CN114397291B - Visual copper ion detection kit and detection method based on cyanine dye aggregate conversion and Click reaction - Google Patents
Visual copper ion detection kit and detection method based on cyanine dye aggregate conversion and Click reaction Download PDFInfo
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- CN114397291B CN114397291B CN202111542688.2A CN202111542688A CN114397291B CN 114397291 B CN114397291 B CN 114397291B CN 202111542688 A CN202111542688 A CN 202111542688A CN 114397291 B CN114397291 B CN 114397291B
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- JPVYNHNXODAKFH-UHFFFAOYSA-N Cu2+ Chemical compound [Cu+2] JPVYNHNXODAKFH-UHFFFAOYSA-N 0.000 title claims abstract description 113
- 229910001431 copper ion Inorganic materials 0.000 title claims abstract description 113
- 238000001514 detection method Methods 0.000 title claims abstract description 57
- 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 44
- 238000006243 chemical reaction Methods 0.000 title claims abstract description 26
- 230000000007 visual effect Effects 0.000 title claims abstract description 24
- 238000012650 click reaction Methods 0.000 title claims abstract description 19
- 239000011550 stock solution Substances 0.000 claims abstract description 66
- 239000000243 solution Substances 0.000 claims abstract description 58
- 239000007850 fluorescent dye Substances 0.000 claims abstract description 51
- 108020004414 DNA Proteins 0.000 claims abstract description 47
- 102000053602 DNA Human genes 0.000 claims abstract description 47
- 239000000975 dye Substances 0.000 claims abstract description 47
- 125000000304 alkynyl group Chemical group 0.000 claims abstract description 21
- 239000007853 buffer solution Substances 0.000 claims abstract description 21
- IVRMZWNICZWHMI-UHFFFAOYSA-N azide group Chemical group [N-]=[N+]=[N-] IVRMZWNICZWHMI-UHFFFAOYSA-N 0.000 claims abstract description 18
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 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 31
- 229910021642 ultra pure water Inorganic materials 0.000 claims description 29
- 239000012498 ultrapure water Substances 0.000 claims description 29
- 235000010378 sodium ascorbate Nutrition 0.000 claims description 23
- 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
- 229960005055 sodium ascorbate Drugs 0.000 claims description 23
- 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 23
- 239000010949 copper Substances 0.000 claims description 21
- 239000002773 nucleotide Substances 0.000 claims description 17
- 125000003729 nucleotide group Chemical group 0.000 claims description 17
- 238000002360 preparation method Methods 0.000 claims description 15
- 230000008859 change Effects 0.000 claims description 13
- 239000011259 mixed solution Substances 0.000 claims description 12
- 238000000034 method Methods 0.000 claims description 11
- 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
- 238000002965 ELISA Methods 0.000 claims description 6
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 6
- 230000010355 oscillation Effects 0.000 claims description 6
- 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
- 125000000852 azido group Chemical group *N=[N+]=[N-] 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
- 125000002355 alkine group Chemical group 0.000 claims description 4
- 238000005859 coupling reaction Methods 0.000 claims description 4
- 230000008878 coupling Effects 0.000 claims description 3
- 238000010168 coupling process Methods 0.000 claims description 3
- 238000004445 quantitative analysis Methods 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 5
- 238000004458 analytical method Methods 0.000 abstract description 2
- 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 14
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 12
- 239000000203 mixture Substances 0.000 description 12
- 239000007787 solid Substances 0.000 description 10
- 238000002835 absorbance Methods 0.000 description 9
- 230000004044 response Effects 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 7
- 108090000790 Enzymes Proteins 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 6
- 238000002211 ultraviolet spectrum Methods 0.000 description 6
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 5
- 229910052802 copper Inorganic materials 0.000 description 5
- -1 3-sulfopropyl Chemical group 0.000 description 4
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 4
- 239000000178 monomer Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003638 chemical reducing agent Substances 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 230000035945 sensitivity Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000012795 verification 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
- 239000000956 alloy Substances 0.000 description 2
- 229910045601 alloy Inorganic materials 0.000 description 2
- 238000001816 cooling Methods 0.000 description 2
- 238000006352 cycloaddition reaction Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical class O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000007774 longterm Effects 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
- 230000008569 process Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 238000005057 refrigeration Methods 0.000 description 2
- 239000000523 sample Substances 0.000 description 2
- 239000007858 starting material Substances 0.000 description 2
- 238000003860 storage Methods 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
- 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
- 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
- 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
- 239000012530 fluid Substances 0.000 description 1
- 231100000869 headache Toxicity 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 230000008693 nausea Effects 0.000 description 1
- 231100000572 poisoning Toxicity 0.000 description 1
- 230000000607 poisoning effect Effects 0.000 description 1
- 229910001414 potassium ion Inorganic materials 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
Classifications
-
- 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
-
- 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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- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Plasma & Fusion (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Optics & Photonics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
A visual copper ion detection kit based on cyanine dye aggregate conversion and Click reaction comprises a deoxyribonucleic acid stock solution, a reducing solution, a copper ion fluorescent probe stock solution and K, wherein the deoxyribonucleic acid stock solution comprises a 3 '-end modified azide group and a 5' -end modified alkynyl group + Buffer solution. The cyanine dye fluorescent probe molecule is dissolved in DMSO to prepare copper ion fluorescent probe stock solution with the concentration of 1-5 mM, and the structural formula of the cyanine dye fluorescent probe molecule is shown as the following formula (I), wherein R is selected from methyl or ethyl. The detection test paper box is convenient to carry, simple to operate, good in repeatability and convenient to store and use for a long time, can be used for efficiently and rapidly detecting copper ions in real time, reduces analysis cost and pollution to the environment, can observe detection results through naked eyes, and can be widely popularized and used.
Description
Technical Field
The invention relates to a copper ion detection test paper box and a detection method, in particular to a visual copper ion detection kit and a detection method based on cyanine dye aggregate conversion and Click reaction.
Background
In nature, copper content is high, and the copper-based alloy has good conductivity, durability and mobility, so that the copper-based alloy is widely applied to light industry, manufacturing industry, national defense industry and the like. Physiologically, copper is one of the trace elements necessary for the organism, and often participates in some important enzyme catalysis processes in the organism, which is beneficial to the growth of the organism to a certain extent. When organisms ingest low doses of copper ions, symptoms such as headache, nausea and the like can occur; when high-concentration copper ions are taken for a long time, the growth of organisms is stopped, and serious diseases such as liver poisoning, acute renal failure and the like even occur. Therefore, developing a method for rapidly detecting copper in the environment is of great importance to human health. Traditional methods for detecting copper ions mainly comprise an atomic spectrometry, a resonance scattering spectrometry, an electrochemical analysis method and the like, and the methods are high in sensitivity and good in accuracy, but the detection process is complex, a large number of professional technicians and expensive equipment are required, and real-time monitoring cannot be performed.
Since 2001, the advent of "click chemistry" has facilitated the detection of copper ions in the environment, primarily by the use of terminal azido and alkynyl groups, in reducing agents and Cu 2+ 1,2,3 triazole is synthesized by cycloaddition reaction in the presence of (a). The method has mild reaction conditions, no side reaction in the reaction process, and high selectivity. Cu (Cu) + Cu can be reduced by sodium ascorbate 2+ And (3) generating. Cu-based + Click chemistry of (c) has been used to visually detect Cu 2+ The detection principle is 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 visual copper ion detection kit and a detection method based on cyanine dye aggregate conversion and Click reaction, wherein the detection test paper box is convenient to carry, simple to operate, good in repeatability and convenient for long-term storage and use, can be used for efficiently, quickly, selectively and highly sensitively detecting copper ions in real time, reduces analysis cost and pollution to the environment, can observe detection results through naked eyes, and can be widely popularized and used.
In order to achieve the aim, the invention provides a visual copper ion detection kit based on cyanine dye aggregate conversion and Click reaction, which comprises a deoxyribonucleic acid stock solution, a reducing solution, a copper ion fluorescent probe stock solution and K, wherein the deoxyribonucleic acid stock solution comprises a 3 '-end modified azide group and a 5' -end modified alkynyl group + 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 an azide group modified at the 3 'end and a nucleotide sequence of deoxyribonucleic acid with an alkyne group modified at the 5' end in ultrapure water to prepare a deoxyribonucleic acid stock solution with the concentration of 100 mu M, oscillating and mixing uniformly, centrifuging for 1min, and refrigerating in a refrigerator for later use;
deoxyribonucleic acid with 3' -end modified azide group the nucleotide sequence is 5' GTGGGTAGG-N=N=N-3 '; 5' -GGGTGGGT-N=N-3 ', 5' -GGGTAGG-N=N-3 ': 5' -GGTGGTGGTGGTT-n=n=n-3 ';
the nucleotide sequence of the 5' -end modified alkynyl group deoxyribonucleic acid is one of 5' -CH (identical to) C-GCGGGTTGGG-3', 5' -CH (identical to) C-GGGTGGGT-3', 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 copper ion fluorescent probe stock solution with the concentration of 1-5 mM, 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 sodium ascorbate solution, ascorbic acid solution and trisodium citrate solution.
Preferably, the concentration of sodium ascorbate solution is 1-10mM.
Preferably, the K + The buffer solution is KCl solution or K 2 SO 4 Solution, K + K in buffer solution + The concentration is 0.1-5mol/L.
The invention also aims to provide a copper ion detection method based on the visual copper ion detection kit for cyanine dye aggregate conversion and Click reaction, which is simple and suitable for wide popularization and application.
In order to achieve the above purpose, the invention also provides a copper ion detection method of the visualized copper ion detection kit based on cyanine dye aggregate conversion and Click reaction, which comprises the following steps:
1) Sequentially adding a deoxyribonucleic acid stock solution, a reducing solution and a sample solution to be tested of 3 '-end modified azido group and 5' -end modified alkynyl group into an ultrapure water system, and vibrating for 2 hours at room temperature;
2) Adding copper ion fluorescent probe stock solution and K into the mixed solution after oscillation is finished + Buffer solution, shaking and mixing evenly, centrifuging, and standing in a dark place for 20min;
3) Observing the color change of the mixed solution, if the sample solution to be detected contains Cu 2+ The two modified nucleic acid sequences are connected to form a new 1,2, 3-triazole coupling nucleic acid sequence, and the solution is blue; if the sample solution to be tested is free of Cu 2+ The alkynyl-modified nucleic acid sequence forms a G-quadruplex, in which case the solution appears pink;
4) Transferring the above mixed solution into an ELISA plate, measuring absorption values of the mixed solution at 580nm and 650nm with an ELISA or ultraviolet spectrophotometer, and measuring Cu 2+ Quantitative analysis was performed on the content of (c).
Preferably, in step 1), the reducing solution is a sodium ascorbate solution.
The detection principle of the kit provided by the invention is as follows: nucleotide sequence of a deoxyribonucleic acid with an azido group modified at the 3 'end and a deoxyribonucleic acid with an alkynyl group modified at the 5' end in ultrapure water/K + In the system, the nucleotide sequence of the two modified deoxyribonucleic acids, the reducing solution and the copper ion cyanine dye probe fluorescent needle form a detection system.
Cyanine dye supramolecular aggregates are sensitive to the environment, and can achieve conversion between different aggregates with concomitant spectral signal conversion and significant color change. Supermolecule aggregation can cascade the recognition signal relative to a single molecule probe. In general, dye molecules self-polymerize due to short-range van der Waals forces, and exist in four forms in solution, monomer, dimer, H-aggregate, J-aggregate, respectively. J-aggregates and H-aggregates are secondary structures formed by head-to-head and face-to-face stacking of dye molecules, as compared to monomers.
As shown in FIG. 1, if copper ions are not present in the solution, the nucleotide sequence modified by the alkynyl group forms a G-quadruplex structure in potassium ions, and after the nucleotide sequence reacts with a monomer of a cyanine dye, the system becomes a light pink visible to the naked eye; if present in solutionCopper ions, cupric ions are reduced to monovalent copper ions under the action of a reducing agent, and Cu is a metal + In the method, two modified guanine (G) -rich sequences are subjected to Click reaction, namely cycloaddition coupling reaction is carried out to connect the modified guanine (G) -rich sequences 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 J-aggregate, and a solution is changed from pink to blue, so that visual detection is realized.
Compared with the prior art, the invention has the following advantages:
the detection test paper box can be used for detecting copper ions in real time with high efficiency, high selectivity and high sensitivity, 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 detection 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 ultraviolet spectrum of copper ions added to the detection system according to the first embodiment of the present invention;
FIG. 3 is a graph of ultraviolet spectra of copper ions with different concentrations added into a detection system according to the first embodiment of the present invention;
FIG. 4 is a graph showing the visual change of color of copper ions with different concentrations added into the detection system according to the first embodiment of the invention;
FIG. 5 is a UV spectrum of copper ions added to the detection system according to the second embodiment of the present invention;
FIG. 6 is a graph of ultraviolet spectra of copper ions with different concentrations added to a detection system according to a second embodiment of the present invention;
FIG. 7 is a graph showing the visual change of color of copper ions with different concentrations added into the detection system according to the second embodiment of the present invention;
FIG. 8 is a UV spectrum of copper ions added to the detection system of the third embodiment of the present invention;
FIG. 9 is a graph of ultraviolet spectra of copper ions with different concentrations added to the detection system according to the third embodiment of the present invention;
FIG. 10 is a graph showing the visual change of color of copper ions added to the detection system according to the third embodiment of the present invention.
Detailed Description
The invention will be further described with reference to the drawings and the specific examples.
The nucleotide sequence of the deoxyribonucleic acid with the 3 '-terminal modified azide group and the nucleotide sequence of the deoxyribonucleic acid with the 5' -terminal modified alkyne group in this example are both purchased from the biological engineering (Shanghai) Co., ltd.
The preparation method of the two cyanine dye fluorescent probe molecules in the following examples is as follows:
MTC cyanine dye fluorescent probe molecules:
0.50g of 2-methylnaphthothiazole and 1.00g of 1, 3-propane sultone (molar ratio is about 1:3) are placed in a thick-wall pressure-resistant pipe and reacted for 5 hours at 170 ℃, and the product is yellow brown. Adding a proper amount of methanol, mashing the solid, washing to remove upper yellow liquid, and repeatedly washing for a plurality of times until the supernatant is nearly colorless. After discarding the aqueous layer, the methanol was evaporated to dryness to give the intermediate as a pale yellow powdery solid, the intermediate yield was calculated to be 64.49%.
Mixing 0.21g of intermediate, 0.60g of phenol and 0.21g of triethyl orthoacetate, heating to 60 ℃ to melt the mixture, adding 0.2mL of triethylamine after fully mixing, raising the temperature to 120 ℃, and reacting for 1.5h, wherein the solution is purple black. After cooling, 5mL of diethyl ether was added, and the excess phenol and starting material were washed off and repeated 3 times. 2mL of methanol was then added to dissolve the MTC solid in the bottle. The solution in the 0.2mL tube was placed in a 1.5mLEP tube, 1mL diethyl ether was added to each tube, and the mixture was allowed to stand for 15min and centrifuged (9000 r/min,1 min). Then 0.2mL of methanol is added into the EP tube, the operation is repeated for about 10 times, the supernatant fluid is light purple, and the crude solid of the MTC is purple black yellow solid. The mobile phase purification adopts methanol: triethylamine: configuration of water=50:1:1 ratio gave MTC pure product in 40.9% yield.
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 taken and placed in a thick-wall pressure-resistant pipe for reaction for 5 hours at 170 ℃, and the product is yellow brown. Adding a proper amount of methanol, mashing the solid, washing to remove upper yellow liquid, and repeatedly washing for a plurality of times until the supernatant is nearly colorless. After discarding the aqueous layer, the methanol was evaporated to dryness to give the intermediate as a pale yellow powdery solid, the intermediate yield was calculated to be 64.49%.
Mixing 0.21g of intermediate, 0.60g of phenol and 0.28g of triethyl orthopropionate, heating to 60 ℃ to melt the three, fully mixing, adding 0.2mL of triethylamine, raising the temperature to 120 ℃, and reacting for 1.5h. After cooling, 5mL of diethyl ether was added, and the excess phenol and starting material were washed off and repeated 3 times. 2mL of methanol was added and the ETC solid in the flask was dissolved. The solution in the 0.2mL tube was placed in a 1.5mL EP tube, 1mL diethyl ether was added to each tube, and the mixture was allowed to stand for 15min and centrifuged (9000 r/min,1 min). Thereafter, 0.2mL of methanol was added to the EP tube, and the above operation was repeated about 10 times to obtain an ETC crude solid. The mobile phase purification adopts methanol: triethylamine: water = 50:1:1 to yield an ETC pure solid in 34.6%.
Example 1
A visual copper ion detection kit based on cyanine dye aggregate conversion and Click reaction, which comprises a deoxyribonucleic acid stock solution of 3 '-end modified azide group (5' -GTGGGTAGG-N=N-3 ') and 5' -end modified alkynyl group (5 '-CH≡C-GCGGGTTGGG-3'), a 2mM sodium ascorbate solution, a 5mM MTC copper ion fluorescent probe stock solution and a 2mol/L KCl 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: dissolving a nucleotide sequence of deoxyribonucleic acid with an azide group modified at the 3 'end and a nucleotide sequence of deoxyribonucleic acid with an alkyne group modified at the 5' end in 105 mu L of ultrapure water and 107 mu L of ultrapure water respectively to prepare a 100 mu M deoxyribonucleic acid stock solution, oscillating and mixing uniformly, centrifuging for 1min, and refrigerating in a refrigerator for later use;
deoxyribonucleic acid with 3' -end modified azide group is 5' -GTGGGTAGG-n=n =n-3 '; the sequence structure of the terminal modified azide is shown as the following formula (II), wherein Oligo is nucleic acid abbreviation;
the nucleotide sequence of the 5' -end modified alkynyl group deoxyribonucleic acid 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 cyanine dye MTC (3, 3' -bis (3-sulfopropyl) -4,5,4,5-2 benzo-9-methyl-thiacarbocyanine dye triethylamine salt) in DMSO to prepare copper ion fluorescent probe stock solution with the concentration of 5mM, oscillating and mixing uniformly, and centrifuging for 1min; 10 mu L of the stock solution of the copper ion fluorescent probe is sucked from 5mM and added into 90 mu L of ultrapure water to prepare 500 mu M stock solution of the MTC fluorescent probe, and the stock solution is stirred and mixed uniformly, centrifuged for 1min and then placed in a refrigerator for refrigeration for standby. The structural formula of the cyanine dye MTC is shown in the following formula (I):
wherein R is selected from methyl.
Dissolving sodium ascorbate in ultrapure water to prepare a reducing agent stock solution with the concentration of 2mM, shaking and mixing uniformly, centrifuging for 1min, and refrigerating in a refrigerator for later use.
KCl was dissolved in ultrapure water to prepare a K having a concentration of 2mol/L + The buffer solution is stirred and mixed evenly, and is placed at room temperature for standby after being centrifuged for 1 min.
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 a deoxyribonucleic acid stock solution of a 3 '-end modified azide group and a 5' -end modified alkynyl group, a sodium ascorbate solution and a sample solution to be tested 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 oscillation is finished + Buffer solution, shaking and mixing evenly, centrifuging, and standing in a dark place for 20min;
3) Observing the color change of the mixed solution, if the sample solution to be detected contains Cu 2+ The two modified nucleic acid sequences are connected to form a new 1,2, 3-triazole coupling nucleic acid sequence, and the solution is blue; if the sample solution to be tested is free of Cu 2+ The alkynyl-modified nucleic acid sequence forms a G-quadruplex, in which case the solution appears pink;
4) Transferring the above mixed solution into an ELISA plate, measuring absorption values of the mixed solution at 580nm and 650nm with an ELISA or ultraviolet spectrophotometer, and measuring Cu 2+ Quantitative analysis was performed on the content of (c).
The following functional verification was performed on the copper ion detection kit prepared in the first embodiment:
copper sulfate is dissolved in ultrapure water to prepare a copper ion standard solution with the concentration of 5mM, and the copper ion standard solution is stirred and mixed uniformly, centrifuged for 1min, and then placed at room temperature, and diluted with water to prepare the copper ion standard solution with the concentration of 500 mu M, 600 mu M, 625 mu M and 1mM for standby.
(1) Response experiment of cyanine dye fluorescent probe molecule on copper ions
Taking out 2 sterile 1.5mL EP tubes, dividing the EP tubes into two groups containing copper ions and containing no copper ions, sequentially adding 70 mu L of ultrapure water and 59 mu L of ultrapure water, respectively, then adding 10 mu L of 100 mu M deoxyribonucleic acid (5 '-GTGGGTAGG-N=N-3' and 5'-CH≡C-GCGGGTTGGG-3') stock solution, shaking and mixing, centrifuging for 1min, adding 0 mu L and 12 mu L of 600 mu M copper ion solution and 5 mu L of 2mM sodium ascorbate solution, shaking and mixing, centrifuging for 1min, and placing 2 samples at room temperature for shaking for 2h; after the completion 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 + Buffer solution, shaking and mixing, centrifuging for 1min, and room temperatureAnd after keeping aside for 20min in dark, measuring ultraviolet absorption luminosity by using an enzyme-labeled instrument 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 obviously between 650nm and 580nm, which indicates that the kit has very good response to copper ions.
(2) Tendency of response change of cyanine dye fluorescent probe molecules to copper ions with different concentrations
After taking 9 sterile 1.5mL EP tubes, 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, 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 respectively added thereto, and after shaking and mixing, the mixture was centrifuged for 1min; 0 μL of 500 μM, 9 μL of 500 μM, 25 μL of 500 μM, 28 μL of 500 μM, 29.6 μL of 625 μM, 10 μL of 5mM, 12.5 μL of 5mM, 15 μL of 5mM and 20 μL of 5mM copper ion standard solution are sequentially added into 9 EP pipes, 5 μL of 2mM sodium ascorbate solution is respectively added into the 9 EP pipes, the mixture is uniformly mixed by oscillation, and after centrifugation for 1min, 9 samples are placed at room temperature for oscillation for 2 hours; then 2. Mu.L of 500. Mu.M stock solution of MTC fluorescent probe and 5. Mu.L of 2mol/L K were added to each + The buffer solution is evenly mixed by vibration, centrifuged for 1min, kept stand at room temperature in dark place for 20min, and then the ultraviolet absorption luminosity is measured by an enzyme-labeled instrument or an ultraviolet spectrophotometer, and the experimental result is shown in figure 3. As can be seen from the graph, the absorbance at 580nm and the absorbance at 650nm have good conversion trends at copper ion concentrations of 0-400. Mu.M.
(3) Visual 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 of ultrapure water and 57 mu L of ultrapure water, respectively adding 10 mu L of 100 mu M of two-tube deoxyribonucleic acid stock solution, shaking and uniformly mixing, and centrifuging for 1min; sequentially adding 0 mu L of 5mM and 12 mu L of 5mM copper ion standard solution into 2 EP pipes, respectively adding 5 mu L of 2mM sodium ascorbate solution into the 2 EP pipes, shaking and mixing uniformly, centrifuging for 1min, and then placing 2 samples at room temperature for shaking for 2h; then 2. Mu.L of 500. Mu.M stock solution of MTC fluorescent probe and 5. Mu.L of 2mol/L K were added to each + Buffer solution, shaking and mixing, centrifuging for 1min, standing at room temperature in dark place for 20min, and directly observingThe color change test results are shown in fig. 4. As can be seen from fig. 4, the color of the system was changed from pink to blue after the addition of the copper ion standard solution. The kit is simple to operate, has obvious results, and can achieve the purpose of rapidly detecting copper ions.
Example two
A visual copper ion detection kit based on cyanine dye aggregate conversion and Click reaction comprises a 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 K 2 SO 4 A 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 cyanine dye ETC (3, 3' -di (3-sulfopropyl) -4,5,4,5-2 benzo-9-ethyl-thiacarbocyanine dye triethylamine salt) in DMSO to prepare copper ion fluorescent probe stock solution with the concentration of 5mM, oscillating and mixing uniformly, and centrifuging for 1min; 10 mu L of the stock solution of the copper ion fluorescent probe is sucked from 5mM of the stock solution of the copper ion fluorescent probe and added into 90 mu L of ultrapure water to prepare 500 mu M of stock solution of the ETC fluorescent probe, the stock solution is uniformly mixed by oscillation, and the stock solution is placed in a refrigerator for refrigeration after centrifugation for 1min for standby. The structural formula of the cyanine dye ETC is shown as the following formula (I):
wherein R is selected from ethyl.
Will K 2 SO 4 Dissolving in ultrapure water to prepare K with concentration of 2mol/L + The buffer solution is stirred and mixed evenly, and is placed at room temperature for standby after being centrifuged for 1 min.
The copper ion detection method and the preparation method of the copper ion standard solution in this embodiment are the same as those in the first embodiment.
The following functional verification was performed on the copper ion detection kit prepared in the second embodiment:
(1) Response experiment of cyanine dye fluorescent probe molecule on copper ions
Taking out 2 sterile 1.5mL EP tubes, dividing the EP tubes into two groups containing copper ions and containing no copper ions, sequentially adding 70 mu L of ultrapure water and 59 mu L of ultrapure water, respectively, then adding 10 mu L of 100 mu M deoxyribonucleic acid (5 '-GGGTGGGT-N=N-3' and 5'-CH≡C-GGGTGGGT-3') stock solution, shaking and mixing, centrifuging for 1min, adding 0 mu L and 12 mu L of 600 mu M copper ion solution and 5 mu L of 2mM sodium ascorbate solution, shaking and mixing, centrifuging for 1min, and then placing 2 samples at room temperature for shaking for 2h; 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 + The buffer solution is evenly mixed by vibration, centrifuged for 1min, kept stand at room temperature in dark place for 20min, and then the ultraviolet absorption luminosity is measured by an enzyme-labeled instrument or an ultraviolet spectrophotometer, and the experimental result is shown in figure 5. As can be seen from the graph, the ultraviolet absorbance changes very obviously between 650nm and 580nm, which indicates that the kit has very good response to copper ions.
(2) Tendency of response change of cyanine dye fluorescent probe molecules to copper ions with different concentrations
9 sterile 1.5mL EP tubes were taken, 69. Mu.L, 49. Mu.L, 56. Mu.L, 54.5. Mu.L, 53. Mu.L, 51. Mu.L, 55.5. Mu.L, 62. Mu.L and 61. Mu.L of ultrapure water were sequentially added thereto, and 10. Mu.L of 100. Mu.M stock solutions of deoxyribonucleic acid (5 '-GGGTGGGT-N=N-3' and 5'-CH≡C-GGGTGGGT-3') were added, respectively, and after shaking and mixing, the mixture was centrifuged for 1min; 0 μL of 500 μM, 10 μL of 1mM, 13 μL of 1mM, 14.5 μL of 1mM, 8 μL of 2mM, 9 μL of 2mM, 13.5 μL of 2mM, 7 μL of 5mM and 8 μL of 5mM copper ion standard solution are sequentially added into 9 EP pipes, 5 μL of 2mM sodium ascorbate solution is respectively added into the 9 EP pipes, the mixture is uniformly mixed by shaking, and after centrifugation for 1min, the 9 samples are placed at room temperature for shaking for 2 hours; 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 + The buffer solution is evenly mixed by vibration, centrifuged for 1min, kept stand at room temperature in dark place for 20min, and then the ultraviolet absorption luminosity is measured by an enzyme-labeled instrument or an ultraviolet spectrophotometer, and the experimental result is shown in figure 6. From the figure, it can be seen thatAs a result, 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) Visual 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 of ultrapure water and 57 mu L of ultrapure water, respectively adding 10 mu L of 100 mu M of two-tube deoxyribonucleic acid stock solution, shaking and uniformly mixing, and centrifuging for 1min; sequentially adding 0 mu L of 5mM and 12 mu L of 5mM copper ion standard solution into 2 EP pipes, respectively adding 5 mu L of 2mM sodium ascorbate solution into the 2 EP pipes, shaking and mixing uniformly, centrifuging for 1min, and then placing 2 samples at room temperature for shaking for 2h; 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 + The buffer solution is stirred and mixed evenly, centrifuged for 1min, kept stand at room temperature in dark place for 20min, and then the experimental result of directly observing the color change is shown in figure 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 rapidly detecting copper ions.
Example III
A visual copper ion detection kit based on cyanine dye aggregate conversion and Click reaction comprises a 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'), a 2mM sodium ascorbate solution, a 5mM MTC copper ion fluorescent probe stock solution and 2mol/L KCl solution;
the preparation method of the deoxyribonucleic acid stock solution of the 3 '-end modified azido group and the 5' -end modified alkynyl group, the preparation method of the copper ion fluorescent probe stock solution, and the preparation process of the sodium ascorbate solution and the KCl solution are the same as in the first embodiment.
The copper ion detection method and the preparation method of the copper ion standard solution in this embodiment are the same as those in the first embodiment.
The following functional verification was performed on the copper ion detection kit prepared in the third embodiment:
(1) Response experiment of cyanine dye fluorescent probe molecule on copper ions
Taking out 2 sterile 1.5mL EP tubes, dividing the EP tubes into two groups containing copper ions and containing no copper ions, sequentially adding 70 mu L of ultrapure water and 59 mu L of ultrapure water, sequentially adding 10 mu L of 100 mu M deoxyribonucleic acid (5 '-GGGTAGG-N=N-3' and 5'-CH≡C-GCGGGTTGGG-3') stock solution into the EP tubes, shaking and mixing the mixture, centrifuging the mixture for 1min, adding 0 mu L and 12 mu L of 600 mu M copper ion solution and 5 mu L of 2mM sodium ascorbate solution, shaking and mixing the mixture, centrifuging the mixture for 1min, and then placing 2 samples at room temperature for shaking for 2h; after the completion 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 + The buffer solution is evenly mixed by vibration, centrifuged for 1min, kept stand at room temperature in a dark place for 20min, and then the ultraviolet absorption luminosity is measured by an enzyme-labeled instrument or an ultraviolet spectrophotometer, and the experimental result is shown in figure 8. As can be seen from the graph, the ultraviolet absorbance changes very obviously between 650nm and 580nm, which indicates that the kit has very good response to copper ions.
(2) Tendency of response change of cyanine dye fluorescent probe molecules to copper ions with different concentrations
After taking 7 sterile 1.5mL EP tubes, 69. Mu.L, 59. Mu.L, 56. Mu.L, 54.5. Mu.L, 61. Mu.L, 54. Mu.L, 57. Mu.L of ultrapure water were sequentially added thereto, 10. Mu.L of 100. Mu.M stock solutions of deoxyribonucleic acid (5 '-GGGTAGG-N=N-3' and 5'-CH≡C-GCGGGTTGGG-3') were respectively added thereto, and after shaking and mixing, the mixture was centrifuged for 1min; adding 0 mu L of 500 mu M, 10 mu L of 1mM, 13 mu L of 1mM, 14.5 mu L of 1mM, 8 mu L of 2mM, 15 mu L of 2mM and 12 mu L of 5mM copper ion standard solution into 7 EP pipes in sequence, adding 5 mu L of 2mM sodium ascorbate solution into the 7 EP pipes respectively, shaking and mixing uniformly, centrifuging for 1min, and placing 7 samples at room temperature for shaking for 2h; then 2. Mu.L of 500. Mu.M stock solution of MTC fluorescent probe and 5. Mu.L of 2mol/L K were added to each + The buffer solution is evenly mixed by vibration, centrifuged for 1min, kept stand at room temperature in dark place for 20min, and then the ultraviolet absorption luminosity is measured by an enzyme-labeled instrument or an ultraviolet spectrophotometer, and 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 good conversion trends at copper ion concentrations of 0-600. Mu.M.
(3) Visual 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 of ultrapure water and 57 mu L of ultrapure water, respectively adding 10 mu L of 100 mu M of two-tube deoxyribonucleic acid stock solution, shaking and uniformly mixing, and centrifuging for 1min; sequentially adding 0 mu L of 5mM and 12 mu L of 5mM copper ion standard solution into 2 EP pipes, respectively adding 5 mu L of 2mM sodium ascorbate solution into the 2 EP pipes, shaking and mixing uniformly, centrifuging for 1min, and then placing 2 samples at room temperature for shaking for 2h; then 2. Mu.L of 500. Mu.M stock solution of MTC fluorescent probe and 5. Mu.L of 2mol/L K were added to each + The buffer solution is stirred and mixed evenly, centrifuged for 1min, kept stand at room temperature in dark place for 20min, and then the experimental result of directly observing the color change is shown in figure 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 rapidly detecting copper ions.
Claims (6)
1. A visual copper ion detection kit based on cyanine dye aggregate conversion and Click reaction is characterized by comprising a deoxyribonucleic acid stock solution, a reducing solution, a copper ion fluorescent probe stock solution and K, wherein the deoxyribonucleic acid stock solution comprises a 3 '-end modified azide group and a 5' -end modified alkynyl group + 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 an azide group modified at the 3 'end and a nucleotide sequence of deoxyribonucleic acid with an alkyne group modified at the 5' end in ultrapure water to prepare a deoxyribonucleic acid stock solution with the concentration of 100 mu M, oscillating and mixing uniformly, centrifuging for 1min, and refrigerating in a refrigerator for later use;
deoxyribonucleic acid with 3' -end modified azide group the nucleotide sequence is 5' GTGGGTAGG-N=N=N-3 '; 5' -GGGTGGGT-N=N-3 ', 5' -GGGTAGG-N=N-3 ': 5' -GGTGGTGGTGGTT-n=n=n-3 ';
the nucleotide sequence of the 5' -end modified alkynyl group deoxyribonucleic acid is one of 5' -CH (identical to) C-GCGGGTTGGG-3', 5' -CH (identical to) C-GGGTGGGT-3', 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 copper ion fluorescent probe stock solution with the concentration of 1-5 mM, 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 conversion and Click reaction according to claim 1, wherein the reducing solution is one of sodium ascorbate solution, ascorbic acid solution and trisodium citrate solution.
3. The visual copper ion detection kit based on cyanine dye aggregate conversion and Click reaction according to claim 2, wherein the concentration of the sodium ascorbate solution is 1-10mM.
4. The visual copper ion detection kit based on cyanine dye aggregate conversion and Click reaction according to claim 1 or 2, wherein the K + The buffer solution is KCl solution or K 2 SO 4 Solution, K + K in buffer solution + The concentration is 0.1-5mol/L.
5. The method for detecting copper ions by using the visual copper ion detection kit based on cyanine dye aggregate conversion and Click reaction according to claim 1, which is characterized by comprising the following steps:
1) Sequentially adding a deoxyribonucleic acid stock solution, a reducing solution and a sample solution to be tested of 3 '-end modified azido group and 5' -end modified alkynyl group into an ultrapure water system, and vibrating for 2 hours at room temperature;
2) Adding copper ion fluorescent probe stock solution and K into the mixed solution after oscillation is finished + Buffer solution, shaking and mixing evenly, centrifuging, and standing in a dark place for 20min;
3) Observing the color change of the mixed solution, if the sample solution to be detected contains Cu 2+ The two modified nucleic acid sequences are connected to form a new 1,2, 3-triazole coupling nucleic acid sequence, and the solution is blue; if the sample solution to be tested is free of Cu 2+ The alkynyl-modified nucleic acid sequence forms a G-quadruplex, in which case the solution appears pink;
4) Transferring the above mixed solution into an ELISA plate, measuring absorption values of the mixed solution at 580nm and 650nm with an ELISA or ultraviolet spectrophotometer, and measuring Cu 2+ Quantitative analysis was performed on the content of (c).
6. The method for detecting copper ions in a visual copper ion detection kit based on cyanine dye aggregate conversion and Click reaction according to claim 5, wherein in step 1), the reducing solution is sodium ascorbate solution.
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| 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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| 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 |
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