CN107561064A - Application of the G tetrads DNA enzymatic in the ultraweak chemiluminescence detection of sulphion - Google Patents
Application of the G tetrads DNA enzymatic in the ultraweak chemiluminescence detection of sulphion Download PDFInfo
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- CN107561064A CN107561064A CN201710608208.5A CN201710608208A CN107561064A CN 107561064 A CN107561064 A CN 107561064A CN 201710608208 A CN201710608208 A CN 201710608208A CN 107561064 A CN107561064 A CN 107561064A
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Abstract
The invention belongs to detection technique field, proposes application of the G tetrads DNA enzymatic in the ultraweak chemiluminescence detection of sulphion, and the G tetrads DNA enzymatic has Catalyzed Synthesis By Peroxidase active.The present invention also proposes a kind of ultraweak chemical luminescence detection method of sulphion using G tetrad DNA enzymatics.Sulphion quick determination method proposed by the present invention based on ultraweak chemoluminescence method, establish, bio-sensing analysis method that is perfect and having developed sulphion pollutant, reference not only can be provided for Environmental Health risk assessment, a kind of new quick, highly sensitive, inexpensive detection method is developed simultaneously, to improving the significant and preferable application value of existing detection technique means.
Description
Technical field
The invention belongs to detection technique field, and in particular to a kind of chemical luminescence detection method for sulphion.
Background technology
Pollutant directly affects ecological environment for the survival of mankind in environment, threatens the health of the mankind.Environment is dirty
Contaminate the monitoring of thing and its preventing and treating has turned into country and has accelerated modernization, adheres to the important step of the strategy of sustainable development, and right
The detection of pollutant is a key in this link in trade effluent.In recent years, the discharge of sour water is to environmental system, ring
Border ecological environment causes serious consequence, and the sulfide that water body contains not only directly affects the health of human body, can also and water body
Or the Cucumber in air chemically reacts, H is generated2S、SO2Deng pernicious gas, air is not only seriously polluted, can also be caused
A series of more serious pollutions such as acid rain.Therefore, analysis detection is carried out to sulfur-containing waste water, to strict control sulfur-containing waste water with low
Mark discharge is significant.
Research shows, sulfur-containing compound existence form in nature is very complicated, sample source it is different and its not same
The difference of sulfur content is huge between product, is difficult to adopt a kind of single assay method.Up to the present, using instrument analytical method such as
Fluorescence spectrophotometry, ultraviolet-visible spectrophotometry, ion selective electrode method, chromatography of ions etc. can realize sulphion
Highly sensitive, selective enumeration method.But due in industrial and agricultural production and its environment monitoring, it is desirable to instrument will be swift in response, and
Can continuous, on-site measurement, and the requirement of field condition detection can not be met using large-scale chromatographic apparatus detection method, limit its
Application in the detection of sewage sulphion.Therefore, seek it is a kind of it is quicker, simple, accurate, sensitive, efficiently detect sulphion
Method have great importance and researching value.
It is excellent that ultraweak chemoluminescence method has that the sample preparation cycle is short, detection is quick, amount of samples is few, response is strong etc.
Point, it is a kind of more satisfactory fast detecting method of sulphion pollutant.Meanwhile as what DNA was recognized gos deep into, researcher hair
Now the DNA with G- tetrad results can be combined with hemin molecules, formed G- tetrads/hemin compounds, be referred to as
G- tetrad DNA enzymatics, the DNA enzymatic have extremely strong catalytic activity, can be catalyzed hydrogen peroxide oxidation luminol and produce chemiluminescence
(Kosman,J.,Juskowiak,B.,2011.Peroxidase-mimicking DNAzymes for biosensing
applications:A review.Anal.Chim.Acta 707,7-17).Based on this property, G- tetrad DNA enzymatics are wide
It is general to be used for bio-sensing analysis field.But up to the present, there has been no sulphion pollutant is entered based on G- tetrads DNA enzymatic
The research report of the ultraweak chemiluminescence detection of row.
The content of the invention
For the weak point of this area, it is an object of the invention to provide G- tetrad DNA enzymatics are ultraweak in sulphion
Application in chemiluminescence detection.
Second object of the present invention is to propose a kind of ultraweak chemiluminescence inspection of sulphion using G- tetrad DNA enzymatics
Survey method.
The technical scheme for realizing above-mentioned purpose of the present invention is：
Application of the G- tetrads DNA enzymatic in the ultraweak chemiluminescence detection of sulphion, the G- tetrads DNA enzymatic have
Catalyzed Synthesis By Peroxidase activity.
Wherein, the sequence of the G- tetrads DNA enzymatic DNA is PW17, PS2.M, one kind in sequence shown in PS2.M2.
The DNA powder specifically used can be PW17DNA, and its nucleotides sequence is classified as：GGGTAGGGCGGGTTGGG.
In practical application, other DNA sequence dnas with continuous G sequence can be used to be replaced, such as:
PS2.M:GTGGGTAGGGCGGGTTGG；
PS2.M2:GGGTAGGGCGGGTTGGGT。
A kind of ultraweak chemical luminescence detection method of sulphion using G- tetrad DNA enzymatics, comprise the following steps：
(1) by G- tetrad DNA enzymatics, luminol solution, sulphion (S2-Ion) mixed merga pass vortex oscillator is uniform；
(2) mixed solution prepared by step (1) is placed in chemiluminescence detection pond, then by H2O2Fast injection to change
Learn in luminescence detecting pool, carry out chemiluminescence signal detection immediately.
Preferably, in the mixed luminescence system obtained by step (2), the concentration of G- tetrad DNA enzymatics is 1-50nM, luminol
Concentration be 1-100nM；H in step (2)2O2The concentration of solution is 1-100mM.
It is highly preferred that in the mixed luminescence system of gained, the molar concentration rate of G- tetrads DNA enzymatic and luminol is 1：5-
10；The molar concentration of sulphion is 0-1000nM.
Wherein, the G- tetrads DNA enzymatic is prepared by the following method：
S1：DNA sample is dissolved with cushioning liquid, and 1-60min is handled in 80-90 DEG C of water-bath, takes out, uses whirlpool
Oscillator mixes, and natural cooling, standby at room temperature；
S2：The DNA solution for taking step S1 to prepare is added in centrifuge tube, adds cushioning liquid thereto, is incubated 1-200min
Afterwards, hemin is added, is mixed with vortex oscillator, 1-200min is placed, obtains G- tetrad DNA enzymatics.
Further, DNA and hemin molar concentration rate is 1 in the DNA Mimetic Peroxidases described in step S1：
0.1-10；DNA concentration is 1-50 μm of ol/L in solution after step S2 addition cushioning liquid.
Wherein, the pH value of the mixed luminescence system of structure is 4-12；The cushioning liquid is by Tris, KClO4、KH2PO4,
K2HPO4, phosphoric acid, potassium phosphate, two kinds or three kinds in citric acid, hydrochloric acid, potassium chloride are prepared and obtained.
Further, the pH value of the mixed luminescence system is 8.0-10.0, and the cushioning liquid is by Tris and KClO4Match somebody with somebody
Make and obtain.
The ultraweak chemical luminescence detection method of described sulphion also includes, and is prepared and mixed using the sulphion of normal concentration
Luminescence system, ultraweak chemiluminescence signal detection is carried out, builds standard curve, bring the detected value of testing sample into calculating,
Quantitative analysis is carried out to the sulphion pollutant of testing sample.
The beneficial effects of the present invention are：
Sulphion quick determination method proposed by the present invention based on ultraweak chemoluminescence method, establish, improve and develop
The bio-sensing analysis method of sulphion pollutant, reference not only can be provided for Environmental Health risk assessment, while develop one
New quick, highly sensitive, the inexpensive detection method of kind, it is significant and preferable to improving existing detection technique means
Application value.
The present invention establishes a kind of based on G- tetrads DNA enzymatic-hydrogen peroxide-luminol chemiluminescence system detection sulphion
The ultraweak chemiluminescence method of pollutant, simple, detection is quick, cost is low, amount of samples with preparing for the chemoluminescence method
Less, the advantages that sensitivity is high.Provide a kind of new detection method for the quick detection of environmental system sulphion pollutant, this
It is significant to improve existing sulphion pollutant monitoring technical elements, while has widened G- tetrad DNA enzymatics in analysisization
Application in field.
Brief description of the drawings
Fig. 1 is the chemiluminescence CL spectrum of different system of determination in the embodiment of the present invention 4；A is represented containing luminol, H2O2、
G- tetrad DNA enzymatic buffer solution systems；B is represented containing luminol, H2O2, sulphion, G- tetrad DNA enzymatic cushioning liquid bodies
System.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.
Unless otherwise specified, the conventional meanses that technological means used in embodiment is well known to those skilled in the art.
Below in conjunction with the accompanying drawings and specific embodiment the invention will be further described.
The preparation of embodiment 1G- tetrad DNA enzymatics
By solid DNA powder (PW17) pH=9.0,20mM Tris-HClO4Cushioning liquid dissolves, and uses vortex oscillator
Mix, be placed in 10min in 85 DEG C of water-bath, take out, stand natural cooling at room temperature.
Take appropriate above-mentioned DNA solution to be added in centrifuge tube, add pH=9.0,20mM Tris-KClO thereto4Solution,
10 μm of ol/L are diluted to, after being incubated 2h, 12 μm of ol/L hemin is added, is mixed with vortex oscillator, place 2h, obtain concentration
For 10 μm of ol/L G- tetrads/hemin compounds, as G- tetrads DNA enzymatic.
Embodiment 2
Detection device is faint chemiluminescence measuring instrument, and manufacturer is Beijing Ya Bo Si Science and Technology Ltd.s.
With 20mM, pH 9.0 Tris-KClO4Buffer preparation G- containing 100nM tetrad DNA enzymatics and 800nM Rumis
The mixed solution of promise, it is designated as A liquid；Prepare 400mM H2O2Solution, it is designated as B liquid；With 100 μM of Tris-KClO4 buffer preparations
The mixed solution of sulphion, it is designated as C liquid.
1st, take the 2940 above-mentioned cushioning liquid of μ L to be added in cell, take the μ L of A liquid 30 to be added in chemiluminescence detection pond,
Then take the μ L of B liquid 30 to be expelled in chemiluminescence detection pond, carry out chemical luminescent detecting (Fig. 1 a lines).
2nd, take the 2910 above-mentioned cushioning liquid of μ L to be added in cell, take the μ L of 30 μ L, C liquid of A liquid 30 to be added to chemistry respectively
In luminescence detecting pool, then take the μ L of B liquid 30 to be expelled in chemiluminescence detection pond, carry out chemical luminescent detecting (Fig. 1 b lines).
From figure 1 it appears that when luminol, hydrogen peroxide, G- tetrad DNA enzymatics in measure system be present, chemistry hair
Optical signal (chemiluminescence intensity, CL intensity) is very strong (a lines), illustrates that G- tetrads DNA enzymatic can effectively be catalyzed Shandong
Minot, hydrogen peroxide system produce chemiluminescence；In the presence of containing sulphion, under chemical luminous system chemiluminescence signal is notable
Drop (b lines), so as to realize the detection to sulphion according to the change of signal.
Influences of the embodiment 3pH to chemiluminescence signal
The mixed luminescence system of different pH value is set, chemiluminescence detection is carried out according to the identical method of embodiment 2.Its
In, cushioning liquid is respectively the Tris-KClO that pH value is 4.0,6.0,8.0,10.0,12.04Buffer solution.
When the pH value for mixing luminescence system is 8.0-12.0, obvious luminous signal can detect.
When the pH value of the mixing luminescence system is 8.0-10.0, luminous signal is stronger.
Embodiment 4
1st, G- tetrads DNA enzymatic and the sulphion of luminol solution, various concentrations prepared by embodiment 1 are shaken using whirlpool
Device is swung to be well mixed；
2nd, the mixed solution obtained by step 1 is placed in chemiluminescence detection pond, then by H2O2Fast injection is to chemiluminescence
In detection cell, chemiluminescence signal detection is carried out.
Wherein, in order to verify influence of the various concentrations sulphion to luminous signal, by the sulphion for adding different volumes
Solution cause the sulphion concentration in mixing system be respectively 0nmol/L, 10nmol/L, 50nmol/L, 100nmol/L,
500nmol/L、1000nmol/L.Simultaneously in mixed luminescence system：The concentration of G- tetrad DNA enzymatics is 1nM, K+Concentration be
20mM (the K in cushioning liquid+), H2O2Concentration be 4mM, the concentration of luminol is 8nM.
The chemiluminescence intensity of different mixing luminescence systems is detected, is shown in Table 1.
Table 1 and chemiluminescence intensity after the effect of various concentrations sulphion
Result of the test embodies sulphion concentration and the good linear relationship of luminous intensity, and available for the inspection of unknown water sample
Survey.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, some improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
SEQUENCE LISTING
<110>North China Electric Power University
<120>Application of the G- tetrads DNA enzymatic in the ultraweak chemiluminescence detection of sulphion
<130> KHP171114527.9
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 17
<212> DNA
<213> PW17
<400> 1
gggtagggcg ggttggg 17
<210> 2
<211> 18
<212> DNA
<213> PS2.M
<400> 2
gtgggtaggg cgggttgg 18
<210> 3
<211> 18
<212> DNA
<213> PS2.M2
<400> 3
gggtagggcg ggttgggt 18
Claims (10)
- Application of the 1.G- tetrads DNA enzymatic in the ultraweak chemiluminescence detection of sulphion, it is characterised in that the G- tetrads DNA enzymatic has Catalyzed Synthesis By Peroxidase active.
- 2. application according to claim 1, it is characterised in that the sequence of the G- tetrads DNA enzymatic DNA is PW17, One kind in sequence shown in PS2.M, PS2.M2.
- 3. a kind of ultraweak chemical luminescence detection method of sulphion using G- tetrad DNA enzymatics, it is characterised in that including as follows Step：(1) by G- tetrad DNA enzymatics, luminol solution, it is uniform that sulphion mixes merga pass vortex oscillator；(2) mixed solution prepared by step (1) is placed in chemiluminescence detection pond, then by H2O2Fast injection is sent out to chemistry In light detection cell, chemiluminescence signal detection is carried out immediately.
- 4. the ultraweak chemical luminescence detection method of sulphion according to claim 3, it is characterised in that obtained by step (2) Mixed luminescence system in, the concentration of G- tetrad DNA enzymatics is 1-50nM, and the concentration of luminol is 1-100nM；In step (2) H2O2The concentration of solution is 1-100mM.
- 5. the ultraweak chemical luminescence detection method of sulphion according to claim 4, it is characterised in that the mixing hair of gained In body of light system, the molar concentration rate of G- tetrads DNA enzymatic and luminol is 1：5-10；The molar concentration of sulphion is 0- 1000nM。
- 6. the ultraweak chemical luminescence detection method of sulphion according to claim 3, it is characterised in that the G- tetrads DNA enzymatic is prepared by the following method：S1：DNA sample is dissolved with cushioning liquid, and 1-60min is handled in 80-90 DEG C of water-bath, takes out, uses vortex oscillation Device mixes, and natural cooling, standby at room temperature；S2：The DNA solution for taking step S1 to prepare is added in centrifuge tube, adds cushioning liquid thereto, after being incubated 1-200min, is added Enter hemin, mixed with vortex oscillator, place 1-200min, obtain G- tetrad DNA enzymatics.
- 7. the ultraweak chemical luminescence detection method of sulphion according to claim 5, it is characterised in that described in step S1 DNA and hemin molar concentration rate is 1 in DNA Mimetic Peroxidases：0.1-10；It is molten after step S2 addition cushioning liquid DNA concentration is 1-50 μm of ol/L in liquid.
- 8. the ultraweak chemical luminescence detection method of sulphion according to claim any one of 3-7, it is characterised in that structure The pH value of mixed luminescence system be 4-12；The cushioning liquid is by Tris, KClO4、KH2PO4, K2HPO4, phosphoric acid, potassium phosphate, Two kinds or three kinds in citric acid, hydrochloric acid, potassium chloride are prepared and obtained.
- 9. the ultraweak chemical luminescence detection method of sulphion according to claim 8, it is characterised in that the mixed luminescence The pH value of system is 8.0-10.0, and the cushioning liquid is by Tris and KClO4Prepare and obtain.
- 10. the ultraweak chemical luminescence detection method of sulphion according to claim any one of 3-9, it is characterised in that profit Prepared with the sulphion of normal concentration and mix luminescence system, carry out ultraweak chemiluminescence signal detection, build standard curve, will The detected value of testing sample brings calculating into, and quantitative analysis is carried out to the sulphion pollutant of testing sample.
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Cited By (1)
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|---|---|---|---|---|
| CN109946294A (en) * | 2019-05-13 | 2019-06-28 | 华北电力大学 | Environmental contaminants sulphion quick visualization colorimetric detection method based on class peroxidase DNA enzymatic |
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| 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 |
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| US20140287531A1 (en) * | 2003-01-08 | 2014-09-25 | Lesley Davenport | G-quadruplex binding assays and compounds therefor |
| CN103305622A (en) * | 2013-06-28 | 2013-09-18 | 上海交通大学 | Method for detecting lead by G-quadruplex fluorescence method formed by using unlabeled functional nucleic acid |
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| CN109946294A (en) * | 2019-05-13 | 2019-06-28 | 华北电力大学 | Environmental contaminants sulphion quick visualization colorimetric detection method based on class peroxidase DNA enzymatic |
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