Study On Piezoelectric Immunosensor For Detection Of Pesticide Residues

The problem of pesticide residues in China's agricultural products (such as vegetables, tea, fruits, etc.) is still very prominent. Excessive pesticide residues and pesticide poisoning incidents have been reported from time to time. Pesticide residues in agricultural products threaten people's health. Rapid determination technology is an effective means of monitoring of pesticide residues, Biosensor-based technologies for determination of pesticide residues have many advantages, such as good-selectivity, fast-speed and high-sensitivity, can realize agricultural origin or market fast detection.Pizeoelectric immunosensor is widely used for the assay of biological analytes. The advantages of this approach include their simple-design, high-sensitivity and low-cost attract substantial research efforts to the developments of some new piezoelectric immunosensors in the recent years. The research is focused on triazophos pesticide residual detection by using piezoelectric immunosensors. The working principle of piezoelectric mass sensor is generally introduced. The immobilizing methods of biomolecule, such as antigen, antibody onto piezoelectric crystal surfaces have been studied, and especially immobilizing them using SAM technique. In addition, electrochemical impedance spectra (EIS) and cyclic voltammetry (CV) were employed to investigate the electrochemical characteristics of immunosensor.The main contents and results are summarized as follows.(1) A self-assembled monolayer-based piezoelectric immunosensor was developed for detection of pesticide triazophos. It was based on the immobilization of triazophos monoclonal antibodies onto a monolayer of 2-Mercaptobenzoic Acid (MBA), self-assembled on an AT-cut quartz crystal's Au electrode surface with N-hydroxysuccinimide (NHS) ester as a reactive intermediate. Two analytical procedures, dip-and-dry and flow-through methods, were investigated. The sensor-to-sensor reproducibility obtained at 5μg/mL and 50μg/mL was 7.1 and 9.8% R.S.D., respectively. The immunosensor could detect the target pesticide in a range of 5-100μg/mL within 40-60 min, there is a good linear relationship between the concentration of triazophos pesticides and the frequency shift. The detection limit was 4.93μg/mL. The immunosensor could meet the general requirements of pesticide residues analysis.(2) A piezoelectric immunosensor coupled with a flow injection system was developed for competitive detection of pesticide triazophos. Surface modifications via three self-assembled monolayers (SAMs) were investigated respectively and a better result was obtained with the SAM of 3-Mercaptopropionic acid (MPA). It was shown that in the range of 5 ng/mL-5μg/mL, there was a good correlative relationship between the concentration of pesticide triazophos and the frequency shift, and the regression equation was y=17.178Ln(x)—147.32 with coefficient of determination of 0.9749. The detection limit was 1.85 ng/mL. Compared with the direct piezoelectric immunosensor, the competitive format based piezoelectric immunosensor got a higher sensitivity and a low detection limit.(3) The performances of biomolecules immobilization usually can be influenced by many factors, such as the effects of SAMs type, buffers, pH, temperature, ionic strength. The nonspecific adsorption and the amount of THBu-OVA immobilized on the surfaces were demonstrated to be controllable by changing the dilution ratio of thioctic acid and thioctamide solution during the fabrication of the mixed SAMs, the optimal ratio of is 3:2. The other optimum conditions for biomolecules immobilization were given as follows:The gold electrode was coated with the THBu-OVA in 0.01 mol/L phosphate-biphosphate buffer (pH 7.4). The electrode was imcubated at 37℃for 2 hours.(4) Electrochemical characteristics of the immobilization and immune response were determined by using cyclic voltammetry and electrochemical impedance analysis. In addition, cyclic voltammetry (CV) was employed to realize the quantitative detection of pesticide residues. The conditions of THBu-OVA immobilization were optimized. Under the optimum conditions, the decrease of peak currents for Fe(CN)63-/Fe(CN)64- redox couple in cyclic voltammetry was related to the logarithm of triazophos concentration between 5 ng/mL and 5μg/mL, and the correlation coefficient was 0.9731.