Method for detecting acute toxicity of water environment by using ATP bioluminescence

An acute toxicity and bioluminescence technology, which is applied in the direction of chemiluminescence/bioluminescence, photometry, and analysis through chemical reactions of materials, to achieve stable test results and easy portability

Inactive Publication Date: 2010-10-20
NANJING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most widely used is the detection of the number of microorganisms in various water bodies, foods, and dairy products, but there are few reports on the use of ATP bioluminescence method for environmental acute toxicity detection

Method used

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  • Method for detecting acute toxicity of water environment by using ATP bioluminescence
  • Method for detecting acute toxicity of water environment by using ATP bioluminescence
  • Method for detecting acute toxicity of water environment by using ATP bioluminescence

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1: the preparation of reagent and instrument:

[0038] 1. Preparation of reagents:

[0039] (1a) Preparation of luciferase:

[0040] The preparation method of luciferase refers to patent 200910109584.5, and the obtained luciferase is used for 10 -5 mol / mL standard ATP for measurement, the luminous intensity should not be lower than 1000RLU / S, and store at -20°C for later use.

[0041] (1b) Preparation of ATP reaction reagent:

[0042] Luciferin was purchased from Progema Company in the United States, 50mg, and stored at -20°C for later use;

[0043] with MgCl 2 ·6H 2 O is formulated into Mg with a concentration of 0.1mol / L 2+ Mother liquor;

[0044] EDTA·2Na is made into EDTA mother liquor with a concentration of 0.5mol / L;

[0045] Prepare a 0.05 mol / L Tris-HCl buffer solution with a pH of 7.8.

[0046] (1c) Preparation of ATP standard:

[0047] Prepare 10 -3 mol / L ATP standard reagent. Draw 0.5mL 10 -3 The standard ATP reagent of mol / L joins in ...

Embodiment 2

[0051] Example 2: Construction of luciferin-luciferase luminescent system.

[0052] (1) Luciferase:

[0053] Add 400uL ATP reaction reagent (described ATP reaction reagent comprises the component of following concentration in measuring glass tube: luciferin 20mg / L, Mg 2+ 10mmol / L, EDTA 1mmol / L, the concentration of Tris-HCl buffer is 25mmol / L), different amounts of luciferase, 25μL 10 -5 mol / L ATP standard reagent, shake gently to make it evenly mixed, immediately add 50 μL of pure water to the measuring tube, immediately cover the lid of the sample chamber, and measure its luminous intensity. Luciferase is the catalyst in the reaction, within a certain range, the greater the amount, the stronger the catalytic ability, see figure 1 . In the higher ATP concentration range, 25uL of enzyme is sufficient.

[0054] (2) Luciferin:

[0055] Dilute luciferin into different concentrations to prepare different ATP reaction reagents. Add 400uL ATP reaction reagent (described ATP re...

Embodiment 3

[0065] Embodiment 3: toxicity test

[0066] Toxic organic samples are typically represented by phenol, prepared as a 3000mg / L phenol dilution, wrapped in aluminum foil and stored at 4°C for later use;

[0067] Heavy metals are typically represented by zinc sulfate, prepared as a 1000 mg / L zinc sulfate dilution, and placed in a reaction test tube at 4°C for later use.

[0068] Add 400 μL of ATP reaction reagent successively to the reaction test tube (ATP reaction reagent contains the components of the following concentrations: luciferin 20 mg / L, Mg 2+ 10mmol / L, EDTA 1mmol / L, Tris-HCl buffer 25mmol / L), 25uL luciferase, 25uLATP standard, shake gently to mix, and finally add 50uL distilled water, quickly cover the lid and put it into the reaction Measurements were carried out in the tank, and this test served as a control group.

[0069] Add 400 μL of ATP reaction reagent successively to the reaction test tube (ATP reaction reagent contains the components of the following concen...

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Abstract

The invention discloses a method for detecting the acute toxicity of water environment by using ATP bioluminescence. The method comprises the following steps: an ATP reaction reagent, luciferase, an ATP standard sample, and a water body to be detected are sequentially added in a reaction test tube, placing the reaction test tube in an ATP fluoroscope testing instrument to detect fluorescence intensity, the detection response time is 30s and the detection is served as an experimental group; the standard sample is replaced by distilled water, the detection is served as a control group; the experimental group and the control group are repeatedly detected for three times; and the toxicity of the water body is judged by calculating relative luminance K. The method of the invention has the characteristic of stable and sensitive test effect; and the effect of a luminescent bacteria test can be reached within 3 min of the testing time at most; and the response on organic toxic substances is more sensitive than the luminescent bacteria. In the invention, a portable ATP handholding instrument with a direct current power supply is adopted, thus the ATP handholding instrument is convenient to be carried and is suitable for site test.

Description

technical field [0001] The invention relates to the technical fields of environmental protection, food safety and the like, in particular to a method for detecting the acute toxicity of toxic and harmful substances in water environment by using the change of ATP luminescence. Background technique [0002] In recent years, sudden environmental pollution accidents are increasing, which directly threaten the national ecological security and human health. To solve such problems, it is necessary to quantitatively evaluate the water environment quality from the perspective of toxicity monitoring. [0003] The current method of controlling toxicity in water bodies is mainly to control a single index of specific toxic chemical substances, and to infer whether there is toxicity in the sample to be tested by judging the test results of the control items. However, there may be a variety of compounds that are harmful to humans or have potential disease-causing risks in the water body. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N21/76G01J1/00
Inventor 肖琳孙旭杨柳燕曹妮妮谭晓辉吴维哲
Owner NANJING UNIV
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