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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

Active Publication Date: 2021-12-03
ANHUI NORMAL UNIV
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Problems solved by technology

[0003] Currently there are methods such as liquid / gas chromatography-mass spectrometry, immunoassay, surface-enhanced Raman spectroscopy, electrochemical and other methods. Involves complex and time-consuming and lengthy detection process, or involves the labeling of external fluorescent signal molecules, so it is very important to develop a fluorescent biosensor with high selectivity, high sensitivity, and simple label-free detection of organophosphorus pesticides

Method used

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  • Fluorescent biosensor, preparation method and application of detecting organophosphorus pesticides
  • Fluorescent biosensor, preparation method and application of detecting organophosphorus pesticides
  • Fluorescent biosensor, preparation method and application of detecting organophosphorus pesticides

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Effect test

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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Abstract

The invention provides a fluorescent biosensor, a preparation method and its application for detecting organophosphorus pesticides, using copper nanoparticles carrying polythymine single-stranded DNA as a template for luminescence, and using tyrosinase to quench the luminescence of copper nanoparticles , adding organophosphorus pesticides to inhibit the activity of tyrosinase, prepared a fluorescent sensor based on copper nanoparticles carrying thymine DNA as a template, tyrosinase and organophosphorus pesticides, and achieved sensitivity and specificity for organophosphorus pesticides sexual detection. Compared with the prior art, the present invention uses unlabeled DNA, which is simple to operate, low in cost, and avoids any chemical labeling and modification. By inhibiting the enzyme activity, a sensor for the detection of organophosphorus pesticides can be prepared. The results show that the sensor can detect organophosphorus pesticides sensitively from 0.1ng / L to 1000ng / L, and has the advantages of simple operation, high sensitivity and low detection limit.

Description

technical field [0001] The invention belongs to the technical field of fluorescent sensor preparation, specifically relates to a fluorescent biosensor, a preparation method and its application for detecting organophosphorus pesticides, and is a copper nanoparticle system based on unmarked single-stranded DNA carrying polythymine as a template Constructed fluorescent biosensors for applications in the detection of organophosphorus pesticides. Background technique [0002] Organophosphorus pesticides (OPs) widely exist in food and the environment and are highly toxic. Ops residues can enter the food chain through air, water and soil, posing health threats to humans and animals, and can cause serious clinical complications including respiratory tract damage and paralysis. Even death, therefore, the development of simple, sensitive, fast, efficient and reliable assay methods is particularly urgent. [0003] At present, methods such as liquid / gas chromatography-mass spectrometry...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/26G01N21/64
CPCC12Q1/26G01N21/6428G01N2021/6432G01N2333/90229
Inventor 王广凤陈纪华
Owner ANHUI NORMAL UNIV
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