Temperature Sensing Detection Method Of Salmonella Typhimurium Based On Photothermal Effect Of Graphene

Salmonella typhimurium is a group of non-adaptive Salmonella with a wide range of hosts.It is distributed in nature widely,and exist in animals such as livestock and rodents.It is one of the main pathogens causing acute gastroenteritis and it can rapidly multiply at room temperature,The bacteria can cause zoonotic diseases and human food poisoning.Among them,the safety incidents of human and animal food poisoning caused by Salmonella typhimurium always has been the most frequent in the country,affecting human life and health seriously,and attracted wide attention from all walks of life.Detection of Salmonella typhimurium has important public health implications.At present,the detection methods of food-borne pathogenic bacteria such as Salmonella typhimurium mainly include:traditional detection methods and new detection methods.The traditional detection method mainly refers to the flat panel counting method,which is accurate in counting,but the operation is cumbersome,which is not conducive to on-site detection.The new detection methods that have been established are complicated to operate,have low detection sensitivity and expensive.Therefore,the detection method with low cost,simple operation,high sensitivity and convenient on-site operation has important practical significance.In this thesis,two new detection methods are established:a thermometer detection method and a temperature sensing method based on the photothermal effect of Graphene oxide（GO）for the detection of Salmonella typhimurium.The detection performance of the two methods was evaluated.Both of these detection methods can achieve quantitative detection of Salmonella typhimurium,which has the advantages of accurate counting,simple operation,low cost,and reusable.This paper mainly includes four parts,the contents are as follows:The first chapter mainly summarizes the hazards of Salmonella typhimurium and the current detection methods of Salmonella typhimurium,and their advantages and disadvantages were compared.Several new detection methods and the currently established detection methods which based on photothermal effects were emphasized.In the second chapter,a method which use the thermometer based on the photothermal effect of GO to detect Salmonella typhimurium was established,and the detection performance of the method was evaluated.By modifying the antibody on the mercury head of the thermometer,different numbers of Salmonella typhimurium can be captured and enriched,and Salmonella typhimurium is bound to the mercury head.After coupling GO with antibody,incubation with a mercury head that binds Salmonella typhimurium,GO is conjugated to the mercury head of the thermometer by specific binding of the antibody to the bacteria.The more bacteria captured,the more GO will be conjugated to the mercury head,and the GO present the strong photothermal effect.The mercury head of the thermometer is placed under the laser at 808 nm.Based on the good photothermal effect of the GO,the mercury head will have obvious temperature changes under the irradiation.The change can be read by the thermometer directly,thus,the standard curve between bacterial counts and temperature changes is established.The paper optimizes the contact conditions of the thermometer mercury head and the bacterial liquid,and the ambient temperature.Under this optimized condition,combined with the laser irradiation power,irradiation time,the combination time of the thermometer and the bacteria liquid optimized.A standard curve between the number of different bacteria and the temperature change is obtained.In the bacteria number range of 10³～10⁹CFU·m L^-1,the linear relationship of the standard curve is good,R² is 0.9922,the signal-to-noise radio（S/N）is low,and the minimum detection limit is 10³CFU·m L^-1.The recoveries of the three samples were between 80 and 117%,the difference between groups was 7.6%,and the difference within the group was 6.8%.In the accelerated storage experiment of 37°C,the recovery rate of the bacteria after storage of the antibody-conjugated thermometer for 48 hours can reach 80%.In the third chapter,a method which use the temperature sensor based on the photothermal effect of GO to detect the Salmonella typhimurium was established.The Salmonella typhimurium antibody was coated on a nitrocellulose membrane while the membrane was blocked with Bovine serum albumin（BSA）.The GO was coupled with a Salmonella typhimurium antibody,and then blocked with BSA to prepare an immunographene.The mixture of immunographene and Salmonella typhimurium was placed in a 200μL centrifuge tube and incubated in a constant temperature and humidity incubator at 37℃for 10 min.The bacteria bind the antibodies in the immunographene specifically.Mixture of immunobending graphene and bacteria is added to the nitrocellulose membrane,reacted 10 min at room temperature,the conjugated immunographene binds to the antibody coated on the membrane specifically,and then,membrane was washed.At the same time,the unbounded immunographene and bacteria were washed away.After drying the membrane at room temperature,the membrane and the temperature sensor probe were adhered with double-sided tape.The more bacteria in the mixture,the more immuno-graphene attached to the membrane,the better the photothermal effect.Use the 808 nm laser to irradiate the sample loading zone,significant temperature changes occurred which based on the good photothermal effect of graphene.The amount of temperature change can be read by a temperature sensor while establishing a standard curve between the temperature change value and the amount of Salmonella typhimurium.Different types of carrier membranes,the concentration of antibody coated on the membrane,the irradiation time and power of the laser,the p H of the immunographene,the concentration of graphene in the immunographene,and the concentration of the antibody in the immunographene were optimized.Non-specific binding is eliminated by optimizing conditions such as the concentration of the blocking solution,the blocking time,and the binding time of the antibody on the membrane to the mixture of the immunographene-bacteria.Experiment result shows:The membrane using Millipore 135 is more suitable.The concentration of antibody coated on the membrane is 0.2 g·L^-1,the irradiation time of the laser is 2 min,the irradiation distance is 2 cm,and the irradiation power is 2.5W(4.98 W·cm^-2).The optimum p H of the immunographene is 8.5,the optimum graphene concentration in the immunographene is 0.8 g·L^-1,and the optimum antibody concentration is 0.02 g·L^-1.BSA blocking concentration was 2%,the blocking time was 2 h,and the optimal binding time of the antibody on the membrane to the immunographene-bacteria mixture at room temperature was 10min.When the bacterial content was between 10⁴～10⁸CFU·m L^-1,the linear relationship was good,R²=0.981,3 S/N=0.314,and the minimum detection limit was 10⁴ CFU·m L^-1.The recoveries of the three samples were92.1±26.30%～110.4±31.96%.In the accelerated storage experiment at 37℃,the coated membrane can be stored for three days,and the bacterial recovery rate after detection for three days can reach 91.45%.It can be stored for 14 days at room temperature（14℃）,and the recovery rate of bacteria on the 14th day can reach89.46%.The storage time was 4 days at 4℃,and the recovery rate of bacteria detected on the 30th day was 93.4%,indicating that the membrane stored at 4℃has good stability and long storage time when the method is used to detect bacteria.In the fourth chapter,the thermometer detection method and temperature sensor detection method of Salmonella typhimurium based on the photothermal effect of graphene oxide were evaluated.Both of these methods can detect Salmonella typhimurium rapidly,specificity,sensitivity,and low in cost.Among them,the method based on temperature sensor is environmentally friendly,easy to carry,and rapidly,it also can be used for on-site inspection.