CN1731150B - A biochip method for detecting dioxin-type chemical species - Google Patents
A biochip method for detecting dioxin-type chemical species Download PDFInfo
- Publication number
- CN1731150B CN1731150B CN 200510019330 CN200510019330A CN1731150B CN 1731150 B CN1731150 B CN 1731150B CN 200510019330 CN200510019330 CN 200510019330 CN 200510019330 A CN200510019330 A CN 200510019330A CN 1731150 B CN1731150 B CN 1731150B
- Authority
- CN
- China
- Prior art keywords
- probe
- chip
- dioxin
- chemical species
- ahr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Images
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a method for detecting the biology chip of the dioxin category chemical substance. It comprises the following steps: first fixing a probe with DRE sequence on the end Cy5 fluorescence label, adding the cell liquid from the small mouse hepatocyte and the mixed liquid of 2,3,7,8-TCDD, then the cell liquid with AhR/ARNT protein can be combined with the DRE sequence, adding T7cutting enzyme so that the combination of AhR/ARNT protein and DRE sequence can hamper the digest of mononucleotide of the end Cy5 fluorescence label, so that the content of 2,3,7,8-TCDD has positiverelativity with the luminescence intensity, at last measuring the luminescence value of the Cy5 luminescence module and ascertaining the content of the cutting enzyme of the sample according to the luminescence value.
Description
Technical field
The present invention relates to environmental chemistry and analyze biological technical field, be specifically related to a kind of method of utilizing the biochip test dioxin-type chemical species.
Background technology
Dioxin-type chemical species extensively exists in environment, environmental contaminants for high anelasticity and high biological concentration, high because of its toxicity, be subjected to showing great attention to of environment circle in recent years always, the research of its detection method has also been become the advanced subject in current Environmental Studies field.The monitoring of dioxin-type chemical species belongs to the trace multicomponent analysis, requires detection method must have higher sensitivity.Because high-resolution gas chromatography mass spectrometry method has characteristics such as high selectivity, high resolving power and high sensitivity, has been acknowledged as the standard method of analysis of dioxin-type chemical species in recent years.Yet, set up this method, need to buy high-resolution gas chromatograph-mass spectrometer and a series of isotope standard, and sample separates, purifying step is loaded down with trivial details, making this be operated in most of laboratories can not carry out, the needs of be difficult to conform supervision and management and monitoring.
Toxicologic study shows, that most of dioxin and analogue thereof all have is carcinogenic, teratogenesis and mutagenicity.They belong to non-genomic toxicity carcinogenic substance from intoxicating mechanism, all to (the aryl hydrocarbon receptor of the aromatic compound receptor protein in the biosome, be abbreviated as AhR) have the height affinity, thereby can selectivity ground in conjunction with specific dna sequence dna and induce the specific gene of body to express.Developed detection specific gene expression product and AhR receptor activation degree such as has directly been detected at bioanalytical method according to this principle.Yet these bioassay methods that adopt cell or animal to cultivate still need certain condition, and incubation time reaches 24-72 hour simultaneously, and whole process reaches several days, can not carry out fast, detect in a large number.
The fluorescence signal detection method is to use maximum chip signal detection methods, and common fluorescent scanning instrument can detect 10 in the zone of 100 microns of radiuses
-18The mol fluorescence molecule meets the requirement that the high-throughout chip signal of high density detects.Utilize the superiority of this technology,, make a kind of fluoroscopic examination chip methods that can be used for a large amount of fast detection dioxin-type chemical species of development become possibility in conjunction with the result of toxicologic study.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing the biochip test dioxin-type chemical species, for providing, environmental monitoring department accurately detects dioxin-type chemical species fast and effectively, to form the multi-level dioxin-type chemical species monitoring system of replenishing mutually with chemical analysis.
In order to achieve the above object, the present invention takes following technical measures: mammiferous aromatic compound receptor protein (aryl hydrocarbon receptor, be abbreviated as AhR) be present in the cell cytosol, can be activated by the dioxin in the environment and analogue thereof, in conjunction with after be transferred to nuclear, move the factor (ARNT) protein combination with the AhR consideration convey, formation can specificity in conjunction with one section special dna sequence dna---the AhR/ARNT heterodimer of Dioxin-responsiveelement (DRE) sequence.According to this principle, in conjunction with the T7 nucleic acid polymerase can catalytic elimination 5 ' mononucleotide characteristic, mark cyanine dye molecule (Cy5) in the DRE sequence, combine with DRE in the presence of dioxin by AhR and ARNT albumen and to hinder of the degraded of T7 nucleic acid polymerase the dna probe that is marked with Cy5, make what of fluorescence intensity be directly proportional, detect the content of dioxin-type chemical species with the content of dioxin-type chemical species.The steps include:
1, the probe that contains the DRE sequence designs sulfydryl modification respectively and Cy5 fluorescence is modified: sequence be respectively 5 ' (SH)-TTTTTTTTTTGGCTCTTCTCACGCAACTCCG-3 ', 5 '-CGGAGTTGCGTGAGAAGAGCC (Cy5)-3 '.
2, the annealing of probe: above-mentioned dna probe is dissolved in the ultrapure water (MiliQ), and final concentration is 10-20 μ M.Handle 5-10min for 90-96 ℃, behind the 50-60 ℃ of annealing 20-30min, dilution is used.
3, probe is fixing: the probe after the annealing adds the slide that sulfhydrylation is modified, the specific action that utilizing between sulfydryl and the sulfydryl interacts forms disulfide bond probe is spent the night under 12-16 ℃ or 35-37 ℃ one hour fixing, form probe array, promptly obtain needed chip.
4, extract the Balb/c mouse liver cytosol that contains AhR and ARNT receptor protein.
5, with Balb/c mouse liver cytosol and each concentration (50,10,5,1,0.5,0.1pg/ μ l) 2,3,7, the 8-tetrachloro is for dibenzo-right-dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, be abbreviated as 2,3,7,8-TCDD) mix, 16-22 ℃ of lucifuge effect is after 1-2 hour, with sample and HEDGK buffer (25mM HEPES, 1mM EDTA, 1mM DTT, 10%Glycerol, 180mM KCl) mix, adding is fixed with on the slide of probe, and 20-25 ℃ of lucifuge reacted 1-2 hour.
6, the washing of chip: with solution TNT (10mM Tris-HCL, pH7.5; 150mM NaCl, 0.05%Tween20) room temperature (20-25 ℃) lucifuge rinsing chip is 5 minutes, uses solution TNW (10mM Tris-HCL, pH7.5 again; 150mM NaCl) room temperature (20-25 ℃) lucifuge rinsing chip twice, the protein of flush away non-specific binding, air-dry then slide.
7, at the endonuclease reaction of sheet: add the T7 exonuclease on the conversion zone at chip, 20 ℃ of lucifuge effects 4 hours.
8, the washing of chip: with solution TNT (10mM Tris-HCL, pH7.5; 150mM NaCl, 0.05%Tween20) room temperature (20-25 ℃) lucifuge rinsing chip is 5 minutes, uses solution TNW (10mM Tris-HCL, pH7.5 again; 150mM NaCl) room temperature (20-25 ℃) lucifuge rinsing chip twice, the protein of flush away non-specific binding, air-dry then slide.
9, the fluoroscopic examination of Cy5: the fluorescent value that adopts GenePix 4000B fluorescent scanning instrument (U.S. Axon company) detection chip, the existence of dioxin-type chemical species makes AhR/ARNT albumen combine with probe and hinders the digestion to the mononucleotide of terminal Cy5 mark of T7 exonuclease, thereby the power of fluorescent value and dioxin-type chemical species content is how much directly related, and further determines the content of dioxin in the testing sample by the numerical value of fluorescence intensity.To 2,3,7, the sensing range of 8-TCDD is 0.1pg/ μ l to 10pg/ μ l.
The present invention compares with other dioxin-type chemical species detection techniques, have the following advantages and effect: hinder of the degraded of T7 exonuclease when utilizing AhR and ARNT albumen in the presence of dioxin-type chemical species, to combine to the DRE sequence with DRE, detect the content of dioxin-type chemical species by the intensity of Cy5 fluorescent value, should have better detection sensitivity based on the detection specific gene expression product of the mechanism of toxication development of dioxin and analogue thereof and the method for deriving thereof equally than what extensively adopt now, and simple to operate, required time shortens greatly.Adopt the high sensitivity of fluorescence detection method and the high flux data production characteristic of chip in addition, can develop into the screening that is suitable for a large amount of samples and the dioxin-type chemical species biological detecting method of the supervision and management under the specified conditions.Therefore, that bioassay method has is quick, easy, detect characteristics such as cost is low, and the detection sensitivity height, is very suitable for the screening of a large amount of samples and the supervision and management under the specified conditions, and application prospect is extensive.
Description of drawings
Fig. 1. at stationary probe is 20fmol/dot, under the excessive situation of AhR/ARNT, detects 2,3,7 of variable concentrations, and 8-TCDD content is to the influence of fluorescent value.Presentation of results is as follows: each sample repeats two points, 1, DMSO negative control; 2, positive control; 3-8 is respectively 2,3,7 of a variable concentrations, and the 8-TCDD test result of samples is followed successively by 50,10,5,1,0.5,0.1pg/ μ l.The point sample amount is every 0.2 μ l.The result shows that do not add 2,3,7, the point of 8-TCDD sample does not almost have fluorescence, is degraded fully by the T7 exonuclease; Add 2,3,7, the point of 8-TCDD has fluorescence, illustrates 2,3,7, and the existence of 8-TCDD can make AhR/ARNT albumen combine with probe really and hinder the degraded of T7 exonuclease to probe; Along with being added to 2,3,7 on the reflecting point, the increase of 8-TCDD content, the brightness increase of point.
Fig. 2. at stationary probe is 20fmol/dot, under the excessive situation of AhR/ARNT, in fluorescence probe intensity and the sample to be checked 2,3,7, the corresponding relation of 8-TCDD concentration.The result shows, fluorescence probe intensity is along with in the sample to be checked 2,3,7, the increase of 8-TCDD concentration and strengthening, and 2,3,7, the concentration of 8-TCDD reaches capacity when reaching 50pg/ μ l.Explanation is 20fmol/dot at stationary probe, and under the excessive situation of AhR/ARNT, to 2,3,7, the upper limit of detection of 8-TCDD is to 50pg/ μ l.
Fig. 3. utilize fluorescence probe in the sample 2,3,7, the linearity curve of 8-TCDD content detection.The result shows, is 20fmol/dot at stationary probe, and under the excessive situation of AhR/ARNT, fluorescence probe can be to 2,3,7, and the scope that the content of 8-TCDD detects is 0.1pg/ μ l to 10pg/ μ l.
Embodiment
A kind of method of utilizing the biochip test dioxin-type chemical species may further comprise the steps:
1, the probe that contains the DRE sequence designs sulfydryl modification respectively and Cy5 fluorescence is modified: sequence be respectively 5 ' (SH)-TTTTTTTTTTGGCTCTTCTCACGCAACTCCG-3 ', 5 '-CGGAGTTGCGTGAGAAGAGCC (Cy5)-3 '.The purpose of sulfydryl modification is to make probe can utilize the specific action between sulfydryl to be fixed on the slide of sulfydryl modification.
2, the annealing of probe: above-mentioned dna probe is dissolved in the MiliQ water, and final concentration is 10-20 μ M.Handle 5-10min for 90-96 ℃, behind the 50-60 ℃ of annealing 20-30min, dilution is used.
3, probe is fixing: the probe after the annealing adds the slide that sulfhydrylation is modified, utilize between sulfydryl and the sulfydryl specific action that forms disulfide bond that probe is spent the night under 12-16 ℃ or 35-37 ℃ one hour fixing, form probe array, promptly obtain needed chip.
4, extract the Balb/c mouse liver cytosol that contains AhR and ARNT receptor protein.
5, with Balb/c mouse liver cytosol and each concentration (50,10,5,1,0.5,0.1pg/ μ l) 2,3,7, the 8-tetrachloro is for dibenzo-right-dioxin (2,3,7,8-tetrachlorodibenzo-p-dioxin, be abbreviated as 2,3,7,8-TCDD) mix, 16-22 ℃ of lucifuge effect is after 1-2 hour, with sample and HEDGK buffer (25mM HEPES, 1mMEDTA, 1mM DTT, 10%Glycerol, 180mM KCl) mix, adding is fixed with on the slide of probe, and 20-25 ℃ of lucifuge reacted 1-2 hour.
6, the washing of chip: with solution TNT (10mM Tris-HCL, pH7.5; 150mM NaCl, 0.05%Tween20) room temperature (20-25 ℃) lucifuge rinsing chip is 5 minutes, uses solution TNW (10mMTris-HCL, pH7.5 again; 150mM NaCl) room temperature (20-25 ℃) lucifuge rinsing chip twice, the protein of flush away non-specific binding, air-dry then slide.
7, at the endonuclease reaction of sheet: add the T7 exonuclease on the conversion zone at chip, 20 ℃ of lucifuge effects 4 hours.
8, the washing of chip: with solution TNT (10mM Tris-HCL, pH7.5; 150mM NaCl, 0.05%Tween20) room temperature (20-25 ℃) lucifuge rinsing chip is 5 minutes, uses solution TNW (10mMTris-HCL, pH7.5 again; 150mM NaCl) room temperature (20-25 ℃) lucifuge rinsing chip twice, the protein of flush away non-specific binding, air-dry then slide.
9, the fluoroscopic examination of Cy5: adopt the fluorescent value of GenePix 4000B fluorescent scanning instrument (U.S. Axon company) detection chip, and further determine the content of dioxin in the testing sample by the numerical value of fluorescence intensity.To 2,3,7, the sensing range of 8-TCDD is 0.1pg/ μ l to 10pg/ μ l.
10, interpretation of result: fluorescence intensity is specifically along with in the sample to be checked 2,3,7, the increase of 8-TCDD concentration and strengthen (Fig. 1,2),, and 2,3,7,8-TCDD concentration is to have better linearity (Fig. 3) in 0.1pg/ μ l to the 10pg/ μ l scope, therefore can determine the content of dioxin-type chemical species in the testing sample according to the Strength Changes of fluorescent value.
Claims (1)
1. method of utilizing the biochip test dioxin-type chemical species, it comprises the following steps:
A, the probe that contains the DRE sequence design sulfydryl modification respectively and Cy5 fluorescence is modified: sequence be respectively 5 ' (SH)-TTTTTTTTTTGGCTCTTCTCACGCAACTCCG-3 ', 5 '-CGGAGTTGCGTGAGAAGAGCC (Cy5)-3 ';
The annealing of B, probe: A step probe is dissolved in the ultrapure water, and final concentration is 10-20 μ M, handles 5-10 minute, 50-60 ℃ annealing after 20-30 minute for 90-96 ℃, and dilution is used;
C, probe fixing: the probe after the annealing adds the slide that sulfhydrylation is modified, utilize the specific action that forms disulfide bond between sulfydryl and the sulfydryl that probe is spent the night under 12-16 ℃ or 35-37 ℃ one hour fixing, form probe array, obtain needed chip;
D, extraction contain the Balb/c mouse liver cytosol of AhR and ARNT receptor protein;
E, be 50,10,5,1,0.5 with Balb/c mouse liver cytosol and concentration, 2,3,7 of 0.1pg/ μ l, the 8-tetrachloro mixes for dibenzo-right-dioxin, adds to be fixed with on the slide of probe 20-25 ℃ of lucifuge reaction 1-2 hour;
The washing of F, chip: use solution TNT lucifuge rinsing at room temperature chip 5 minutes, use solution TNW lucifuge rinsing chip twice under room temperature again, the protein of flush away non-specific binding, air-dry then slide;
G, at the endonuclease reaction of sheet: add the T7 exonuclease on the conversion zone at chip, 20 ℃ of lucifuge effects 4 hours;
The washing of H, chip: identical with the F step;
The fluoroscopic examination of I, Cy5: the fluorescent value that adopts GenePix 4000B fluorescent scanning instrument detection chip, the existence of dioxin-type chemical species makes AhR/ARNT albumen combine with probe and hinders the digestion to the mononucleotide of terminal Cy5 mark of T7 exonuclease, and determine the content of dioxin in the testing sample by the numerical value of fluorescence intensity, to 2,3,7, the sensing range of 8-TCDD is 0.1pg/ μ l to 10pg/ μ l.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510019330 CN1731150B (en) | 2005-08-23 | 2005-08-23 | A biochip method for detecting dioxin-type chemical species |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 200510019330 CN1731150B (en) | 2005-08-23 | 2005-08-23 | A biochip method for detecting dioxin-type chemical species |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1731150A CN1731150A (en) | 2006-02-08 |
| CN1731150B true CN1731150B (en) | 2010-04-28 |
Family
ID=35963546
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 200510019330 Expired - Fee Related CN1731150B (en) | 2005-08-23 | 2005-08-23 | A biochip method for detecting dioxin-type chemical species |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1731150B (en) |
Families Citing this family (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101957319B (en) * | 2010-07-22 | 2011-11-09 | 合肥学院 | Chemical preparation method of CaMoO4: Tb3+fluorescent probe for detecting trace amount of TNT (Tri-Nitro-Toluene) |
| CN102520154B (en) * | 2011-12-08 | 2014-01-08 | 环境保护部华南环境科学研究所 | Method for detecting dioxins in environment |
| CN102643921B (en) * | 2012-05-09 | 2014-11-05 | 湖南大学 | Fluorescent biosensing method for analyzing and detecting mutual action of organic micromolecules and combined protein based on T7 exonuclease inhibition |
| CN104048947B (en) * | 2014-05-23 | 2017-02-22 | 孟令启 | Method for detecting dioxin content in food |
| CN108444960B (en) * | 2018-02-09 | 2020-04-10 | 清华大学 | Fluorescence correlation spectrum detection method |
| CN109212187B (en) * | 2018-09-06 | 2022-01-11 | 东华大学 | ELISA-like kit for detecting polycyclic aromatic hydrocarbons and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19811327A1 (en) * | 1998-03-16 | 1999-09-30 | Karlsruhe Forschzent | Bioassay for compounds that bind to Ah receptors, especially dioxins and related compounds |
| CN1341859A (en) * | 2000-09-07 | 2002-03-27 | 中国科学院水生生物研究所 | Dioxian detection method |
| CN1548945A (en) * | 2003-05-15 | 2004-11-24 | 中国科学院生态环境研究中心 | Biological measuring method for dioxins compound in monitoring environment samples |
| EP1508793A1 (en) * | 2002-05-28 | 2005-02-23 | National Institute of Advanced Industrial Science and Technology | Method for measuring trace amount of material by use of quarts resonator |
-
2005
- 2005-08-23 CN CN 200510019330 patent/CN1731150B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19811327A1 (en) * | 1998-03-16 | 1999-09-30 | Karlsruhe Forschzent | Bioassay for compounds that bind to Ah receptors, especially dioxins and related compounds |
| CN1341859A (en) * | 2000-09-07 | 2002-03-27 | 中国科学院水生生物研究所 | Dioxian detection method |
| EP1508793A1 (en) * | 2002-05-28 | 2005-02-23 | National Institute of Advanced Industrial Science and Technology | Method for measuring trace amount of material by use of quarts resonator |
| CN1548945A (en) * | 2003-05-15 | 2004-11-24 | 中国科学院生态环境研究中心 | Biological measuring method for dioxins compound in monitoring environment samples |
Non-Patent Citations (2)
| Title |
|---|
| 黎雯，徐盈，吴文忠.利用离体大白鼠肝癌细胞的EROD 诱导指示二恶英的复合毒性效应.动物学报47 1.2001,47(1),64-70. |
| 黎雯，徐盈，吴文忠.利用离体大白鼠肝癌细胞的EROD 诱导指示二恶英的复合毒性效应.动物学报47 1.2001,47(1),64-70. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1731150A (en) | 2006-02-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Sittampalam et al. | High-throughput screening: advances in assay technologies | |
| US6511815B1 (en) | Method for quantitating competitive binding of molecules to proteins utilizing fluorescence polarization | |
| CN1731150B (en) | A biochip method for detecting dioxin-type chemical species | |
| US11169152B2 (en) | Function-based probes for environmental microbiome analysis and methods of making and using the same | |
| CN102031284B (en) | A kind of lead ion detection chip based on picodna enzyme, making and using method | |
| CN108660191A (en) | A kind of digitlization multiple nucleic acid detection method based on coding microball reactor | |
| Cronk et al. | High-throughput screening | |
| Zhang et al. | Advances in CE‐mediated microanalysis: An update | |
| Li et al. | Targeted quantification of functional enzyme dynamics in environmental samples for microbially mediated biogeochemical processes | |
| CN110658270B (en) | Method for quantitatively analyzing interaction between small molecule and protein kinase | |
| Yang et al. | A new label-free fluorescent sensor for human immunodeficiency virus detection based on exonuclease III-assisted quadratic recycling amplification and DNA-scaffolded silver nanoclusters | |
| Hajare et al. | Review on: High-throughput screening is an approach to drug discovery | |
| Ghosh et al. | Discriminating lineages of Batrachochytrium dendrobatidis using quantitative PCR | |
| CN112147114A (en) | Method for determining interaction of compound and target in living cell by using fluorescence labeling compound | |
| Zhou et al. | Multiplex detection of quality indicator molecule targets in urine using programmable hairpin probes based on a simple double-T type microchip electrophoresis platform and isothermal polymerase-catalyzed target recycling | |
| Raddaoui et al. | Dendrimer‐Based Signal Amplification of Click‐Labelled DNA in Situ | |
| CN103163010A (en) | Method for relative quantitative analysis of protein phosphorylation modification level | |
| Barra et al. | Integrated approaches for detecting the occurrence and effects of endocrine disrupting substances in surface waters | |
| CN1304600C (en) | Method for high-flux detecting environmental dioxin-like chemical substances | |
| Kim et al. | Recent methodological advances towards single-cell proteomics | |
| Ford et al. | Label-free quantitative phosphoproteomics for algae | |
| US20220098643A1 (en) | Affinity labeling of dna-linked ligands for high throughput ligand binding assays and the uses thereof | |
| CN116626189B (en) | Analysis method for DNA damage in On-DNA chemical reaction based On LC-MS | |
| CN116773805A (en) | Kit and method for detecting cell damage degree | |
| RU2812699C1 (en) | Method for determining chloramphenicol in aqueous environment using 3,6-bis[(trimethylsilyl)ethynyl]-9h-carbazole as molecular recognition element |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20100428 Termination date: 20100823 |