CN1731150A - A biochip method for detecting dioxin-like chemicals - Google Patents
A biochip method for detecting dioxin-like chemicals Download PDFInfo
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Abstract
本发明公开了一种检测二恶英类化学物质的生物芯片方法。其步骤是：首先，在玻片上固定末端Cy5荧光标记的含DRE序列的探针，加入从小鼠肝细胞提取的胞液与2，3，7，8－TCDD的混合液，则胞液中含有的AhR/ARNT蛋白可与DRE序列结合，再加入T7核酸外切酶，由于AhR/ARNT蛋白与DRE序列的结合阻碍了T7核酸外切酶对末端Cy5标记的单核苷酸的消化，使得2，3，7，8－TCDD的含量与荧光强度呈正相关，最后检测Cy5荧光分子的荧光值，根据荧光值的多少来确定样品中二恶英类化学物质的含量。本发明能快速准确的检测二恶英，检测成本低，应用前景广泛。The invention discloses a biochip method for detecting dioxin-like chemical substances. The steps are as follows: firstly, fix the end Cy5 fluorescence-labeled probe containing DRE sequence on the glass slide, add the mixture of cytosol extracted from mouse hepatocytes and 2,3,7,8-TCDD, then the cytosol contains The AhR/ARNT protein can be combined with the DRE sequence, and then T7 exonuclease is added, because the combination of the AhR/ARNT protein and the DRE sequence hinders the digestion of the end Cy5-labeled single nucleotide by the T7 exonuclease, making 2 , The content of 3,7,8-TCDD is positively correlated with the fluorescence intensity. Finally, the fluorescence value of Cy5 fluorescent molecule is detected, and the content of dioxin-like chemical substances in the sample is determined according to the fluorescence value. The invention can quickly and accurately detect dioxin, has low detection cost and wide application prospect.
Description
Technical field
The present invention relates to environmental chemistry and analyze biological technical field, be specifically related to a kind of biochip method that detects 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 biochip method that detects 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 utilize dredging interacts between base and the sulfydryl 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 biochip method that detects 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, a kind of biochip method that detects 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, and behind 5-10min, 50-60 ℃ of annealing of the 90-96 ℃ of processing 20-30min, 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.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101957319A (en) * | 2010-07-22 | 2011-01-26 | 合肥学院 | Chemical preparation method of CaMoO4:Tb3+ fluorescent probe for trace TNT detection |
| CN102520154A (en) * | 2011-12-08 | 2012-06-27 | 环境保护部华南环境科学研究所 | Method for detecting dioxins in environment |
| CN102643921A (en) * | 2012-05-09 | 2012-08-22 | 湖南大学 | Fluorescent biosensing method for analyzing and detecting mutual action of organic micromolecules and combined protein based on T7 exonuclease inhibition |
| CN104048947A (en) * | 2014-05-23 | 2014-09-17 | 孟令启 | Method for detecting dioxin content in food |
| CN108444960A (en) * | 2018-02-09 | 2018-08-24 | 清华大学 | Fluorescence correlation spectroscopy detection method |
| CN109212187A (en) * | 2018-09-06 | 2019-01-15 | 东华大学 | An enzyme-linked immunosorbent assay kit for detecting polycyclic aromatic hydrocarbons and its application |
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| 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 |
| AU2003234851A1 (en) * | 2002-05-28 | 2003-12-12 | National Institute Of Advanced Industrial Science And Technology | Method for measuring trace amount of material by use of quarts resonator |
| CN1223846C (en) * | 2003-05-15 | 2005-10-19 | 中国科学院生态环境研究中心 | Biological measuring method for dioxins compound in monitoring environment samples |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101957319A (en) * | 2010-07-22 | 2011-01-26 | 合肥学院 | Chemical preparation method of CaMoO4:Tb3+ fluorescent probe for trace TNT detection |
| CN102520154A (en) * | 2011-12-08 | 2012-06-27 | 环境保护部华南环境科学研究所 | Method for detecting dioxins in environment |
| CN102520154B (en) * | 2011-12-08 | 2014-01-08 | 环境保护部华南环境科学研究所 | A method for detecting dioxin-like substances in the environment |
| CN102643921A (en) * | 2012-05-09 | 2012-08-22 | 湖南大学 | Fluorescent biosensing method for analyzing and detecting mutual action of organic micromolecules and combined protein based on T7 exonuclease inhibition |
| 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 |
| CN104048947A (en) * | 2014-05-23 | 2014-09-17 | 孟令启 | Method for detecting dioxin content in food |
| CN104048947B (en) * | 2014-05-23 | 2017-02-22 | 孟令启 | Method for detecting dioxin content in food |
| CN108444960A (en) * | 2018-02-09 | 2018-08-24 | 清华大学 | Fluorescence correlation spectroscopy detection method |
| CN109212187A (en) * | 2018-09-06 | 2019-01-15 | 东华大学 | An enzyme-linked immunosorbent assay kit for detecting polycyclic aromatic hydrocarbons and its application |
| CN109212187B (en) * | 2018-09-06 | 2022-01-11 | 东华大学 | ELISA-like kit for detecting polycyclic aromatic hydrocarbons and application thereof |
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