| Brucellosis is a natural source infectious disease, which are widely distributed throughout the world. Huge losses were caused in agriculture, and human healthiness was also threatened. The methods of bacteriology and serology were often employed to detect the Brucellosis, while they need a long period of time and complicated procedures. It is significant to quickly and accurately detect low concentrations of Brucella antibody for early detection of brucellosis and reducing the threat of brucellosis on agricultural production and human healthiness.Biosensor technology is a new technology which was rapidly developed and attached importance to all over the world. Due to the low cost, good reproducibility and easy production, etc., the biosensor electrode based on the screen printing technology is particularly suitable for use in making of the disposable sensor to be used on field detection. In this dissertation, combined with the serological mechanism of antigen-antibody agglutination reaction and relevant biosensor technology, several different immunosensor were constructed. The methods to pretreat the sensor electrode, to bind antigens on the electrode surface efficiently and to process the responding signals were studied. The feasibility to detect the Brucella antibodies rapidly and sensitively were also investigated. The relevant results are as follows:(1) In order to improve the electrochemical properties of the electrode surface, the screen-printed gold electrodes were pretreated with chemical (in piranha solution) and electrochemical (in Sulfuric acid by cyclic voltammetry) methods together (Method 1), and the screen-printed gold electrodes and carbon electrodes were only pretreated with electrochemical methods(Method 2). With the aid of Characterizing the electrode surface properties by cyclic voltammetry, the effects of two different pretreatment methods on electrode surface were investigated. The results showed that the electrochemical treatment (Method 2) could improve the electrochemical properties of the screen-printed electrodes and the carbon electrodes, which were helpful to improve the detection accuracy and sensitivity of the sensor.(2) To improve the measurement sensitivity of Brucella antibody, and to meet quantitative and rapid detection requirements, the method to bind antigens on the surface of screen-printed gold electrode after Cysteamine modified on the activated electrode was used. After characterized the reaction of immune electrode by cyclic voltammetry, a linear relationship was found between the variations of oxidation peak current value and the concentrations of Brucella antibody in the concentration range of 10-5～10-3IU/mL, and the detection limit was 2.8×10-5IU/mL. Transfomed the cyclic voltammetry curves by semi-differential conversion, the relationship was built between the variations of semi-differential curves and the concentrations. Within the concentration range of 10-5～10-3IU/mL, there was a linear relationship between the variations of semi-differential oxidation peak current and the concentrations, and the detection limit was 2.7x10-6IU/mL. Within the concentration range of 10-2～lIU/mL, the correlation coefficient was 0.9992 between the variations of semi-differential oxidation peak current and the concentrations. The results showed that the detection range and the limit were improved obviously by the semi-differential conversion.(3) To further improve the detection sensitivity and discrimination of Brucella antibody, the potential step method was used to measure the immunization electrode constructed before, therefore the quantitative detection of different concentrations of antibody was realized. By detection of the series concentrations of Brucella antibody with the differential pulse voltammetry and the square wave voltammetry, the nonlinear correlation between the peak current values of the tested curves and corresponding logarithm concentrations of antibody were established. When the antibody concentration range was 10-5IU/mL～102IU/mL, five different Brucella antibody concentrations, such as 10-5IU/mL、10-3IU/mL、 10-1IU/mL、101IU/mL and 102IU/mL, could be well distinguished by the nonlinear curve. And, the detection limit were 2.1486×10-4IU/mL and 1.1076×10-4IU/mL separately. The data showed that the square wave voltammetry could detect the minor detection limit than the differential pulse voltammetry.(4) In order to find a simple and effective binding method, the glutaraldehyde cross-linking method were employed to bind the Brucella antigens on the screen-printed gold electrode surface, and the immunosensor for detecting Brucella antibody was constructed. Three electrochemical test methods, which were the cyclic voltammetry, differential pulse voltammetry and square wave voltammetry, were used to characterize the immunosensor with different concentrations of Brucella antibodies. With the comparison of the results, it was showed that the fitted curve of cyclic voltammetry could clearly distinguish four Brucella antibody concentrations of 10-5IU/mL、10-3IU/mL, 10-1IU/mL and 101IU/mL, and the reproducibility of the immune electrodes was the best. The method of square wave voltammetry gained the minimum detection limit of 1.93×10-7IU/mL and the residual sum of squares of 2.07x 10-5, which made two antibody concentrations of 10-5IU/mL and 10-3IU/mL could be distinguished.(5) To explore the feasibility of using impedance test technology for immunosensor, the immune electrodes was directly adsorbed Brucella antigens for detecting Brucella antibody, and the equivalent circuit of this immunosensor system was also proposed. Based on all above, the Zview software was used to fit the impedance data, and the basic parameters of the circuit were gained. By comparing these parameters, it was found that the variation of electron transfer resistance was the maximum when the antibodies were being binded to Brucella surface of the immunosensor. When Brucella antibody concentration was from 10-4IU/mL to 1IU/mL, there was a linear relationship between the changes of electron transfer impedance and the logarithm concentrations of Brucella antibody with the correlation coefficient of 0.9874.(6) The immunosensors with the screen-printed carbon electrode modified with gold nanoparticles were discussed, and the quantitative detection of Brucella antibody was achieved by the electrochemical impedance spectroscopy. There was a linear relationship between the Brucella antibody concentrations and the changes of electron transfer impedance value. Meanwhile, the immune reactions were compared between three types of immunosensor reacted to 10-5IU/mL、10-3IU/mL and 10-1IU/mL three different antibody solutions respectively. The three types of immunosensor were constructed by the screen-printed carbon electrode, the activated screen-printed carbon electrode and the screen-printed carbon electrode modified with gold nanoparticles separately. By comparing the relationship between the changes of impedance value and the logarithm concentrations, it was showed that the impedance increment of gold nanoparticles modified screen-printed carbon electrode caused by immunoreaction is the maximum. It is indicated that the gold nanoparticles modified screen-printed carbon electrode is of a larger specific surface area and very good biocompatibility, which could increase the amount of the bound antigens and make more immunoreactive to antibodies. |
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