Construction And Application Of Surface Imprinted Electrochemical Sensors In The Detection Of Foodborne Pathogenic Bacteria

Foodborne pathogens could cause various diseases and pose a huge threat to public health.E.coli and Staphylococcus aureus are two common pathogenic bacteria which can induce food poisoning,mainly manifested as diarrhea,vomiting,etc.,which are can cause death.Therefore,accurate and rapid detection of foodborne pathogens is of great scientific significance and application prospect for the control and prevention of foodborne disease outbreaks.In order to achieve simple,fast,sensitive and cheap bacterial detection,a more suitable detection method needs to be found.Among of other methods,the electrochemical biosensor technology has the advantages of fast response speed,high sensitivity,simple operation,low cost,and easy miniaturization.It has attracted widespread attention in bacterial detection.The impedance method does not require the use of an analyte as an enzymatic substrate or forms an electroactive substance when it is used for bacterial detection,and is therefore widely used in bacterial detection.Another important part of the sensor is the identification element.As far as recognition elements are concerned,antibodies are the most commonly used natural biological receptor in sensors and have a high affinity for target antigens.However,the acquisition of antibodies requires animal experiments,which is expensive and not very stable,and requires harsh operating conditions.Compared with other recognition systems,molecular imprinting polymer?MIPs?are easy to prepare,have predictable structures,high physical stability,high versatility,low cost,and have the same selective detection capabilities as antibodies.In this thesis,pathogenic bacteria cells were used as a template,and a pathogenic bacteria surface imprinting membrane was prepared by electrochemical polymerization.Using this as a probe for pathogenic bacteria capture,combined with electrochemical impedance technology,pathogenic bacteria recognition and electrochemical impedance detection based on surface imprinted polymer was constructed.The label-free electrochemical sensing platform is suitable for highly specific and rapid detection of a variety of pathogenic bacteria.The main work includes the following:Neutral potassium chloride solution was used as the electrolyte solution,and pyrrole and E.coli O157:H7 were copolymerized and fixed on the surface of the glassy carbon electrode by cyclic voltammetry,and then E.coli O157:H7 was removed in situ to form a no cavity bacterial surface imprinting membrane to specifically detect E.coli O157:H7.On this basis,the experimental conditions were optimized to build a high sensitivity and high specificity electrochemical sensor system for the detection of E.coli O157:H7.The mixed film of E.coli O157:H7 and polypyrrole could be immersed in the mixed solution of HAc/SDS at room temperature for 4 hours to removed E.coli O157:H7 and formed on the mixed film to detected E.coli O157:H7 recognition site.This sensor is easy to operate under mild conditions and shows rapid?1 hour?and highly sensitive?10³ CFU/m L?analytical performance.In the concentration range of10³ to 10⁸ CFU/m L,there is a linear relationship between?R/R and the logarithm of the concentration of E.coli O157:H7.The linear regression equation is expressed as?R/R???=17.99log₁₀C-56.21,and the correlation coefficient is 0.9883.In addition,the sensor shows significant specificity,Staphylococcus aurous?S.aurous?,Listeria monocytogenes,Salmonella,and parahemolytic Vibrio as the interfering bacteria,the results shows that the impedance response intensity of E.coli O157:H7 is about 5 times higher than that of interfering bacteria.and the impedance response intensity of E.coli O157:H7 is also higher than that of E.coli O6 for different serotypes.When S.aureus and Salmonella are used as the template bacteria,the sensor also shows good specificity.So the sensor has good specificity and versatility.When used to evaluate E.coli O157:H7 in real samples?Drinking water,juice and milk?,the sensor recovery range fluctuates from 96.0%to 107.9%with a relative standard deviation?RSD?of less than4%.It shows that the sensor has good accuracy and practicality.In order to further shorten the recognition time and improve the sensor regeneration performance,we using poly-3-thiopheneacetic acid as the functional monomer and S.aureus as the template molecule,pathogenic bacteria surface imprinted biosensor capable of specifically detecting S.aureus was constructed by electropolymerization and template removal.S.aureus and 3-thiopheneacetic acid were copolymerized on the surface of the gold electrode by cyclic voltammetry,then the modified electrode was immersed in the HAc/SDS mixed solution,and then at 37°C at a constant speed of 400rpm for 2 h to removed S.aureus in the hybrid membrane.Because the surface of poly3-thiopheneacetic acid is rough and the surface area is large,the probability of contact between bacteria and recognition sites is increased,and the detection time of bacteria is greatly shortened,only 10 minutes.The limit of quantification of S.aureus detected was as low as 10³ CFU/m L.The sensor has good specificity and versatility.E.coli O157:H7,Listeria monocytogenes,and S.Paratyphi B as the interfering bacteria,the results show that the impedance response intensity of S.aurous is about 5 times higher than that of interfering bacteria.When E.coli O157:H7 and Listeria monocytogenes are used as the template bacteria,and other bacteria are used as interfering bacteria,the sensor also shows good specificity.When applied to the detection of S.aureus in actual samples,the recovery rate of S.aureus was between103.6%and 122.5%,and the relative standard deviation was less than 2%.The most worth mentioning is that the bacterial blotting membrane can be reused at least 3 times during use when using Staphylococcus aureus as the template bacteria.This phenomenon has been repeatedly verified and can be achieved in different batches of samples.Therefore,the sensor has good stability and can be reused.