CN105259314B - Lead ion visual detection method and detection kit - Google Patents
Lead ion visual detection method and detection kit Download PDFInfo
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- CN105259314B CN105259314B CN201510707611.4A CN201510707611A CN105259314B CN 105259314 B CN105259314 B CN 105259314B CN 201510707611 A CN201510707611 A CN 201510707611A CN 105259314 B CN105259314 B CN 105259314B
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Abstract
The invention discloses a lead ion visual detection method and detection kit. The detection kit comprises a test strip detection platform, a buffer system and nucleotide sequences A, B C and D. By means of the characteristics that the A, B, C and D are partially complementary in sequence and the A has a lead ion specific recognition element, A-B heteroduplex and lead ion recognition are utilized, the A and lead ions form a G-tetramer structure, and the A is dissociated out; then the B-chain obtained through replacement is dripped onto a sample pad of the test strip for detection, and the B forms a macroscopic strip in the detection area by continuing to be combined with the C. Neither traditional 8-17DNAzyme nor RNA synthesis nor antibodies are needed, the detection result is directly macroscopic, operation is easy, cost is low, and the lead ion visual detection method and detection kit are suitable for being used and popularized in units. The lead detection limit of the method is 1 nM which is lower than 72 nM, the maximum allowable limit, ruled by the USEPA, of lead ions in drinking water.
Description
Technical field
The invention belongs to environmental analytical chemistry, is related to a kind of lead ion detection method and detection kit.
Background technology
Lead ion is serious to harm, is the important indicator of environmental monitoring.Its major toxicity effect is to cause anaemia, god
Jing dysfunctions and injury of kidney, reproductive system damage etc..Environmental Protection Agency specifies the maximum allowable of Pb in Drinking Water ion
Amount must not exceed 72 nM.At present, the common detection methods of Environmental Trace lead ion mainly have atomic absorption method, atomic fluorescence
Spectroscopic methodology, inductively coupled plasma emission spectrography, electrochemical method etc..But these methods are cumbersome, need trouble
Pre-treatment, special analytical technology personnel and costliness instrument, be unfavorable for live quick analysis detection.In recent years, utilize
The nuclease (8-17DNAzyme) sensitive to lead ion receives much concern (D. come the method for detecting lead ion as recognition component
Mazumdar, J. Liu, G.Lu, J. Zhou and Y. Lu, Chem. Commun., 2010, 46, 1416-
1418).But potential shortcoming needs DNA of the synthesis containing RNA to detect, stability is poor, relatively costly.Therefore, compel
It is essential and will selects a kind of new lead ion recognition component, the synthesis of RNA need not be related to.
It is reported that, there is a kind of DNA nucleic acid can be with lead ion specific recognition（T. Li, S. Dong and E. Wang,J. Am. Chem.Soc.,2010, 132, 13156-13157）, the DNA sequences and lead ion have very strong affine
Power, can be folded into the G tetramers.If using the nucleic acid DNA as lead ion recognition component, exploitation it is a kind of it is simple to operate, into
This is cheap, can quick detection the visible detection method of lead ion and detection kit are significant in the environment.
The content of the invention
In order to solve the deficiencies in the prior art, it is an object of the invention to set up a kind of visualization lead ion detection method and
Detection kit.
The technical solution used in the present invention is：
A kind of lead ion Visual retrieval kit, including nucleotide sequence, buffer system and ELISA test strip platform, its core
Acid sequence is made up of nucleotide sequence A, B, C, D；Wherein
Sequence A：With a regions and b regions, a and b regions collectively form lead ion specific recognition element, with lead
G tetramer structures can be formed after ion identification；
Sequence B：With a* regions, c regions and b* regions, the a* regions and b* regions of wherein B a areas respectively with A
Domain and b regional complementarities, the c regions of B are located at the centre in a* regions and b* regions, raised, not complementary with A；One end of B is modified with spy
Different in nature affine small-molecule substance；
Sequence C：With a regions and c* regions, a the and c* regions of wherein C a* and c regional complementarities respectively with B；The one of C
End sulfydryl modification；
Sequence D：With a* regions and c regions；A* the and c regions of D are mutual with a regions of C and c* regions respectively
Mend.
Preferably, 1-5 base is contained in the c regions of nucleotide sequence B.
Preferably, the specific affine small-molecule substance described in nucleotide sequence B includes biotin, digoxin, Cy3.
Preferably, what the detection zone of ELISA test strip platform was fixed is the part or antibody that can be combined with small-molecule substance；
That quality control region is fixed is nucleotide sequence D.
Preferably, buffer system does not generate precipitation with lead ion reaction.
A kind of lead ion Visual retrieval kit, including nucleotide sequence, buffer system and ELISA test strip platform, its core
Acid sequence is made up of nucleotide sequence A, B, C, D；Wherein
Nucleotide sequence A：5'-GGGTGGGT（a）GGGTGGGT（b）-3'（SEQ ID NO：1）；
Nucleotide sequence B：5'-biotin-AAAAAA-ACCCACCC（b*）TAT（c）ACCCACCC（a*）-3'（SEQ ID
NO：2）；
Nucleotide sequence C：5'-SH-AAAAAA-GGGTGGGT（a）ATA（c*）-3'（SEQ ID NO：3）；
Nucleotide sequence D：5'-biotin-AAAAAA-TAT（c）ACCCACCC（a*）-3'（SEQ ID NO：4）.
Preferably, nucleotide sequence C is fixed on gold nano grain, and is injected in the gold standard pad of ELISA test strip platform.
Preferably, what the detection zone of ELISA test strip platform was fixed is Streptavidin；That quality control region is fixed is strepto- parent
With element-biotin-nucleic acid D.
Preferably, buffer system is 10 mM Tris-Ac buffer solutions, pH=8.0.
A kind of lead ion visible detection method, carries out lead ion detection using the kit of any one described above.
The invention has the beneficial effects as follows：
（1）Lead ion recognition component is common DNA sequences, without using traditional 8-17 DNAzyme, need not
It is related to synthesize RNA, without using antibody.
（2）Directly naked eyes are visible for testing result, can obtain without the need for detecting instrument and use data.
（3）It is simple to operate, it is with low cost, it is suitable to unit and promotes the use of.
（4）Detection of the inventive method to lead is limited to 1 nM, specifies Pb in Drinking Water ion less than Environmental Protection Agency
72 nM of maximum magnitude, detection sensitivity is high, selective good.
Description of the drawings
Fig. 1 is the schematic diagram of detection method of the present invention；
Fig. 2 is the Pb to variable concentrations2+The result figure of detection；
Fig. 3 is specificity experiments result figure.
Specific embodiment
A kind of lead ion Visual retrieval kit, including nucleotide sequence, buffer system and ELISA test strip platform, its core
Acid sequence is made up of nucleotide sequence A, B, C, D；Wherein
Sequence A：With a regions and b regions, a and b regions collectively form lead ion specific recognition element, with lead
G tetramer structures can be formed after ion identification；
Sequence B：With a* regions, c regions and b* regions, the a* regions and b* regions of wherein B a areas respectively with A
Domain and b regional complementarities, the c regions of B are located at the centre in a* regions and b* regions, raised, not complementary with A；One end of B is modified with spy
Different in nature affine small-molecule substance；
Sequence C：With a regions and c* regions, a the and c* regions of wherein C a* and c regional complementarities respectively with B；The one of C
End sulfydryl modification；
Sequence D：With a* regions and c regions；A* the and c regions of D are mutual with a regions of C and c* regions respectively
Mend.
Preferably, 1-5 base is contained in the c regions of nucleotide sequence B.
It is furthermore preferred that 3 bases are contained in the c regions of nucleotide sequence B.
Preferably, the specific affine small-molecule substance described in nucleotide sequence B includes biotin, digoxin, Cy3.
Preferably, nucleotide sequence B is connected with specific affine small molecule by linking arm, and sequence C passes through linking arm and mercapto
Base connects.
Preferably, linking arm is the base sequence of multiple A, and its base number is 3-12.
It is furthermore preferred that linking arm is the base sequence of 6 A.
Preferably, what the detection zone of ELISA test strip platform was fixed is the part or antibody that can be combined with small-molecule substance；
That quality control region is fixed is nucleotide sequence D.
Preferably, the method for quality control region fixed nucleic acid sequence D is that nucleotide sequence D is injected in after the quality control region of test strips
It is crosslinked 5 minutes under ultraviolet；Or the one end with biotin of nucleotide sequence D is modified, quality control region will be raw using Streptavidin
The nucleic acid D of thing element modification is fixed.
Preferably, buffer system does not generate precipitation with lead ion reaction.
A kind of lead ion Visual retrieval kit, including nucleotide sequence, buffer system and ELISA test strip platform, its core
Acid sequence is made up of nucleotide sequence A, B, C, D；Wherein
Nucleotide sequence A：5'-GGGTGGGT（a）GGGTGGGT（b）-3'（SEQ ID NO：1）；
Nucleotide sequence B：5'-biotin-AAAAAA-ACCCACCC（b*）TAT（c）ACCCACCC（a*）-3'（SEQ ID
NO：2）；
Nucleotide sequence C：5'-SH-AAAAAA-GGGTGGGT（a）ATA（c*）-3'（SEQ ID NO：3）；
Nucleotide sequence D：5'-biotin-AAAAAA-TAT（c）ACCCACCC（a*）-3'（SEQ ID NO：4）.
Preferably, nucleotide sequence C is fixed on gold nano grain, and is injected in the gold standard pad of ELISA test strip platform.
Preferably, what the detection zone of ELISA test strip platform was fixed is Streptavidin；That quality control region is fixed is strepto- parent
With element-biotin-nucleic acid D.
Preferably, the method for quality control region fixed nucleic acid sequence D is that nucleotide sequence D is injected in after the quality control region of test strips
It is crosslinked 5 minutes under ultraviolet；Or the one end with biotin of nucleotide sequence D is modified, quality control region will be raw using Streptavidin
The nucleic acid D of thing element modification is fixed.
Preferably, buffer system is 10 mM Tris-Ac buffer solutions, pH=8.0.
A kind of lead ion visible detection method, carries out lead ion detection using the kit of any of the above-described.
Below in conjunction with the accompanying drawings (see Fig. 1), further illustrate the Cleaning Principle of the present invention：
A kind of lead ion visible detection method, comprises the following steps：（Shown in Fig. 1）
（1）Nucleic acid A and lead ion（Pb2+）With high-affinity, the G tetramers can be formed.A is first hybridized with B, and one end of B is repaiied
Decorations biotin（biotin）.When there is Pb2+In the presence of, A and Pb2+Recognize and interact, form the G tetramers, so as to A is solved from B
From getting off.The B chains for cementing out drip and are detected in the sample pad of test strips.
（2）ELISA test strip platform includes 4 parts：Sample pad, gold standard pad, nitrocellulose filter and blotting paper, this four
Individual part is from left to right in turn secured on offset plate.One end of nucleic acid C is modified with sulfydryl (- SH), is fixed on gold nano grain
(AuNPs) on, and it is injected in gold standard pad.Drawing on nitrocellulose filter has two detection zones：Detection zone and quality control region, wherein
That detection zone is fixed is Streptavidin (SA)；What quality control region was fixed is Streptavidin (SA)-biotin (biotin)-nucleic acid
D。
（3）The B chains for cementing out are dripped after the sample pad of test strips, are moved forward by capillar migration effect, are passed through
During gold standard pad, combined with C, continued to move along, B-C compounds can be detected the SA captures in area（By SA-biotin phase interactions
With）, to assemble in detection zone, the gold nano grain of aggregation forms macroscopic red area.Excessive gold nano particle modification
C can move forward, hybridize with the D for being fixed on quality control region, assemble in quality control region, it is visible that the gold nano grain of aggregation forms naked eyes
Red area.When there is Pb2+In the presence of, can all there is redness, and detection zone red line in the detection zone of test strips and quality control region
The depth of bar color and Pb2+Concentration is proportionate；Without Pb2+In the presence of, only there is redness in quality control region, and detection zone does not have
There is redness.
With reference to embodiment, technical scheme, but not limited to this are further illustrated.
Embodiment 1
A kind of lead ion Visual retrieval agent box includes following component：
（1）Nucleotide sequence A, B, C, D；A one end modified biological element（biotin）；The one terminal modified sulfydryl of C（-SH）；D
One end modified biological element（biotin）.Particular sequence is as follows：
Nucleotide sequence A：5'-GGGTGGGT（a）GGGTGGGT（b）-3'（SEQ ID NO：1）；
Nucleotide sequence B：5'-biotin-AAAAAA-ACCCACCC（b*）TAT（c）ACCCACCC（a*）-3'（SEQ ID
NO：2）；
Nucleotide sequence C：5'-SH-AAAAAA-GGGTGGGT（a）ATA（c*）-3'（SEQ ID NO：3）；
Nucleotide sequence D：5'-biotin-AAAAAA-TAT（c）ACCCACCC（a*）-3'（SEQ ID NO：4）.
（2）Lead ion standard liquid.
（3）ELISA test strip platform：Comprising sample pad, gold standard pad, nitrocellulose filter and blotting paper, this four parts from
It is left-to-right to be in turn secured on offset plate.C modifies gold nano grain, is sprayed in gold standard pad, and the detection zone of nitrocellulose filter is fixed
Be Streptavidin（SA）；That quality control region is fixed is SA-biotin-D；Test strips are sealed with hermetic bag.
（4）10 mM Tris-Ac buffer solutions（pH=8.0）.
Lead ion visible detection method, is carried out in accordance with the following steps：
The B of the A and 500 nM of (1) 500 nM is with 10 mM Tris-Ac buffer solutions（pH=8.0）Dissolving, room
It is warm fully to mix, react 30 minutes, form A-B double-strands.
(2) add variable concentrations lead ion standard liquid, room temperature reaction 45 minutes, then by reactant liquor be added dropwise in
In the sample pad of test strips, detected.
(3) after test strips flow 10 minutes, directly detect by an unaided eye detection zone and whether quality control region red line occurs
Bar, so that judge that system whether there is Pb2+, wherein detection zone redness line color the depth and Pb2+Concentration is proportionate.
Detection to variable concentrations lead ion：
Lead ion standard liquid is prepared, concentration is respectively 1 nM, 10 nM, 100 nM, 500 nM and 1 μM, and room temperature is protected
Deposit.The lead ion solution of variable concentrations is added separately in the reaction system described in embodiment 1, after fully reacting, will be anti-
Answer liquid（100 μL）It is added drop-wise in the sample pad of test strips, detection zone and the Quality Control of test strips are observed in room temperature detection after 10 minutes
The color change in area.
In the presence of 1 nM lead ions be can be seen that from the testing result of Fig. 2, can observe in the detection zone of test strips
Obvious red area, its detection are limited to 1 nM, specify the maximum allowable of Pb in Drinking Water ion less than Environmental Protection Agency
Measure 72 nM.Also, with the increase (nM from 1 nM to 500) of plumbum ion concentration, the color of detection zone also gradually increases.
When plumbum ion concentration is more than 500 nM, color change tends to saturation.Meanwhile, quality control region is all taken on a red color in all cases,
Illustrate that test strips system running is normal, credible result.
Specificity experiments:
The standard liquid of 1 μM of different ions is prepared, they are Hg respectively2+、Cu2+、Fe3+、Mn2+、Cr3+、Co2+、Ag+And Cd2+。
1 μM of different ion standard liquid and 100 nM lead ion standard liquids are added separately in embodiment 1
In described reaction system, after fully reacting, by reactant liquor（100 μL）It is added drop-wise in the sample pad of ELISA test strip platform, room
Temperature detector is surveyed, and observes the detection zone of test strips and the color change of quality control region after 10 minutes.
1 μM of Hg be can be seen that from the testing result of Fig. 32+、Cu2+、Fe3+、Mn2+、Cr3+、Co2+、Ag+And Cd2+
On detection without impact, there is not red area in the detection zone of test strips；Only just can be in test strips after lead ion is added
Detection zone there is red area.This proves that detection of the method to lead ion has specificity well.
<110>Guangdong Prov. Inst. of Ecological Environment ＆ Soil Science
<120>A kind of lead ion visible detection method and detection kit
<130>
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 16
<212> DNA
<213>Artificial combination sequence
<400> 1
gggtgggtgg gtgggt 16
<210> 2
<211> 25
<212> DNA
<213>Artificial combination sequence
<400> 2
aaaaaaaccc accctatacc caccc 25
<210> 3
<211> 17
<212> DNA
<213>Artificial combination sequence
<400> 3
aaaaaagggt gggtata 17
<210> 4
<211> 17
<212> DNA
<213>Artificial combination sequence
<400> 4
aaaaaatata cccaccc 17
Claims (10)
1. a kind of lead ion Visual retrieval kit, including nucleotide sequence, buffer system and ELISA test strip platform, its feature
It is：Nucleotide sequence is made up of nucleotide sequence A, B, C, D；Wherein
Sequence A：With a regions and b regions, a and b regions collectively form lead ion specific recognition element, with lead ion
G tetramer structures can be formed after identification；
Sequence B：With a* regions, c regions and b* regions, the a* regions and b* regions of wherein B a regions and b respectively with A
Regional complementarity, the c regions of B are located at the centre in a* regions and b* regions, raised, not complementary with A；One end of B is modified with specificity
Affine small-molecule substance；
Sequence C：With a regions and c* regions, a the and c* regions of wherein C a* and c regional complementarities respectively with B；Use one end of C
Sulfydryl modification；
Sequence D：With a* regions and c regions；A* the and c regions of D a regions and c* regional complementarities respectively with C.
2. kit according to claim 1, it is characterised in that：Contain 1-5 base in the c regions of nucleotide sequence B.
3. kit according to claim 1, it is characterised in that：Specific affine small molecule described in nucleotide sequence B
Material includes biotin, digoxin, Cy3.
4. kit according to claim 1, it is characterised in that：What the detection zone of ELISA test strip platform was fixed be can be with
The part or antibody of small-molecule substance combination that specificity is affine；That quality control region is fixed is nucleotide sequence D.
5. kit according to claim 1, it is characterised in that：Buffer system does not generate precipitation with lead ion reaction.
6. a kind of lead ion Visual retrieval kit, including nucleotide sequence, buffer system and ELISA test strip platform, its feature
It is：Nucleotide sequence is made up of nucleotide sequence A, B, C, D；Wherein
Nucleotide sequence A：5'-GGGTGGGT-GGGTGGGT-3'；
Nucleotide sequence B：5'-biotin-AAAAAA-ACCCACCC-TAT-ACCCACCC-3'；
Nucleotide sequence C：5'-SH-AAAAAA-GGGTGGGT-ATA-3'；
Nucleotide sequence D：5'-biotin-AAAAAA-TAT-ACCCACCC-3'.
7. kit according to claim 6, it is characterised in that：Nucleotide sequence C is fixed on gold nano grain, and is sprayed
In the gold standard pad of ELISA test strip platform.
8. kit according to claim 6, it is characterised in that：What the detection zone of ELISA test strip platform was fixed is strepto-
Avidin；That quality control region is fixed is Streptavidin-nucleic acid D.
9. kit according to claim 6, it is characterised in that：Buffer system is 10 mM Tris-Ac buffer solutions, pH=
8.0。
10. a kind of lead ion visible detection method, it is characterised in that using the kit of any one described in claim 1-9
To carry out lead ion detection.
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| CN107012208B (en) * | 2017-03-08 | 2020-09-01 | 广东省生态环境技术研究所 | Label-free lead ion visual detection method and detection kit |
| CN107884580B (en) * | 2017-10-13 | 2019-12-20 | 广东省生态环境技术研究所 | Detection method and detection kit for pathogenic listeria monocytogenes |
| CN107966438B (en) * | 2017-10-27 | 2020-11-24 | 中国农业大学 | Zinc-based high-salt-resistance sensor of functional nucleic acid and application thereof |
| WO2020177059A1 (en) * | 2019-03-04 | 2020-09-10 | Nano And Advanced Materials Institute Limited | System and method for detecting heavy metals and bacteria in an analyte |
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| CN102703441B (en) * | 2012-04-28 | 2013-09-25 | 陕西师范大学 | Hairpin type nucleic acid fluorescent probe and application of probe in lead ion detection |
| CN103245652A (en) * | 2013-05-16 | 2013-08-14 | 上海交通大学 | Method for detecting lead ions by forming G-quadruplex quenching fluorescent light by using functional nucleic acid |
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| CN103773855B (en) * | 2014-01-02 | 2015-12-09 | 广东省生态环境与土壤研究所 | A kind of method for quick of mercury ion and detection kit |
| CN103773856B (en) * | 2014-01-02 | 2016-05-18 | 广东省生态环境与土壤研究所 | A kind of super sensitivity detection method and detection kit of mercury ion |
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