CN114397291A - Visual copper ion detection kit and detection method based on cyanine dye aggregate transition and Click reaction - Google Patents
Visual copper ion detection kit and detection method based on cyanine dye aggregate transition and Click reaction Download PDFInfo
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- CN114397291A CN114397291A CN202111542688.2A CN202111542688A CN114397291A CN 114397291 A CN114397291 A CN 114397291A CN 202111542688 A CN202111542688 A CN 202111542688A CN 114397291 A CN114397291 A CN 114397291A
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- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 title claims abstract description 127
- 229910001431 copper ion Inorganic materials 0.000 title claims abstract description 116
- 238000001514 detection method Methods 0.000 title claims abstract description 50
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 title claims abstract description 43
- 238000012650 click reaction Methods 0.000 title claims abstract description 18
- 230000000007 visual effect Effects 0.000 title claims description 14
- 230000007704 transition Effects 0.000 title claims description 6
- 239000000243 solution Substances 0.000 claims abstract description 67
- 239000011550 stock solution Substances 0.000 claims abstract description 61
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 54
- 239000000975 dye Substances 0.000 claims abstract description 46
- 108020004414 DNA Proteins 0.000 claims abstract description 44
- 102000053602 DNA Human genes 0.000 claims abstract description 44
- 125000000304 alkynyl group Chemical group 0.000 claims abstract description 24
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 claims abstract description 22
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims abstract description 21
- 239000007853 buffer solution Substances 0.000 claims abstract description 19
- 238000006243 chemical reaction Methods 0.000 claims abstract description 15
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 claims abstract description 4
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 claims abstract description 4
- 238000002156 mixing Methods 0.000 claims description 40
- PPASLZSBLFJQEF-RKJRWTFHSA-M sodium ascorbate Substances [Na+].OC[C@@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RKJRWTFHSA-M 0.000 claims description 23
- 235000010378 sodium ascorbate Nutrition 0.000 claims description 22
- 229960005055 sodium ascorbate Drugs 0.000 claims description 22
- PPASLZSBLFJQEF-RXSVEWSESA-M sodium-L-ascorbate Chemical compound [Na+].OC[C@H](O)[C@H]1OC(=O)C(O)=C1[O-] PPASLZSBLFJQEF-RXSVEWSESA-M 0.000 claims description 22
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 22
- 239000012498 ultrapure water Substances 0.000 claims description 22
- 125000003729 nucleotide group Chemical group 0.000 claims description 17
- 239000002773 nucleotide Substances 0.000 claims description 16
- 230000010355 oscillation Effects 0.000 claims description 16
- 238000000034 method Methods 0.000 claims description 15
- 230000008859 change Effects 0.000 claims description 13
- 238000002360 preparation method Methods 0.000 claims description 13
- 239000011259 mixed solution Substances 0.000 claims description 12
- 150000007523 nucleic acids Chemical class 0.000 claims description 11
- 238000010521 absorption reaction Methods 0.000 claims description 9
- 239000012488 sample solution Substances 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 7
- QWENRTYMTSOGBR-UHFFFAOYSA-N 1H-1,2,3-Triazole Chemical compound C=1C=NNN=1 QWENRTYMTSOGBR-UHFFFAOYSA-N 0.000 claims description 5
- 108091081406 G-quadruplex Proteins 0.000 claims description 5
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
- 229910052939 potassium sulfate Inorganic materials 0.000 claims description 4
- 230000008878 coupling Effects 0.000 claims description 3
- 238000010168 coupling process Methods 0.000 claims description 3
- 230000009466 transformation Effects 0.000 claims description 3
- 235000010323 ascorbic acid Nutrition 0.000 claims description 2
- 239000011668 ascorbic acid Substances 0.000 claims description 2
- 229960005070 ascorbic acid Drugs 0.000 claims description 2
- 239000001509 sodium citrate Substances 0.000 claims description 2
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical compound [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 claims description 2
- 229940038773 trisodium citrate Drugs 0.000 claims description 2
- 238000012360 testing method Methods 0.000 abstract description 4
- 238000004458 analytical method Methods 0.000 abstract description 2
- 230000007774 longterm Effects 0.000 abstract description 2
- 238000003860 storage Methods 0.000 abstract description 2
- 238000011897 real-time detection Methods 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 30
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 16
- 239000012086 standard solution Substances 0.000 description 13
- 239000007787 solid Substances 0.000 description 10
- 238000002835 absorbance Methods 0.000 description 9
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 6
- 230000004044 response Effects 0.000 description 6
- 238000002211 ultraviolet spectrum Methods 0.000 description 6
- 239000010949 copper Substances 0.000 description 5
- 238000010586 diagram Methods 0.000 description 5
- -1 3-sulfopropyl Chemical group 0.000 description 4
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 229910052802 copper Inorganic materials 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 239000003638 chemical reducing agent Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012800 visualization Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- FSSPGSAQUIYDCN-UHFFFAOYSA-N 1,3-Propane sultone Chemical compound O=S1(=O)CCCO1 FSSPGSAQUIYDCN-UHFFFAOYSA-N 0.000 description 2
- OUXMJRMYZCEVKO-UHFFFAOYSA-N 2-methylbenzo[e][1,3]benzothiazole Chemical compound C1=CC=C2C(N=C(S3)C)=C3C=CC2=C1 OUXMJRMYZCEVKO-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000003139 buffering effect Effects 0.000 description 2
- 238000005119 centrifugation Methods 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000006352 cycloaddition reaction Methods 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000005360 mashing Methods 0.000 description 2
- 102000039446 nucleic acids Human genes 0.000 description 2
- 108020004707 nucleic acids Proteins 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- NDQXKKFRNOPRDW-UHFFFAOYSA-N 1,1,1-triethoxyethane Chemical compound CCOC(C)(OCC)OCC NDQXKKFRNOPRDW-UHFFFAOYSA-N 0.000 description 1
- FGWYWKIOMUZSQF-UHFFFAOYSA-N 1,1,1-triethoxypropane Chemical compound CCOC(CC)(OCC)OCC FGWYWKIOMUZSQF-UHFFFAOYSA-N 0.000 description 1
- 208000009304 Acute Kidney Injury Diseases 0.000 description 1
- 206010019233 Headaches Diseases 0.000 description 1
- 206010019851 Hepatotoxicity Diseases 0.000 description 1
- 206010028813 Nausea Diseases 0.000 description 1
- 208000033626 Renal failure acute Diseases 0.000 description 1
- 238000005411 Van der Waals force Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 201000011040 acute kidney failure Diseases 0.000 description 1
- 208000012998 acute renal failure Diseases 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 238000004220 aggregation Methods 0.000 description 1
- 238000000559 atomic spectroscopy Methods 0.000 description 1
- 150000001540 azides Chemical class 0.000 description 1
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000000539 dimer Substances 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 238000000840 electrochemical analysis Methods 0.000 description 1
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical class O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 230000007686 hepatotoxicity Effects 0.000 description 1
- 231100000304 hepatotoxicity Toxicity 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 229910001414 potassium ion Inorganic materials 0.000 description 1
- 238000005057 refrigeration Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
- G01N21/77—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator
- G01N21/78—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated by observing the effect on a chemical indicator producing a change of colour
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N21/643—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" non-biological material
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Abstract
A visualized copper ion detection kit based on cyanine dye aggregate conversion and Click reaction comprises deoxyribonucleic acid stock solution with 3 '-end modified azide groups and 5' -end modified alkynyl groups, reducing solution, copper ion fluorescent probe stock solution and K+And (4) buffer solution. The cyanine dye fluorescent probe molecules are dissolved in DMSO to prepare 1-5 mM copper ion fluorescent probe stock solution, and the structural formula of the cyanine dye fluorescent probe molecules is shown as the following formula (I), wherein R is selected from methyl or ethyl. This detect test paper box conveniently carries, easy operation, good reproducibility, convenient long-term storage use, can high-efficiently carry out real-time detection to the copper ion fast, reduce the analysis cost and to the pollution of environment, the testing result just can observe through the naked eye, can extensively use widely.
Description
Technical Field
The invention relates to a copper ion detection test paper box and a detection method, in particular to a visualized copper ion detection kit based on cyanine dye aggregate conversion and Click reaction and a detection method.
Background
In nature, copper has a high content, and has good conductivity, durability and easy migration, and thus is widely used in the light industry, manufacturing industry, defense industry, and the like. Physiologically, copper is one of the essential trace elements of organisms, often participates in some important enzyme catalysis processes in the organisms, and is beneficial to the growth of the organisms to a certain extent. When the organism takes low dose of copper ions, symptoms such as headache, nausea and the like can occur; when a high concentration of copper ions is taken for a long time, growth of an organism is stopped, and serious diseases such as hepatotoxicity and acute renal failure occur. Therefore, the development of a method for rapidly detecting copper in the environment has great significance to human health. The traditional methods for detecting copper ions mainly comprise atomic spectroscopy, resonance scattering spectroscopy, electrochemical analysis and the like, and the methods have high sensitivity and good accuracy, but the detection process is complicated, a large amount of professional technicians and expensive equipment and instruments are needed, and real-time monitoring cannot be carried out.
Since 2001, the advent of "click chemistry" has provided the convenience of people to detect copper ions in the environment, primarily by using terminal azido and alkynyl groups, in reducing agents and Cu2+In the presence of (a), 1,2,3 triazole is synthesized by cycloaddition reaction. The method has mild reaction conditions, no side reaction in the reaction process and high selectivity. Cu+Cu can be reduced by sodium ascorbate2+And (4) generating. Based on Cu+Has been used for visual detection of Cu2+Most of the detection principles are based on colorimetric methods, the operation is complex, the repeatability is poor, and the sensitivity of the methods needs to be further improved.
Disclosure of Invention
The invention aims to provide a visualized copper ion detection kit and a detection method based on cyanine dye aggregate transformation and Click reaction, the detection kit is convenient to carry, simple to operate, good in repeatability, convenient to store and use for a long time, capable of efficiently, quickly, highly selectively and highly sensitively detecting copper ions in real time, reducing analysis cost and pollution to the environment, and capable of being observed by naked eyes, and being widely popularized and used.
In order to achieve the above object, the present inventionThe kit comprises deoxyribonucleic acid stock solution with 3 '-end modified azide group and 5' -end modified alkynyl group, reducing solution, copper ion fluorescent probe stock solution and K+A buffer solution;
the preparation method of the deoxyribonucleic acid stock solution with the 3 '-end modified azide group and the 5' -end modified alkynyl group comprises the following steps: respectively dissolving a nucleotide sequence of deoxyribonucleic acid with 3 '-end modified azide group and a nucleotide sequence of deoxyribonucleic acid with 5' -end modified alkynyl group in ultrapure water to prepare deoxyribonucleic acid stock solution with the concentration of 100 mu M, uniformly mixing by oscillation, centrifuging for 1min, and refrigerating in a refrigerator for later use;
the nucleotide sequence of the deoxyribonucleic acid with the 3' end modified azide group is one of 5' -GTGGGTAGG-N-3 ', 5' -GGGTGGGT-N-3 ', 5' -GGGTAGG-N-3 ', 5' -GGTGGTGGTGGTT-N-3 ';
the nucleotide sequence of the deoxyribonucleic acid with the 5' end modified alkynyl group is one of 5' -CH (identical to) C-GCGGGTTGGG-3 ', 5' -CH (identical to) C-GGGTGGGT-3' and 5' -CH (identical to) C-GTGGTGGTGGTGG-3 ';
the preparation method of the copper ion fluorescent probe stock solution comprises the following steps: dissolving cyanine dye fluorescent probe molecules in DMSO to prepare 1-5 mM copper ion fluorescent probe stock solution, wherein the structural formula of the cyanine dye fluorescent probe molecules is shown as the following formula (I):
wherein R is selected from methyl or ethyl.
Preferably, the reducing solution is one of a sodium ascorbate solution, an ascorbic acid solution and a trisodium citrate solution.
Preferably, the concentration of the sodium ascorbate solution is 1-10 mM.
Preferably, said K+The buffer solution is KCl solution or K2SO4Solution of, K+K in buffer solution+The concentration is 0.1-5 mol/L.
The invention also aims to provide a copper ion detection method of the visual copper ion detection kit based on cyanine dye aggregate conversion and Click reaction, which is simple and suitable for wide popularization and application.
In order to achieve the above object, the present invention further provides a copper ion detection method of a visualized copper ion detection kit based on cyanine dye aggregate conversion and Click reaction, comprising the following steps:
1) sequentially adding deoxyribonucleic acid stock solution with 3 '-end modified azide groups and 5' -end modified alkynyl groups, reducing solution and sample solution to be detected into an ultrapure water system, and oscillating for 2 hours at room temperature;
2) adding copper ion fluorescent probe stock solution and K into the mixed solution after the oscillation is finished+Shaking and mixing the buffer solution uniformly, centrifuging, and standing in a dark place for 20 min;
3) observing the color change of the mixed solution, if the sample solution to be detected contains Cu2+The two modified nucleic acid sequences are connected into a new 1,2, 3-triazole coupling nucleic acid sequence, and the solution is blue; if the sample solution to be tested does not contain Cu2+The alkynyl-modified nucleic acid sequence forms a G-quadruplex, and the solution is pink;
4) transferring the mixed solution into an enzyme label plate, and measuring the absorption values of the mixed solution at 580nm and 650nm by using an enzyme label or an ultraviolet spectrophotometer to the Cu2+The content of (b) was quantitatively analyzed.
Preferably, in step 1), the reducing solution is a sodium ascorbate solution.
The detection principle of the kit of the invention is as follows: a nucleotide sequence of deoxyribonucleic acid with 3 'end modified with azide group and a nucleotide sequence of deoxyribonucleic acid with 5' end modified with alkynyl group in ultra-pure water/K+In the system, two modified nucleotide sequences of deoxyribonucleic acid, reducing solution and a copper ion cyanine dye probe fluorescent probe form a detection system.
Cyanine dye supramolecular aggregates are sensitive to the environment and allow for conversion between different aggregates with concomitant spectral signal conversion and significant color change. Supramolecular aggregation can cascade recognition signals relative to single molecule probes. In general, dye molecules self-polymerize due to short-range van der Waals forces and exist in solution in four forms, monomer, dimer, H-aggregate, and J-aggregate, respectively. In contrast to monomers, J-aggregates and H-aggregates are secondary structures formed by head-to-head and face-to-face stacking of dye molecules.
As shown in figure 1, if no copper ions exist in the solution, the alkyne group modified nucleotide sequence forms a G-quadruplex structure in potassium ions, and the system becomes light pink visible to naked eyes after the alkyne group modified nucleotide sequence reacts with a monomer of a cyanine dye; if copper ions are present in the solution, the divalent copper ions are reduced to monovalent copper ions under the action of a reducing agent, and the monovalent copper ions are reduced to monovalent copper ions in the presence of Cu+In the method, two modified guanine (G) -rich sequences are subjected to Click reaction, namely cycloaddition coupling reaction to be connected into a new 1,2, 3-triazole coupled deoxyribonucleic acid sequence, the newly formed sequence cannot form a G-quadruplex, a monomer of a cyanine dye is converted into a J-aggregate, a solution is changed from pink to blue, and visual detection is realized.
Compared with the prior art, the invention has the following advantages:
the detection test paper box can efficiently, quickly, selectively and sensitively detect copper ions in real time, has the advantages of convenience in carrying, simplicity in operation, good repeatability and convenience in long-term storage and use, and has a good application prospect; the detection method is simple, the detection result can be observed by naked eyes, and the method has important significance for detecting the copper ions in tap water and the environment and is suitable for wide popularization and application.
Drawings
FIG. 1 is a schematic diagram of a copper ion detection kit and a detection method according to the present invention;
FIG. 2 is a diagram of an ultraviolet spectrum of a detection system with copper ions added in accordance with a first embodiment of the present invention;
FIG. 3 is a UV spectrum of a detection system with copper ions of different concentrations according to a first embodiment of the present invention;
FIG. 4 is a diagram illustrating the visual change of the color of copper ions with different concentrations added to the detection system according to the first embodiment of the present invention;
FIG. 5 is a UV spectrum of a copper ion added to the detection system according to example two of the present invention;
FIG. 6 is a UV spectrum of a detection system according to example two of the present invention with copper ions added at different concentrations;
FIG. 7 is a diagram showing the visual change of the color of copper ions added to different concentrations in the detection system according to the second embodiment of the present invention;
FIG. 8 is a UV spectrum of a copper ion added to the detection system of example III of the present invention;
FIG. 9 is a UV spectrum of a detection system according to a third embodiment of the present invention with copper ions of different concentrations;
FIG. 10 is a diagram showing the visual change of color of copper ions added to different concentrations in the detection system according to the third embodiment of the present invention.
Detailed Description
The invention is further described with reference to the following figures and specific examples.
In this example, the nucleotide sequence of the deoxyribonucleic acid with the azide group modified at the 3 '-end and the nucleotide sequence of the deoxyribonucleic acid with the alkynyl group modified at the 5' -end were obtained by bioengineering (Shanghai) GmbH.
The preparation method of two cyanine dye fluorescent probe molecules in the following examples is as follows:
MTC cyanine dye fluorescent probe molecule:
0.50g of 2-methylnaphthothiazole and 1.00g of 1, 3-propane sultone (the molar ratio is about 1:3) are placed in a thick-wall pressure-resistant tube and react for 5 hours at 170 ℃, and the product is yellow brown. Adding a proper amount of methanol, mashing the solid, washing and removing the upper layer of yellow liquid, and repeatedly washing for multiple times until the supernatant is nearly colorless. After discarding the aqueous layer, the intermediate was obtained as a pale yellow powder solid after the methanol had evaporated to dryness, calculated as an intermediate yield of 64.49%.
Taking 0.21g of intermediate, 0.60g of phenol and 0.21g of triethyl orthoacetate, mixing and heating to 60 ℃ to melt the intermediate, adding 0.2mL of triethylamine after fully mixing, raising the temperature to 120 ℃, and reacting for 1.5h to obtain a solution which is purple black. After cooling, 5mL of ether was added and the excess phenol and starting material were washed off and repeated 3 times. Then 2mL of methanol was added to dissolve the MTC solid in the bottle. The solution in 0.2mL tube was placed in 1.5mL LEP tube, 1mL ether was added to each tube, and after standing for 15min, centrifugation was carried out (9000r/min,1 min). Then 0.2mL of methanol was added into the EP tube, and the above operation was repeated about 10 times until the supernatant was light purple to obtain crude MTC solid as a purple black yellowish solid. Mobile phase purification with methanol: triethylamine: the MTC pure product is obtained by preparing water in a ratio of 50:1:1, and the yield is 40.9%.
ETC cyanine dye fluorescent probe molecule:
0.50g of 2-methylnaphthothiazole and 1.00g of 1, 3-propane sultone (the molar ratio is about 1:3) are put into a thick-wall pressure-resistant tube and react for 5 hours at 170 ℃, and the product is yellow brown. Adding a proper amount of methanol, mashing the solid, washing and removing the upper layer of yellow liquid, and repeatedly washing for multiple times until the supernatant is nearly colorless. After discarding the aqueous layer, the intermediate was obtained as a pale yellow powder solid after the methanol had evaporated to dryness, calculated as an intermediate yield of 64.49%.
Taking 0.21g of intermediate, 0.60g of phenol and 0.28g of triethyl orthopropionate, mixing and heating to 60 ℃ to melt the intermediate, adding 0.2mL of triethylamine after fully mixing, raising the temperature to 120 ℃, and reacting for 1.5 h. After cooling, 5mL of ether was added and the excess phenol and starting material were washed off and repeated 3 times. Then 2mL of methanol was added to dissolve the ETC solid in the bottle. The solution in 0.2mL tube was put in 1.5mL EP tube, 1mL ether was added to each tube, and after standing for 15min, centrifugation was carried out (9000r/min,1 min). Then, 0.2mL of methanol was added to the EP tube, and the above operation was repeated about 10 times to obtain a crude ETC solid. Mobile phase purification with methanol: triethylamine: 50 parts of water: 1:1, the ETC pure solid is obtained, and the yield is 34.6%.
Example one
A visual copper ion detection kit based on cyanine dye aggregate conversion and Click reaction comprises deoxyribonucleic acid stock solution of 3 '-end modified azide groups (5' -GTGGGTAGG-N ═ N-3 ') and 5' -end modified alkynyl groups (5 '-CH ≡ C-GCGGGTTGGG-3'), sodium ascorbate solution of 2mM, MTC copper ion fluorescent probe stock solution of 5mM and KCl solution of 2 mol/L;
the preparation method of the deoxyribonucleic acid stock solution with the 3 '-end modified azide group and the 5' -end modified alkynyl group comprises the following steps: dissolving a nucleotide sequence of deoxyribonucleic acid with 3 '-end modified azide group and a nucleotide sequence of deoxyribonucleic acid with 5' -end modified alkynyl group in 105 muL and 107 muL of ultrapure water respectively to prepare deoxyribonucleic acid stock solution with the concentration of 100 muM, oscillating, uniformly mixing, centrifuging for 1min, and refrigerating in a refrigerator for later use;
the nucleotide sequence of the deoxyribonucleic acid with the 3' end modified azide group is 5' -GTGGGTAGG-N-3 '; the sequence structure of the end-modified azide is shown as the following formula (II), wherein Oligo is a nucleic acid abbreviation;
the nucleotide sequence of the deoxyribonucleic acid with the 5' end modified alkynyl group is 5' -CH ≡ C-GCGGGTTGGG-3 '; the sequence structure of the terminal modified alkynyl is shown as the following formula (III), wherein Oligo is a nucleic acid abbreviation;
the preparation method of the copper ion fluorescent probe stock solution comprises the following steps: dissolving a cyanine dye MTC (3, 3' -bis (3-sulfopropyl) -4,5,4,5-2 benzo-9-methyl-thia-cyanine dye triethylamine salt) in DMSO to prepare a copper ion fluorescent probe stock solution with the concentration of 5mM, uniformly mixing the copper ion fluorescent probe stock solution with the DMSO by oscillation, and centrifuging the mixture for 1 min; sucking 10 mu L of the stock solution of the copper ion fluorescent probe from 5mM, adding the 10 mu L of the stock solution of the copper ion fluorescent probe into 90 mu L of ultrapure water to prepare 500 mu M of stock solution of the MTC fluorescent probe, oscillating, uniformly mixing, centrifuging for 1min, and then placing the mixture in a refrigerator for refrigeration for later use. The structural formula of the cyanine dye MTC is shown as the following formula (I):
wherein R is selected from methyl.
Dissolving sodium ascorbate in ultrapure water to prepare reducing agent stock solution with concentration of 2mM, shaking, mixing, centrifuging for 1min, and refrigerating in refrigerator.
KCl is dissolved in ultrapure water to prepare K with the concentration of 2mol/L+And (5) buffering the solution, uniformly mixing the solution by oscillation, centrifuging the solution for 1min, and then placing the solution at room temperature for later use.
A copper ion detection method of a visual copper ion detection kit based on cyanine dye aggregate conversion and Click reaction comprises the following steps:
1) sequentially adding deoxyribonucleic acid stock solution with 3 '-end modified azide groups and 5' -end modified alkynyl groups, sodium ascorbate solution and sample solution to be detected into an ultrapure water system, and oscillating for 2 hours at room temperature;
2) adding copper ion fluorescent probe stock solution and K into the mixed solution after the oscillation is finished+Shaking and mixing the buffer solution uniformly, centrifuging, and standing in a dark place for 20 min;
3) observing the color change of the mixed solution, if the sample solution to be detected contains Cu2+The two modified nucleic acid sequences are connected into a new 1,2, 3-triazole coupling nucleic acid sequence, and the solution is blue; if the sample solution to be tested does not contain Cu2+The alkynyl-modified nucleic acid sequence forms a G-quadruplex, and the solution is pink;
4) transferring the mixed solution into an enzyme label plate, and measuring the absorption values of the mixed solution at 580nm and 650nm by using an enzyme label or an ultraviolet spectrophotometer to the Cu2+The content of (b) was quantitatively analyzed.
The functionality of the copper ion detection kit prepared in this example is verified as follows:
dissolving copper sulfate in ultrapure water to prepare a copper ion standard solution with the concentration of 5mM, uniformly mixing by oscillation, placing at room temperature after centrifuging for 1min, and diluting with water to obtain the copper ion standard solution with the concentrations of 500. mu.M, 600. mu.M, 625. mu.M and 1mM for later use.
(1) Response experiment of cyanine dye fluorescent probe molecule to copper ion
Taking out 2 sterile 1.5mL EP tubes, dividing the tubes into two groups containing copper ions and two groups containing no copper ions, sequentially adding 70 μ L and 59 μ L of ultrapure water, then adding 10 μ L of 100 μ M deoxyribonucleic acid (5 '-GTGGGTAGG-N ═ N-3' and 5 '-CH ≡ C-GCGGGTTGGG-3') stock solutions, shaking and mixing uniformly, centrifuging for 1min, adding 0 μ L and 12 μ L of 600 μ M copper ion solutions and 5 μ L of 2mM sodium ascorbate solution, shaking and mixing uniformly, centrifuging for 1min, and placing 2 samples at room temperature and shaking for 2 h; after the end of the shaking, 2. mu.L of 500. mu.M stock solution of MTC fluorescent probe and 5. mu.L of 2mol/L K were added to 2 samples+And (3) oscillating and uniformly mixing the buffer solution, centrifuging for 1min, standing for 20min in the dark at room temperature, and measuring the ultraviolet absorption luminosity by using a microplate reader or an ultraviolet spectrophotometer, wherein the experimental result is shown in figure 2. As can be seen from the graph, the ultraviolet absorbance changes very significantly between 650nm and 580nm, indicating that the kit responds well to copper ions.
(2) Response change trend of cyanine dye fluorescent probe molecules to copper ions with different concentrations
9 sterile 1.5mL EP tubes were taken, 69. mu.L, 50. mu.L, 44. mu.L, 41. mu.L, 40. mu.L, 59. mu.L, 56.5. mu.L, 54. mu.L and 49. mu.L of ultrapure water were sequentially added thereto, 10. mu.L of 100. mu.M stock solutions of deoxyribonucleic acid (5 '-GTGGGTAGG-N ═ N-3' and 5 '-CH ≡ C-GCGGGTTGGG-3') were further added thereto, and the mixture was shaken, mixed and centrifuged for 1 min; sequentially adding 0 μ L of 500 μ M, 9 μ L of 500 μ M, 25 μ L of 500 μ M, 28 μ L of 500 μ M, 29.6 μ L of 625 μ M, 10 μ L of 5mM, 12.5 μ L of 5mM, 15 μ L of 5mM and 20 μ L of 5mM copper ion standard solution into 9 EP tubes, respectively adding 5 μ L of 2mM sodium ascorbate solution into 9 EP tubes, shaking and mixing, centrifuging for 1min, and then placing 9 samples at room temperature and shaking for 2 h; then 2. mu.L of 500. mu.M stock solution of MTC fluorescent probe and 5. mu.L of 2mol/L K were added+And (3) oscillating and uniformly mixing the buffer solution, centrifuging for 1min, standing for 20min in the dark at room temperature, and measuring the ultraviolet absorption luminosity by using a microplate reader or an ultraviolet spectrophotometer, wherein the experimental result is shown in figure 3. As can be seen from the graph, the absorbance at 580nm and the absorbance at 650nm are concentrated in the copper ionThe degree of 0-400 mu M has good transformation tendency.
(3) Visualization experiment of cyanine dye fluorescent probe molecules on copper ions with different concentrations
Taking 2 sterile 1.5mL EP tubes, sequentially adding 74 mu L and 57 mu L of ultrapure water, respectively adding 10 mu L of 100 mu M two-tube deoxyribonucleic acid stock solution, shaking, mixing uniformly, and centrifuging for 1 min; sequentially adding 0 mu L of 5mM and 12 mu L of 5mM copper ion standard solution into 2 EP tubes, respectively adding 5 mu L of 2mM sodium ascorbate solution into the 2 EP tubes, uniformly mixing by oscillation, centrifuging for 1min, and placing 2 samples at room temperature for 2h by oscillation; then 2. mu.L of 500. mu.M stock solution of MTC fluorescent probe and 5. mu.L of 2mol/L K were added+The buffer solution was shaken and mixed, centrifuged for 1min, and directly observed for color change at room temperature in the dark for 20min, as shown in FIG. 4. As can be seen from FIG. 4, the color of the system is changed from pink to blue after the standard solution of copper ions is added. The kit is simple to operate, has obvious results, and can achieve the purpose of quickly detecting copper ions.
Example two
A visualized copper ion detection kit based on cyanine dye aggregate conversion and Click reaction comprises deoxyribonucleic acid stock solution of 3 '-end modified azide group (5' -GGGTGGGT-N ═ N-3 ') and 5' -end modified alkynyl group (5 '-CH ≡ C-GGGTGGGT-3'), 2mM sodium ascorbate solution, 5mM ETC copper ion fluorescent probe stock solution and 2mol/L K2SO4A solution;
the preparation method of the deoxyribonucleic acid stock solution with the 3 '-end modified azide group and the 5' -end modified alkynyl group and the preparation process of the sodium ascorbate solution are the same as those in the first embodiment.
The preparation method of the copper ion fluorescent probe stock solution comprises the following steps: dissolving a cyanine dye ETC (3, 3' -bis (3-sulfopropyl) -4,5,4,5-2 benzo-9-ethyl-thia-cyanine dye triethylamine salt) in DMSO to prepare a copper ion fluorescent probe stock solution with the concentration of 5mM, uniformly mixing the copper ion fluorescent probe stock solution with the DMSO by oscillation, and centrifuging the mixture for 1 min; sucking 10 mu L from 5mM copper ion fluorescent probe stock solution, adding into 90 mu L ultrapure water to prepare 500 mu M ETC fluorescent probe stock solution, shaking, mixing, centrifuging for 1min, and refrigerating in a refrigerator for later use. The structural formula of the cyanine dye ETC is shown as the following formula (I):
wherein R is selected from ethyl.
Will K2SO4Dissolving in ultrapure water to prepare K with the concentration of 2mol/L+And (5) buffering the solution, uniformly mixing the solution by oscillation, centrifuging the solution for 1min, and then placing the solution at room temperature for later use.
The method for detecting copper ions and the method for preparing the copper ion standard solution in this embodiment are the same as those in the first embodiment.
The functionality of the copper ion detection kit prepared in the second embodiment is verified as follows:
(1) response experiment of cyanine dye fluorescent probe molecule to copper ion
Taking out 2 sterile 1.5mL EP tubes, dividing the tubes into two groups containing copper ions and two groups containing no copper ions, sequentially adding 70 μ L and 59 μ L of ultrapure water, then adding 10 μ L of stock solutions of 100 μ M deoxyribonucleic acid (5'-GGGTGGGT-N ═ N-3' and 5 '-CH ≡ C-GGGTGGGGGT-3') into the tubes, shaking and mixing the tubes, centrifuging the tubes for 1min, adding 0 μ L and 12 μ L of 600 μ M copper ion solution and 5 μ L of 2mM sodium ascorbate solution, shaking and mixing the tubes, centrifuging the tubes for 1min, and placing 2 samples at room temperature and shaking for 2 h; after the end of the shaking, 2. mu.L of 500. mu.M stock solution of ETC fluorescent probe and 5. mu.L of 2mol/L K were added to 2 samples+And (3) oscillating and uniformly mixing the buffer solution, centrifuging for 1min, standing for 20min in the dark at room temperature, and measuring the ultraviolet absorption luminosity by using a microplate reader or an ultraviolet spectrophotometer, wherein the experimental result is shown in figure 5. As can be seen from the graph, the ultraviolet absorbance changes very significantly between 650nm and 580nm, indicating that the kit responds well to copper ions.
(2) Response change trend of cyanine dye fluorescent probe molecules to copper ions with different concentrations
9 sterile 1.5mL EP tubes were taken, and 69. mu.L, 49. mu.L, 56. mu.L, 54.5. mu.L, 53. mu.L, 51. mu.L, and,Adding 100 μ M stock solutions of deoxyribonucleic acid (5'-GGGTGGGT-N ═ N-3' and 5 '-CH ≡ C-GGGTGGGT-3') to 55.5 μ L, 62 μ L and 61 μ L of ultrapure water, shaking, mixing, and centrifuging for 1 min; sequentially adding 0 μ L of 500 μ M, 10 μ L of 1mM, 13 μ L of 1mM, 14.5 μ L of 1mM, 8 μ L of 2mM, 9 μ L of 2mM, 13.5 μ L of 2mM, 7 μ L of 5mM and 8 μ L of 5mM copper ion standard solution into 9 EP tubes, respectively adding 5 μ L of 2mM sodium ascorbate solution into the 9 EP tubes, uniformly shaking, centrifuging for 1min, and then placing 9 samples at room temperature and shaking for 2 h; then, 2. mu.L of 500. mu.M stock solution of ETC fluorescent probe and 5. mu.L of 2mol/L K were added to each well+And (3) oscillating and uniformly mixing the buffer solution, centrifuging for 1min, standing for 20min in the dark at room temperature, and measuring the ultraviolet absorption luminosity by using a microplate reader or an ultraviolet spectrophotometer, wherein the experimental result is shown in figure 6. As can be seen from the graph, the absorbance at 580nm and the absorbance at 650nm have a good conversion tendency at a copper ion concentration of 0 to 600. mu.M.
(3) Visualization experiment of cyanine dye fluorescent probe molecules on copper ions with different concentrations
Taking 2 sterile 1.5mL EP tubes, sequentially adding 74 mu L and 57 mu L of ultrapure water, respectively adding 10 mu L of 100 mu M two-tube deoxyribonucleic acid stock solution, shaking, mixing uniformly, and centrifuging for 1 min; sequentially adding 0 mu L of 5mM and 12 mu L of 5mM copper ion standard solution into 2 EP tubes, respectively adding 5 mu L of 2mM sodium ascorbate solution into the 2 EP tubes, uniformly mixing by oscillation, centrifuging for 1min, and placing 2 samples at room temperature for 2h by oscillation; then, 2. mu.L of 500. mu.M stock solution of ETC fluorescent probe and 5. mu.L of 2mol/L K were added to each well+The buffer solution was shaken and mixed, centrifuged for 1min, and directly observed for color change at room temperature in the dark for 20min, as shown in FIG. 7. As can be seen from the figure, the color of the system is changed from pink to blue after the copper ion standard solution is added. The kit is simple to operate, has obvious results, and can achieve the purpose of quickly detecting copper ions.
EXAMPLE III
A visual copper ion detection kit based on cyanine dye aggregate conversion and Click reaction comprises deoxyribonucleic acid stock solution of 3 '-end modified azide group (5' -GGGTAGG-N ═ N-3 ') and 5' -end modified alkynyl group (5 '-CH ≡ C-GCGGGTTGGG-3'), sodium ascorbate solution of 2mM, MTC copper ion fluorescent probe stock solution of 5mM and KCl solution of 2 mol/L;
the preparation method of the deoxyribonucleic acid stock solution with the 3 '-end modified azide group and the 5' -end modified alkynyl group, the preparation method of the copper ion fluorescent probe stock solution, and the preparation processes of the sodium ascorbate solution and the KCl solution are the same as in the first embodiment.
The method for detecting copper ions and the method for preparing the copper ion standard solution in this embodiment are the same as those in the first embodiment.
The functionality of the copper ion detection kit prepared in the third embodiment is verified as follows:
(1) response experiment of cyanine dye fluorescent probe molecule to copper ion
Taking out 2 sterile 1.5mL EP tubes, dividing the tubes into two groups containing copper ions and not containing copper ions, sequentially adding 70 μ L and 59 μ L of ultrapure water, respectively, then adding 10 μ L of 100 μ M deoxyribonucleic acid (5 '-GGGTAGG-N ═ N-3' and 5 '-CH ≡ C-GCGGGTTGGG-3') stock solutions, shaking and mixing uniformly, centrifuging for 1min, adding 0 μ L and 12 μ L of 600 μ M copper ion solutions and 5 μ L of 2mM sodium ascorbate solution, shaking and mixing uniformly, centrifuging for 1min, and placing 2 samples at room temperature and shaking for 2 h; after the end of the shaking, 2. mu.L of 500. mu.M stock solution of MTC fluorescent probe and 5. mu.L of 2mol/L K were added to 2 samples+And (3) oscillating and uniformly mixing the buffer solution, centrifuging for 1min, standing for 20min in the dark at room temperature, and measuring the ultraviolet absorption luminosity by using a microplate reader or an ultraviolet spectrophotometer, wherein the experimental result is shown in figure 8. As can be seen from the graph, the ultraviolet absorbance changes very significantly between 650nm and 580nm, indicating that the kit responds well to copper ions.
(2) Response change trend of cyanine dye fluorescent probe molecules to copper ions with different concentrations
After 69. mu.L, 59. mu.L, 56. mu.L, 54.5. mu.L, 61. mu.L, 54. mu.L and 57. mu.L of ultrapure water were sequentially added to 7 sterile 1.5mL EP tubes, 10. mu.L of 100. mu.M deoxyribonucleic acid (5 '-GGGTAGG-N ═ N-3' and 5 '-CH ≡ C-GCGGGTTGGG-3') was added to each tubeShaking, mixing, and centrifuging for 1 min; sequentially adding 0 μ L of 500 μ M, 10 μ L of 1mM, 13 μ L of 1mM, 14.5 μ L of 1mM, 8 μ L of 2mM, 15 μ L of 2mM and 12 μ L of 5mM copper ion standard solution into 7 EP tubes, respectively adding 5 μ L of 2mM sodium ascorbate solution into 7 EP tubes, shaking and mixing uniformly, centrifuging for 1min, and placing 7 samples at room temperature and shaking for 2 h; then 2. mu.L of 500. mu.M stock solution of MTC fluorescent probe and 5. mu.L of 2mol/L K were added+And (3) oscillating and uniformly mixing the buffer solution, centrifuging for 1min, standing for 20min in the dark at room temperature, and measuring the ultraviolet absorption luminosity by using a microplate reader or an ultraviolet spectrophotometer, wherein the experimental result is shown in figure 9. As can be seen from the graph, the absorbance at 580nm and the absorbance at 650nm have a good conversion tendency at a copper ion concentration of 0 to 600. mu.M.
(3) Visualization experiment of cyanine dye fluorescent probe molecules on copper ions with different concentrations
Taking 2 sterile 1.5mL EP tubes, sequentially adding 74 mu L and 57 mu L of ultrapure water, respectively adding 10 mu L of 100 mu M two-tube deoxyribonucleic acid stock solution, shaking, mixing uniformly, and centrifuging for 1 min; sequentially adding 0 mu L of 5mM and 12 mu L of 5mM copper ion standard solution into 2 EP tubes, respectively adding 5 mu L of 2mM sodium ascorbate solution into the 2 EP tubes, uniformly mixing by oscillation, centrifuging for 1min, and placing 2 samples at room temperature for 2h by oscillation; then 2. mu.L of 500. mu.M stock solution of MTC fluorescent probe and 5. mu.L of 2mol/L K were added+The buffer solution was shaken and mixed, centrifuged for 1min, and directly observed for color change at room temperature in the dark for 20min, as shown in FIG. 10. As can be seen from the figure, the color of the system is changed from pink to blue after the copper ion standard solution is added. The kit is simple to operate, has obvious results, and can achieve the purpose of quickly detecting copper ions.
Claims (6)
1. A visualized copper ion detection kit based on cyanine dye aggregate conversion and Click reaction is characterized by comprising deoxyribonucleic acid stock solution with 3 '-end modified azide groups and 5' -end modified alkynyl groups, reducing solution, copper ion fluorescent probe stock solution and K+A buffer solution;
the preparation method of the deoxyribonucleic acid stock solution with the 3 '-end modified azide group and the 5' -end modified alkynyl group comprises the following steps: respectively dissolving a nucleotide sequence of deoxyribonucleic acid with 3 '-end modified azide group and a nucleotide sequence of deoxyribonucleic acid with 5' -end modified alkynyl group in ultrapure water to prepare deoxyribonucleic acid stock solution with the concentration of 100 mu M, uniformly mixing by oscillation, centrifuging for 1min, and refrigerating in a refrigerator for later use;
the nucleotide sequence of the deoxyribonucleic acid with the 3' end modified azide group is one of 5' -GTGGGTAGG-N-3 ', 5' -GGGTGGGT-N-3 ', 5' -GGGTAGG-N-3 ', 5' -GGTGGTGGTGGTT-N-3 ';
the nucleotide sequence of the deoxyribonucleic acid with the 5' end modified alkynyl group is one of 5' -CH (identical to) C-GCGGGTTGGG-3 ', 5' -CH (identical to) C-GGGTGGGT-3' and 5' -CH (identical to) C-GTGGTGGTGGTGG-3 ';
the preparation method of the copper ion fluorescent probe stock solution comprises the following steps: dissolving cyanine dye fluorescent probe molecules in DMSO to prepare 1-5 mM copper ion fluorescent probe stock solution, wherein the structural formula of the cyanine dye fluorescent probe molecules is shown as the following formula (I):
wherein R is selected from methyl or ethyl.
2. The visual copper ion detection kit based on cyanine dye aggregate transition and Click reaction as claimed in claim 1, wherein the reducing solution is one of sodium ascorbate solution, ascorbic acid solution, trisodium citrate solution.
3. The visual copper ion detection kit based on cyanine dye aggregate transition and Click reaction according to claim 2, wherein the concentration of the sodium ascorbate solution is 1-10 mM.
4. The visual copper ion detection kit based on cyanine dye aggregate transition and Click reaction as claimed in claim 1 or 2, wherein the K is+The buffer solution is KCl solution or K2SO4Solution of, K+K in buffer solution+The concentration is 0.1-5 mol/L.
5. The method for detecting copper ions by using the visualized copper ion detection kit based on cyanine dye aggregate transition and Click reaction as claimed in claim 1, which comprises the following steps:
1) sequentially adding deoxyribonucleic acid stock solution with 3 '-end modified azide groups and 5' -end modified alkynyl groups, reducing solution and sample solution to be detected into an ultrapure water system, and oscillating for 2 hours at room temperature;
2) adding copper ion fluorescent probe stock solution and K into the mixed solution after the oscillation is finished+Shaking and mixing the buffer solution uniformly, centrifuging, and standing in a dark place for 20 min;
3) observing the color change of the mixed solution, if the sample solution to be detected contains Cu2+The two modified nucleic acid sequences are connected into a new 1,2, 3-triazole coupling nucleic acid sequence, and the solution is blue; if the sample solution to be tested does not contain Cu2+The alkynyl-modified nucleic acid sequence forms a G-quadruplex, and the solution is pink;
4) transferring the mixed solution into an enzyme label plate, and measuring the absorption values of the mixed solution at 580nm and 650nm by using an enzyme label or an ultraviolet spectrophotometer to the Cu2+The content of (b) was quantitatively analyzed.
6. The method for detecting copper ions by using the visualized copper ion detection kit based on cyanine dye aggregate transformation and Click reaction as claimed in claim 5, wherein in step 1), the reducing solution is sodium ascorbate solution.
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| CN101482508A (en) * | 2009-01-21 | 2009-07-15 | 苏州纳米技术与纳米仿生研究所 | High-sensibility detection method for trace metal ion |
| CN108414488A (en) * | 2018-03-16 | 2018-08-17 | 江西省农业科学院农产品质量安全与标准研究所 | A kind of specificity fluorescent probe, method and kit for detecting copper ion |
| CN110357896A (en) * | 2019-08-22 | 2019-10-22 | 山西大同大学 | A kind of compound and preparation and its application in detection bivalent cupric ion and strong acid pH |
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| CN101482508A (en) * | 2009-01-21 | 2009-07-15 | 苏州纳米技术与纳米仿生研究所 | High-sensibility detection method for trace metal ion |
| CN108414488A (en) * | 2018-03-16 | 2018-08-17 | 江西省农业科学院农产品质量安全与标准研究所 | A kind of specificity fluorescent probe, method and kit for detecting copper ion |
| CN110357896A (en) * | 2019-08-22 | 2019-10-22 | 山西大同大学 | A kind of compound and preparation and its application in detection bivalent cupric ion and strong acid pH |
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