| Organophosphorus pesticide is a kind of broad-spectrum efficient pesticides, and is applied widely in farmland to control the pests. Organophosphorus pesticide has the important position in the proportion of pesticide using occupies. While organophosphorus pesticide is degradation easily, because of excessive use, it can cause harm to people’s physical health. According to the structure of the organophosphorus pesticides, select the containing double-ethoxy structure the organophosphorus pesticide, the establishment of a new sensitive and rapid chemiluminescence immunoassay method for the target pesticides to be tested for the environment. Synthesis of a novel light emitting marker, haptens, and light emitting markers Determination of purity and luminescent properties of the magnetic fine particles introduced into the immune response, to facilitate the separation after the reaction is extracted, and on this basis, the initial establishment of the detection of a class of organicpesticide residues heterogeneous chemiluminescence immunoassay method, demonstrated the feasibility of using a single half of the antigen to detect multi-residue heterogeneous chemiluminescence immunoassay.1.The luminescent marked compound were reacted to form Diethylphosphonoacetic Acid and ABEI, which marker contain both O,O-dimethyl and have characteristic luminescence. By silica gel column chromatography, wet packed column chromatography system of ethyl acetate:petroleum ether:triethylamine=2:5:0.1. Wet packed column, each 5 ml collection tube and collection 60 tubes, and thin layer chromatography analysis on each tube, drawn tube section 26-48, respectively, containing the desired product was collected to a rotary evaporation flask Savepressure concentrated spin dry product. The level of the product mass spectra (MS-IT-TOF) molecular ion peak 455.2056 theoretical molecular ion peak [M+H]+455.2054 match, and its UV absorption spectrum with 260nm and 280nm at an amide bond absorption peak phase ABEI than ultraviolet absorption peak blue shift.2.Light emitting marker has good light emitting characteristics, which shows a good linear relationship between the concentration of 10-160mg/L, and the standard curve equation was Y=268.263X+5837.667, R2 was 0.998. Luminous intensity has reached more than 11,000 at the concentration of 20mg/L, there is a strong light-emitting ability. And it exhibit good light-emitting characteristics at transmittance of light 300 nm and the reflected light of 365 nm. In the dynamic test, the maximum emission value from the start of the reaction to simply 4S, after the rapid decline, flatten when 45S is a fast reaction in preliminary competition immune response in the assay, after chlorpyrifos luminous intensity compared with non-Add to chlorpyrifos luminous intensity decreased significantly, chemiluminescence immunoassay, indicating that this marker can be used for subsequent rapid chemiluminescence immunoassay detection of organophosphorus pesticides.3.The four magnetic particles and anti-diethoxy antibodies coupled comparison of experimental results show that the amino-and carboxy-terminal coupled to magnetic particles are about 33%, while the isothiocyanate root end of the magnetic particles and antibodies coupled rate40%, the gold magnetic particles with antibodies coupled rate of 53.7%. Conjugate stability experimental results show that the decomposition rate of less than 4℃ under the four magnetic particles antibody conjugates stored at room temperature to be much larger, And bigger than the decomposition rate of the conjugate stored at room temperature in the 60 day almost 70%.While at 4℃ under of gold magnetic particles-antibody conjugates stability is better than the other three, and the decomposition rate of less than 10% within 30 day, good stability of the other three 20 day, but as time the extension of the decomposition rate becomes large; 60 day the decomposition of the four magnetic particles are more than 25%.4. Single-Factor Experiment were used to select the most suitable reaction condition including the pH of carbonate buffer solution, the time of immune response, the concentration of hydrogen peroxide and the concentration of photohapten and GoldMag-antibodies conjugates. Standard substance of chlorpyrifos, phoxim, triazophos were used to draw standard curve in optimization condition. Chlorpyrifos between 10 to 320 ng/mL showed a good linear correlation coefficient of 0.99496, detection limit is 3.6ng/mL, linear equation Y=4488.363-34.486X, The triazophos between 5 to 32Ong/mL a good linear correlation coefficient is 0.99596, the The detection limit 2.1ng/mL linear equation was Y=17960.477-41.191X., phoxim between 10 to 320 ng/mL showed a good linear correlation coefficient of 0.99159, the prosecution the limit 5.4ng/mL the linear equation Y=16468.860-39.354X, three pesticides and the relative standard deviation of less than 8%. The cross-reactivity test results showed that the the cross with double-ethoxy structure pesticides reaction rate in more than 90%, while the cross-reactivity of the double-methoxy structure having a similar structure pesticide below 10%, and other structures of the pesticide no cross the reaction described CLIA for double ethoxy structure pesticide has good selectivity and with high specificity. The type of sample matrix after diluted 2-fold, the matrix effect of the luminous intensity for the CLIA almost any continue diluted 4 times or 8 times, is consistent with a 2-fold dilution results.Blank spike recoveries, select Low Medium High concentration of 20 ng/mL and 100 ng/mL and 300 ng/mL, results show that the three concentrations blank recoveries were 106.6%,99.8% and 100.9% and the relative standard deviation within 5%, the description the CLIA has good precision and recovery.Experimental results show that the established detection with dual-ethoxy structure of organophosphorus pesticides multi-residues in non-homogeneous chemical luminescent immunoassay method is feasible. |
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