the surface of the NC,other nonspecific sites were blocked,GO-Au-antibody-bacteria complex were added to the NC.The temperature contrast caused by GO was greater with increased number of Salmonella.Coating buffer,blocking buffer,washing buffer and laser irradiation time were optimized Sensitivity of the test strip methods were tested.The results showed that the optimum conditions as follows: 6%methanol was chosen as coating buffer,4% BSA + 0.6% Tween-20 was chosen as blocking buffer,0.4% Tween-20 was chosen as washing buffer.The optimal laser irradiation time was20 s.Thestandardcurve equation: y=0.754x-2.09,R2 = 0.971.The limit of detection of this strip method was 2600 CFU/m L.In the fourth chapter,paper-based detection method of Salmonella using the photo-thermal effect of MBs was established.Antibody to Salmonella was coated on the surface of NC.Nonspecific sites were blocked.Salmonella antibody-modified 10 nm MBs were added to the sample of Salmonella.Reaction for a while,the complex was added to the NC.The temperature contrast caused by MB was greater with increased number of Salmonella.On the basis of the test strip,coating buffer,blocking buffer,washing buffer and the MB-antibody dosage were optimized.The sensitivity of the test strip was tested.The results showed that the optimum conditions as follows: 6% methanol was chosen as coating buffer,2% BSA was chosen as blocking buffer,0.4%Tween-20 was chosen as washing buffer.The optimal amount of antibody was 20 μL.The standard curve equation was: y = 1.42x-3.588,R2 = 0.960,and the minimum detection limit of the test stripwas 800 CFU/m L.In the fifth chapter,food-borne pathogen detection methods based on the photo-thermal effect of nanomaterials were summarized.These three methods achieved rapid detection of food-borne pathogens.It is simple,fast,less of samples and no large-scale equipment compared with the traditional methods.