Aptamer And Antibody Functionalized Gold Nanoparticles As Probes In Dry-Reagent Strip Biosensor For Hg (Ⅱ) And Pyrethriods Pesticide Detection

A dry-reagent strip for the detection of Hg(II) and pyrethriods has been studied based on the aptamer and antibody functionalized gold nanoparticles. The major results present as following.一、 Aptamer functionalized gold nanoparticles as probes in dry-reagent strip biosensor for Hg(II) detectionSingle strand DNA aptamer selected through systematic evolution of ligands by exponential enrichment (SELEX), which presented specific characterization for mercury recognition, was modified and used as probe in the dry-reagent strip study. Combining with colloidal gold chromatography technology, a rapid method for Hg2+ detection was established. The contents and results are as follows.1.preparing different size of colloidal goldSeveral different colloidal gold particles were made with sodium citrate gradient reduction, size of which is 10nm,15nm,20nm,40nm respectively. The preparing conditions were optimized by the centrifugal speed, the results show that the best time of centrifugal condition is 15min and the speed is 10000rpm, 1000rpm,8000rpm,6000rpm respectively.2.Detection principle outline and the Control and Test line packaging.We use competition principle to design T line package material of the strip. On the basis of base pairing combination, complementary sequence is immobilized in the strip as probe, which will bind aptamer N5 competition with Hg2+ and conduct to different color grades. The results show that the Hg2+ can’t compete with the complementary sequence N5-C-5B but compete with the MB-1-5 well. So the MB-1-5 chose as the test line package material.For control line, the complementary sequence of aptamer non-specificity to Hg2+is immobilized in the strip as probe, which will bind the aptamer lead to colorable emergence.3.Test strip optimizationFirst, optimization of the coating location of C/T line on test strip, the results show that the best location for test line is 9mm away from the lower edge of NC membrane and the distance between control line and test line distance is 4-5mm.Second, the optimizing coating volume of aptamer-functionalized gold nanoparticles and C/T line of the test strip. The activated aptamer solution was added into 5ml gold NP solution, then the solution was concentrated 5-fold and coated on the conjugate pad with gradient volume; 1.0OD MB-1-5 was mixed with streptavidin, then the solution was diluted to 500μL by adding PBS and ready for the test line coating. The result indicates that the best coating volume of the strip is 3μL aptamer-functionalized gold nanoparticles solution and 1.5-2.0μL MB-1-5 combined with streptavidin solution. The cut-off level of strip biosensor is 3μg/mL for Hg2+.Third, right colloidal gold choosing between different particle sizes. Different particle size of colloidal gold conjugated with aptamer for the strip test. Comparing the chromogenic results and the detection sensitivity of the test strip, the best particle size of colloidal gold is selected to functionlize aptamer. The results show that the detection effect of 15-20nm colloidal gold is the best. The experiment chose 15nm colloidal gold.二、Antibody functionalized gold nanoparticles as probes in dry-reagent strip biosensor for detection pyrethroid pesticideCombining the advantage of monoclonal antibody and gold immunochromatographic assay, this study developes a test strip for rapid detection of pyrethroid insecticides using the broad specificity monoclonal antibody (mAb) against a group of pyrethroid insecticides.As described above, a final test strip was achieved for pyrethroid insecticides detection, based on the monoclonal antibody(mAb) marked by 15 to 20nm colloidal gold The final concentration of antibody functionalied gold solution is 1.5mg/ml,2μL coated on the conjugate pad. Test line is coated with immunogen PBA-OVA and control line with goat anti mouse IgG. To improve the detection sensitivity, the coating concentration of C/T line is optimized. The results show that the optimum coating concentration of the C/T line is as follow:T line,0.25mg/ml,0.8μL; C line, 0.1mg/ml,0.8μL. The cut-off level of strip biosensor is 0.1μg/mL for beta-cypermethrin.