| In the previous years, with the frequent occurence of the melamine, clenbuterol, and the pathogenic bacteria pollution incidents, People pay more and more attention to health and health care. In the event of bacterial contamination, a outrageous toxic pathogens which is called Brucella. It is a prokaryotic bacterium with a strong toxicity, it causes human and livestock zoonotic brucellosis, it may cause persistent fever, female abortion, even can lead to permanent disability, or even loss of labor capacity. However, the bacteria are widely existed throughout the world. It is mainly distrubuted in the field of animal husbandry, also in the industrial of meat, dairy. It is aware of us that the food we need in daily life is full of health risks.In order to solve this difficult problems which are caused by Brucellosis, firstly, we need to cut off all the possible ways of transmission from the source of infection, and ban contaminated meat, containing bacteria in milk or milk products which has been found into the market. How to know quickly whether the bacteria exited in the meat, milk, dairy products, and how to have a quantitative understanding of the number of bacteria in the food. This study in order to achieve high precision and high selectivity of Brucella, low cost, fast unbiased quantitative detection,the results of this paper will be elaborated as following: (1) In order to improve the electrochemical properties of the electrode surface, the gold electrode was pretreated by physical pretreatment, ultrasonic pretreatment and chemical pretreatment,the pretreatment of gold electrode by physical pretreatment, ultrasonic pretreatment and electrochemical method were compared, The change of value of oxidation peak current was investigated by cyclic voltammetry (CV) in two pretreatment methods. It was found that the second pretreatment has better effect on improving the electrochemical performance of gold electrode, which was helpful to improve the precision and sensitivity of detection.(2)In order to realize the immunosensor for high selectivity, rapidity and further enhances the immune sensor for quantitative detection of Brucella antigen accuracy and sensitivity. The excellent electrical conductivity of graphene oxide and chitosan as the material of the modified electrode was selected,the conductive composite films of graphene oxide and chitosan (GO-CS) were prepared by molecular self assembly method, the GO-CS conductive composite film was bond to gold electrode by electrodeposition, better binding properties of protein and chitosan, then modified the brucella antibody bound to immune electrode.By the synergistic effect of GO-CS two on the function, the electrochemical immunosensor for the detection of Brucella antigen is found. The immune electrode was characterized by cyclic voltammetry (CV),The bacteria antigen in 4× 103CFU/mL-4× 107CFU/mL concentration range of peak current change values showed a good linear relationship with the concentration of Brucella antigen, Linear regression equation:y=2.04211gC+1.3916,then further derive C= 0.2082 x 100.4897?I(where ?I is the variation of the current, C represents the antigen concentration).Correlation coefficient r=0.9898,In this range, the concentration of the solution to be tested can be well distinguished, and the detection limit is 8.623 ×102CFU/mL.(3) In order to detect the sensitivity and detection limit of Brucella antigen sensor further enhance, On the one hand, the synergistic effect of graphene oxide and chitosan, after making the immune electrode. On the other hand, the method of square wave voltammetry (SWV) and differential pulse voltammetry (DPV) was used to test the immune electrode.The relationship between the peak current and the logarithmic concentration of the bacteria was established, for DPV method, When the concentration of the bacterial antigen was in the 4 × 103 CFU/mL-4 × 107 CFU/mL range, there was a good linear relationship, and the linear regression equation was y=3.04511gC-1.3997,Further deduced C= 2.8817×100.3284?I, (where AI is the variation of the current, C represents the antigen concentration), correlation coefficient r=0.9856, The detection limit is 5.452 × 101 CFU/mL, In this section, the concentration of the solution to be tested can be well distinguished. For SWV method, there was a significant linear relationship when the concentration of the bacterial antigen was in the 4×102CFU/mL-4×107CFU/mL range, Linear regression equation is y=2.76561gC+0.0391, then further derive C= 0.9679×10a3616?I(where AI is the variation of the current, C represents the antigen concentration), the correlation coefficient r=0.9890, the detection limit is 3.386× 101 CFU/mL. The concentration of the solution to be tested can be well distinguished in this section. Through the comparison of the two, the SWV method has lower detection limit compared to the DPV method.(4) On the basis of the gold electrode, the GO-CS conductive polymer films was prepared by molecular self assembly method, and by electrodeposition method modified gold electrode surface, and then bound on brucella antibody,after making the immunosensor,Using EIS method to detect the Brucella antigen concentration gradient increased gradually. And put forward the corresponding equivalent circuit of immune sensor.the measured data are processed by Zview software, and obtained all the basic parameters, through data comparison analysis, only the charge transfer impedance(Rct) significant change with the reaction of antibody and antigen, the other three parameters can be ignored. Make a relationship between the charge transfer impedance variation of the Rct(?Rct) and the logarithmic concentration of Brucella antigen,The results showed that there was a significant linear relationship between the bacterial antigens in the4×102CFU/mL-4×107CFU/mLconcentration range, the regression equation was y=490.94491gC-562.9266, correlation coefficient r=0.9943,Further derived C= 14.0158×100.0021?Rct (where the ?Rct represent for variation of transfer impedance and C represents the antigen concentration). In this section,the concentration of the solution to be tested can be well distinguished, and the detection limit is 2.528 ×10~1 CFU/mL. |
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