Fluorescent biosensor, preparation method and application of detecting organophosphorus pesticides
A biosensor, organophosphorus pesticide technology, applied in biochemical equipment and methods, fluorescence/phosphorescence, microbial determination/inspection, etc., can solve the problems of time-consuming and lengthy detection process, complicated instruments, expensive and other problems, and avoid chemical labeling. And the effect of modification, simple operation and low cost
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Embodiment 1
[0046] A method for preparing a fluorescent biosensor, comprising the following steps:
[0047] 1) Add 2 μL of PolyT30-DNA sequence to 88 μL of MOPS (10 mM (3-(N-morpholino)propanesulfonic acid) buffer solution at pH=7.6), add sodium ascorbate solution (8.0 mM)37 After incubating at ℃ for 10 min, add 50 μL of CuSO 4 Solution (800μM) was incubated at 37°C for 15min;
[0048] 2) Mix 4U / mL tyrosinase solution with 100ng / L organophosphorus pesticide, and culture it at 4°C for 30 minutes to obtain a mixed solution of tyrosinase and organophosphorus pesticide, add tyrosinase and 10 μL of organophosphorus pesticide mixed solution was incubated at 20°C-50°C for 10-30min to obtain a PolyT30-DNA / Cu NPs-tyrosinase-organophosphorus pesticide system, and to prepare a fluorescent biosensor with an "on" signal.
Embodiment 2
[0050] A fluorescent biosensor is prepared by the above method.
[0051] Determine the optimal concentration of tyrosinase concentration:
[0052] 1) Add 2 μL of PolyT30-DNA solution (1 μM) to 93 μL of MOPS (10 mM (3-(N-morpholino)propanesulfonic acid) pH=7.6 buffer solution), then add 50 μL of sodium ascorbate solution (8.0mM), after incubating at 37°C for 10min, add 50μL of CuSO 4 solution (800 μM), incubated at 37°C for 15 minutes, and prepared luminescent PolyT30-DNA copper nanoparticles;
[0053] 2) Dilute the tyrosinase solution, configure it into 0.01, 0.1, 0.5, 1, 1.5, 2, 2.5, 3 and 4 U / mL tyrosinase solution, and add the prepared solution to the system prepared in step 1) respectively. 5 μL of tyrosinase solutions with different concentrations, and the maximum quenching value was reached when the tyrosinase concentration was 4 U / mL. See Figure 4A .
Embodiment 3
[0055] Application of a fluorescent biosensor to detect organophosphorus pesticides.
[0056] The specific detection method is:
[0057] 1) Add 2 μL of PolyT30-DNA solution (1 μM) to 88 μL of MOPS (10 mM (3-(N-morpholino) propanesulfonic acid) buffer solution at pH = 7.6), and add 50 μL of sodium ascorbate solution (8.0mM), after incubating at 37°C for 10min, add 50μL of CuSO 4 solution (800 μM), incubated at 37°C for 15 minutes, and prepared luminescent PolyT30-DNA copper nanoparticles;
[0058] 2) After mixing 5 μL of 4U / mL tyrosinase and 5 μL of organophosphorus pesticides of different concentrations and incubating at 4°C for 30 minutes, they were added to the luminescent copper nanoparticles prepared in step 1), and incubated at 37°C for 30 minutes to detect different Concentration fluorescence intensity, as the concentration of organophosphorus pesticides increases, the fluorescence intensity of copper nanoparticles will gradually become stronger, and the linear relatio...
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