CN107607513A - A kind of method of testing of DNA sensor based on the serobilas of G tetra- to Pb2+ concentration - Google Patents
A kind of method of testing of DNA sensor based on the serobilas of G tetra- to Pb2+ concentration Download PDFInfo
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- CN107607513A CN107607513A CN201711022555.6A CN201711022555A CN107607513A CN 107607513 A CN107607513 A CN 107607513A CN 201711022555 A CN201711022555 A CN 201711022555A CN 107607513 A CN107607513 A CN 107607513A
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Abstract
The invention belongs to DNA sensor technical field, and in particular to a kind of DNA sensor based on the serobilas of G tetra- is to Pb2+The method of testing of concentration.The present invention only passes through the detection Pb of " hybrid detection " step2+, there is extraordinary sensitivity and selectivity；The serobila DNA of G tetra- are more stable compared to for metal ion auxiliary enzymes and RNA cracking deoxyribonucleases, it is not necessary to complicated preparation process；The cost just more economic, convenient and very cheap as the PPIX of fluorescence signal indicating member and that there is the characteristics of very effective fluorescent characteristic, these are label-free fundamentally to reduce detection compared to for the scheme using various shearing enzymes.
Description
Technical field
The invention belongs to DNA sensor technical field, and in particular to a kind of DNA sensor based on the serobilas of G- tetra- is to Pb2+
The method of testing of concentration.
Background technology
In aquatic ecosystem, lead ion is a kind of one of environmental contaminants of most toxicity, and its serious threat people
The life and health of class, therefore, develop with hypersensitivity, the sensor of detection lead ion can be quantified with extremely important
Meaning.However, traditional detection method, such as atomic absorption spectrum（AAS）Or inductivity coupled plasma mass spectrometry（ICP-MS）
Deng, due to instrument in itself it is expensive, preprocessing process is cumbersome etc. limits it and be used at the scene with real-time detection.Researcher is led to
Cross and be continually striving to, huge advance is had been achieved in the detection method of lead ion.Some of electrochemical assays also obtain
Practical application, but its processing procedure is relative complex, requires higher to operating condition, therefore, it is difficult to promote the use of.
Over the last number of years, the nucleic acid of functionalization becomes a kind of strong detection instrument and is widely used.
Particularly two kinds RNA cracking deoxyribonuclease -8-17E DNAzyme and GR-5 DNAzyme based on lead ion, are obtained
Generally use.Their action principle is all to utilize Pb2+Booster action excite enzyme active catalytic crack have ribonucleotide
Sour rA DNA, detected according to the change of obtained fluorescence signal.But these detection platforms largely could not be in reality
It is applied in border, because enzyme is costly, processing procedure is also relative complex, it is crucial that RNA molecule less stable.Remove
Outside this, Pb2+This phenomenon of the change of the serobila conformations of G- tetra- can be induced to be also applied to Pb2+Detection.Such as
Dong seminars introduce a Pb2+The DNA enzymatic of the serobilas of induced synthesis G- tetra- is as detection platform, but they need to use
Hemin and H2O2As co-factor, this may also limit its application in practice.Again for example, Li Xiang et al. is devised in addition
One scheme, they are with graphene oxide（GO）As quencher, the DNA of rich G bases is adsorbed by π-π effects, modification is existed
FRET effects occur between fluorophor and GO on DNA, by its fluorescent quenching, when adding Pb2+The shape of the serobilas of G- tetra- is induced afterwards
Into the DNA for making to be modified with fluorophor departs from GO surfaces and recovers the fluorescence intensity of itself.Based on the principle that he reaches
Detection Pb2+Purpose, although having preferable Detection results, but DNA modification and GO preparation process it is more complicated and
Cost is higher, is equally unfavorable for practical application.And Zhang et al. utilizes Pb2+The serobilas of G- tetra- of induced synthesis and fluorophor it
Between quenching effect come to Pb2+Detected, although this method has reached 0.4nM test limit, but also do not avoid pair
DNA fluorescence labeling.
The content of the invention
The present invention is to make probe with PS2.M, is realized first with Pb2+The change of G- tetra- stranded structures is induced to be quenched G- tetra-
The experimental design of serobila-PPIX complex fluorescences, and only just completed to Pb by " mixing-detection " step2+Detection.This
Detection method is workable, and cost is cheap, practical, disclosure satisfy that routine testing requirement, while also has extraordinary sensitive
Degree and selectivity.
The technical solution adopted by the present invention is that a kind of DNA sensor based on the serobilas of G- tetra- is to Pb2+The test side of concentration
Method, comprise the following steps：
1. preparing the mixed solution containing DNA probe, the mixed solution for having DNA probe includes 50mM pH 6.0 Tris-HCl
Cushioning liquid, 50mM KCl, 10mM NaCl, 0.5 μM of DNA probe, 0 ~ 2000nM Pb2+；It is placed in 95 DEG C of water-bath and heats 10
Minute, it is slowly cooled to room temperature afterwards；The sequence of the DNA probe such as SEQ ID NO:Shown in 1；
2. different Pb after 1. being handled to step2+Isometric 0.5 μM is added in the mixed solution containing DNA probe under concentration
PPIX solution, 1h is reacted in the dark, its fluorescence spectrum, excitation wave are finally measured in fluophotometer with particle fluorescence cuvette
Length is set as 410nm, scanning range 550nm-750nm, determines the fluorescence intensity level at 632nm；PPIX structural formula is such as
Under：
；
3. draw various concentrations Pb2+The standard working curve of lower fluorescence intensity level；
4. by the Pb to be measured of preparation2+The mixed solution containing DNA probe of concentration is placed in 95 DEG C of water-bath and heated 10 minutes,
It is slowly cooled to room temperature afterwards, adds isometric 0.5 μM of PPIX solution, 1h is reacted in the dark, finally with particle fluorescence colorimetric
Ware measures its fluorescence spectrum in fluophotometer, and excitation wavelength is set as 410nm, scanning range 550nm-750nm, measure
Fluorescence intensity level at 632nm；According to the standard working curve of step 3., you can draw the Pb of solution to be measured2+Concentration value.
The step 1. in 0 ~ 2000nM Pb2+Respectively 0,5,10,15,20,30,40,50,70,90,100,200,
500、1000、2000 nM。
Pb2+The stable stranded structures of G- tetra- are compared to K+The stable stranded structures of G- tetra- have shorter M-O（M represents gold
Belong to atom）And O -- O bond, this just imply that the serobilas of G- tetra- have overall compact structure, also illustrates Pb2+Have more to the serobilas of G- tetra-
High affinity, such result will result in Pb2+Substitute K+To stablize the serobilas of G- tetra-, so that the structure and work(of the serobilas of G- tetra-
Delicate change can occur.Design such as Fig. 1, we freely roll up by the use of rich G DNA sequence dna PS2.M as probe when it is in
When curved and PPIX effect it is very weak, hardly with fluorescence, and in K+Under the conditions of existing, it can fold to be formed
The stranded structures of G- tetra- and with PPIX with reference to and send very strong fluorescence signal；When being gradually added into Pb2+When, it will result in G- tetra-
Change of the serobila in structure, PPIX is forced to depart from the stranded structures of G- tetra-, so that its fluorescence intensity gradually reduces.
In the present invention, we have investigated influence of the PPIX concentration to detection architecture, as a result as shown in Figure 2.We with
CDNA:CPPIXValue as reference, when ratio between the two is 3 times that 3 i.e. DNA concentration is PPIX, addition target dna after
Fluorescence intensity it is weaker, illustrate the serobilas of G- tetra- do not combined with enough PPIX；When ratio is 2, its fluorescence intensity does not have yet
Having and reach maximum, when it is that DNA concentration is equal with PPIX that both ratio, which is 1, its fluorescence intensity has reached maximum, this
When be further added by PPIX concentration i.e. when both concentration ratios reach 1:When 2, its fluorescence intensity is not remarkably reinforced yet, and illustrates G- tetra-
Serobila and PPIX are 1:1 combination.
Beneficial effects of the present invention：Only pass through the detection Pb of " mixing-detection " step2+, have extraordinary sensitivity and
Selectivity；The serobila DNA of G- tetra- are more stable compared to for metal ion auxiliary enzymes and RNA cracking deoxyribonucleases, no
Need the preparation process of complexity；It is just more economical, convenient compared to for the scheme using various shearing enzymes, and as glimmering
The PPIX of optical signal indicating member is very cheap and has the characteristics of very effective fluorescent characteristic, these are label-free fundamentally
Reduce the cost of detection.
Brief description of the drawings
Fig. 1 is the Pb based on the serobilas of G- tetra-2+The experimental principle figure of sensor；
Fig. 2 is the C of four kinds of different ratiosDNA:CPPIXThe fluorescence intensity change in the presence of target dna；
Fig. 3 is K+Influence of the concentration to detecting system（There is no Pb2+Under the conditions of）：A. in different K+Fluorescence light under concentration conditions
Spectrum；B. different K+Fluorescence intensity block diagram under concentration conditions at 632nm；
Fig. 4 is selectivity test result figure；
Fig. 5 is the testing result figure of sensitivity:A. in different Pb2+Fluorescence spectrum under concentration；B. in different Pb2+Under concentration
Fluorescence intensity change curve map at 632nm；C. the linearly interval of detecting system.
Embodiment
（1）NaCl is weighed, with deionized water dissolving and 10mL is settled to, is configured to 1M concentration, preserved at 4 DEG C, is used
When be diluted to required concentration.
（2）The Tris-HCl cushioning liquid of pH=6.0 is prepared, 0.0169g plumbi nitras is then weighed, with matched somebody with somebody cushioning liquid
It is gradually dissolved and be settled to 50mL, be configured to 1mM concentration, be kept in dark place at 4 DEG C, the used time is diluted to required concentration.
（3）The serobila DNA of G- tetra- will be contained（0.5μM）Tris-HCl buffer solutions（50mM, include 50mM KCl, 10mM
NaCl）It is placed in constant temperature water bath, is kept for 10 minutes, be injected into afterwards in 1cm quartz colorimetric utensil at different temperatures,
Measure the absorbance at 295nm respectively with ultraviolet specrophotometer.
（4）By mixed liquor（Tris-HCl cushioning liquid comprising 50mM pH6.0,0.5 μM of DNA, various concentrations
KCl, 10mM NaCl, it is 1mL to make cumulative volume）It is placed in 95 DEG C of water-bath and heats 10 minutes, is slowly cooled to room temperature afterwards（25
℃）, 0.5 μM of 1mL PPIX solution is added, 1h is reacted in the dark, is finally surveyed with particle fluorescence cuvette in fluophotometer
Obtain its fluorescence spectrum.Excitation wavelength is set as 410nm, scanning range 550nm-750nm, and the fluorescence determined at 632nm is strong
Angle value.
We have been 6 different K respectively+The experiment of concentration, as a result as shown in figure 3, in no K+Under conditions of, its fluorescence
Intensity is very faint；Work as K+When concentration is less than 50mM, detecting system fluorescence intensity is with K+The increase of concentration and drastically strengthen
, after concentration is higher than 50mM, fluorescence intensity, which has, a little to be reduced, and from the point of view of the result in figure, 50mM is K+Optimal concentration.
The drafting of working curve；
1. preparing the mixed solution containing DNA probe, the mixed solution for having DNA probe includes 50mM pH 6.0 Tris-HCl
Cushioning liquid, 50mM KCl, 10mM NaCl, 0.5 μM of DNA probe, 0 ~ 2000nM Pb2+；It is placed in 95 DEG C of water-bath and heats 10
Minute, it is slowly cooled to room temperature afterwards；The sequence of the DNA probe such as SEQ ID NO:Shown in 1；
2. different Pb after 1. being handled to step2+Isometric 0.5 μM is added in the mixed solution containing DNA probe under concentration
PPIX solution, 1h is reacted in the dark, its fluorescence spectrum, excitation wave are finally measured in fluophotometer with particle fluorescence cuvette
Length is set as 410nm, scanning range 550nm-750nm, determines the fluorescence intensity level at 632nm；PPIX structural formula is such as
Under：
；
3. draw various concentrations Pb2+The standard working curve of lower fluorescence intensity level（F and F0Respectively Pb2+When in the presence of and in the absence of
The fluorescence intensity of mixed solution）；The linearly interval of detecting system is 0 ~ 110 nM；
4. by the Pb to be measured of preparation2+The mixed solution containing DNA probe of concentration is placed in 95 DEG C of water-bath and heated 10 minutes,
It is slowly cooled to room temperature afterwards, adds isometric 0.5 μM of PPIX solution, 1h is reacted in the dark, finally with particle fluorescence colorimetric
Ware measures its fluorescence spectrum in fluophotometer, and excitation wavelength is set as 410nm, scanning range 550nm-750nm, measure
Fluorescence intensity level at 632nm；According to the standard working curve of step 3., you can draw the Pb of solution to be measured2+Concentration value.
The step 1. in 0 ~ 2000nM Pb2+Respectively 0,5,10,15,20,30,40,50,70,90,100,200,
500、1000、2000 nM。
Sequence table
<110>He'nan Normal University
<120>A kind of method of testing of DNA sensor based on the serobilas of G- tetra- to Pb2+ concentration
<130> 2017
<141> 2017-10-27
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 18
<212> DNA
<213>Artificial sequence (1)
<400> 1
gtgggtaggg cgggttgg 18
Claims (2)
1. a kind of DNA sensor based on the serobilas of G- tetra- is to Pb2+The method of testing of concentration, it is characterised in that comprise the following steps：
1. preparing the mixed solution containing DNA probe, the mixed solution for having DNA probe includes 50mM pH 6.0 Tris-HCl
Cushioning liquid, 50mM KCl, 10mM NaCl, 0.5 μM of DNA probe, 0 ~ 2000nM Pb2+；It is placed in 95 DEG C of water-bath and heats 10
Minute, room temperature is cooled to afterwards；The sequence of the DNA probe such as SEQ ID NO:Shown in 1；
2. different Pb after 1. being handled to step2+Isometric 0.5 μM is added in the mixed solution containing DNA probe under concentration
PPIX solution, 1h is reacted in the dark, its fluorescence spectrum, excitation wave are finally measured in fluophotometer with particle fluorescence cuvette
Length is set as 410nm, scanning range 550nm-750nm, determines the fluorescence intensity level at 632nm；PPIX structural formula is such as
Under：
；
3. draw various concentrations Pb2+The standard working curve of lower fluorescence intensity level；
4. by the Pb to be measured of preparation2+The mixed solution containing DNA probe of concentration is placed in 95 DEG C of water-bath and heated 10 minutes,
Room temperature is cooled to afterwards, is added isometric 0.5 μM of PPIX solution, is reacted 1h in the dark, finally existed with particle fluorescence cuvette
Its fluorescence spectrum is measured in fluophotometer, excitation wavelength is set as 410nm, scanning range 550nm-750nm, determined
Fluorescence intensity level at 632nm；According to the standard working curve of step 3., you can draw the Pb of solution to be measured2+Concentration value.
2. the DNA sensor as claimed in claim 1 based on the serobilas of G- tetra- is to Pb2+The method of testing of concentration, it is characterised in that institute
0 ~ 2000nM Pb in stating step 1.2+Respectively 0,5,10,15,20,30,40,50,70,90,100,200,500,1000,2000
nM。
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108801997A (en) * | 2018-06-11 | 2018-11-13 | 岭南师范学院 | A kind of no quencher fluorescence probe CP-DNA and preparation method thereof, application and detection micro Pb2+Method |
| CN109799215A (en) * | 2018-12-25 | 2019-05-24 | 北京农业质量标准与检测技术研究中心 | A kind of Pb based on G- tetrad DNA2+Fluorescence sense detection method |
| CN111487227A (en) * | 2020-01-17 | 2020-08-04 | 商丘师范学院 | Be used for detecting Pb in human serum2+Fluorescence-enhanced sensor of concentration |
| CN112175950A (en) * | 2020-09-23 | 2021-01-05 | 农业农村部环境保护科研监测所 | Preparation method of stable circular DNAzyme and application of stable circular DNAzyme in Pb2+ detection |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
-
2017
- 2017-10-27 CN CN201711022555.6A patent/CN107607513A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
Non-Patent Citations (1)
| Title |
|---|
| 侯瑞鹏: "基于G-四链体的核酸、铅离子和汞离子传感器的设计", 《中国优秀硕士学位论文全文数据库 信息科技辑》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108801997A (en) * | 2018-06-11 | 2018-11-13 | 岭南师范学院 | A kind of no quencher fluorescence probe CP-DNA and preparation method thereof, application and detection micro Pb2+Method |
| CN109799215A (en) * | 2018-12-25 | 2019-05-24 | 北京农业质量标准与检测技术研究中心 | A kind of Pb based on G- tetrad DNA2+Fluorescence sense detection method |
| CN109799215B (en) * | 2018-12-25 | 2021-09-28 | 北京农业质量标准与检测技术研究中心 | Pb based on G-quadruplex DNA2+Fluorescence sensing detection method |
| CN111487227A (en) * | 2020-01-17 | 2020-08-04 | 商丘师范学院 | Be used for detecting Pb in human serum2+Fluorescence-enhanced sensor of concentration |
| CN112175950A (en) * | 2020-09-23 | 2021-01-05 | 农业农村部环境保护科研监测所 | Preparation method of stable circular DNAzyme and application of stable circular DNAzyme in Pb2+ detection |
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