Method for high-throughput rapid detection and evaluation of neurotoxicity of water sample
A neurotoxic, high-throughput technology, applied in the field of detection and analysis, which can solve the problems of lack of toxicity assessment system, high cost, and long cycle.
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Embodiment 1
[0054] This example verifies the sensitivity of the method of the present invention.
[0055] The methomyl solution was serially diluted 3 times (3×) with methanol, and the concentrations were 0.01, 0.033, 0.1, 0.33, 1, 3.3, 10, 33, 100, 330, 1000 mg / L. Use pH 6.8 phosphate buffer to prepare electric eel AChE, 0.5g / L DTNB and 0.5g / L ATCH solutions with concentrations of 0.067U / mL, respectively.
[0056] Take a 96-well plate, add 10 μL of methomyl solution of each dilution concentration to the plate in turn, each concentration is 3 parallel, and set up 3 wells and add 10 μL of methanol as a solvent control, and place the 96-well plate in a fume hood for 15 minutes to allow methanol Completely volatilize until there is no liquid substance visible to the naked eye, then add 150 μL of the prepared electric eel AChE solution and 50 μL of the prepared DTNB solution to each well in sequence, and after standing at 37°C for 15 minutes, add 50 μL of the prepared ATCH solution to each we...
Embodiment 2
[0060] In this embodiment, different sample addition steps are compared.
[0061] According to the method of Example 1, the concentration of methomyl gradient solution of 0.0132mg / L, 0.04mg / L, 0.132mg / L, 0.4mg / L, 1.32mg / L and 4mg / L, and AChE, DTNB and ATCH were prepared respectively. solution.
[0062] Take two 96-well plates respectively, and set them as plate 1 and plate 2. Add 10 μL of methomyl solution of each dilution concentration to plate 1 in turn, and each concentration has 3 parallels, and add 10 μL of methanol in 3 wells as a solvent control, and place the 96-well plate in a fume hood for 15 minutes until the methanol is completely volatilized to nothing. Liquid substances visible to the naked eye, then add 50 μL of prepared DTNB solution, 50 μL of prepared ATCH solution and 150 μL of prepared electric eel AChE solution to each well in turn, and leave it at room temperature in the dark for 15 minutes, then measure the absorbance at 415 nm wavelength with a micropla...
Embodiment 3
[0065] The method of the invention is used to detect and evaluate the neurotoxicity of a certain municipal sewage sample.
[0066] Collect 2L of a municipal sewage sample after biochemical and advanced treatment, first adjust the pH of the water sample to 3 with hydrochloric acid, then filter the water sample with a glass fiber membrane with a pore size of 1.6μm, and filter the water sample with 6cc The HLB column was used for solid-phase extraction, and the eluent was successively 10 mL of methanol and 10 mL of ethyl acetate, blown with nitrogen to dryness, and then redissolved with 0.8 mL of methanol to obtain a sample with a concentration factor of 2500.
[0067] The concentrated samples were serially diluted 2 to 3 times with methanol to obtain samples with concentration multiples of 2500, 1000, 500, 250 and 100, respectively. The preparation of gradient dilution solution of methomyl is the same as in Example 1. Use pH 6.8 phosphate buffer to prepare electric eel AChE, 0...
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