Fluorescent biosensor, preparation method and application of fluorescent biosensor to detect organic phosphorus pesticide

Abstract

The invention provides a fluorescent biosensor, a preparation method and an application of the fluorescent biosensor to detect an organic phosphorus pesticide. The luminous effect of copper nanoparticles which take polythymine-carrying single-chain DNA as a template is used, tyrosinase is used to quench the copper nanoparticles, and the organic phosphorus pesticide is added to inhibit the activityof tyrosinase, thereby preparing the fluorescent biosensor which is constructed based on copper nanoparticles which take polythymine-carrying DNA as a template, tyrosinase and the organic phosphoruspesticide. The fluorescent biosensor can detect the organic phosphorus pesticide with sensitivity and specificity. Compared with a fluorescent biosensor in the prior art, the fluorescent biosensor adopts unmarked DNA, is simple to operate and low in cost, and is free of any chemical mark and modification. Through enzymatic activity inhibition, a sensor capable of detecting an organic phosphorus pesticide can be prepared. The result shows that the sensor can detect the organic phosphorus pesticide with the sensitivity being 0.1 ng / L to 1000 ng / L. The fluorescent biosensor is simple to operate,high in sensitivity and low in 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...

Examples

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...

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...

Examples

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...

Images