CN101825630A - A kind of method that detects diarrhetic shellfish poison - Google Patents
A kind of method that detects diarrhetic shellfish poison Download PDFInfo
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- CN101825630A CN101825630A CN201010187786A CN201010187786A CN101825630A CN 101825630 A CN101825630 A CN 101825630A CN 201010187786 A CN201010187786 A CN 201010187786A CN 201010187786 A CN201010187786 A CN 201010187786A CN 101825630 A CN101825630 A CN 101825630A
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Abstract
The invention describes a kind of novel capillary electrophoresis electrochemical immune analysis method of measuring diarrhetic shellfish poison in the shellfish sample.Diarrhetic shellfish poison in the shellfish sample separates in kapillary with limited antibody competition reaction back with enzyme mark diarrhetic shellfish poison, enzyme mark diarrhetic shellfish poison after the separation and enzyme mark diarrhetic shellfish poison-antibody complex carry out the styletable Electrochemical Detection with entering electrochemical investigating pond behind the substrate reactions respectively in reaction tube, calculate the content of diarrhetic shellfish poison in the shellfish sample according to the reduction degree at enzyme mark diarrhetic shellfish poison-antibody complex peak.This method has been simplified the sample preparation process, and selectivity is good, and the accuracy height is the Perfected process that detects diarrhetic shellfish poison in the shellfish sample.
Description
Technical field
The present invention relates to capillary electrophoresis technique, specifically a kind of capillary electrophoresis electrochemical immune analysis method that detects diarrhetic shellfish poison in the shellfish sample.
Background technology
In recent years, increasing marine environmental pollution causes the quantity and the scale of harmful algal outburst constantly to increase, not only havoc marine fishery resources and the aquaculture of a large amount of red-tide toxins that produce by red tide plankton or shellfish poison, deterioration marine environment, and can be tired by the food chain lamination that makes progress, be detrimental to health.
Diarrhetic shellfish poison comprises 15 kinds of compounds at least mainly from dinoflagellate fin Trentepohlia and Prorocentrum biology, has wherein determined structure for 13 kinds.Diarrhetic shellfish poison is the derivative of fatty acid with a plurality of cyclic ethers, is unsaturated hydrocarbon, and its typical chemical structural formula as depicted in figs. 1 and 2.
Diarrhetic shellfish poison can be divided three classes:
1) polyethers toxin: as okadaic acid and derivant dinophysistoxin-1 thereof, dinophysistoxin-2, dinophysistoxin-3;
2) big acyclic polyether lactone toxin: as scallop toxin and derivant thereof;
3) merge the polyethers toxin: as the Patinopecten yessoensis toxin etc.
In these compounds, the toxicity of okadaic acid and dinophysistoxin-3 is the strongest.Diarrhetic shellfish poison is distributed widely in global littoral sea, can be at the shellfish cylinder accumulation, by the edible back of people produce diarrhoea, vomiting, feel sick, toxicity symptoms such as stomachache and headache, also have the chronic effect of TPA.At present, there are strict requirements to the content of diarrhetic shellfish poison in many countries, and as Canada's regulation, the content of the diarrhetic shellfish poison in the food must not surpass 200ng/g.
The diarrhetic shellfish poison classes of compounds is various, and chemical property and complex structure, the kind of determination and analysis shellfish poison and content be difficulty relatively.The shellfish poison detection method of generally using in the world is biological mouse method, high performance liquid chromatography and Immunological Method at present.Biological mouse method is the diarrhetic shellfish poison that at high temperature extracts algae or Bei Tizhong with hydrochloric acid, then mouse is carried out lumbar injection, according to the death time judgement toxicity size of mouse; But in leaching process, might cause the thaumatropy of shellfish poison compound and change toxicity, and have that error is big, poor reproducibility, shortcoming that comparability is low.High performance liquid chromatography is the method the most accurately and effectively of present Analysis and Identification shellfish poison, its principle be with the shellfish tissue after pre-treatment, separate with high performance liquid chromatography, the connexus spectrometer is finished structure and is identified and content analysis; Shortcoming is the pre-treatment trouble, instrument and analytical reagent costliness, and complicated operation needs special analytical technology personnel.Immunological Method is the method for at present popular simple fast detecting diarrhetic shellfish poison, utilize the association reaction of antibody to diarrhetic shellfish poison, make detection kit according to enzyme-linked immune analytic method, the analyzing and testing that is used for diarrhetic shellfish poison, this method is easy fast, and used instrument is simple, be easy to be applied to field monitoring, and domestic finished product kit on sale, but detection sensitivity is lower, and reagent costs an arm and a leg.
Capillary Electrophoresis is one of marine biotoxins separation detection technology that grew up in recent years, plays a significant role in the compartment analysis of saxitoxin so that it is quick, sensitive, the consumption sample size is few.Interfering material is many in the shellfish sample matrices, and the analysis to diarrhetic shellfish poison often causes interference.Can get rid of interference by capillary electrophoresis separation, electrochemical enzymatic linked immune analysis, selectively detect at the target toxin.But so far, device and the detection method based on the detection diarrhetic shellfish poison of capillary electrophoresis electrochemical enzyme-linked immuno assay yet there are no report.
Summary of the invention
The purpose of this invention is to provide a kind of method that detects diarrhetic shellfish poison, this method has been simplified the sample preparation process, running program is simple, selectivity good, sensitivity is quick, is specially adapted to the fast detecting of diarrhetic shellfish poison in the shellfish sample.
For achieving the above object, the technical solution used in the present invention is:
A kind of method that detects diarrhetic shellfish poison in the shellfish sample, operate as follows:
(1) shellfish sample preparation: shellfish is removed shell, and shellfish meat embathes, after the homogenate, transfer pH with hydrochloric acid, centrifugal, the supernatant that inclines adds methanol extraction, collect upper layer of extraction liquid, reduction vaporization extract to methyl alcohol volatilization finishes, with lauryl sodium sulfate micellar solution dissolving precipitate;
(2) in the shellfish sample solution of above-mentioned processing, add excessive enzyme mark diarrhetic shellfish poison and a certain amount of antibody and hatch reaction;
(3) in separation capillary, be divided into different district's bands behind the above-mentioned hatching reaction mixture sample introduction, enter in turn in the reaction kapillary, hydrogen peroxide oxidation substrate on enzyme mark diarrhetic shellfish poison-antibody complex and the residual enzyme mark diarrhetic shellfish poison in the enzymatic damping fluid of mark, generation has the material of electrochemical activity, enters electrochemical investigating pond and detects.
Described damping fluid is the BR damping fluid, by phosphoric acid, acetic acid, that boric acid adds superoxol is formulated.
Described substrate is an o-aminophenol; Described enzyme is a horseradish peroxidase.
Advantage of the present invention:
1, the present invention combines Capillary Electrophoresis, Electrochemical Detection and immunoassay, both had the high separating efficiency of Capillary Electrophoresis, the high sensitivity of electrochemical analysis, the high selectivity and the selectivity that have immunoassay again, be used for the detection of shellfish sample diarrhetic shellfish poison, needing in the shellfish sample pre-treatments to avoid the loaded down with trivial details program of ion exchange column, simplified operation steps.
2, the capillary electrophoresis electrochemical enzyme-linked immunosorbent assay is applied to the detection of diarrhetic shellfish poison, foreshorten to a few minutes by the routine immunization analysis more than 1 day detection time, and easy and simple to handle quick, reagent consumption is few.
3, utilize immunoreactive selectivity, the diarrhetic shellfish poison immune system of different structure after capillary electrophoresis separation, can be realized that the capillary electrophoresis electrochemical enzyme-linked immunosorbent assay measures multiple diarrhetic shellfish poison compound simultaneously.
Description of drawings
Fig. 1 is the chemical constitution of diarrhetic shellfish poison acid ingredient, okadaic acid: R
1=H, R
2=CH
3, R
3=H, dinophysistoxin-1:R
1=H, R
2=CH
3, R
3=CH
3, dinophysistoxin-2:R
1=H, R
2=H, R
3=CH
3, dinophysistoxin-3:R
1=acyl group, R
2=CH
3, R
3=CH
3
Fig. 2 is the chemical constitution of diarrhetic shellfish poison neutral compound, clam toxin-1:R=CH
2OH, clam toxin-2:R=CH
3, clam toxin-3:R=CHO, clam toxin-6:R=COOH;
Fig. 3 is the Capillary Electrophoresis spectrogram of enzyme in the one embodiment of the invention+mark diarrhetic shellfish poison;
Fig. 4 is the Capillary Electrophoresis spectrogram that excessive enzyme is marked diarrhetic shellfish poison and enzyme mark diarrhetic shellfish poison-antibody complex in the one embodiment of the invention, peak 1 is the electrophoresis peak that excessive enzyme mark diarrhetic shellfish poison catalytic substrate forms, and peak 2 is the enzyme mark electrophoresis peak that diarrhetic shellfish poison-the antibody complex catalytic substrate forms;
Fig. 5 be in the one embodiment of the invention diarrhetic shellfish poison in the shellfish sample and enzyme mark diarrhetic shellfish poison with the reacted Capillary Electrophoresis spectrogram of a certain amount of antibody competition.
Embodiment
Below, its role is to further illustrate content of the present invention, the reader is more readily understood, but does not constitute qualification or restriction the protection domain of requirement of the present invention for implementing concrete example of the present invention.
Experiment condition:
MPI-A type Capillary Electrophoresis numerical control high voltage power supply, MPI-A type capillary electrophoresis electrochemical analyser (Xi'an Rui Mai Analytical Instrument Co., Ltd), three-electrode system: platinum dish working electrode, platinum filament auxiliary electrode and silver/silver chloride contrast electrode, PHS-3D type pH meter (Shanghai thunder magnetic scientific instrument company).O-aminophenol, phosphoric acid, boric acid, acetic acid, NaOH, horseradish peroxidase (the full bio tech ltd of Shanghai snow), diarrhetic shellfish poison diagnostic kit (the U.S., Abraxis company), other reagent are pure for analyzing, all damping fluids and sample solution all need filter with 0.22 μ m miillpore filter before use, and are facing with before carrying out the ultrasonic degas processing.
Specific operation process:
Shellfish (scallop or oyster) is removed shell, and the meat distilled water of shellfish embathes 3 times, homogenate 10min, the ultrasonic 10min of ice-water bath, transfer to pH 3.4～4.0 with 6mol/L hydrochloric acid, centrifugal 5min, supernatant liquor inclines, adding methyl alcohol stirs evenly, ultrasonic extraction 5min, centrifugal 5min collects upper layer of extraction liquid, reduction vaporization extract to methyl alcohol volatilization finishes under 35～50 ℃ of water-baths, with lauryl sodium sulfate micellar solution dissolving precipitate;
In the shellfish sample solution of above-mentioned processing, add excessive enzyme mark diarrhetic shellfish poison and a certain amount of antibody, at 37 ℃ of hatching reaction 30min, the diarrhetic shellfish poison in the shellfish sample and a certain amount of antibody of enzyme mark diarrhea sheufish-poison competitive:
Diarrhetic shellfish poison+enzyme mark diarrhetic shellfish poison (excessive)+antibody (a certain amount of)=diarrhetic shellfish poison-antibody complex+enzyme mark diarrhetic shellfish poison-antibody complex+residual enzyme mark diarrhetic shellfish poison;
Above-mentioned hatching reaction mixture is placed the capillary inlet end, add 14kV voltage at the separation capillary two ends, behind the electrokinetic injection 5s, diarrhetic shellfish poison-antibody complex in the mixed liquor, enzyme mark diarrhetic shellfish poison-antibody complex and residual enzyme mark diarrhetic shellfish poison do not coexist according to migration rate and are divided into different district's bands in the separation capillary and enter in turn in the reaction kapillary, in the reaction kapillary, be marked at the hydrogen peroxide oxidation substrate o-aminophenol in the horseradish peroxidase enzyme catalytic damping fluid on the diarrhetic shellfish poison, generation has the amino phenoxazine of material 3-of electrochemical activity, enters electrochemical investigating pond and detects;
Enzyme mark diarrhetic shellfish poison-antibody complex is different with the concentration that residual enzyme is marked the horseradish peroxidase on the diarrhetic shellfish poison, the concentration that catalyzing hydrogen peroxide oxidation o-aminophenol generates the amino phenoxazine of oxidation product 3-is just different, produce different electrochemical signals, can carry out qualitative and quantitative analysis to the diarrhetic shellfish poison that enzyme is marked in diarrhetic shellfish poison-antibody complex and the shellfish sample thus.
Its experimental result:
As shown in Figure 3, enzyme mark diarrhetic shellfish poison to cathodic migration, enters electrochemical investigating pond and detects under the 14kV high-voltage electric field from capillary inlet end sample introduction, and the electrophoresis peak appears near the 100s.
As shown in Figure 4, excessive enzyme mark diarrhetic shellfish poison and antibody-solutions are at 37 ℃ of hatching reaction 30min, form enzyme mark diarrhetic shellfish poison-antibody complex, detect carrying out electrophoretic separation behind the mixed solution 14kV electrokinetic injection 5s, peak 1 is the electrophoresis peak that excessive enzyme mark diarrhetic shellfish poison catalytic substrate forms, peak 2 is the enzyme mark electrophoresis peak that diarrhetic shellfish poison-the antibody complex catalytic substrate forms, and two electrophoresis peak-to-peak shapes are better, can reach baseline separation.
As shown in Figure 5, in above-mentioned excessive enzyme mark diarrhetic shellfish poison and antibody mixed solution, add the scallop sample, diarrhetic shellfish poison in the scallop sample is with a certain amount of antibody of enzyme mark diarrhea sheufish-poison competitive, generate unlabelled diarrhetic shellfish poison-antibody complex, make the concentration of hatching enzyme mark diarrhetic shellfish poison-antibody complex in the mixed solution of back reduce, excessive enzyme mark diarrhetic shellfish poison concentration increases, therefore, compare with Fig. 4, the electrophoresis peak (peak 1) of enzyme mark diarrhetic shellfish poison raises among Fig. 5, the electrophoresis peak (peak 2) of enzyme mark diarrhetic shellfish poison-antibody complex reduces, and illustrates in the scallop sample and contains diarrhetic shellfish poison.
Claims (4)
1. method that detects diarrhetic shellfish poison in the shellfish sample is characterized in that:
(1) shellfish sample preparation: the shellfish sample is removed shell, the meat distilled water of shellfish embathes, homogenate, transfer to pH 2.0～5.0 with hydrochloric acid, centrifugal, supernatant liquor inclines, add methanol extraction, collect upper layer of extraction liquid, reduction vaporization extract to methyl alcohol volatilization finishes under hot bath, with lauryl sodium sulfate micellar solution dissolving precipitate;
(2) in the shellfish sample of above-mentioned processing, add excessive enzyme mark diarrhetic shellfish poison and a certain amount of antibody and hatch reaction;
(3) in separation capillary, be divided into different district's bands behind the above-mentioned hatching reaction mixture sample introduction, enter in turn in the reaction kapillary, hydrogen peroxide oxidation substrate on enzyme mark diarrhetic shellfish poison-antibody complex and the residual enzyme mark diarrhetic shellfish poison in the enzymatic damping fluid of mark, generation has the material of electrochemical activity, enters electrochemical investigating pond and detects.
2. according to the described method that detects diarrhetic shellfish poison in the shellfish sample of claim 1, it is characterized in that: described damping fluid is the BR damping fluid, its compound method is: get phosphoric acid, acetic acid, boric acid dissolved dilution, with adding the superoxol constant volume behind the NaOH accent pH.
3. according to the method for diarrhetic shellfish poison in the described detection of the claim 1 shellfish sample, it is characterized in that: described substrate is an o-aminophenol.
4. according to the method for diarrhetic shellfish poison in the described detection of the claim 1 shellfish sample, it is characterized in that: described enzyme is a horseradish peroxidase.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108949848A (en) * | 2018-08-08 | 2018-12-07 | 浙江海洋大学 | A method of it is fermented using marine bacteria and prepares sponge acid |
| CN110736777A (en) * | 2019-09-19 | 2020-01-31 | 江南大学 | electrochemical-ELISA immunosensor based on rolling circle amplification DNA enzyme and covalent organic framework |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1892225A (en) * | 2005-07-06 | 2007-01-10 | 国家海洋环境监测中心 | Enzymelinked immunity detection reagent-case of red-tide toxin diarrhea shellfish poison |
| CN1979169A (en) * | 2005-12-05 | 2007-06-13 | 曹际娟 | Diarrhea sheufish-poison competitive enzyme-linked immune quantitative detection reagent box, its preparation and use |
| CN101131390A (en) * | 2007-10-15 | 2008-02-27 | 国家海洋环境监测中心 | Immune colloidal gold test paper strip for fast detecting diarrhetic shellfish poison and method for making the same |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1892225A (en) * | 2005-07-06 | 2007-01-10 | 国家海洋环境监测中心 | Enzymelinked immunity detection reagent-case of red-tide toxin diarrhea shellfish poison |
| CN1979169A (en) * | 2005-12-05 | 2007-06-13 | 曹际娟 | Diarrhea sheufish-poison competitive enzyme-linked immune quantitative detection reagent box, its preparation and use |
| CN101131390A (en) * | 2007-10-15 | 2008-02-27 | 国家海洋环境监测中心 | Immune colloidal gold test paper strip for fast detecting diarrhetic shellfish poison and method for making the same |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108949848A (en) * | 2018-08-08 | 2018-12-07 | 浙江海洋大学 | A method of it is fermented using marine bacteria and prepares sponge acid |
| CN108949848B (en) * | 2018-08-08 | 2021-08-20 | 浙江海洋大学 | Method for preparing sponge acid by using marine bacteria fermentation |
| CN110736777A (en) * | 2019-09-19 | 2020-01-31 | 江南大学 | electrochemical-ELISA immunosensor based on rolling circle amplification DNA enzyme and covalent organic framework |
| CN110736777B (en) * | 2019-09-19 | 2020-12-01 | 江南大学 | electrochemical-ELISA immunosensor based on rolling circle amplification DNA enzyme and covalent organic framework |
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