CN110568036A - Method for electrochemically detecting mercury ions based on nucleic acid dye - Google Patents
Method for electrochemically detecting mercury ions based on nucleic acid dye Download PDFInfo
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- CN110568036A CN110568036A CN201910761801.2A CN201910761801A CN110568036A CN 110568036 A CN110568036 A CN 110568036A CN 201910761801 A CN201910761801 A CN 201910761801A CN 110568036 A CN110568036 A CN 110568036A
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- 238000000034 method Methods 0.000 title claims abstract description 20
- 229910052753 mercury Inorganic materials 0.000 title claims abstract description 8
- 239000000980 acid dye Substances 0.000 title claims abstract description 7
- -1 mercury ions Chemical class 0.000 title claims abstract description 7
- 108020004707 nucleic acids Proteins 0.000 title claims abstract description 7
- 102000039446 nucleic acids Human genes 0.000 title claims abstract description 7
- 150000007523 nucleic acids Chemical class 0.000 title claims abstract description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 12
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 12
- 108020003215 DNA Probes Proteins 0.000 claims abstract description 10
- 239000003298 DNA probe Substances 0.000 claims abstract description 10
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 9
- 239000010931 gold Substances 0.000 claims abstract description 9
- 229910052737 gold Inorganic materials 0.000 claims abstract description 9
- 239000002105 nanoparticle Substances 0.000 claims abstract description 9
- JGBUYEVOKHLFID-UHFFFAOYSA-N gelred Chemical compound [I-].[I-].C=1C(N)=CC=C(C2=CC=C(N)C=C2[N+]=2CCCCCC(=O)NCCCOCCOCCOCCCNC(=O)CCCCC[N+]=3C4=CC(N)=CC=C4C4=CC=C(N)C=C4C=3C=3C=CC=CC=3)C=1C=2C1=CC=CC=C1 JGBUYEVOKHLFID-UHFFFAOYSA-N 0.000 claims abstract description 7
- BQPIGGFYSBELGY-UHFFFAOYSA-N mercury(2+) Chemical compound [Hg+2] BQPIGGFYSBELGY-UHFFFAOYSA-N 0.000 claims abstract description 7
- 239000000975 dye Substances 0.000 claims abstract description 6
- 238000004070 electrodeposition Methods 0.000 claims description 5
- 238000011534 incubation Methods 0.000 claims description 5
- 238000001903 differential pulse voltammetry Methods 0.000 claims description 4
- 238000005259 measurement Methods 0.000 claims description 2
- 239000000243 solution Substances 0.000 claims 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims 2
- 239000007853 buffer solution Substances 0.000 claims 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 abstract description 11
- 238000001514 detection method Methods 0.000 abstract description 10
- 239000000523 sample Substances 0.000 abstract description 8
- 238000007650 screen-printing Methods 0.000 abstract description 3
- 230000009471 action Effects 0.000 abstract description 2
- 230000002687 intercalation Effects 0.000 abstract description 2
- 238000009830 intercalation Methods 0.000 abstract description 2
- 238000011084 recovery Methods 0.000 abstract description 2
- 238000012360 testing method Methods 0.000 abstract description 2
- 108020004414 DNA Proteins 0.000 abstract 2
- 102000053602 DNA Human genes 0.000 abstract 1
- 238000002372 labelling Methods 0.000 abstract 1
- 230000009870 specific binding Effects 0.000 abstract 1
- 230000001960 triggered effect Effects 0.000 abstract 1
- 238000004458 analytical method Methods 0.000 description 5
- UGZAJZLUKVKCBM-UHFFFAOYSA-N 6-sulfanylhexan-1-ol Chemical compound OCCCCCCS UGZAJZLUKVKCBM-UHFFFAOYSA-N 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 4
- 230000035945 sensitivity Effects 0.000 description 4
- 229910001385 heavy metal Inorganic materials 0.000 description 3
- 229910004042 HAuCl4 Inorganic materials 0.000 description 2
- 238000000970 chrono-amperometry Methods 0.000 description 2
- 238000004140 cleaning Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 230000007613 environmental effect Effects 0.000 description 2
- 238000003912 environmental pollution Methods 0.000 description 2
- 238000001095 inductively coupled plasma mass spectrometry Methods 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 1
- 238000001479 atomic absorption spectroscopy Methods 0.000 description 1
- 238000001391 atomic fluorescence spectroscopy Methods 0.000 description 1
- 239000012472 biological sample Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 210000002249 digestive system Anatomy 0.000 description 1
- 239000003651 drinking water Substances 0.000 description 1
- 235000020188 drinking water Nutrition 0.000 description 1
- 238000000840 electrochemical analysis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 239000002440 industrial waste Substances 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 description 1
- 238000005065 mining Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000008399 tap water Substances 0.000 description 1
- 235000020679 tap water Nutrition 0.000 description 1
- 239000013076 target substance Substances 0.000 description 1
- 239000002351 wastewater Substances 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
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/308—Electrodes, e.g. test electrodes; Half-cells at least partially made of carbon
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/48—Systems using polarography, i.e. measuring changes in current under a slowly-varying voltage
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54373—Apparatus specially adapted for solid-phase testing involving physiochemical end-point determination, e.g. wave-guides, FETS, gratings
- G01N33/5438—Electrodes
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
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Abstract
A method for electrochemically detecting mercury ions based on nucleic acid dye mainly utilizes a silk-screen printing carbon electrode modified by gold nanoparticles as a detection element, and modifies a DNA probe on the surface of the electrode through the action of a stable Au-S bond. Based on T-T base pairs and Hg2+Specific binding property for capturing Hg in system2+Simultaneously, the structural change of the DNA probe is triggered. Then the intercalation of the nucleic acid dye GelRed on double-stranded DNA promotes the combination of the dye and DNA molecules, thereby causing the electrochemical signal on the surface of the electrode to be increased. Based on the method, Hg in a water sample can be adjusted2+And (4) detecting without marks. The method has simple operation, no need of marking, low cost, and can be used for labeling detection in actual water sampleThe test result shows that the recovery rate is good, and the application prospect is good.
Description
Technical Field
The invention relates to quantitative detection of mercury ions by using an electrochemical biosensor, and belongs to the technical field of biosensing.
Background
With the development of human society and industry, the problem of environmental pollution is increasingly highlighted, and the survival and development of human beings are seriously threatened. Heavy metal pollution refers to environmental pollution caused by heavy metals and their compounds, mainly caused by artificial factors such as mining, industrial waste gas and water discharge, etc. Wherein the mercury ion (Hg)2+) Is a water-soluble and high-risk heavy metal ion, can not only destroy the ecological environment, but also cause no harm to aquatic organisms and human healthDamage can be reversed. Hg is a mercury vapor2+It can seriously damage various organs of the human body, including the brain, kidneys, central nervous system and digestive system. Hg in drinking water according to the United states Environmental Protection Agency (EPA) regulations2+Should not exceed 10 nM. Conventional Hg2+The analysis method includes Atomic Absorption Spectroscopy (AAS), Atomic Fluorescence Spectroscopy (AFS), High Performance Liquid Chromatography (HPLC), inductively coupled plasma mass spectrometry (ICP-MS), and the like. These highly sensitive, highly accurate and highly accurate analytical methods have been widely used for Hg in environmental and biological samples2+Detection of (3). However, these analytical methods have some disadvantages such as the need for expensive equipment and complicated operating procedures. Furthermore, these techniques cannot be used for field testing due to the large scale instrumentation required. In comparison, the electrochemical analysis method has the advantages of simple equipment and process, high sensitivity, quick analysis and the like.
In recent years, biosensors based on DNA molecules that specifically recognize a target substance have been vigorously developed because they exhibit advantages such as rapidity, high sensitivity, and high selectivity. Ono and Togashi discovered Hg in 20042+And specific mismatch between T-T base pairs in DNA molecules, for the detection of Hg by DNA biosensors2+A basis is provided.
Disclosure of Invention
The invention aims to construct a label-free electrochemical biosensor based on nucleic acid dye, which is used for quickly and sensitively detecting mercury ions.
The gold nanoparticles are modified on the surface of the disposable silk-screen printing carbon electrode by using an electrochemical deposition technology, so that the purposes of improving the conductivity of the silk-screen printing carbon electrode and facilitating modification of a DNA probe are achieved. Then modifying the DNA probe with one end modified with-SH to the surface of the electrode through the action of Au-S bond to capture Hg in the solution2+The purpose of enhancing electrochemical signals is achieved by virtue of the intercalation effect of a nucleic acid dye GelRed and a DNA double strand. Therefore, the mercury ion detection device can achieve high selectivity, no mark and high sensitivity detection of mercury ions in water.
The technical scheme of the invention is as follows:
(1) And modifying the gold nanoparticles on the screen-printed carbon electrode by an electrochemical deposition method.
(2) dripping a DNA probe solution with the concentration of 1 mu M on the surface of the electrode in (1), and incubating at room temperature overnight.
(3) Immersing the electrode in (2) into Hg with a certain concentration2+Incubate in solution for 30 minutes.
(4) And (3) immersing the electrode in a GelRed dye solution with the concentration of 5 x, incubating for 10 minutes, detecting an electrochemical signal by a Differential Pulse Voltammetry (DPV) method by using an electrochemical workstation, and analyzing the measurement result.
Effects of the invention
Compared with the prior art, the invention has the following advantages:
(1) The sensitivity is high. The method can detect nM Hg2+Can reach Hg in the actual water environment2+And (5) content standard.
(2) The cost is low. The method only needs a single DNA probe molecule and does not need to be marked, thereby greatly reducing the detection cost.
(3) The operation is simple. The detection of the sample can be completed in a short time without other special sample pretreatment processes.
Detailed Description
Example 1
After cleaning the screen-printed carbon electrode, the carbon electrode was placed in 5mM HAuCl4Electrochemical deposition of gold nanoparticles was performed using a chronoamperometry method in a solution (containing 50mM HCl) set at a potential of-0.6V for a time of 150 s. After the surface of the screen-printed carbon electrode modified with gold nanoparticles was incubated overnight with 1. mu.M DNA probe solution, the electrode was blocked by incubating in 1mM 6-mercapto-1-hexanol (MCH) solution for 1 hour. Immersing the prepared electrode into a tap water sample (the water sample is centrifuged for 20 minutes by a centrifuge at 10000 r/min, then filtered by a 0.22 mu M filter membrane, and 5nM Hg is added2+) And then incubation is continued for 30 minutes; after incubation in a GelRed dye solution at a concentration of 5X for 10 minutes, the electrochemical signals were detected by Differential Pulse Voltammetry (DPV) using an electrochemical workstation, and the results were analyzed.
Example 2
After cleaning the screen-printed carbon electrode, the carbon electrode was placed in 5mM HAuCl4Electrochemical deposition of gold nanoparticles was performed using a chronoamperometry method in a solution (containing 50mM HCl) set at a potential of-0.6V for a time of 150 s. After the surface of the screen-printed carbon electrode modified with gold nanoparticles was incubated overnight with 1. mu.M DNA probe solution, the electrode was blocked by incubating in 1mM 6-mercapto-1-hexanol (MCH) solution for 1 hour. Immersing the prepared electrode into a water sample of an east wind channel (centrifuging the water sample at 10000 rpm for 20 minutes by a centrifugal machine, filtering by a 0.22 mu M filter membrane, adding 5nM Hg2+) And then incubation is continued for 30 minutes; after incubation in a GelRed dye solution at a concentration of 5X for 10 minutes, the electrochemical signals were detected by Differential Pulse Voltammetry (DPV) using an electrochemical workstation, and the results were analyzed.
Through the analysis of actual samples, the electrochemical biosensor detects Hg in a water sample2+Feasibility was evaluated. For Hg in two water samples2+The spiked assay was performed at 5nM, and the results show Hg for two actual water samples2+the recovery rate is good. The results are shown in Table 1.
TABLE 1 addition of Hg to actual water samples2+Detection of (2)
Claims (5)
1. A method for electrochemically detecting mercury ions based on nucleic acid dyes is characterized by comprising the following four steps:
(1) And modifying the gold nanoparticles on the screen-printed carbon electrode by an electrochemical deposition method.
(2) Dripping a DNA probe solution with the concentration of 1 mu M on the surface of the electrode in (1), and incubating at room temperature overnight.
(3) And (3) immersing the electrode in a mercury ion solution with a certain concentration for incubation for 30 minutes.
(4) and (3) immersing the electrode in a GelRed dye solution with the concentration of 5 x, incubating for 10 minutes, detecting an electrochemical signal by a Differential Pulse Voltammetry (DPV) method by using an electrochemical workstation, and analyzing the measurement result.
2. The method of claim 1, wherein the selected DNA probe sequence is 5' -SH- (CH)2)6-GGTCTGTCTTCCCCCCCCCCCCTTGTCTGT-3’。
3. The method of claim 1, wherein the electrode is a screen-printed carbon electrode modified by electrochemically deposited gold nanoparticles.
4. The method of claim 1, wherein the dye used to enhance the electrochemical signal is GelRed.
5. The method of claim 1, wherein the Tris-HCl (Tris-hydroxymethyl-aminomethane-hydrochloric acid) buffer solution has a pH of 7.4.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201910761801.2A CN110568036A (en) | 2019-08-16 | 2019-08-16 | Method for electrochemically detecting mercury ions based on nucleic acid dye |
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| CN201910761801.2A CN110568036A (en) | 2019-08-16 | 2019-08-16 | Method for electrochemically detecting mercury ions based on nucleic acid dye |
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Citations (9)
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|---|---|---|---|---|
| CN1558957A (en) * | 2001-07-25 | 2004-12-29 | Posco | Detection method of nucleic acid hybridization |
| CN102375021A (en) * | 2010-08-25 | 2012-03-14 | 中国科学院大连化学物理研究所 | Electrochemical method employing DNA as probe to detect environmental pollutant |
| CN102706940A (en) * | 2012-06-15 | 2012-10-03 | 湖南大学 | Electrochemical sensor capable of detecting trace mercury in water body, and preparation method and application thereof |
| CN105675676A (en) * | 2016-01-22 | 2016-06-15 | 湖南大学 | Mercuric ion or cysteine detecting electrochemical DNA biosensor and manufacturing method and application thereof |
| CN105758918A (en) * | 2016-04-08 | 2016-07-13 | 青岛科技大学 | Preparation and application method of electrochemical reduction graphene oxide and nanogold modified electrode based DNA sensor |
| CN105784822A (en) * | 2016-04-19 | 2016-07-20 | 青岛科技大学 | Preparation and application methods for electrochemical DNA sensor based on chitosan-graphene/ gold nanoparticle composite membrane |
| CN106706733A (en) * | 2016-11-14 | 2017-05-24 | 江苏大学 | Preparation method of electrochemical adapter sensor for detecting fumonisin B1 ( FB1) |
| CN107367540A (en) * | 2017-09-05 | 2017-11-21 | 重庆大学 | A kind of aptamers electrochemical sensor and the method for detecting chlopyrifos |
| CN113295745A (en) * | 2021-05-18 | 2021-08-24 | 长春理工大学 | Electrochemical DNA sensor and preparation method and application thereof |
-
2019
- 2019-08-16 CN CN201910761801.2A patent/CN110568036A/en active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1558957A (en) * | 2001-07-25 | 2004-12-29 | Posco | Detection method of nucleic acid hybridization |
| CN102375021A (en) * | 2010-08-25 | 2012-03-14 | 中国科学院大连化学物理研究所 | Electrochemical method employing DNA as probe to detect environmental pollutant |
| CN102706940A (en) * | 2012-06-15 | 2012-10-03 | 湖南大学 | Electrochemical sensor capable of detecting trace mercury in water body, and preparation method and application thereof |
| CN105675676A (en) * | 2016-01-22 | 2016-06-15 | 湖南大学 | Mercuric ion or cysteine detecting electrochemical DNA biosensor and manufacturing method and application thereof |
| CN105758918A (en) * | 2016-04-08 | 2016-07-13 | 青岛科技大学 | Preparation and application method of electrochemical reduction graphene oxide and nanogold modified electrode based DNA sensor |
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| CN107367540A (en) * | 2017-09-05 | 2017-11-21 | 重庆大学 | A kind of aptamers electrochemical sensor and the method for detecting chlopyrifos |
| CN113295745A (en) * | 2021-05-18 | 2021-08-24 | 长春理工大学 | Electrochemical DNA sensor and preparation method and application thereof |
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