CN108627559B - Immunoassay method based on the Prussian blue self energizing electrochemical sensor building of aluminium- - Google Patents

Immunoassay method based on the Prussian blue self energizing electrochemical sensor building of aluminium- Download PDF

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CN108627559B
CN108627559B CN201810438328.XA CN201810438328A CN108627559B CN 108627559 B CN108627559 B CN 108627559B CN 201810438328 A CN201810438328 A CN 201810438328A CN 108627559 B CN108627559 B CN 108627559B
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prussian blue
aluminium
electrode
electrochemical sensor
self energizing
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CN108627559A (en
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唐点平
余镇重
蔡国能
任蓉蓉
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Fuzhou University
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Fuzhou University
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28Electrolytic cell components
    • G01N27/30Electrodes, e.g. test electrodes; Half-cells
    • G01N27/36Glass electrodes
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28Electrolytic cell components
    • G01N27/30Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3271Amperometric enzyme electrodes for analytes in body fluids, e.g. glucose in blood
    • G01N27/3272Test elements therefor, i.e. disposable laminated substrates with electrodes, reagent and channels
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28Electrolytic cell components
    • G01N27/30Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3277Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction being a redox reaction, e.g. detection by cyclic voltammetry
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/28Electrolytic cell components
    • G01N27/30Electrodes, e.g. test electrodes; Half-cells
    • G01N27/327Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
    • G01N27/3275Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction
    • G01N27/3278Sensing specific biomolecules, e.g. nucleic acid strands, based on an electrode surface reaction involving nanosized elements, e.g. nanogaps or nanoparticles

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Abstract

The invention discloses a kind of immunoassay methods based on the Prussian blue self energizing electrochemical sensor building of aluminium-, it is first to prepare Prussian blue modified electrode, again by itself and aluminium electrode, digital multimeter, reaction tank series connection is built into the self energizing electrochemical sensor, glucose oxidase and antibody mutual load are formed into enzyme mark nano-probe on nanogold particle again, it is formed into immuno-sandwich structure with sample to be tested on ELISA Plate again, glucose catalysis is set to generate a large amount of hydrogen peroxide using the immuno-sandwich structure, hydrogen peroxide is injected in self energizing electrochemical sensor again, the current signal of Prussian blue color and sensor can be significantly changed to Prussian blue oxidation by it, to by reading the current value generated, or Prussian blue color change is observed to measure the concentration of object.The method of the present invention equipment is simple, easy to carry, and can be used for the detection of Multiple Antibodies, provides a kind of simple, sensitive and stable method for portable inspectiont.

Description

Immunoassay method based on the Prussian blue self energizing electrochemical sensor building of aluminium-
Technical field
The invention belongs to biochemical analysis technical fields, and in particular to one kind is based on the Prussian blue self energizing electrification of aluminium- Learn the immunoassay method of sensor building.
Background technique
With the development of society, environmental monitoring, food safety and medical diagnosis on disease are increasingly valued by people, therefore send out Quick, easy, the sensitive detection method of exhibition is still one of hot spot studied now.In modern analysis detection architecture, big portion The standard detecting method divided is based on foundation such as optics, chromatography, mass spectrums, these detection methods have accurate, sensitive, stable etc. Advantage.But expensive equipment, complicated operation etc. limit its quick detection and in terms of further answer With especially in remote districts.Therefore, design a kind of equipment it is simple, it is easily operated, stablize practical detection method have it is important Meaning.
For self energizing electrochemical sensor as a kind of portable sensor, the electrode mainly modified by two forms one A primary battery structure.The sensor itself can generate energy output, be not necessarily to other external energy, and the output of its energy and inspection The concentration for surveying object in liquid has tight association.After this sensor is suggested, many researchs have been carried out.By in electricity The different material of extremely upper modification, is successfully realized the detection to substances such as glucose, lactic acid, cholesterol.But it is traditional certainly at these It energizes in electrochemical sensor, detection signal is often output power, and detection device is often also the electrochemical operation of profession It stands, is unfavorable for its universal and portability.On the other hand, most of self energizing electrochemical sensor is typically split into cathode and anode Two parts are detected, this is also unfavorable for its further integration.Therefore, simpler, small-sized self energizing is designed Electrochemical sensor, and it is applied to quick, portable immunoassay field with important research meaning.
Summary of the invention
Based on background above, the purpose of the present invention is to provide one kind to be based on the Prussian blue self energizing electrochemical sensing of aluminium- The immunoassay method of device building.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of immunoassay method based on aluminium-Prussian blue self energizing electrochemical sensor building, specifically include as Lower step:
(1) preparation of Prussian blue modified electrode: by clean FTO glass electrode, platinum electrode and saturated calomel electrode Respectively as working electrode, to electrode and reference electrode, be placed in containing 0.01-0.5 mol/L HCl, 0.01-0.5 mol/L KCl、0.1-10 mmol/L K3[Fe(CN6)]、0.1-10 mmol/L FeCl3Electrolyte in, 0.1-0.6 V perseverance electricity Pressure electropolymerization 300-400 seconds, is then washed with water, dries, and Prussian blue modified electrode is made;
(2) building of the Prussian blue self energizing electrochemical sensor of aluminium-: by the electricity of Prussian blue modification made from step (1) Pole, aluminium electrode, digital multimeter series connection and a two interelectrode small-sized detection cell series connection, are made the Prussian blue confession of the aluminium- It can electrochemical sensor;
(3) preparation of the nano-probe of glucose oxidase and antibody mutual load: by glucose oxidase and antibody according to It is supported on nanogold particle by electrostatic adsorption, the enzyme mark nano-probe is made;
(4) formation of antibody-antigen-antibody interlayer type immune complex: in 96 hole polystyrene micropores of high-affinity 30-80 μ L antibody is added in plate, closes overnight, sequentially adds 30-80 μ L object, 30-80 μ L enzyme mark nano-probe shape At antibody-antigen-antibody interlayer type immune complex;
(5) it is detected using the Prussian blue self energizing electrochemical sensor of aluminium-: gained interlayer type immune complex is used After phosphate buffer solution washing, phosphate buffer (pH=6) of the 50-150 μ L containing 50 mmol/L glucose is added and is reacted, Then reaction solution is injected into small-sized detection cell, the current signal in digital multimeter is read after 25 s, or directly observe The color change of Prussian blue electrode measures the concentration of object.
The size of FTO glass electrode described in step (1) is 1 × 5 cm2;The aluminium electrode is to be pasted onto FTO by aluminium foil Foil electrode is formed in glass electrode;
Prussian blue film is the circle of 6 mm of diameter on gained Prussian blue modified electrode, and blue, UV, visible light is presented Optical absorption peak is at about 700 nm.
Small-sized detection cell described in step (2) is prepared by dimethyl silicone polymer (PDMS), and cross section is 6 mm's of diameter Circle, volume are 40 μ L.
The partial size of nanogold particle described in step (3) is 16 nm.
The present invention is immunoreacted with the single chamber primary battery of the Prussian blue bipolar electrode building of aluminium-in conjunction with antigen-antibody, and building is certainly Energize electrochemical immunosensor.Its technical principle is to be catalyzed glucose by introducing glucose oxidase on immunological probe It decomposes and generates hydrogen peroxide, and hydrogen peroxide is injected in detection cell.It is Prussian blue to be reduced in the presence of there is no object Colourless Prussia is white, and the Prussian blue primary battery of aluminium-releases lower current signal;In the presence of object, due to peroxidating The presence of hydrogen, Prussia is white can be Prussian blue by the reoxidized generation of hydrogen peroxide, and increases the current signal of detection significantly By force, wherein the variation of current signal and Prussian blue color are positively correlated with target concentration in a certain range, therefore can With by digital multimeter come quantitative detection, can also be according to Prussian blue color change come qualitative detection.
Advantages of the present invention is as follows:
(1) material feedstock required for the present invention is easy to get, and preparation process is simple, low in cost.
(2) it is compared with conventional detection devices, digital multimeter can be used to quantify as signal reader for the present invention Detection carries out qualitative detection with Prussian blue color change, accesses without extra power, greatlies simplify detection device, It is easy to operate, it is suitable for portable inspectiont.
(3) present invention makes signal enhancing by introducing glucose oxidase catalysis breakdown of glucose generation hydrogen peroxide, It can be used for the detection of Multiple Antibodies object, it is versatile, it is with a wide range of applications.
(4) detection method has the characteristics that high sensitivity, stability are strong, selectivity is good, the range of linearity is wide, and can Detection for Multiple Antibodies.
Detailed description of the invention
Fig. 1 is that the present invention is based on the signals of the immunoassay method of the Prussian blue self energizing electrochemical sensor building of aluminium- Figure;
Fig. 2 is the component pictorial diagram for constructing self energizing electrochemical sensor of the present invention;
Fig. 3 is by the standard working curve that does in embodiment 1 to alpha-fetoprotein;
Fig. 4 is the corresponding Prussian blue electrode color of standard sample of various concentration in example example 1.
Specific embodiment
In order to make content of the present invention easily facilitate understanding, With reference to embodiment to of the present invention Technical solution is described further, but the present invention is not limited only to this.
Embodiment 1
1. the preparation of Prussian blue modified electrode
Using clean FTO glass electrode, platinum electrode and saturated calomel electrode as working electrode, to electrode and ginseng Than electrode, it is placed in containing 0.1 mol/L HCl, 0.1 mol/L KCl, 5 mmol/L K3[Fe(CN6)]、5 mmol/L FeCl3 Electrolyte in, then electropolymerization 360 seconds under the constant voltage of 0.4 V are washed with water, dry, Prussian blue modification electricity is made Pole.
2. self energizing electrochemical sensor constructs
By the Prussian blue modified electrode prepared, aluminium electrode, digital multimeter series connection and a two interelectrode small-sized inspections Pond series connection is surveyed, the Prussian blue self energizing electrochemical sensor of the aluminium-is made.
3. the preparation of enzyme labelled antibody nano-probe
Firstly, 5 mL nano-Au solutions sodium carbonate is adjusted pH to 8.5, the first tire of 50 μ L, 0.5 mg/mL are added The glucose oxidase of protein antibodies and 200 μ L, 0.5 mg/mL, 4 DEG C of incubated overnights.Then by above-mentioned solution 4 DEG C, It is centrifuged 20 minutes under the conditions of 14000 rmp, the precipitating that centrifugation obtains is dispersed in 2 mL again and contains 1.0 wt % BSA and 0.1 In the PBS (pH=7.4) of wt % sodium azide, be stored in 4 DEG C it is spare.
4. the preparation of interlayer type immune complex
Firstly, the alpha-fetoprotein antibody of 50 μ L, 10 μ g/mL are added in the high affine plate in 96 holes, hatching overnight at 4 DEG C, Add 300 μ L, the BSA of 1.0 wt % is closed 1 hour.After being washed with PBS solution, it is sequentially added into 50 μ L alpha-fetoproteins The enzyme mark nano-probe of the series of concentrations standard sample of specific antigen and 50 μ L reacts 30 minutes respectively, removes supernatant.
5. the detection pair alpha-fetoprotein specific antigen
The PBS buffer solution (pH=6) that 100 μ L contain 50 mM glucose is added in gained interlayer type immune complex, instead It answers 30 minutes.Then, it in the detection cell for taking 40 μ L reaction solutions injection sensor, reacts 25 seconds, reads the electricity in digital multimeter The variation of flow valuve or the Prussian blue electrode color of observation.Standard working curve of its gained electric current to alpha-fetoprotein specific antigen As shown in figure 3, corresponding Prussian blue electrode color change is as shown in Figure 4.
As seen from Figure 3, the range of linearity is 5-200 ng/mL, and detection is limited to 1.67 ng/mL.
The foregoing is merely presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with Modification, is all covered by the present invention.

Claims (3)

1. a kind of immunoassay method based on the Prussian blue self energizing electrochemical sensor building of aluminium-, it is characterised in that: described Method includes the following steps:
(1) preparation of Prussian blue modified electrode: clean FTO glass electrode, platinum electrode and saturated calomel electrode are distinguished As working electrode, to electrode and reference electrode, be placed in containing 0.01-0.5 mol/L HCl, 0.01-0.5 mol/L KCl, 0.1-10 mmol/L K3[Fe(CN6)]、0.1-10 mmol/L FeCl3Electrolyte in, under the constant voltage of 0.1-0.6 V It electropolymerization 300-400 seconds, is then washed with water, dries, Prussian blue modified electrode is made;
(2) building of the Prussian blue self energizing electrochemical sensor of aluminium-: by Prussian blue modified electrode, aluminium made from step (1) Electrode, digital multimeter series connection and a two interelectrode small-sized detection cell series connection, are made the Prussian blue self energizing electrification of the aluminium- Learn sensor;
(3) preparation of the nano-probe of glucose oxidase and antibody mutual load: by glucose oxidase and antibody by quiet Enzyme mark nano-probe is made on nanogold particle in Electro Sorb used load;
(4) formation of antibody-antigen-antibody interlayer type immune complex: in 96 porous polystyrene microporous plates of high-affinity Be added 30-80 μ L antibody, overnight close, sequentially add 30-80 μ L object, 30-80 μ L enzyme mark nano-probe formed it is anti- Body-Ag-Ab interlayer type immune complex;
(5) it is detected using the Prussian blue self energizing electrochemical sensor of aluminium-: gained antibody-antigen-antibody interlayer type is exempted from After epidemic disease compound is washed with phosphate buffer solution, the phosphoric acid buffer containing 50 mmol/L glucose of 50-150 μ L, pH=6 is added Liquid is reacted, and then reaction solution is injected into the Prussian blue self energizing electrochemical sensor of aluminium-, and number ten thousand is read after 25 s With the current signal on table, or observe the color change of Prussian blue electrode directly to measure the concentration of object.
2. the immunoassay side according to claim 1 based on the Prussian blue self energizing electrochemical sensor building of aluminium- Method, it is characterised in that: the circle that the Prussian blue film of gained is 6 mm of diameter in step (1).
3. the immunoassay side according to claim 1 based on the Prussian blue self energizing electrochemical sensor building of aluminium- Method, it is characterised in that: the partial size of nanogold particle described in step (3) is 16 nm.
CN201810438328.XA 2018-05-09 2018-05-09 Immunoassay method based on the Prussian blue self energizing electrochemical sensor building of aluminium- Expired - Fee Related CN108627559B (en)

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CN109239135B (en) * 2018-10-24 2021-04-27 福州大学 Portable biological immunoassay method constructed based on flexible air pressure sensor
CA3120917A1 (en) * 2018-11-27 2020-06-04 Module Innovations Private Limited Method for testing of antibiotic susceptibility in microorganisms
CN110068570B (en) * 2019-04-03 2021-05-25 济南大学 Preparation of electrochemical sensor with instant visual colorimetric display
CN110261453A (en) * 2019-05-20 2019-09-20 武汉中科志康生物科技有限公司 A kind of avian influenza virus electrochemical sensor based on screen printing electrode

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102053161A (en) * 2009-10-29 2011-05-11 南京大学 Disposable multi-channel electrochemical immunosensor with high sensitivity
CN102411022A (en) * 2011-08-03 2012-04-11 中国科学院化学研究所 Electrochemical sensor based on primary battery and preparation method thereof
CN106104264A (en) * 2014-01-21 2016-11-09 加利福尼亚大学董事会 Saliva biosensor and biological fuel cell

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102053161A (en) * 2009-10-29 2011-05-11 南京大学 Disposable multi-channel electrochemical immunosensor with high sensitivity
CN102411022A (en) * 2011-08-03 2012-04-11 中国科学院化学研究所 Electrochemical sensor based on primary battery and preparation method thereof
CN106104264A (en) * 2014-01-21 2016-11-09 加利福尼亚大学董事会 Saliva biosensor and biological fuel cell

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
A bi-functional device for self-powered electrochromic window and self-rechargeable transparent battery applications;Jinmin Wang et al.;《NATURE COMMUNICATIONS》;20140923;第5卷 *
Self-powering amperometric sensor and biosensor;Lai Peng Wong et al.;《Journal of Electroanalytical Chemistry》;20120306;第671卷;80-84 *

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