CN1341859A - Dioxian detection method - Google Patents

Abstract

The present invention discloses a method for quickly-detecting dioxain in the environment sample and food, including the following steps: freeze-drying biological sample, grinding into powder, culturing cell, inoculating cell into kit, adding sample extract and culture medium of dioxain into the kit, then removing culture medium and adding fluorogenic substrate 7-ethoxy-3-isophenoxazolone into the kit, making reaction for 1hr., adding ethyl alcohol and stopping reaction, determining fluorescence intensity of fluorescent material 7-hydroxy-3-isophenoxazolone and calculating enzymatic activity, plotting dioxain curve and calculating toxicity concentration of dioxain compound in the sample.

Description

The method that dioxin detects

The present invention relates to the detection of dioxin pollution thing, more specifically relate to the biological detecting method that a kind of effective fast quantification detects the dioxin pollution thing.

Many chloros dibenzo dioxin (PCDDs) and many chloros dibenzofurans (PCDFs) because their similaritys on chemical constitution and toxicology all are collectively referred to as dioxin compounds in the world, are called for short dioxin (Dioxin).Because this compounds has the chemical stability of strong toxicity and height, makes it to become important environmental contaminants highly visible.Because nearly 210 kinds of the heterochromatic bodies of dioxin, toxicity difference is big and exist with the ultratrace level usually in environment and food, so dioxin detects the difficult point that is regarded as modern analysis.At present, analytical approach is that isotopic dilution-organic solvent extraction-multilayer chromatographic column purifying-high-resolution chromatogram mass spectrometer (HRGC-Ms) detects the most accurately and effectively.Yet, set up this cover method and not only need to buy expensive high-resolution chromatograph-mass spectrometer, but also there are problems such as tediously long, the time-consuming and cost height of sample analysis process, can't satisfy the needs of dioxin fast quantification screening in various environmental samples and the import and export food, also not meet China's national situation.Therefore, be necessary to set up that a cover tallies with the national condition and in what promote the dioxin contamination thing is carried out the method that fast quantification detects.Utilize chemical method to detect dioxin in China, its expense is minimum to be 5000 yuan/sample, uses chemical method, and a people once can only analyze 5 samples at most, and complicated operation, and required time is a week.

The purpose of this invention is to provide the method that a kind of dioxin detects, easy to implement the method, easy and simple to handle, can be fast, sensitive, detect the quantitative screening of dioxin contamination thing in food exactly.

In order to achieve the above object, the present invention adopts following technical measures:

According to the intoxicating mechanism of dioxin, promptly combine with Ah acceptor height, and the characteristics of interior certain enzyme-specific of selectivity ground inducing cell, set up the biological method of measuring dioxin under the isolated condition.But toxic equivalent by the dioxin in the vigor quantitative measurement sample of measuring enzyme, thereby the monitoring of using as dioxin contamination, in addition, with the most malicious dioxin isomeride 2,3,7,8-TCDD is a model compound, utilize typical curve that the dioxin in the sample is carried out quantitatively and with chemical method the biological detection result being confirmed, will further prove the accuracy and the reliability of this biological detecting method.

It detects step:

1. cellular incubation makes cell grow up to fine and close individual layer;

2. cell is seeded in the kit with certain density;

3. add the nutrient culture media that 100 μ l contain sample extracting solution and dioxin standard solution respectively at kit, cultivated 72 hours;

4. remove nutrient culture media, in kit, add fluorogenic substrate ERF (the different phenoxazine oxazolone of 7-ethoxy-3-) reaction 1 hour;

5. add the ethanol cessation reaction, measure the fluorescence intensity of fluorescence-causing substance RF (the different phenoxazine oxazolone of 7-hydroxyl-3-) and calculate enzyme alive;

6. draw docs-effect dioxin typical curve, and calculate the toxic concentration of dioxin compounds in the sample thus.

The present invention compared with prior art, have the following advantages and effect: easy to implement the method, simple to operate, testing cost is low, its expense only needs 100 yuan/sample, can be used for the detection of gross sample, required time is 24-72 hour, can be widely used in the fast quantification screening of dioxin contamination thing in environmental sample and the food.

Embodiment:

At first be with grind into powder after the biological sample freeze drying, take by weighing the 1-5g sample powder, extract 24 hours, with after concentrated near the doing of extract, cross a sandwich post again, collect eluent and also concentrate near doing with the toluene Soxhlet; Next is a cellular incubation, makes cell grow up to fine and close individual layer; The 3rd is that cell is seeded in the kit with certain density, adds the nutrient culture media that 100 μ l contain biological sample extract and dioxin standard in kit respectively, cultivates 72 hours; The 4th is to remove nutrient culture media, adds fluorogenic substrate A reaction 1 hour in kit; The 5th is to add reagent B cessation reaction, measures the fluorescence intensity of fluorescence-causing substance C and calculate enzyme alive: the 6th is to draw docs-effect dioxin typical curve, and by the toxic concentration that calculates dioxin compounds in the sample.

The detection of dioxin in the silver carp liver in each oxidation pond of Yan Jia lake, Hubei Ya Erhu area

Biological detecting method of the present invention is answered the detection of dioxin in the silver carp liver in each oxidation pond of Yan Jia lake, Hubei Ya Erhu area, and compare with international standard chemical process, find to utilize the result that biological method detects and the result of chemical method analysis to have good correlativity and consistance (seeing Table 1).4# pond fish liver from the 1# pond fish liver of high concentration to low concentration, concentration differs greatly, and biological detecting method can be with these dry straight reflecting, and illustrate that biological detecting method measures that dioxin has good accuracy and reliability in the biological sample.

In each oxidation pond of Yan Jia lake, table 1 Hubei Ya Erhu area in the silver carp liver dioxin

Chemical analysis and biological detection result

Sampled point	The 1# pool	The 2# pool	The 3# pool	The 4# pool
					Chemical analysis results (TEQng/kg)	??300	??111	??18.5	??8.33
Biological detection result (TEQng/kg)	??336	??105	??29.4	??18.4

Claims (1)

1, a kind of method of dioxin detection, this method comprises the following steps:

A, with grind into powder after the biological sample freeze drying, take by weighing powder, toluene Soxhlet extracting 24 hours;

B, cellular incubation make cell grow up to fine and close individual layer;

C, with cell inoculation in kit, in kit, add the nutrient culture media that 100 μ l contain sample extracting solution and dioxin respectively, cultivated 72 hours;

D, removal nutrient culture media, adding fluorogenic substrate in kit is the different phenoxazine oxazolone of 7-ethoxy-3-, reacts 1 hour;

E, adding ethanol cessation reaction, the mensuration fluorescence is the fluorescence intensity of the different phenoxazine oxazolone of 7-hydroxyl-3-and calculates enzyme and live;

F, draw docs-effect dioxin curve, calculate the toxic concentration of dioxin compounds in the sample.