CN114047166A - DNA hydrogel-based heavy metal ion rapid detection method - Google Patents
DNA hydrogel-based heavy metal ion rapid detection method Download PDFInfo
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- CN114047166A CN114047166A CN202111200506.3A CN202111200506A CN114047166A CN 114047166 A CN114047166 A CN 114047166A CN 202111200506 A CN202111200506 A CN 202111200506A CN 114047166 A CN114047166 A CN 114047166A
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- 238000000034 method Methods 0.000 claims abstract description 14
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- 239000007864 aqueous solution Substances 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 claims description 7
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical group NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 claims description 5
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 5
- 239000000178 monomer Substances 0.000 claims description 5
- 229920000642 polymer Polymers 0.000 claims description 5
- 150000003384 small molecules Chemical class 0.000 claims description 5
- 239000000243 solution Substances 0.000 claims description 5
- ILJSQTXMGCGYMG-UHFFFAOYSA-N triacetic acid Chemical compound CC(=O)CC(=O)CC(O)=O ILJSQTXMGCGYMG-UHFFFAOYSA-N 0.000 claims description 5
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 claims description 4
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 claims description 4
- 239000003999 initiator Substances 0.000 claims description 3
- 229960000549 4-dimethylaminophenol Drugs 0.000 claims description 2
- LHIJANUOQQMGNT-UHFFFAOYSA-N aminoethylethanolamine Chemical group NCCNCCO LHIJANUOQQMGNT-UHFFFAOYSA-N 0.000 claims description 2
- 229910001870 ammonium persulfate Inorganic materials 0.000 claims description 2
- 108010061446 deoxynucleotidase Proteins 0.000 claims description 2
- 238000006116 polymerization reaction Methods 0.000 claims description 2
- USHAGKDGDHPEEY-UHFFFAOYSA-L potassium persulfate Chemical compound [K+].[K+].[O-]S(=O)(=O)OOS([O-])(=O)=O USHAGKDGDHPEEY-UHFFFAOYSA-L 0.000 claims description 2
- ZXKINMCYCKHYFR-UHFFFAOYSA-N aminooxidanide Chemical compound [O-]N ZXKINMCYCKHYFR-UHFFFAOYSA-N 0.000 claims 1
- 238000009833 condensation Methods 0.000 claims 1
- 230000005494 condensation Effects 0.000 claims 1
- 230000018044 dehydration Effects 0.000 claims 1
- 238000006297 dehydration reaction Methods 0.000 claims 1
- 108020004414 DNA Proteins 0.000 abstract description 22
- 229910021645 metal ion Inorganic materials 0.000 abstract description 10
- 230000027455 binding Effects 0.000 abstract description 6
- 102000016911 Deoxyribonucleases Human genes 0.000 abstract description 5
- 108010053770 Deoxyribonucleases Proteins 0.000 abstract description 5
- 239000000463 material Substances 0.000 abstract description 5
- 108091027757 Deoxyribozyme Proteins 0.000 abstract description 3
- 230000001419 dependent effect Effects 0.000 abstract description 3
- 230000009870 specific binding Effects 0.000 abstract description 3
- 108091034117 Oligonucleotide Proteins 0.000 abstract description 2
- 102000035195 Peptidases Human genes 0.000 abstract description 2
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- 239000004365 Protease Substances 0.000 abstract description 2
- 238000004458 analytical method Methods 0.000 abstract description 2
- 230000003197 catalytic effect Effects 0.000 abstract description 2
- 229910052751 metal Inorganic materials 0.000 abstract description 2
- 239000002184 metal Substances 0.000 abstract description 2
- 102000039446 nucleic acids Human genes 0.000 abstract description 2
- 108020004707 nucleic acids Proteins 0.000 abstract description 2
- 150000007523 nucleic acids Chemical class 0.000 abstract description 2
- 238000011002 quantification Methods 0.000 abstract description 2
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- 238000005199 ultracentrifugation Methods 0.000 description 3
- 239000012498 ultrapure water Substances 0.000 description 3
- AVXURJPOCDRRFD-UHFFFAOYSA-N Hydroxylamine Chemical class ON AVXURJPOCDRRFD-UHFFFAOYSA-N 0.000 description 2
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- 235000020188 drinking water Nutrition 0.000 description 2
- 238000012544 monitoring process Methods 0.000 description 2
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 230000002411 adverse Effects 0.000 description 1
- 238000003968 anodic stripping voltammetry Methods 0.000 description 1
- 238000001479 atomic absorption spectroscopy Methods 0.000 description 1
- 238000001391 atomic fluorescence spectroscopy Methods 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
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- 238000011161 development Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 1
- RVPVRDXYQKGNMQ-UHFFFAOYSA-N lead(2+) Chemical compound [Pb+2] RVPVRDXYQKGNMQ-UHFFFAOYSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
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- 238000002360 preparation method Methods 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
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Images
Classifications
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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
- G01N21/6402—Atomic fluorescence; Laser induced fluorescence
Abstract
The invention discloses a method for rapidly detecting heavy metal ions by using DNA hydrogel, which is based on metal ion-dependent deoxyribonuclease and has high selectivity, specific binding capacity and good stability. DNase is a functional nucleic acid with the catalytic function of proteases and the binding function of antibodies. It consists of a loop region for binding a specific metal ion and hybridizing to its oligonucleotide substrate. Upon binding to the metal cofactor, the deoxyribozyme will be activated and the substrate will be split into two strands. The bound fluorescent material on the strand was activated and the increase in fluorescent signal was measured for sensitive lead quantification with a detection limit of 1 nM. Meanwhile, the DNA-based hydrogel detection method also shows high selectivity to different metal ions, which is very important for the analysis of complex samples.
Description
Technical Field
The invention belongs to the technical field of heavy metal pollution detection, and particularly relates to a DNA hydrogel-based heavy metal ion rapid detection method.
Background
Heavy metal pollution is an increasingly challenging problem for water environments, soil and drinking water safety. Heavy metal ions are difficult to degrade by microorganisms, accumulate and strongly interact with cellular proteins, so that enzyme is inactivated, and chronic poisoning is caused. Lead, for example, is one of the most toxic heavy metal ions, and is easily accumulated in the environment and hardly degraded. Trace lead exposure can cause serious harm and long-term lead ions have adverse effects on humans, especially children. This includes severe effects on the cardiovascular, reproductive and other metabolic organs and damage to the central nervous system. The maximum concentration of lead ions in drinking water was specified to be 10. mu.g/L (48 nm). Thus, monitoring of heavy metal ions in water or soil has been a significant challenge.
Therefore, the development of a rapid, convenient, high-sensitivity and high-selectivity heavy metal ion monitoring sensor is widely demanded. At present, methods for detecting heavy metal ions include inductively coupled plasma mass spectrometry, X-ray fluorescence spectrometry, atomic absorption spectrometry, atomic fluorescence spectrometry and anodic stripping voltammetry, and although these apparatuses can quantitatively detect heavy metal ions, these methods mostly require apparatuses and professionals with complicated centers. These techniques are only suitable for centralized laboratories and not for real-time testing. Therefore, a high-precision and high-sensitivity real-time detection method for heavy metal ions is urgently needed.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention provides a DNA hydrogel-based method for rapidly detecting heavy metal ions. The invention develops a label-free fluorescence sensing strategy, in the presence of heavy metal ions, the tail part of a DNA (deoxyribonucleic acid) enzyme chain is cut and released from a substrate chain, then the increase of a fluorescence signal is measured, the fluorescence sensing strategy is used for sensitive lead quantification, and the detection limit is 1 nM. Meanwhile, the DNA-based hydrogel detection method also shows high selectivity to different metal ions, which is very important for the analysis of complex samples. The material of the hydrogel is as follows: 5' -terminal base modified DNA strand (abbreviated SA, ST), GR-5DNA polymerase chain. The metal ion dependent deoxyribonuclease has high selectivity, specific binding capacity and good stability. DNase is a functional nucleic acid with the catalytic function of proteases and the binding function of antibodies. It consists of a loop region for binding a specific metal ion and hybridizing to its oligonucleotide substrate. Upon binding to the metal cofactor, the deoxyribozyme will be activated and the substrate will be split into two strands. The fluorescent material combined on the chain is activated, so that the function of detecting the content of the heavy metal is achieved.
In order to solve the technical problems, the invention provides the following technical scheme:
the invention provides a DNA hydrogel-based heavy metal ion rapid detection method, which comprises the following steps:
s1, mixing an acrylamide-DNA chain comprising an A chain and a T chain with 2-10% acrylamide monomer aqueous solution respectively; placing the mixed solution in a vacuum drier for 10-30 minutes to remove air, and then adding 1.4% of ammonium polysulfate and tetramethylethylenediamine aqueous solution; the reaction tube was immediately placed in a vacuum desiccator to remove air and polymerized for 15 minutes to produce polyacrylamide-deoxyribonucleic acid conjugates P-SA and P-ST; after the reaction, the polymer product is purified by an ultracentrifuge filter to remove unpolymerized monomers and other small molecules for further use;
s2, generating DNA hydrogel for detecting heavy metal ions, and mixing the DNA hydrogel with the molar ratio of 100-500 microns: 100-500 μm: a solution of 100-well 500 μm P-SA, P-ST and GR-5 deoxynucleotidase chains was mixed with an equal amount of substrate chains in triacetic acid buffer.
In a preferred embodiment of the present invention, the hydroxyamides of chain A and chain T are used in step S1, and the hydroxyamides are obtained by dehydrating and condensing hydroxyethylethylenediamine and DNA chain with DCC and DMAP.
As a preferable embodiment of the present invention, N is required for the mixed solution in step S12Protection to remove O in mixed liquid2。
In a preferred embodiment of the present invention, the initiator in step S1 is at least one of ammonium persulfate and potassium persulfate, and the amount of the initiator is 0.5% to 3%, and the accelerator is tetramethylethylenediamine, so as to promote polymerization.
As a preferred embodiment of the present invention, the step S1 is performed by purifying with an ultracentrifugal filter having a molecular weight of 50-100 kDa.
In a preferred embodiment of the present invention, the mixed solution is placed in a vacuum drier at room temperature of 15 to 35 ℃ in step S2.
As a preferable embodiment of the present invention, the mixed solution mixed in step S2 is heated at 30-70 ℃ for 1-10 minutes, then shaken vigorously 2-20 times, and then stored at 1-9 ℃.
Compared with the prior art, the invention has the following beneficial effects:
1. the DNA hydrogel prepared by utilizing the metal ion-dependent deoxyribonuclease with high selectivity, specific binding capacity and good stability can be used for rapidly detecting heavy metal ions. The method is simple and effective, is simple and convenient to operate, requires short preparation time and requires simple equipment.
2. The selected materials are all materials with good biocompatibility and low biotoxicity.
3. The prepared DNA hydrogel provides the capability of efficiently and quickly detecting heavy metal ions, and can realize the detection effect on different heavy metal ions.
4. In the presence of heavy metal ions, the deoxyribozyme cleaves a substrate chain to release a part rich in a fluorescence signal, and the fluorescence signal is obviously enhanced, so that the heavy metal ions are rapidly detected.
Drawings
FIG. 1 is a schematic diagram of DNA hydrogel for detecting heavy metal ions.
FIG. 2 is a graph showing the fluorescence intensity of 100nM lead ions detected at different pH.
FIG. 3 is a graph showing fluorescence intensities of heavy metal ions at different concentrations (1nM to 1mM) at pH 6.
FIG. 4 is a graph showing fluorescence intensities of different heavy metal ions at a concentration of 100nM, at pH 6.
Detailed Description
The preferred embodiments of the present invention will be described in conjunction with the accompanying drawings, and it will be understood that they are described herein for the purpose of illustration and explanation and not limitation.
Example 1
The hydroxyamide-DNA strands including the A chain and the T chain (300-700 μm, dissolved in ultrapure water) were mixed with 5% acrylamide monomer aqueous solutions, respectively. The mixed solution was placed in a vacuum desiccator at room temperature (. apprxeq.15-35 ℃) for 10-30 minutes to remove air, and then 1.4% ammonium polysulfate and tetramethylethylenediamine aqueous solution were added. The reaction tube was immediately placed in a vacuum desiccator to remove air and polymerized for 15 minutes to produce polyacrylamide-deoxyribonucleic acid conjugates P-SA and P-ST. After the reaction, the polymer product is purified by ultracentrifugation filters with molecular mass 50-100kDa to remove unpolymerized monomers and other small molecules for further use.
To generate a DNA hydrogel for the detection of heavy metal ions, a solution of the P-SA oxygen nuclear enzyme chain (molar ratio 100-500 μm) was mixed with an equal amount of substrate chain in triacetic acid buffer. In order to ensure the uniformity of the hydrogel, the mixed solution is heated at 30-70 ℃ for 1-10 minutes and then vigorously shaken 2-20 times. Thereafter, the final mixture is stored at 1-9 ℃.
Fluorescence intensity of lead ions with 100nM concentration at normal temperature under different pH environments. Fluorescence intensity was measured at different concentrations (1nM-1mM) of lead ion in the environment of pH6, as shown in FIG. 3. The DNA-based hydrogel pairs including Cu were subsequently investigated under the same analytical conditions (pH6)2+、Hg2+、Co2+、Ni2+、Cd2+And Mn2+Several other divalent metal ion responses, such as fig. 4.
Example 2:
the hydroxyamide-DNA strands including the A chain and the T chain (300-700 μm, dissolved in ultrapure water) were mixed with 10% acrylamide monomer aqueous solutions, respectively. The mixed solution was placed in a vacuum desiccator at room temperature (. apprxeq.15-35 ℃) for 10-30 minutes to remove air, and then 1.4% ammonium polysulfate and tetramethylethylenediamine aqueous solution were added. The reaction tube was immediately placed in a vacuum desiccator to remove air and polymerized for 15 minutes to produce polyacrylamide-deoxyribonucleic acid conjugates P-SA and P-ST. After the reaction, the polymer product is purified by ultracentrifugation filters with molecular mass 50-100kDa to remove unpolymerized monomers and other small molecules for further use.
To generate a DNA hydrogel for the detection of heavy metal ions, a solution of the P-ST deoxynucleotidyl enzyme chain (molar ratio 100-500 μm) was mixed with an equal amount of substrate chain in triacetic acid buffer. In order to ensure the uniformity of the hydrogel, the mixed solution is heated at 30-70 ℃ for 1-10 minutes and then vigorously shaken 2-20 times. Thereafter, the final mixture is stored at 1-9 ℃.
Fluorescence intensity of lead ions with 100nM concentration at normal temperature under different pH environments. The fluorescence intensity of different concentrations (1nM-1mM) of lead ions was measured at pH 6. The DNA-based hydrogel pairs including Cu were subsequently investigated under the same analytical conditions (pH6)2+、Hg2+、Co2+、Ni2+、Cd2+And Mn2+But several other divalent metal ions.
Example 3:
the hydroxyamide-DNA strands including the A chain and the T chain (300-700 μm, dissolved in ultrapure water) were mixed with 8% acrylamide monomer aqueous solutions, respectively. The mixed solution was placed in a vacuum desiccator at room temperature (. apprxeq.15-35 ℃) for 10-30 minutes to remove air, and then 1.4% ammonium polysulfate and tetramethylethylenediamine aqueous solution were added. The reaction tube was immediately placed in a vacuum desiccator to remove air and polymerized for 15 minutes to produce polyacrylamide-deoxyribonucleic acid conjugates P-SA and P-ST. After the reaction, the polymer product is purified by ultracentrifugation filters with molecular mass 50-100kDa to remove unpolymerized monomers and other small molecules for further use.
To generate DNA hydrogels for the detection of heavy metal ions, a solution of GR-5 deoxynucleotidyl enzyme chain (molar ratio 100-500 μm) was mixed with an equal amount of substrate chain in triacetic acid buffer. In order to ensure the uniformity of the hydrogel, the mixed solution is heated at 30-70 ℃ for 1-10 minutes and then vigorously shaken 2-20 times. Thereafter, the final mixture is stored at 1-9 ℃.
Fluorescence intensity of lead ions with 100nM concentration at normal temperature under different pH environments. The fluorescence intensity of different concentrations (1nM-1mM) of lead ions was measured at pH 6. The DNA-based hydrogel pairs including Cu were subsequently investigated under the same analytical conditions (pH6)2+、Hg2+、Co2+、Ni2+、Cd2+And Mn2+But several other divalent metal ions.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A heavy metal ion rapid detection method based on DNA hydrogel is characterized by comprising the following steps:
s1, mixing the hydroxyamide-DNA chain including the A chain and the T chain with 2-10% acrylamide monomer aqueous solution respectively; placing the mixed solution in a vacuum drier for 10-30 min, and adding N2Replacing air in the mixed solution, and then adding 1.4% of ammonium polysulfate and tetramethylethylenediamine aqueous solution; the reaction tube was immediately placed in a vacuum desiccator to remove air and polymerized for 15 minutes to produce polyacrylamide-deoxyribonucleic acid conjugates P-SA and P-ST; after the reaction, the polymer product is purified by an ultracentrifuge filter to remove unpolymerized monomers and other small molecules for further use;
s2, generating DNA hydrogel for detecting heavy metal ions, and mixing the DNA hydrogel with the molar ratio of 100-500 microns: 100-500 μm: a solution of 100-well 500 μm P-SA, P-ST and GR-5 deoxynucleotidase chains was mixed with an equal amount of substrate chains in triacetic acid buffer.
2. The method for rapidly detecting heavy metal ions based on a DNA hydrogel of claim 1, wherein the hydroxyamide of A chain and T chain is used in step S1, and the method is obtained by performing dehydration condensation on hydroxyethyl ethylenediamine and DNA chain by DCC and DMAP.
3. The method for rapidly detecting heavy metal ions based on DNA hydrogel of claim 1, wherein the step S1 is performed by using an ultracentrifugal filter with molecular mass of 50-100 kDa.
4. The method for rapidly detecting heavy metal ions based on DNA hydrogel of claim 1, wherein N is required in step S12Protection to remove O from air2。
5. The method for rapidly detecting heavy metal ions based on DNA hydrogel of claim 1, wherein the initiator in step S1 is at least one of ammonium persulfate and potassium persulfate, the dosage is 0.5% -3%, and the accelerator is tetramethylethylenediamine, so as to promote polymerization.
6. The method for rapidly detecting heavy metal ions based on a DNA hydrogel of claim 1, wherein the mixed solution is placed in a vacuum drier at room temperature of 15-35 ℃ in step S2.
7. The method for rapidly detecting heavy metal ions based on a DNA hydrogel of claim 1, wherein the mixed solution mixed in step S2 is heated at 30-70 ℃ for 1-10 minutes, then vigorously shaken for 2-20 times, and then stored at 1-9 ℃.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507877A (en) * | 2011-11-16 | 2012-06-20 | 厦门出入境检验检疫局检验检疫技术中心 | Deoxyribozyme hydrogel based copper ion detection sensor |
| CN103852436A (en) * | 2014-03-14 | 2014-06-11 | 厦门大学 | Method for detecting lead ion through high-specificity DNA hydrogel |
| CN111705113A (en) * | 2020-06-24 | 2020-09-25 | 上海海洋大学 | Functional nucleic acid fluorescence sensor and application thereof in lead ion detection |
| CN112941152A (en) * | 2020-10-12 | 2021-06-11 | 中国工程物理研究院材料研究所 | Harmful metal ion detection method based on pure DNA functional hydrogel |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102507877A (en) * | 2011-11-16 | 2012-06-20 | 厦门出入境检验检疫局检验检疫技术中心 | Deoxyribozyme hydrogel based copper ion detection sensor |
| CN103852436A (en) * | 2014-03-14 | 2014-06-11 | 厦门大学 | Method for detecting lead ion through high-specificity DNA hydrogel |
| CN111705113A (en) * | 2020-06-24 | 2020-09-25 | 上海海洋大学 | Functional nucleic acid fluorescence sensor and application thereof in lead ion detection |
| CN112941152A (en) * | 2020-10-12 | 2021-06-11 | 中国工程物理研究院材料研究所 | Harmful metal ion detection method based on pure DNA functional hydrogel |
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