CN105259314A - Lead ion visual detection method and detection kit - Google Patents
Lead ion visual detection method and detection kit Download PDFInfo
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- CN105259314A CN105259314A CN201510707611.4A CN201510707611A CN105259314A CN 105259314 A CN105259314 A CN 105259314A CN 201510707611 A CN201510707611 A CN 201510707611A CN 105259314 A CN105259314 A CN 105259314A
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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 field, relate 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 causes anaemia, nervous function to be lacked of proper care and injury of kidney, reproductive system damage etc.Environmental Protection Agency specifies that the maximum magnitude of Pb in Drinking Water ion must not more than 72nM.At present, the common detection methods of Environmental Trace lead ion mainly contains atomic absorption method, atomic fluorescence spectrometry, inductively coupled plasma emission spectrography, electrochemical method etc.But these method complex operations, need the instrument of troublesome pre-treatment, special analytical technology personnel and costliness, are unfavorable for that on-the-spot express-analysis detects.In recent years, utilize the nuclease to lead ion sensitivity (8-17DNAzyme) as recognition component to the method detecting lead ion receive much concern (D.Mazumdar, J.Liu, G.Lu, J.ZhouandY.Lu,
chem.Commun., 2010,46,1416-1418).But potential shortcoming needs synthesis, and the DNA containing RNA detects, and poor stability, cost is higher.Therefore, in the urgent need to selecting a kind of novel lead ion recognition component, without the need to relating to the synthesis of RNA.
It is reported, have a kind of DNA nucleic acid can with lead ion specific recognition (T.Li, S.DongandE.Wang,
j.Am.Chem.Soc.,2010,132,13156-13157), this DNA sequence dna and lead ion have very strong affinity, can be folded into the G tetramer.If using this nucleic acid DNA as lead ion recognition component, develop a kind of simple to operate, with low cost, visible detection method that can detect lead ion in the environment fast and detection kit significant.
Summary of the invention
In order to solve the deficiencies in the prior art, the object of the invention is to set up a kind of visual lead ion detection method and detection kit.
The technical solution used in the present invention is:
A kind of lead ion Visual retrieval kit, comprise nucleotide sequence, buffer system and ELISA test strip platform, its nucleotide sequence is made up of nucleotide sequence A, B, C, D; Wherein
Sequence A: there is a region and b region, a and b region forms lead ion specific recognition element jointly, can form G tetramer structure with after lead ion identification;
Sequence B: there is a* region, c region and b* region, wherein the a* region of B and b* region respectively with a region and the b regional complementarity of A, the c region of B is positioned at the centre in a* region and b* region, protruding, complementary with A; One end of B is modified with the affine small-molecule substance of specificity;
Sequence C: there is a region and c* region, wherein a and the c* region of C respectively with a* and the c regional complementarity of B; One end sulfydryl modification of C;
Sequence D: there is a* region and c region; A* and the c region of D respectively with a region and the c* regional complementarity of C.
Preferably, 1-5 base is contained in the c region of nucleotide sequence B.
Preferably, the specificity described in nucleotide sequence B is affine, and small-molecule substance comprises 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 fixing is nucleotide sequence D.
Preferably, buffer system is not reacted to generate with lead ion and is precipitated.
A kind of lead ion Visual retrieval kit, comprise nucleotide sequence, buffer system and ELISA test strip platform, its nucleotide sequence is made up of nucleotide sequence A, B, C, D; Wherein
Nucleotide sequence A:5'-GGGTGGGT(a) GGGTGGGT(b)-3'(SEQIDNO:1);
Nucleotide sequence B:5'-biotin-AAAAAA-ACCCACCC(b*) TAT(c) ACCCACCC(a*)-3'(SEQIDNO:2);
Nucleotide sequence C:5'-SH-AAAAAA-GGGTGGGT(a) ATA(c*)-3'(SEQIDNO:3);
Nucleotide sequence D:5'-biotin-AAAAAA-TAT(c) ACCCACCC(a*)-3'(SEQIDNO:4).
Preferably, nucleotide sequence C is fixed on gold nano grain, and is injected on the gold mark pad of ELISA test strip platform.
Preferably, what the detection zone of ELISA test strip platform was fixing is Streptavidin; That quality control region is fixing is Streptavidin-biotin-nucleic acid D.
Preferably, buffer system is 10mMTris-Ac damping fluid, pH=8.0.
A kind of lead ion visible detection method, utilizes the kit of any one described above to carry out lead ion detection.
The invention has the beneficial effects as follows:
(1) lead ion recognition component is common DNA sequence dna, without the need to using traditional 8-17DNAzyme, without the need to relating to synthesis RNA, also without the need to using antibody.
(2) the direct naked eyes of testing result are visible, can obtain usage data without the need to detecting instrument.
(3) simple to operate, with low cost, be suitable for unit and promote the use of.
(4) detection of the inventive method to lead is limited to 1nM, and specify the maximum magnitude 72nM of Pb in Drinking Water ion lower than Environmental Protection Agency, detection sensitivity is high, and selectivity is good.
Accompanying drawing explanation
Fig. 1 is the schematic diagram of detection method of the present invention;
Fig. 2 is the Pb to variable concentrations
2+the result figure detected;
Fig. 3 is specificity experiments result figure.
Embodiment
A kind of lead ion Visual retrieval kit, comprise nucleotide sequence, buffer system and ELISA test strip platform, its nucleotide sequence is made up of nucleotide sequence A, B, C, D; Wherein
Sequence A: there is a region and b region, a and b region forms lead ion specific recognition element jointly, can form G tetramer structure with after lead ion identification;
Sequence B: there is a* region, c region and b* region, wherein the a* region of B and b* region respectively with a region and the b regional complementarity of A, the c region of B is positioned at the centre in a* region and b* region, protruding, complementary with A; One end of B is modified with the affine small-molecule substance of specificity;
Sequence C: there is a region and c* region, wherein a and the c* region of C respectively with a* and the c regional complementarity of B; One end sulfydryl modification of C;
Sequence D: there is a* region and c region; A* and the c region of D respectively with a region and the c* regional complementarity of C.
Preferably, 1-5 base is contained in the c region of nucleotide sequence B.
Preferred, 3 bases are contained in the c region of nucleotide sequence B.
Preferably, the specificity described in nucleotide sequence B is affine, and small-molecule substance comprises biotin, digoxin, Cy3.
Preferably, nucleotide sequence B is connected by the affine Small molecular of linking arm and specificity, and sequence C is connected with sulfydryl by linking arm.
Preferably, linking arm is the base sequence of multiple A, and its base number is 3-12.
Preferred, 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 fixing is nucleotide sequence D.
Preferably, the method for quality control region fixed nucleic acid sequence D is be cross-linked 5 minutes under ultraviolet light after nucleotide sequence D being injected in the quality control region of test strips; Or modified by the one end with biotin of nucleotide sequence D, quality control region utilizes Streptavidin to be fixed by the nucleic acid D of biotin modification.
Preferably, buffer system is not reacted to generate with lead ion and is precipitated.
A kind of lead ion Visual retrieval kit, comprise nucleotide sequence, buffer system and ELISA test strip platform, its nucleotide sequence is made up of nucleotide sequence A, B, C, D; Wherein
Nucleotide sequence A:5'-GGGTGGGT(a) GGGTGGGT(b)-3'(SEQIDNO:1);
Nucleotide sequence B:5'-biotin-AAAAAA-ACCCACCC(b*) TAT(c) ACCCACCC(a*)-3'(SEQIDNO:2);
Nucleotide sequence C:5'-SH-AAAAAA-GGGTGGGT(a) ATA(c*)-3'(SEQIDNO:3);
Nucleotide sequence D:5'-biotin-AAAAAA-TAT(c) ACCCACCC(a*)-3'(SEQIDNO:4).
Preferably, nucleotide sequence C is fixed on gold nano grain, and is injected on the gold mark pad of ELISA test strip platform.
Preferably, what the detection zone of ELISA test strip platform was fixing is Streptavidin; That quality control region is fixing is Streptavidin-biotin-nucleic acid D.
Preferably, the method for quality control region fixed nucleic acid sequence D is be cross-linked 5 minutes under ultraviolet light after nucleotide sequence D being injected in the quality control region of test strips; Or modified by the one end with biotin of nucleotide sequence D, quality control region utilizes Streptavidin to be fixed by the nucleic acid D of biotin modification.
Preferably, buffer system is 10mMTris-Ac damping fluid, pH=8.0.
A kind of lead ion visible detection method, utilizes the kit of above-mentioned any one to carry out lead ion detection.
Below in conjunction with accompanying drawing (see Fig. 1), further illustrate 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 (Pb
2+) there is high-affinity, the G tetramer can be formed.A is first hybridized with B, one end modified biological element (biotin) of B.When there being Pb
2+when existing, A and Pb
2+identify and interact, form the G tetramer, thus A disintegrating down from B.The B chain cemented out drips and detects in the sample pad of test strips.
(2) ELISA test strip platform comprises 4 parts: sample pad, gold mark pad, nitrocellulose filter and thieving paper, these four parts are from left to right fixed on offset plate successively.One end of nucleic acid C is modified with sulfydryl (-SH), is fixed on gold nano grain (AuNPs), and is injected on gold mark pad.Nitrocellulose filter is drawn and has two surveyed areas: detection zone and quality control region, that wherein detection zone is fixing is Streptavidin (SA); That quality control region is fixing is Streptavidin (SA)-biotin (biotin)-nucleic acid D.
(3) the B chain cemented out drips after the sample pad of test strips, moved forward by capillar migration effect, when gold mark pad, be combined with C, continue to move forward, the SA that B-C compound can be detected district catches (being interacted by SA-biotin), and assemble in detection zone, the gold nano grain of gathering forms macroscopic red area.The C of excessive gold nano particle modification can move forward, and hybridizes with the D being fixed on quality control region, and assemble in quality control region, the gold nano grain of gathering forms macroscopic red area.When there being Pb
2+when existing, all can there is redness in the detection zone of test strips and quality control region, and the depth of the red line color in detection zone and Pb
2+concentration is proportionate; And there is no Pb
2+when existing, only have quality control region to occur red, and detection zone does not have redness.
Below in conjunction with embodiment, further illustrate technical scheme of the present invention, but be not limited thereto.
embodiment 1
A kind of lead ion Visual retrieval agent box comprises following composition:
(1) nucleotide sequence A, B, C, D; A one end modified biological element (biotin); The one terminal modified sulfydryl (-SH) of C; D one end modified biological element (biotin).Concrete sequence is as follows:
Nucleotide sequence A:5'-GGGTGGGT(a) GGGTGGGT(b)-3'(SEQIDNO:1);
Nucleotide sequence B:5'-biotin-AAAAAA-ACCCACCC(b*) TAT(c) ACCCACCC(a*)-3'(SEQIDNO:2);
Nucleotide sequence C:5'-SH-AAAAAA-GGGTGGGT(a) ATA(c*)-3'(SEQIDNO:3);
Nucleotide sequence D:5'-biotin-AAAAAA-TAT(c) ACCCACCC(a*)-3'(SEQIDNO:4).
(2) lead ion standard solution.
(3) ELISA test strip platform: comprise sample pad, gold mark pad, nitrocellulose filter and thieving paper, these four parts are from left to right fixed on offset plate successively.C modifies gold nano grain, and be sprayed on gold mark pad, that the detection zone of nitrocellulose filter is fixing is Streptavidin (SA); That quality control region is fixing is SA-biotin-D; Test strips sealing bag seals.
(4) 10mMTris-Ac damping fluid (pH=8.0).
Lead ion visible detection method, carries out in accordance with the following steps:
(1) B of A and 500nM of 500nM dissolves with 10mMTris-Ac damping fluid (pH=8.0), and room temperature fully mixes, and reacts 30 minutes, forms A-B double-strand.
(2) add the lead ion standard solution of variable concentrations, room temperature reaction 45 minutes, then reactant liquor is dripped in the sample pad of test strips, detect.
(3) after test strips flows 10 minutes, whether directly detect by an unaided eye detection zone and quality control region there are red lines, thus judge whether system exists Pb
2+, the wherein depth of the red line color in detection zone and Pb
2+concentration is proportionate.
detection to variable concentrations lead ion:
Preparation lead ion standard solution, concentration is respectively 1nM, 10nM, 100nM, 500nM and 1 μM, room temperature preservation.The lead ion solution of variable concentrations is added in the reaction system described in embodiment 1 respectively, after abundant reaction, be added drop-wise in the sample pad of test strips by reactant liquor (100 μ L), room temperature detects, and observes the detection zone of test strips and the color change of quality control region after 10 minutes.
As can be seen from the testing result of Fig. 2, when 1nM lead ion exists, can observe obvious red area in the detection zone of test strips, its detection is limited to 1nM, specifies the maximum magnitude 72nM of Pb in Drinking Water ion lower than Environmental Protection Agency.Further, along with the increase (from 1nM to 500nM) of plumbum ion concentration, the color of detection zone also increases gradually.When plumbum ion concentration is more than 500nM, color change is tending towards saturated.Meanwhile, quality control region all takes on a red color in all cases, illustrates that test strips system running is normal, credible result.
specificity experiments:
The standard solution of preparation 1 μM of different ions, they are Hg respectively
2+, Cu
2+, Fe
3+, Mn
2+, Cr
3+, Co
2+, Ag
+and Cd
2+.
1 μM of different ion standard solution and 100nM lead ion standard solution are added in the reaction system described in embodiment 1 respectively, after abundant reaction, reactant liquor (100 μ L) is added drop-wise in the sample pad of ELISA test strip platform, room temperature detects, and observes the detection zone of test strips and the color change of quality control region after 10 minutes.
As can be seen from the testing result of Fig. 3, the Hg of 1 μM
2+, Cu
2+, Fe
3+, Mn
2+, Cr
3+, Co
2+, Ag
+and Cd
2+on detection not impact, not there is red area in the detection zone of test strips; Only have and just can occur red area in the detection zone of test strips after adding lead ion.This proves that the detection of the method to lead ion has good specificity.
<110> Guangdong Prov. Inst. of Ecological Environment & Soil Science
<120> lead ion visible detection method and detection kit
<130>
<160>4
<170>PatentInversion3.5
<210>1
<211>16
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<213> artificial combination sequence
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gggtgggtgggtgggt16
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aaaaaaacccaccctatacccaccc25
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Claims (10)
1. a lead ion Visual retrieval kit, comprises nucleotide sequence, buffer system and ELISA test strip platform, it is characterized in that: nucleotide sequence is made up of nucleotide sequence A, B, C, D; Wherein
Sequence A: there is a region and b region, a and b region forms lead ion specific recognition element jointly, can form G tetramer structure with after lead ion identification;
Sequence B: there is a* region, c region and b* region, wherein the a* region of B and b* region respectively with a region and the b regional complementarity of A, the c region of B is positioned at the centre in a* region and b* region, protruding, complementary with A; One end of B is modified with the affine small-molecule substance of specificity;
Sequence C: there is a region and c* region, wherein a and the c* region of C respectively with a* and the c regional complementarity of B; One end sulfydryl modification of C;
Sequence D: there is a* region and c region; A* and the c region of D respectively with a region and the c* regional complementarity of C.
2. kit according to claim 1, is characterized in that: 1-5 base is contained in the c region of nucleotide sequence B.
3. kit according to claim 1, is characterized in that: the specificity described in nucleotide sequence B is affine, and small-molecule substance comprises biotin, digoxin, Cy3.
4. kit according to claim 1, is characterized in that: 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 fixing is nucleotide sequence D.
5. kit according to claim 1, is characterized in that: buffer system is not reacted to generate with lead ion and precipitated.
6. a lead ion Visual retrieval kit, comprises nucleotide sequence, buffer system and ELISA test strip platform, it is characterized in that: nucleotide sequence is made up of nucleotide sequence A, B, C, D; Wherein
Nucleotide sequence A:5'-GGGTGGGT(a) GGGTGGGT(b)-3'(SEQIDNO:1);
Nucleotide sequence B:5'-biotin-AAAAAA-ACCCACCC(b*) TAT(c) ACCCACCC(a*)-3'(SEQIDNO:2);
Nucleotide sequence C:5'-SH-AAAAAA-GGGTGGGT(a) ATA(c*)-3'(SEQIDNO:3);
Nucleotide sequence D:5'-biotin-AAAAAA-TAT(c) ACCCACCC(a*)-3'(SEQIDNO:4).
7. kit according to claim 6, is characterized in that: nucleotide sequence C is fixed on gold nano grain, and is injected on the gold mark pad of ELISA test strip platform.
8. kit according to claim 6, is characterized in that: what the detection zone of ELISA test strip platform was fixing is Streptavidin; That quality control region is fixing is Streptavidin-biotin-nucleic acid D.
9. kit according to claim 6, is characterized in that: buffer system is 10mMTris-Ac damping fluid, pH=8.0.
10. a lead ion visible detection method, is characterized in that, utilizes the kit of any one described in claim 1-9 to carry out lead ion detection.
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