CN113358716A - Histamine electrochemical sensor based on aptamer, preparation method thereof and application thereof in river crab detection - Google Patents
Histamine electrochemical sensor based on aptamer, preparation method thereof and application thereof in river crab detection Download PDFInfo
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- NTYJJOPFIAHURM-UHFFFAOYSA-N Histamine Chemical compound NCCC1=CN=CN1 NTYJJOPFIAHURM-UHFFFAOYSA-N 0.000 title claims abstract description 114
- 229960001340 histamine Drugs 0.000 title claims abstract description 56
- 108091023037 Aptamer Proteins 0.000 title claims abstract description 52
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 238000001514 detection method Methods 0.000 title abstract description 15
- 229910052797 bismuth Inorganic materials 0.000 claims abstract description 24
- JCXGWMGPZLAOME-UHFFFAOYSA-N bismuth atom Chemical compound [Bi] JCXGWMGPZLAOME-UHFFFAOYSA-N 0.000 claims abstract description 24
- LSGOVYNHVSXFFJ-UHFFFAOYSA-N vanadate(3-) Chemical compound [O-][V]([O-])([O-])=O LSGOVYNHVSXFFJ-UHFFFAOYSA-N 0.000 claims abstract description 24
- 239000000523 sample Substances 0.000 claims abstract description 21
- 238000000034 method Methods 0.000 claims abstract description 18
- 239000010931 gold Substances 0.000 claims abstract description 13
- 239000002057 nanoflower Substances 0.000 claims abstract description 12
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 11
- 229910052737 gold Inorganic materials 0.000 claims abstract description 11
- 238000004070 electrodeposition Methods 0.000 claims abstract description 9
- 238000001027 hydrothermal synthesis Methods 0.000 claims abstract description 9
- 229910000033 sodium borohydride Inorganic materials 0.000 claims abstract description 9
- 239000012279 sodium borohydride Substances 0.000 claims abstract description 9
- 239000011521 glass Substances 0.000 claims abstract description 7
- 239000003446 ligand Substances 0.000 claims abstract description 7
- 230000009467 reduction Effects 0.000 claims abstract description 7
- 239000002245 particle Substances 0.000 claims abstract description 3
- 229910002915 BiVO4 Inorganic materials 0.000 claims description 15
- 239000000243 solution Substances 0.000 claims description 15
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- 239000012498 ultrapure water Substances 0.000 claims description 10
- 239000005457 ice water Substances 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 7
- 238000001035 drying Methods 0.000 claims description 7
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 claims description 6
- 239000002077 nanosphere Substances 0.000 claims description 6
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- PIEPQKCYPFFYMG-UHFFFAOYSA-N tris acetate Chemical compound CC(O)=O.OCC(N)(CO)CO PIEPQKCYPFFYMG-UHFFFAOYSA-N 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 229910020700 Na3VO4 Inorganic materials 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 3
- 238000001816 cooling Methods 0.000 claims description 3
- 239000001301 oxygen Substances 0.000 claims description 3
- 229910052760 oxygen Inorganic materials 0.000 claims description 3
- 239000002244 precipitate Substances 0.000 claims description 3
- 238000001179 sorption measurement Methods 0.000 claims description 3
- IHIXIJGXTJIKRB-UHFFFAOYSA-N trisodium vanadate Chemical compound [Na+].[Na+].[Na+].[O-][V]([O-])([O-])=O IHIXIJGXTJIKRB-UHFFFAOYSA-N 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- 229910004042 HAuCl4 Inorganic materials 0.000 claims description 2
- 239000000356 contaminant Substances 0.000 claims 2
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 claims 1
- 230000004048 modification Effects 0.000 claims 1
- 238000012986 modification Methods 0.000 claims 1
- 239000011734 sodium Substances 0.000 claims 1
- 229910052708 sodium Inorganic materials 0.000 claims 1
- 230000009471 action Effects 0.000 abstract description 2
- AMQJEAYHLZJPGS-UHFFFAOYSA-N N-Pentanol Chemical compound CCCCCO AMQJEAYHLZJPGS-UHFFFAOYSA-N 0.000 description 9
- 239000000872 buffer Substances 0.000 description 8
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 6
- 230000035945 sensitivity Effects 0.000 description 6
- 239000000284 extract Substances 0.000 description 5
- UGZAJZLUKVKCBM-UHFFFAOYSA-N 6-sulfanylhexan-1-ol Chemical compound OCCCCCCS UGZAJZLUKVKCBM-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- 238000000605 extraction Methods 0.000 description 4
- 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 description 3
- 241000238557 Decapoda Species 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000000835 electrochemical detection Methods 0.000 description 3
- 239000011780 sodium chloride Substances 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 238000002965 ELISA Methods 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 150000001412 amines Chemical class 0.000 description 2
- 238000005251 capillar electrophoresis Methods 0.000 description 2
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- 150000002978 peroxides Chemical class 0.000 description 2
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- 238000002798 spectrophotometry method Methods 0.000 description 2
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- 230000003321 amplification Effects 0.000 description 1
- 208000003455 anaphylaxis Diseases 0.000 description 1
- 230000000035 biogenic effect Effects 0.000 description 1
- 238000000861 blow drying Methods 0.000 description 1
- 238000006664 bond formation reaction Methods 0.000 description 1
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- 239000003153 chemical reaction reagent Substances 0.000 description 1
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- 238000006114 decarboxylation reaction Methods 0.000 description 1
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- 230000008021 deposition Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- ZOMNIUBKTOKEHS-UHFFFAOYSA-L dimercury dichloride Chemical class Cl[Hg][Hg]Cl ZOMNIUBKTOKEHS-UHFFFAOYSA-L 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000007791 liquid phase Substances 0.000 description 1
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- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
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- 230000027756 respiratory electron transport chain Effects 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- YNJBWRMUSHSURL-UHFFFAOYSA-N trichloroacetic acid Chemical compound OC(=O)C(Cl)(Cl)Cl YNJBWRMUSHSURL-UHFFFAOYSA-N 0.000 description 1
- 238000004506 ultrasonic cleaning Methods 0.000 description 1
- 238000001291 vacuum drying Methods 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
- 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
-
- 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
-
- 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
- G01N27/3275—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
- G01N27/3278—Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles
Abstract
The invention discloses a histamine electrochemical sensor based on an aptamer, a preparation method thereof and application thereof in river crab detection, wherein the sensor is used for modifying flower-shaped nanogold on the surface of conductive glass based on an electrodeposition method, and simultaneously connecting a sulfhydrylation modified histamine aptamer on the surface of AuNFs/ITO based on Au-S bonding action to construct a histamine electrochemical sensing interface; preparing bismuth vanadate nano flower balls by hydrothermal synthesis; based on a sodium borohydride reduction method, nano gold particles are introduced to the surface of the bismuth vanadate nano flower ball, ligand DNA matched with histamine aptamer is self-assembled, and the electrochemical probe DNA/Au @ BiVO is obtained4Namely said aptamer-based electrochemical sensor of histamine.
Description
Technical Field
The invention relates to an electrochemical sensor and a preparation method and application thereof, in particular to a histamine electrochemical sensor based on an aptamer, a preparation method thereof and application thereof in river crab detection.
Background
Histamine (Histamine) is a biogenic amine, one of the self-active substances. The longer the aquatic products such as fish, shrimp and crab are stored, the higher the content of histamine formed by decarboxylation of histidine in the protein under the action of microorganisms is, so that the content of histamine is one of the signs for evaluating the freshness of the aquatic products. When a human body ingests excessive histamine, anaphylactic reaction can be caused, and the life can be threatened when the excessive histamine is serious, so that the quick high-sensitivity real-time detection of the histamine has important practical significance.
At present, the main methods for detecting histamine include high performance liquid chromatography, spectrophotometry, capillary electrophoresis, enzyme-linked immunosorbent assay, and the like. Because histamine does not have specific ultraviolet absorption groups and fluorescence characteristics, the high performance liquid chromatography generally needs derivatization and complex pretreatment, the process is complicated, and instruments are expensive and are only suitable for laboratory operation; the pretreatment of the sample by the azo reagent color-developing spectrophotometry is relatively complex, the detection limit is high, and the sensitivity is low; although the capillary electrophoresis method has the advantages of simple instrument, high separation speed, high sensitivity and the like, the operation is complex, and the biological amine can be directly measured by the capillary zone electrophoresis without derivatization process; enzyme linked immunosorbent assay, in order to improve the detection sensitivity, a double-antibody sandwich method is commonly used, so that the detection cost is higher.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide an aptamer-based histamine electrochemical sensor which is high in detection speed and high in sensitivity. Another object of the present invention is to provide a method for manufacturing the sensor. The invention also aims to disclose the application of the sensor in river crab detection, which can be well used for detecting histamine in river crabs.
The technical scheme is as follows: the histamine electrochemical sensor based on the aptamer is characterized in that firstly, flower-shaped nanogold is modified on the surface of conductive glass based on an electrodeposition method, and meanwhile, a sulfhydrylation modified histamine aptamer is connected to the surface of AuNFs/ITO based on Au-S bonding effect to construct a histamine electrochemical sensing boundaryKneading; preparing bismuth vanadate nano flower balls by hydrothermal synthesis; based on a sodium borohydride reduction method, nano gold particles are introduced to the surface of the bismuth vanadate nano flower ball, ligand DNA matched with histamine aptamer is self-assembled, and the electrochemical probe DNA/Au @ BiVO is obtained4Namely said aptamer-based electrochemical sensor of histamine.
The histamine electrochemical sensor based on the aptamer comprises the following components:
5’-SH-(CH2)6-GCCTGTTGTGAGCCTCCTAACATTTCTATGCTGCAGCCAACTTTTCCATACTTCCAGCTTACCATTTATCCATGCTTATTCTTGTCTCCC-3’。
the histamine electrochemical sensor based on the aptamer comprises the following ligand DNA:
5’-SH-(CH2)6-GGGAGACAAGAATAAGCATGGATAAATGGTAAGCTGGAAGTATGGAAAAGTTGGCTGCAGCATAGAAATGTTAGGAGGCTCACAACAGGC-3’
the preparation method of the histamine electrochemical sensor based on the aptamer comprises the following steps:
(1) preparing flower-shaped nano gold modified electrodes AuNFs/ITO by constant potential electrodeposition;
(2) preparing an aptamer/AuNFs/ITO;
(3) preparation of bismuth vanadate BiVO4Nano flower ball;
(4) preparation of DNA/Au @ BiVO4An electrochemical probe.
The preparation method comprises the following steps of (1) specifically cleaning the conductive glass ITO to remove surface pollutants, and then carrying out HAuCl treatment on the cleaned conductive glass ITO4Is electrodeposited in an aqueous solution of (1), N is introduced before the electrodeposition2Removing dissolved oxygen to prepare the flower-shaped nano gold modified electrode AuNFs/ITO.
The preparation method comprises the step (2) of washing AuNFs/ITO ultrapure water, and then carrying out N2Drying, placing the aptamer in Tris-acetate buffer solution containing the aptamer and incubating at room temperature in a dark place, self-assembling the sulfhydryl-modified aptamer on the surface of AuNFs/ITO through Au-S bonding, and then placing the aptamer in MCH solution to remove nonspecific adsorption active sites; respectively using PB buffer solution and ultrapure water to rinse the prepared aptamer/AuNFs/ITO, and placing the aptamer/AuNFs/ITO into PB buffer solution for preservationAnd storing for later use.
The preparation method comprises the following steps of (3) preparing the bismuth vanadate nanospheres by hydrothermal synthesis: first Bi (NO)3)3·5H2O and Na3VO4·12H2Dispersing O in water by ultrasonic, sealing in a hydrothermal reaction kettle, reacting at 140 ℃ and 180 ℃ for 6-10 hours, naturally cooling, respectively cleaning the obtained product with ethanol and ultrapure water, centrifugally separating, and drying in vacuum overnight to obtain the bismuth vanadate nano flower ball.
The preparation method comprises the following steps of (4) modifying nano gold on the surface of bismuth vanadate by adopting a sodium borohydride reduction method: stirring the mixed solution containing bismuth vanadate and chloroauric acid under the condition of ice-water bath, adding sodium borohydride solution prepared by the ice-water bath, keeping the ice-water bath, stirring, centrifuging, taking precipitate, and drying in vacuum to obtain Au @ BiVO4(ii) a Then, in a solution containing Au @ BiVO4Adding a mixed solution containing DNA and TCEP into the buffer solution, and incubating in a dark place to prepare DNA/Au @ BiVO4The electrochemical probe of (1).
The electrochemical sensor for histamine based on the aptamer is applied to histamine detection of aquatic products.
The application is that the aquatic product is river crab.
The invention relates to an aptamer-based histamine electrochemical sensor which can be used for detecting histamine in aquatic products such as river crabs and the like. In the presence of histamine, aptamers specifically bind to histamine to cause a conformational change, unbound aptamers hybridize to the coordinating DNA, and the electrochemical probe DNA/Au @ BiVO is applied4The sensor surface is captured. The bismuth vanadate nano-flower ball has good peroxide mimic enzyme activity, and electrochemical detection of histamine is realized by detecting reduction current of hydrogen peroxide. The higher the concentration of histamine, the fewer electrochemical probes captured by the sensing surface, and the lower the electrochemical signal will be. The sensor has good selectivity due to the specific recognition of histamine by histamine aptamer; the flower-shaped nanogold and the bismuth vanadate nanospheres can double amplify electrochemical signals, so that the sensor has high sensitivity, and fig. 1 is a schematic diagram. The sensor is used for detecting histamine in river crab, and the result and high efficiency liquidPhase chromatography (GB 5009.208-2016) was consistent.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: (1) the histamine aptamer is used as a recognition group, so that the sensor is high in anti-interference capacity and high in selectivity. (2) When the flower-shaped nanogold is used as a carrier to be connected with the electrochemical probe, the electron transfer rate can be increased, and an electrochemical signal can be amplified; the bismuth vanadate nanosphere with the peroxide mimic enzyme activity is used as an electrochemical probe and can also play a role in signal amplification, so that the sensor has high sensitivity and the detection limit is 8.4 multiplied by 10-10mol/L. (3) Expensive instruments and equipment are not required, and portability and miniaturization can be realized. (4) The electrochemical detection is simple to operate and low in cost.
Drawings
FIG. 1 is a schematic view of detection;
FIG. 2 (A) shows DPV detection curves with histamine concentrations of 0, 1, 2, 5, 20, 50, 100, 200nM, respectively; (B) is a standardized curve for histamine determination.
Detailed Description
Example 1
Materials: ITO, aptamer, TCEP (tris (2-carboxyethyl) phosphine), MCH (6-mercapto-1-hexanol), ligand DNA
Histamine aptamers:
5’-SH-(CH2)6-GCCTGTTGTGAGCCTCCTAACATTTCTATGCTGCAGCCAACTTTTCCATACTTCCAGCTTACCATTTATCCATGCTTATTCTTGTCTCCC-3’
ligand DNA:
5’-SH-(CH2)6-GGGAGACAAGAATAAGCATGGATAAATGGTAAGCTGGAAGTATGGAAAAGTTGGCTGCAGCATAGAAATGTTAGGAGGCTCACAACAGGC-3’
A. preparation of aptamer/AuNFs/ITO
Preparing AuNFs/ITO by constant potential electrodeposition: conducting ultrasonic cleaning of conductive glass ITO (5 multiplied by 10mm) with acetone, ethanol and ultrapure water for 20 minutes in sequence to remove possible pollutants on the surface of the ITO; then at a concentration of 0.5mM HAuCl4Is electrodeposited in an aqueous solution of (1), N is introduced before the electrodeposition2Removing dissolved oxygen for at least 30 min, and precipitating at a deposition potential of 0.3VThe product time is 1200s, and the flower-shaped nano gold modified electrode (AuNFs/ITO) is prepared. ② preparing the aptamer/AuNFs/ITO: after AuNFs/ITO ultra pure water cleaning, N2Blow-drying, incubation in 25mM Tris-acetate buffer (pH 8.0, 0.5mM TCEP added to reduce disulfide bond formation) containing 10. mu.M aptamer overnight at room temperature in the absence of light, self-assembly of thiol-modified aptamer to AuNFs/ITO surface by Au-S bonding, and soaking in 1mM MCH solution for 2 hours to remove non-specific adsorption active sites. The prepared aptamer/AuNFs/ITO was rinsed with pH8.0PB and ultrapure water, respectively, and then stored in a 10mM PB buffer (pH 8.0) in a refrigerator at 4 ℃ for further use.
B、DNA/Au@BiVO4Preparation of electrochemical probes
(ii) bismuth vanadate (BiVO)4) And (5) preparing the nano flower balls. Hydrothermal synthesis of bismuth vanadate nanospheres, first 0.1mmol of Bi (NO)3)3·5H2O and 0.2mmol Na3VO4·12H2Dispersing O in 40mL of water by ultrasonic, sealing in an 80mL hydrothermal reaction kettle, and reacting at 160 ℃ for 8 hours. Naturally cooling to room temperature, respectively washing the obtained product twice with ethanol and ultrapure water, centrifugally separating (10000rpm,5 minutes), and vacuum drying at 60 ℃ overnight to obtain the bismuth vanadate nanospheres.
②DNA/Au@BiVO4And (3) preparing an electrochemical probe. Firstly, modifying nano gold on the surface of bismuth vanadate by adopting a sodium borohydride reduction method. Stirring in 200mL of a solution containing 0.1mgmL of water in an ice-water bath-1Adding 5mL of 10mM sodium borohydride solution prepared by an ice water bath into the mixed solution of bismuth vanadate and 0.1mM chloroauric acid for 0.5 hour, keeping the ice water bath, stirring for 1 hour, centrifuging at 10000rpm for 5 minutes, taking the precipitate, drying at 60 ℃ in vacuum overnight to obtain Au @ BiVO4. Secondly, at 1mLAu @ BiVO4(0.1mgmL-125mM Tris-acetate buffer, pH8.0) was added to 100. mu.L of a mixture containing 15. mu.M DNA, 0.5mM TCEP and incubated overnight in the dark. The prepared DNA/Au @ BiVO4The electrochemical probe of (1) was washed twice with 10mM, pH8.0PB buffer, and then dispersed in 1mL of a buffer (10mM Tris-HCl, 1mM EDTA, 50mM NaCl, pH 7.4) and stored at 4 ℃ until use.
C. Electrochemical detection of histamine
mu.L of PBS buffer (10mM, pH8.0) containing histamine at various concentrations was added dropwise to the aptamer/AuNFs/ITO surface, and after 15 minutes of reaction, the surface was rinsed 2 times with PB buffer to remove histamine that may have not reacted completely. Then dripping 10 mu L of DNA/Au @ BiVO on the surface of the electrode4Electrochemical probes (10mM Tris-HCl, 1mM EDTA, 50mM NaCl, pH 7.4) were incubated at room temperature for 1.5 hours, followed by 3 washes with PB buffer (pH 8.0) to remove excess probe. The electrode was used as a working electrode, a platinum wire electrode as a counter electrode, a saturated calomel electrode as a reference electrode, and a gas electrode containing 1.0mM H2O2And 0.5mM TMB in 0.1M PBS (pH 6.5) electrolyte, with scanning potentials ranging from 0.5 to-0.1V, the results of the measurements are shown in FIG. 2.
Example 2
River crab sample detection (histamine extraction according to methods of GB 5009.208-2016):
taking 100g of the edible part of the river crab, fully mashing the edible part of the river crab by a mashing machine, respectively putting the mashed river crab into a clean container by dividing into two parts, sealing and marking, and preserving at the temperature of minus 20 ℃. Accurately weighing 10g (accurate to 0.01g) of uniformly ground river crab 2 sample, adding 20mL of 10% trichloroacetic acid solution, soaking for 2-3 h, oscillating for 2min, uniformly mixing, filtering by using filter paper, accurately sucking 2.0mL of filtrate into a separating funnel, dropwise adding sodium hydroxide solution to adjust the pH value to 10-12, adding 3mL of n-amyl alcohol, shaking for extraction for 5min, standing for layering, and transferring the n-amyl alcohol extract (upper layer) into a 10mL graduated test tube. N-pentanol extraction is carried out three times, the extracts are combined and diluted to the scale with n-pentanol. Sucking 2.0mL of n-amyl alcohol extract into a separating funnel, adding 3mL of hydrochloric acid solution, shaking for extraction, standing for layering, and transferring the hydrochloric acid extract (lower layer) into a 10mL graduated test tube. Extracting for three times, combining the extracting solutions, and diluting to 20 mL. Finally the pH was adjusted to 8.0 with PB buffer stock.
Dripping 10 mu L of river crab extract on the surface of the aptamer/AuNFs/ITO, reacting for 15 minutes, and then rinsing for 2 times by using PB buffer solution to remove histamine which may not be completely reacted. Then dripping 10 mu L of DNA/Au @ BiVO on the surface of the electrode4Electrochemical probes (10mM Tris-HCl, 1mM EDTA, 50mM NaCl, pH 7.4), incubated for 1.5 hours at room temperature, followed by incubation with PExcess probe was removed by 3 washes in buffer B (pH 8.0). Followed by a reaction in the presence of 1.0mM H2O2And 0.5mM TMB in 0.1M PBS (pH 6.5) electrolyte, against a standard curve, the results of which are shown in Table 1.
TABLE 1 detection of histamine in river crab samples
Claims (10)
1. The histamine electrochemical sensor based on the aptamer is characterized in that firstly, flower-shaped nanogold is modified on the surface of conductive glass based on an electrodeposition method, and meanwhile, a histamine aptamer subjected to sulfhydrylation modification is connected to the surface of AuNFs/ITO based on Au-S bonding effect to construct a histamine electrochemical sensing interface; preparing bismuth vanadate nano flower balls by hydrothermal synthesis; based on a sodium borohydride reduction method, nano gold particles are introduced to the surface of the bismuth vanadate nano flower ball, ligand DNA matched with histamine aptamer is self-assembled, and the electrochemical probe DNA/Au @ BiVO is obtained4Namely said aptamer-based electrochemical sensor of histamine.
2. The electrochemical histamine-based sensor as claimed in claim 1, wherein said histamine aptamer is:
5’-SH-(CH2)6-GCCTGTTGTGAGCCTCCTAACATTTCTATGCTGCAGCCAACTTTTCCATACTTCCAGCTTACCATTTATCCATGCTTATTCTTGTCTCCC-3’。
3. the electrochemical sensor of histamine based on an aptamer according to claim 1, wherein the ligand DNA is:
5’-SH-(CH2)6-GGGAGACAAGAATAAGCATGGATAAATGGTAAGCTGGAAGTATGGAAAAGTTGGCTGCAGCATAGAAATGTTAGGAGGCTCACAACAGGC-3’。
4. a method for preparing an aptamer-based histamine electrochemical sensor according to claim 1, comprising the steps of:
(1) preparing flower-shaped nano gold modified electrodes AuNFs/ITO by constant potential electrodeposition;
(2) preparing an aptamer/AuNFs/ITO;
(3) preparation of bismuth vanadate BiVO4Nano flower ball;
(4) preparation of DNA/Au @ BiVO4An electrochemical probe.
5. The method according to claim 4, wherein the step (1) is specifically to clean the conductive glass ITO to remove surface contaminants, and then to remove the surface contaminants in HAuCl4Is electrodeposited in an aqueous solution of (1), N is introduced before the electrodeposition2Removing dissolved oxygen to prepare the flower-shaped nano gold modified electrode AuNFs/ITO.
6. The method according to claim 4, wherein step (2) is carried out by washing AuNFs/ITO ultra pure water and then adding N2Drying, placing the aptamer in Tris-acetate buffer solution containing the aptamer and incubating at room temperature in a dark place, self-assembling the sulfhydryl-modified aptamer on the surface of AuNFs/ITO through Au-S bonding, and then placing the aptamer in MCH solution to remove nonspecific adsorption active sites; and respectively using PB buffer solution and ultrapure water to rinse the prepared aptamer/AuNFs/ITO, and placing the aptamer/AuNFs/ITO into PB buffer solution for storage for later use.
7. The preparation method according to claim 4, wherein the step (3) is specifically to prepare bismuth vanadate nanospheres by hydrothermal synthesis: first Bi (NO)3)3·5H2O and Na3VO4·12H2Dispersing O in water by ultrasonic, sealing in a hydrothermal reaction kettle, reacting at 140 ℃ and 180 ℃ for 6-10 hours, naturally cooling, respectively cleaning the obtained product with ethanol and ultrapure water, centrifugally separating, and drying in vacuum overnight to obtain the bismuth vanadate nano flower ball.
8. The preparation method according to claim 4, wherein in the step (4), sodium is modified on the surface of the bismuth vanadate by adopting a sodium borohydride reduction methodRice gold: stirring the mixed solution containing bismuth vanadate and chloroauric acid under the condition of ice-water bath, adding sodium borohydride solution prepared by the ice-water bath, keeping the ice-water bath, stirring, centrifuging, taking precipitate, and drying in vacuum to obtain Au @ BiVO4(ii) a Then, in a solution containing Au @ BiVO4Adding a mixed solution containing DNA and TCEP into the buffer solution, and incubating in a dark place to prepare DNA/Au @ BiVO4The electrochemical probe of (1).
9. Use of an aptamer-based electrochemical sensor of claim 1 for histamine determination in aquatic products.
10. The use of claim 9, wherein the aquatic product is a river crab.
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