CN104931570B - A kind of preparation method of the heavy metal ion electrochemical sensor based on aptamer and application - Google Patents
A kind of preparation method of the heavy metal ion electrochemical sensor based on aptamer and application Download PDFInfo
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- CN104931570B CN104931570B CN201510305386.1A CN201510305386A CN104931570B CN 104931570 B CN104931570 B CN 104931570B CN 201510305386 A CN201510305386 A CN 201510305386A CN 104931570 B CN104931570 B CN 104931570B
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- heavy metal
- metal ion
- aptamer
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- 229910001385 heavy metal Inorganic materials 0.000 title claims abstract description 39
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 34
- 238000002360 preparation method Methods 0.000 title claims abstract description 11
- 150000002500 ions Chemical class 0.000 claims abstract description 30
- 238000001514 detection method Methods 0.000 claims abstract description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-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 4
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims description 18
- 239000011324 bead Substances 0.000 claims description 12
- 108010090804 Streptavidin Proteins 0.000 claims description 10
- 239000011780 sodium chloride Substances 0.000 claims description 10
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Inorganic materials [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 10
- 239000000243 solution Substances 0.000 claims description 10
- 229960002685 biotin Drugs 0.000 claims description 9
- 235000020958 biotin Nutrition 0.000 claims description 9
- 239000011616 biotin Substances 0.000 claims description 9
- 238000001903 differential pulse voltammetry Methods 0.000 claims description 8
- 229910021397 glassy carbon Inorganic materials 0.000 claims description 8
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 6
- 238000004140 cleaning Methods 0.000 claims description 5
- 239000012153 distilled water Substances 0.000 claims description 5
- -1 potassium ferricyanide Chemical compound 0.000 claims description 5
- 239000000843 powder Substances 0.000 claims description 5
- ANRHNWWPFJCPAZ-UHFFFAOYSA-M thionine Chemical compound [Cl-].C1=CC(N)=CC2=[S+]C3=CC(N)=CC=C3N=C21 ANRHNWWPFJCPAZ-UHFFFAOYSA-M 0.000 claims description 5
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 claims description 4
- 229910052697 platinum Inorganic materials 0.000 claims description 4
- 239000012488 sample solution Substances 0.000 claims description 4
- FOIXSVOLVBLSDH-UHFFFAOYSA-N Silver ion Chemical compound [Ag+] FOIXSVOLVBLSDH-UHFFFAOYSA-N 0.000 claims description 2
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 claims 2
- PNEYBMLMFCGWSK-UHFFFAOYSA-N aluminium oxide Inorganic materials [O-2].[O-2].[O-2].[Al+3].[Al+3] PNEYBMLMFCGWSK-UHFFFAOYSA-N 0.000 claims 1
- 229910052593 corundum Inorganic materials 0.000 claims 1
- 238000005498 polishing Methods 0.000 claims 1
- 229910001845 yogo sapphire Inorganic materials 0.000 claims 1
- 230000009870 specific binding Effects 0.000 abstract description 3
- 229910052753 mercury Inorganic materials 0.000 abstract description 2
- 229910052709 silver Inorganic materials 0.000 abstract description 2
- 150000001455 metallic ions Chemical class 0.000 abstract 2
- QSHDDOUJBYECFT-UHFFFAOYSA-N mercury Chemical compound [Hg] QSHDDOUJBYECFT-UHFFFAOYSA-N 0.000 abstract 1
- 239000004332 silver Substances 0.000 abstract 1
- 108020004414 DNA Proteins 0.000 description 4
- 102000053602 DNA Human genes 0.000 description 4
- 238000012986 modification Methods 0.000 description 4
- 230000004048 modification Effects 0.000 description 4
- 238000013461 design Methods 0.000 description 3
- 229910052793 cadmium Inorganic materials 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 229920002477 rna polymer Polymers 0.000 description 2
- 208000030527 Minamata disease Diseases 0.000 description 1
- 208000009507 Nervous System Mercury Poisoning Diseases 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229910052785 arsenic Inorganic materials 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000027455 binding Effects 0.000 description 1
- BDOSMKKIYDKNTQ-UHFFFAOYSA-N cadmium atom Chemical compound [Cd] BDOSMKKIYDKNTQ-UHFFFAOYSA-N 0.000 description 1
- 229910052804 chromium Inorganic materials 0.000 description 1
- 238000004737 colorimetric analysis Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000002848 electrochemical method Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 229910052737 gold Inorganic materials 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 229910000765 intermetallic Inorganic materials 0.000 description 1
- 238000002372 labelling Methods 0.000 description 1
- 229910052745 lead Inorganic materials 0.000 description 1
- 238000000034 method Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 229910052759 nickel Inorganic materials 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013077 target material Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
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- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analyzing Materials By The Use Of Magnetic Means (AREA)
Abstract
The present invention relates to a kind of preparation method and application of the heavy metal ion electrochemical sensor based on aptamer, belong to technical field of analytical chemistry.Based on the specific binding of particular bases in aptamer and metallic ion, relate to not homotactic aptamer, significantly improve the selectivity of sensor to metallic ion, be adapted to detect the mercury in water quality and silver; Detection for analyzing mercury ion is limited to 0.33nmol/L, and the range of linearity is 1 ~ 200nmol/L.
Description
Technical field
The present invention relates to a kind of preparation method of the heavy metal ion electrochemical sensor based on aptamer, belong to sensing assays technical field.
Background technology
Aptamer aptamer is one section of DNA (deoxyribonucleic acid) DNA or RNA (ribonucleic acid) sequence, can go out have by in-vitro screening a kind of material of particular sequence, it can with the combination of plurality of target material high specific, high selectivity.It is a kind of novel identification molecule, has many advantages, as can chemosynthesis, good stability, there is no toxicity.Large quantity research through researcher shows, the base " T " in aptamer and " C " can specific identification heavy metal ion Hg
2+and Ag
+, thus form stable " T-Hg
2+-T " and " C-Ag
+-C " structure.The structure that researcher utilizes this special, devises many detection Hg
2+and Ag
+system, as fluorescence, galvanochemistry, electrochemiluminescence and colourimetry.
Heavy metal, a kind of definition is wherein exactly that the proportion element that is greater than 5 is all referred to as heavy metal, mainly comprise Au, Ag, Cu, Pb, Ni, Co, Cr, Hg, Cd, As etc. 45 kinds, these heavy metals can not be decomposed in water body, can be converted into the stronger metallic compound of toxicity under the effect of microorganism.Biology extracts heavy metal from environment, and through the biological magnification of food chain, enrichment in higher level biosome, then enters human body by food, is detrimental to health.The minamata disease and the Itai-itai diseases that occur in Japan are caused by heavy metal Hg and cadmium pollution respectively.Therefore, the detection of heavy metal seems particularly important.
The present invention utilizes " T-Hg
2+-T " and " C-Ag
+-C " structure, designs not homotactic aptamer, adopts electrochemical method, realize the detection of heavy metal ion.In order to accelerate analysis speed, the present invention adopts magnetic glassy carbon electrode, is fixed the magnetic bead SA-Fe of Streptavidin modification by magnetic glassy carbon electrode
3o
4thus effectively enhance the stability of electrode, Streptavidin and biotin have stronger specific binding capacity, aptamer is carried out biotin labeling, by the binding ability between Streptavidin and biotin, effectively aptamer can be fixed to electrode surface, thus increase the stability of electrode.
Summary of the invention
An object of the present invention designs not homotactic aptamer, builds a kind of quick, sensitive electrochemical sensor.
Two of object of the present invention is the detections electrochemical sensor of structure being used for heavy metal ion.
technical scheme of the present invention is as follows:
1. the preparation method based on the heavy metal ion electrochemical sensor of aptamer
(1) by diameter be the magnetic glassy carbon electrode of the 4mm Al of 1.0,0.3,0.05 μm
2o
3burnishing powder is polished, and then rinses electrode surface well rear thieving paper with distilled water and dries; Electrode is placed in 5mmol/L potassium ferricyanide solution, in-0.2 ~ 0.6V scanning, spike potential difference is less than 110mV; Drip the magnetic bead SA-Fe of 6 μ L, 1 ~ 5mg/mL
3o
4, leave standstill and dry;
(2) electrode surface modified in step (1) drips the aptamer Bio-ssDNA of 6 μ L, 200nmol/L, incubated at room 30min, then washes working electrode with pH=7.4Tris-HCl-NaCl buffer solution, washes away unnecessary ssDNA;
(3) working electrode that step (2) is modified is immersed in the heavy metal ion of variable concentrations, keeps 30min; (4) working electrode surface modified in step (3) drips the thionine of 3 μ L, 1.0mmol/L in electrode surface, after electrode surface is dry, with Tris-HCl-NaCl solution cleaning twice, finally places it in 4 DEG C of refrigerators and saves backup;
Described magnetic bead SA-Fe
3o
4modify with Streptavidin;
Described aptamer Bio-ssDNA is with biotin labeled;
2. the detection method of heavy metal ion
(1) electrochemical workstation is used to test with three-electrode system, using the electrochemical sensor based on aptamer of structure as working electrode, take saturated calomel electrode as contrast electrode, be to electrode with platinum electrode, test in the PBS buffer solution of 10mL, 50mmol/L, pH5.80 ~ 8.49;
(2) detect with differential pulse voltammetry DPV heavy metal ion, arranging scanning potential range is-0.4 ~ 0V, sweeps speed for 0.1V/s, has scanned the size of rear record peak current, and preserved data;
(3) according to the size of the peak current of gained and the relation of concentration of heavy metal ion, drawing curve;
(4) heavy metal ion is replaced to detect sample solutions to be measured.
Described heavy metal ion can be mercury ion Hg
2+or silver ion Ag
+.
useful achievement of the present invention
(1) magnetic bead that Streptavidin is modified is introduced in the building process of electrochemical sensor, utilize magnetic glassy carbon electrode, thus add the stability of modified electrode.
(2) utilize Streptavidin and biotin specific binding to react, add the stability of electrode.
(3) " T-Hg is utilized
2+-T " and " C-Ag
+-C " structure, the aptamer of design particular sequence, thus enhance the selectivity of electrode pair heavy metal ion, add the specificity of sensor.
(4) electrochemical sensor prepared of the present invention is for the detection of heavy metal ion, and have higher selectivity and antijamming capability, detectability is low, and the range of linearity is wide.Detection for analyzing mercury ion is limited to 0.33nmol/L, the range of linearity 1 ~ 200nmol/L.
Described basic reagent and material all can be bought in Reagent Company.
Embodiment
embodiment 1a kind of preparation method of the heavy metal ion electrochemical sensor based on aptamer
(1) by diameter be the magnetic glassy carbon electrode of the 4mm Al of 1.0,0.3,0.05 μm
2o
3burnishing powder is polished, and then rinses electrode surface well rear thieving paper with distilled water and dries.Then electrode is placed in 5mmol/L potassium ferricyanide solution, in-0.2 ~ 0.6V scanning, spike potential difference is less than 110mV; Then the magnetic bead SA-Fe of 6 μ L, 1mg/mL is dripped
3o
4, leave standstill and dry;
(2) drip the aptamer Bio-ssDNA of 6 μ L, 200nmol/L, incubated at room 30min, then rinse electrode with pH=7.4Tris-HCl-NaCl, wash away unnecessary Bio-ssDNA;
(3) working electrode of modification is immersed in the heavy metal ion of variable concentrations, keeps 30min;
(4) drip thionine and the electrode surface of 3 μ L, 1.0mmol/L, after electrode surface is dry, with Tris-HCl-NaCl solution cleaning twice, finally places it in 4 DEG C of refrigerators and save backup;
Described magnetic bead SA-Fe
3o
4modify with Streptavidin; Described aptamer Bio-ssDNA is with biotin labeled.
embodiment 2a kind of preparation method of the heavy metal ion electrochemical sensor based on aptamer
(1) by diameter be the magnetic glassy carbon electrode of the 4mm Al of 1.0,0.3,0.05 μm
2o
3burnishing powder is polished, and then rinses electrode surface well rear thieving paper with distilled water and dries.Then electrode is placed in 5mmol/L potassium ferricyanide solution, in-0.2 ~ 0.6V scanning, spike potential difference is less than 110mV; Then the magnetic bead SA-Fe of 6 μ L, 3mg/mL is dripped
3o
4, leave standstill and dry;
(2) drip the aptamer Bio-ssDNA of 6 μ L, 200nmol/L, incubated at room 30min, then rinse electrode with pH=7.4Tris-HCl-NaCl, wash away unnecessary Bio-ssDNA;
(3) working electrode of modification is immersed in the heavy metal ion of variable concentrations, keeps 30min;
(4) drip thionine and the electrode surface of 3 μ L, 1.0mmol/L, after electrode surface is dry, with Tris-HCl-NaCl solution cleaning twice, finally places it in 4 DEG C of refrigerators and save backup;
Described magnetic bead SA-Fe
3o
4modify with Streptavidin; Described aptamer Bio-ssDNA is with biotin labeled.
embodiment 3a kind of preparation method of the heavy metal ion electrochemical sensor based on aptamer
(1) by diameter be the magnetic glassy carbon electrode of the 4mm Al of 1.0,0.3,0.05 μm
2o
3burnishing powder is polished, and then rinses electrode surface well rear thieving paper with distilled water and dries.Then electrode is placed in 5mmol/L potassium ferricyanide solution, in-0.2 ~ 0.6V scanning, spike potential difference is less than 110mV; Then the magnetic bead SA-Fe of 6 μ L, 5mg/mL is dripped
3o
4, leave standstill and dry;
(2) drip the aptamer Bio-ssDNA of 6 μ L, 200nmol/L, incubated at room 30min, then rinse electrode with pH=7.4Tris-HCl-NaCl, wash away unnecessary Bio-ssDNA;
(3) working electrode of modification is immersed in the heavy metal ion of variable concentrations, keeps 30min;
(4) drip thionine and the electrode surface of 3 μ L, 1.0mmol/L, after electrode surface is dry, with Tris-HCl-NaCl solution cleaning twice, finally places it in 4 DEG C of refrigerators and save backup;
Described magnetic bead SA-Fe
3o
4modify with Streptavidin; Described aptamer Bio-ssDNA is with biotin labeled.
embodiment 4electrochemical sensor is used for heavy metal Hg
2+detection
(1) electrochemical workstation is used to test with three-electrode system, using the electrochemical sensor based on aptamer of structure as working electrode, take saturated calomel electrode as contrast electrode, be to electrode with platinum electrode, test in the PBS buffer solution of 10mL, 50mmol/L, pH5.80 ~ 8.49;
(2) use differential pulse voltammetry DPV to Hg
2+detect, arranging scanning potential range is-0.4 ~ 0V, sweeps speed for 0.1V/s, has scanned the size of rear record peak current, and preserved data;
(3) according to size and the Hg of the peak current of gained
2+the relation of concentration, drawing curve;
(4) sample solutions to be measured is replaced Hg
2+detect.Its detection is limited to 0.33nmol/L, the range of linearity 1 ~ 200nmol/L.
embodiment 5electrochemical sensor is used for heavy metal Ag
+detection
(1) electrochemical workstation is used to test with three-electrode system, using the electrochemical sensor based on aptamer of structure as working electrode, take saturated calomel electrode as contrast electrode, be to electrode with platinum electrode, test in the PBS buffer solution of 10mL, 50mmol/L, pH5.80 ~ 8.49;
(2) use differential pulse voltammetry DPV to Ag
+detect, arranging scanning potential range is-0.4 ~ 0V, sweeps speed for 0.1V/s, has scanned the size of rear record peak current, and preserved data;
(3) according to size and the Ag of the peak current of gained
+the relation of concentration, drawing curve;
(4) sample solutions to be measured is replaced Ag
+detect.
Claims (3)
1. the preparation method based on the heavy metal ion electrochemical sensor of aptamer, it is characterized in that, comprise the following steps: diameter is the magnetic glassy carbon electrode Al2O3 burnishing powder polishing of 1.0,0.3,0.05 μm of 4mm by (1), then rinses electrode surface well rear thieving paper with distilled water and dries; Electrode is placed in 5mmol/L potassium ferricyanide solution, in-0.2 ~ 0.6V scanning, spike potential difference is less than 110mV; Drip the magnetic bead SA-Fe3O4 of 6 μ L, 1 ~ 5mg/mL, leave standstill and dry;
(2) electrode surface modified in step (1) drips the aptamer Bio-ssDNA of 6 μ L, 200nmol/L, incubated at room 30min, then washes working electrode with pH=7.4Tris-HCl-NaCl buffer solution, washes away unnecessary Bio-ssDNA;
(3) working electrode that step (2) is modified is immersed in the heavy metal ion of variable concentrations, keeps 30min; (4) working electrode surface modified in step (3) drips the thionine of 3 μ L, 1.0mmol/L in electrode surface, after electrode surface is dry, with Tris-HCl-NaCl solution cleaning twice, finally places it in 4 DEG C of refrigerators and saves backup;
Described magnetic bead SA-Fe3O4 Streptavidin is modified;
Described aptamer Bio-ssDNA is with biotin labeled.
2. preparation method according to claim 1 prepares a kind of heavy metal ion electrochemical sensor based on aptamer, and it is characterized in that, for the detection of heavy metal ion, step is as follows:
(1) electrochemical workstation is used to test with three-electrode system, using the electrochemical sensor based on aptamer of structure as working electrode, take saturated calomel electrode as contrast electrode, be to electrode with platinum electrode, test in the PBS buffer solution of 10mL, 50mmol/L, pH5.80 ~ 8.49;
(2) detect with differential pulse voltammetry DPV heavy metal ion, arranging scanning potential range is-0.4 ~ 0V, sweeps speed for 0.1V/s, has scanned the size of rear record peak current, and preserved data;
(3) according to the size of the peak current of gained and the relation of concentration of heavy metal ion, drawing curve;
(4) heavy metal ion is replaced to detect sample solutions to be measured.
3. the preparation method of a kind of heavy metal ion electrochemical sensor based on aptamer according to claim 1, it is characterized in that, described heavy metal ion can be mercury ion Hg
2+or silver ion Ag
+.
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Cited By (1)
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| CN107228941A (en) * | 2017-06-30 | 2017-10-03 | 中国农业科学院农业质量标准与检测技术研究所 | It is a kind of to detect the aptamer kit and method of mercury and silver ion simultaneously |
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| CN105424777B (en) * | 2015-12-07 | 2018-01-16 | 江苏大学 | A kind of method for detecting two kinds of mycotoxins simultaneously by magnetic control electrochemical aptamer sensor |
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| CN107402249B (en) * | 2016-05-21 | 2019-07-09 | 云南民族大学 | A method of metal ion is detected based on hybridization chain reaction signal amplification technique |
| CN105866101B (en) * | 2016-05-23 | 2018-11-30 | 中国科学院生态环境研究中心 | A kind of metal mercury ions detection method based on aptamer label |
| CN106443003A (en) * | 2016-09-18 | 2017-02-22 | 暨南大学 | Fluorescent quenching test paper strip based on aptamer specific recognition and preparation method and application thereof |
| CN106841350B (en) * | 2017-02-07 | 2019-03-26 | 济南大学 | Electrochemical sensor and preparation method thereof based on aptamer detection mercury ion |
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| CN104931570A (en) | 2015-09-23 |
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