CN102621133A - Method for detecting 1,8-diaminonaphthalene based on optical DNA (Deoxyribonucleic Acid) biosensor - Google Patents
Method for detecting 1,8-diaminonaphthalene based on optical DNA (Deoxyribonucleic Acid) biosensor Download PDFInfo
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- CN102621133A CN102621133A CN2012101114369A CN201210111436A CN102621133A CN 102621133 A CN102621133 A CN 102621133A CN 2012101114369 A CN2012101114369 A CN 2012101114369A CN 201210111436 A CN201210111436 A CN 201210111436A CN 102621133 A CN102621133 A CN 102621133A
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Abstract
The invention discloses a method for detecting 1,8-diaminonaphthalene based on an optical DNA (Deoxyribonucleic Acid) biosensor, which belongs to the technical field of analytical chemistry detection and DNA biosensors, and relates to detection of 1,8-diaminonaphthalene on the basis of catalyzing oxidation of luminol with hydrogen peroxide for generating chemiluminescence by using a peroxydase-like G-DNA. The method comprises the following specific step of: making a hairpin DNA containing a G-DNA structure and serving as a raw material react with 1,8-diaminonaphthalene to form a G-DNA with peroxydase-like catalytic activity under the actions of K<+> and hemin,; and detection of the 1,8-diaminonaphthalene through the change of chemiluminescence generated by the luminol oxidized with the hydrogen peroxide is realized. The optical DNA biosensor method has the advantages of easiness, convenience, short detection period, quick response, high stability, high sensitivity, and the like.
Description
Technical field
The invention belongs to analytical chemistry and DNA biosensor technique field, relate to a kind ofly based on 1 of optical dna biology sensor, the detection method of 8-diaminonaphthalene can realize to 1 the detection of 8-diaminonaphthalene fast and effectively.
Background technology
Aromatic amine is one type of important environmental contaminants, has been classified as one of environmental contaminants of preferential monitoring.The aromatic amine purposes is very extensive in industry; As important chemical material and fine-chemical intermediate; Often be used to art production process, especially printing and dyeing industries such as printing and dyeing, pharmacy, medicine, agricultural chemicals, plastics, rubber, weaving, ceramic glazing and paint, waste water often contains the aromatic amine intermedium of high concentration; And the part dyestuff finally also can discharge the aromatic amine pollutant through complicated chemical reaction and microbial action, thereby causes the pollution of environment.In addition, aromatic amine compound gets into that the activation through the enzyme in the body can cause damage to DNA behind the human body, causes dna mutation, thereby brings out malignant disease (as causing carcinoma of urinary bladder, carcinoma of ureter, kidney) such as cancer, and is very harmful to human beings'health.
The analyzing detecting method of aromatic amine has a variety of; Like vapor-phase chromatography, high performance liquid chromatography, capillary electrophoresis, gas chromatography-mass spectrography, LC/MS etc.; The multiple aromatic amine pollutant of these methods quantitative measurement simultaneously; Have characteristics such as efficient and sensible, but have apparatus expensive, need professional and technical personnel's operation, shortcoming such as sample preparation is loaded down with trivial details.Along with the development of electrochemical techniques and DNA biosensor technique, the DNA biology sensor also is used to detect the aromatic amine pollutant as a kind of novel detection technique.Wang etc. use the carbon paste electrode biology sensor that natural calf thymus DNA is modified, and have realized the amino bitter edible plant of 2-, 1-amino anthracene, 2-amino anthracene, 9, the detection of aromatic amine pollutants such as 10-diamido phenanthrene, the amino pyrrole of 1-.The silk screen graphite that employing n DNA such as Chiti and the dna fragmentation that contains 23 bases that contains synthetic the are modified sensor that prints electrode, to aromatic amine 2-amino naphthalenes, 2-amino anthracene, 1, compounds such as 2-diamino-anthraquinone, acridine yellow detect.Prabhakar etc. use polypyrrole-PVC sulphonate/indium oxide film electrode sensor that natural calf thymus DNA is modified, and with cyclic voltammetry aromatics 2-amino anthracene are detected.Yet; As the molecular recognition main body mostly the detection of aromatic amine compound is to adopt the method for dna modification electrode sensor with DNA; Though these DNA biology sensors have higher sensitivity to the aromatic amine pollutant, its weak point is that the electrode preparation time is longer, and cost is higher relatively.Discover that G-DNA is the dna fragmentation of the catalytic activity of one type of type of having peroxidase, under the oxydol existence condition G-DNA structure can catalysis the hydrogen peroxide oxidation luminol produce chemiluminescence, it is fast to have a response time, characteristics such as selectivity height.The catalytic activity of application G-DNA class peroxidase such as Li has successfully realized heavy metal ion Ag
+And Pb
2+Detection, the formation of micromolecule such as application fibrin ferment and ATP can induce G-DNA structure such as Xiao etc. have realized the detection to the target dna chain based on this mechanism, Liu has also successfully realized the detection to it.Up to the present; Application G-DNA catalysis luminol chemiluminescence does not also appear in the newspapers to the detection of aromatic amine compounds; Compare with dna modification solid electrode formula sensor, method, this method need not dna modification is arrived electrode surface, the cycle that shortens greatly; Reduced cost simultaneously, the response speed of detection also obviously improves.
In sum, setting up, improve and developing aromatic amine Analysis of contaminant method is the hot research problem of analytical chemistry and environmental chemistry, also is one of key link of comprehensive environmental improvement, has important Research Significance.Develop simultaneously a kind of new highly sensitive, detection method is to improving the also significant and using value preferably of existing detection technique means cheaply.
Summary of the invention
The object of the present invention is to provide a kind of fast detecting 1, the method for 8-diaminonaphthalene based on the optical dna biology sensor.
Technical scheme of the present invention is following:
A kind of based on 1 of optical dna biology sensor, the detection method of 8-diaminonaphthalene may further comprise the steps:
1) solid hairpin DNA is used pH=8.0,20mM Tris-NaClO
4The buffer solution dissolving also is diluted to finite concentration, and with vortex oscillation device mixing, be statically placed in 6h under the room temperature, and is subsequent use;
2) get an amount of above-mentioned steps 1) the hairpin dna solution of preparation is added in the frozen pipe, and certain density 1 to wherein adding, 8-diaminonaphthalene solution is placed 30min;
3) to above-mentioned steps 2) add certain density K in the system
+And hemin solution, place 1h;
4) in above-mentioned step 3) system, add certain density H
2O
2And luminol solution, carry out chemical luminescent detecting.
Preferably, above-mentioned steps 1) described in hairpin dna double chain part base logarithm be 9 pairs.
Preferably, above-mentioned steps 2) described in hairpin DNA concentration be 100nM.
Preferably, above-mentioned steps 3) described in K
+Concentration is 10mM.
Preferably, above-mentioned steps 3) described in hemin concentration be 1 μ M.
Preferably, above-mentioned steps 4) described in H
2O
2Concentration is 300 μ M.
Preferably, above-mentioned steps 4) described in luminol concentration be 50 μ M.
Preferably, the pH value of solution value is 8.0 above-mentioned steps 1-4).
Compared with prior art patent art has the following advantages:
1. the hairpin dna sequence dna that the present invention adopted is the dna sequence dna of synthetic, can be optimized dna sequence dna;
2. the coupling base-pair of hairpin dna double chain " stem " part that the present invention adopted is 9 pairs, and to 1, the 8-diaminonaphthalene detects has higher sensitivity;
3. optical dna biosensor technique of the present invention is in solution system, to measure, and shorter experimental period than solid electrode formula DNA biology sensor, reliability is high, and cost is lower;
4. the present invention adopts chemoluminescence method to measure, and has simple to operate, characteristics such as response speed fast, strong interference immunity.
Description of drawings
Accompanying drawing is the chemiluminescence spectra that different systems are measured: Tris-NaClO
4Buffer solution system (-) contains G-DNA, luminol, H
2O
2, K
+, hemin Tris-NaClO
4Buffer solution system (---), contain G-DNA, luminol, H
2O
2, K
+, hemin and 1, the Tris-NaClO of 8-diaminonaphthalene
4Buffer solution system ().
Embodiment
Following embodiment is done more detailed description to the present invention, but said enforcement is not construed as limiting the invention.
Embodiment 1
1) with the 20mM Tris-NaClO of pH=8.0
4Buffer solution with vortex oscillation device mixing, and is statically placed in 6h under the room temperature with solid hairpin DNA dissolving, forms the hairpin dna structure with this understanding;
2) get an amount of above-mentioned steps 1) the hairpinDNA solution of preparation is added in the frozen pipe, and certain density 1 to wherein adding, 8-diaminonaphthalene solution is placed 30min, in above-mentioned system, adds certain density K then
+And hemin solution, place 1h, form the G-DNA structure of type of having peroxidase activity with this understanding;
Embodiment 2
Control mensuration system hairpin DNA concentration is 100nM, K
+Concentration is 10mM, and hemin concentration is 1 μ M, H
2O
2Concentration is 300 μ M, and luminol concentration is 50 μ M, respectively with use 20mM Tris-NaClO
4The variable concentrations 1 of (pH=8.0) buffer preparation, 8-diaminonaphthalene (6nmol/L, 30nmol/L, 150nmol/L, 300nmol/L and 600nmol/L) solution effects is measured the system chemiluminescence intensity.1 of above-mentioned variable concentrations, 8-diaminonaphthalene solution carry out the chemiluminescence experiment respectively, and chemiluminescence intensity is seen table 1.
Table 1 and variable concentrations 1, chemiluminescence intensity after the effect of 8-diaminonaphthalene
Embodiment 3
With concentration is that concentration is 100nM hairpin DNA in the 50nM hairpin DNA replacement embodiment 2, and other conditions are with embodiment 2, and experiment shows that this optical dna biology sensor is to 1, and the 8-diaminonaphthalene also has response.Variable concentrations 1,8-diaminonaphthalene solution chemiluminescence intensity is with this understanding seen table 2.
Table 1 and variable concentrations 1, chemiluminescence intensity after the effect of 8-diaminonaphthalene
Claims (7)
1. one kind based on 1 of optical dna biology sensor, and the detection method of 8-diaminonaphthalene is characterized in that may further comprise the steps:
1) solid hairpin DNA is used pH=8.0,20mM Tris-NaClO
4The buffer solution dissolving also is diluted to finite concentration, and with vortex oscillation device mixing, be statically placed in 6h under the room temperature, and is subsequent use;
2) get an amount of above-mentioned steps 1) the hairpin dna solution of preparation is added in the frozen pipe, and certain density 1 to wherein adding, 8-diaminonaphthalene solution is placed 30min;
3) to above-mentioned steps 2) add certain density K in the system
+And hemin solution, place 1h;
4) in above-mentioned step 3) system, add certain density H
2O
2And luminol solution, carry out chemical luminescent detecting.
2. as claimed in claim 1 based on 1 of optical dna biology sensor, the detection method of 8-diaminonaphthalene is characterized in that: the Tris-NaClO of preparation hairpinDNA solution
4Buffer concentration is 20mM, pH=8.0.
3. as claimed in claim 1 based on 1 of optical dna biology sensor, the detection method of 8-diaminonaphthalene is characterized in that: hairpin DNA and 1, the 8-diaminonaphthalene reaction time is 30min, then with K
+And the hemin solution effects time is 1h.
4. as claimed in claim 1 based on 1 of optical dna biology sensor, the detection method of 8-diaminonaphthalene is characterized in that: hairpin DNA concentration is 100nM, K
+Concentration is 10mM, and hemin concentration is 1 μ M, H
2O
2Concentration is 300 μ M, and luminol concentration is 50 μ M.
5. as claimed in claim 1 based on 1 of optical dna biology sensor, the detection method of 8-diaminonaphthalene is characterized in that: the base of the two strands of hairpin DNA " stem " part coupling is 9 pairs.
According to claim 1 or claim 2 based on 1 of optical dna biology sensor, the detection method of 8-diaminonaphthalene is characterized in that: comprise in the employed hairpin dna sequence dna and can form the G-DNA sequence fragment.
7. like the described detection method of claim 1-4, it is characterized in that: amino naphthalenes is 1, the 8-diaminonaphthalene.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104062288A (en) * | 2014-07-09 | 2014-09-24 | 北京师范大学 | Chemiluminiscence-based detection method of naphthylamine compound |
| CN106841080A (en) * | 2017-04-01 | 2017-06-13 | 北京农业质量标准与检测技术研究中心 | Application of the DNA Mimetic Peroxidases in 1,8 diaminonaphthalenes are detected |
| CN116297758A (en) * | 2023-02-03 | 2023-06-23 | 中国科学院地理科学与资源研究所 | Electrochemical DNA sensor with high sensitivity identification and application thereof |
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| JP2009036689A (en) * | 2007-08-03 | 2009-02-19 | Jfe Steel Kk | METHOD FOR EASY ANALYSIS OF TRACE AMOUNT OF Se IN ENVIRONMENTAL WATER |
| CN101606057A (en) * | 2007-02-06 | 2009-12-16 | 凸版印刷株式会社 | Biomolecule detecting method and biomolecule detection chip |
| CN102021226A (en) * | 2009-09-11 | 2011-04-20 | 中国科学技术大学 | Luminol direct bonded nano gold nucleic acid analyzing probe and application thereof |
| CN102375021A (en) * | 2010-08-25 | 2012-03-14 | 中国科学院大连化学物理研究所 | Electrochemical method employing DNA as probe to detect environmental pollutant |
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2012
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101606057A (en) * | 2007-02-06 | 2009-12-16 | 凸版印刷株式会社 | Biomolecule detecting method and biomolecule detection chip |
| JP2009036689A (en) * | 2007-08-03 | 2009-02-19 | Jfe Steel Kk | METHOD FOR EASY ANALYSIS OF TRACE AMOUNT OF Se IN ENVIRONMENTAL WATER |
| CN102021226A (en) * | 2009-09-11 | 2011-04-20 | 中国科学技术大学 | Luminol direct bonded nano gold nucleic acid analyzing probe and application thereof |
| CN102375021A (en) * | 2010-08-25 | 2012-03-14 | 中国科学院大连化学物理研究所 | Electrochemical method employing DNA as probe to detect environmental pollutant |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104062288A (en) * | 2014-07-09 | 2014-09-24 | 北京师范大学 | Chemiluminiscence-based detection method of naphthylamine compound |
| CN104062288B (en) * | 2014-07-09 | 2018-02-02 | 北京师范大学 | A kind of detection method of the naphthylamine compound based on chemoluminescence method |
| CN106841080A (en) * | 2017-04-01 | 2017-06-13 | 北京农业质量标准与检测技术研究中心 | Application of the DNA Mimetic Peroxidases in 1,8 diaminonaphthalenes are detected |
| CN106841080B (en) * | 2017-04-01 | 2019-11-01 | 北京农业质量标准与检测技术研究中心 | Application of the DNA Mimetic Peroxidase in detection 1,8- diaminonaphthalene |
| CN116297758A (en) * | 2023-02-03 | 2023-06-23 | 中国科学院地理科学与资源研究所 | Electrochemical DNA sensor with high sensitivity identification and application thereof |
| CN116297758B (en) * | 2023-02-03 | 2024-03-19 | 中国科学院地理科学与资源研究所 | Electrochemical DNA sensor with high sensitivity identification and application thereof |
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| CN102621133B (en) | 2016-08-03 |
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