CN104459124A - Preparation method and application of electrochemical immunosensor based on HS-beta-CD-Ag-GOD conjugate - Google Patents

Abstract

The invention belongs to the technical fields of immunoassay and biosensing and discloses a preparation method and application of an electrochemical immunosensor based on an HS-beta-CD-Ag-GOD conjugate. The electrochemical immunosensor is used for rapidly detecting a hog cholera virus antigen CSFV. The manufacturing scheme comprises the following steps: modifying a working electrode a bare platinum carbon electrode by using MWCNTs-CD-Fc-Ab1, and sequentially adding bovine serum albumin, hog cholera virus antigen CSFV and an Ab2-HS-beta-CD-Ag-GOD conjugate. The HS-beta-CD-Ag-GOD conjugate can convert glucose into gluconic acid, two protons and two electrons are transferred to oxygen molecules, hydrogen peroxide is generated, and an HS-beta-CD-Ag nanometer material serves as a mimic enzyme, the reduction of the hydrogen peroxide is catalyzed, the dual amplification of an electrochemical signal is realized, the high sensitivity is realized, and the detection limit can be low to 0.33pg/mL.

Description

A kind of preparation method of the electrochemical immunosensor based on HS-β-CD-Ag-GOD conjugate and application

Technical field

The invention belongs to immunoassay and biosensor technique field, be specifically related to a kind of preparations and applicatio of the electrochemical immunosensor based on HS-β-CD-Ag-GOD conjugate, develop the sandwich type electrochemical immunosensor of a kind of Rapid Detection of Classical Swine Fever Virus antigens c SFV.

Background technology

CSFV antigens c SFV, or claim hog cholera, is a kind of acute, hot, high degree in contact sexually transmitted disease, principal character is high temperature, blood capillary sex change and cause systemic bleeding, necrosis, infraction.Due to the pathogenic of its height with can cause dead widely, cause serious economic loss to pig industry.Many countries formulate veterinary law and regulation, and CSFV is classified as one of Notifiable disease, namely must the disease that reports of list.There is epidemic situation or suspicious epidemic situation, report as early as possible, at once urgent veterinary hygienic epidemic prevention measure is taked to epidemic place and contiguous pasture thereof.

Current domestic and international most testing agency is all detected CSFV antigens c SFV in use euzymelinked immunosorbent assay (ELISA).Because its sense cycle is long, the shortcomings such as reagent needed for program complexity is various can not meet modern measure requirement far away.Along with the development of modern science and technology, particularly immunology biological chemistry, molecular biological development, electrochemical immunosensor has caused studies interest widely.

The present invention adopts sandwich type electrochemical immunosensor, in conjunction with dual amplifying technique, improve the detection sensitivity of sensor, in addition, electrochemical immunosensor so that its selectivity is good, structure is simple, easy and simple to handle, be easy to miniaturization, can the advantage such as continuous, rapid automatized detection analysis, can be widely used in the middle of prevention detects.

Summary of the invention

The object of the invention is to avoid that the instrument and equipment of traditional detection method is complicated, operating process is loaded down with trivial details, testing staff requires the shortcomings such as high, testing cost is high, provide the preparation method of sandwich type electrochemical immunosensor of a kind of highly sensitive, high specificity, favorable reproducibility, detection CSFV antigens c SFV easy and simple to handle and cheap and easy to get.

To achieve these goals, the present invention is realized by following measures.Technical scheme of the present invention, comprises the following steps.

1. the preparation method based on the electrochemical immunosensor of HS-β-CD-Ag-GOD conjugate

(1) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, EtOH Sonicate cleans, cleaner with ultrapure water, then electrode is placed in 5 mmolL ^-1in potassium ferricyanide solution, scan under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 80 mV;

(2) by pH 7.0, the MWCNTs-CD-Fc-Ab of the phosphate buffered solution dispersion of 0.1 mol/L ₁modify in glassy carbon electrode surface, drying at room temperature, described MWCNTs-CD-Fc-Ab ₁concentration be 1.0 ~ 3.0 mgmL ^-1;

(3) painting 3 μ L, 100 μ gmL are dripped ^-1bovine serum albumin solution to electrode surface, dry under wet condition at 4 DEG C;

(4) painting 6 μ L, 0.001 ~ 5.0 ngmL is dripped ^-1cSFV antigens c SFV standard solution to electrode surface, dry under wet condition at 4 DEG C;

(5) Ab of painting 6 μ L is dripped ₂-CD-Ag-GOD solution, to electrode surface, dries under wet condition at 4 DEG C, obtained a kind of electrochemical immunosensor based on HS-β-CD-Ag-GOD conjugate.

2. Ab described above ₂the preparation method of-HS-β-CD-Ag-GOD conjugate solution

(1) preparation of HS-β-CD-Ag nano material

By the AgNO of 1 mL, 0.005-0.015 mol/L ₃join in the high purity water of 97 mL with the trisodium citrate of 1 mL, 0.02-0.04 mol/L, stir, concussion dropping 1 mL, the NaBH of 1.5-2.0 mg/mL ₄, fully stir 20 min, add the sulfhydrylation beta cyclodextrin HS-β-CD of 0.0238 g, magnetic agitation 24 h, rotating speed is centrifugal 10 min under the condition of 11000 rpm, constant temperature 60 DEG C of vacuum drying;

(2) preparation of the glucose oxidase ADA-GOD of adamantanecarboxylic acid functionalization

Take 25-41 mg adamantanecarboxylic acid ADA in 40 mL ultrapure waters, add 200 μ L, the NaOH solution of 0.5-1.5 mol/mL, 100 mL are diluted to after solution clarification, 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC is added in the adamantanecarboxylic acid ADA solution of 1-3 mL, 10 mg N-hydroxysuccinimide NHS and 2 mL, pH is the PBS buffer solution of 7.4, stirring at room temperature 0.5 h, add 200 μ L, the glucose oxidase of 0.5-1.5 mg/mL, constant temperature 4 DEG C spends the night, rotating speed is centrifugal 8 min under the condition of 8000 rpm, preserve under the condition of-20 DEG C, the glucose oxidase ADA-GOD solution of the adamantanecarboxylic acid functionalization of obtained 100-300 μ g/mL,

(3) the detection antibody A DA-Ab of adamantanecarboxylic acid functionalization ₂preparation

Take 25-41 mg adamantanecarboxylic acid ADA in 40 mL ultrapure waters, add 200 μ L, the NaOH solution of 0.5-1.5 mol/mL, 100 mL are diluted to after solution clarification, the PBS buffer solution that 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC, 10 mg N-hydroxysuccinimide NHS and 2 mL, pH are 7.4 is added, stirring at room temperature 0.5 h in the adamantanecarboxylic acid ADA solution of 1-3 mL, add 200 μ L, 0.5-1.5 mg/mL detects antibody A b ₂, constant temperature 4 DEG C spends the night, and rotating speed is centrifugal 8 min under the condition of 8000 rpm, adds 1 mL, and pH is the PBS buffer solution of 7.4, preserves under the condition of-20 DEG C, the detection antibody A DA-Ab of the adamantanecarboxylic acid functionalization of obtained 100-300 μ g/mL ₂solution;

(4) Ab ₂the preparation of-HS-β-CD-Ag-GOD conjugate solution

By the detection antibody A DA-Ab of adamantanecarboxylic acid functionalization ₂be diluted to 40-60 μ g/mL with the glucose oxidase ADA-GOD of adamantanecarboxylic acid functionalization, get 200 μ L, 40-60 μ g/mL ADA-Ab ₂with 200 μ L, 40-60 μ g/mL ADA-GOD, add 200 μ L, 3-5 mg/mL HS-β-CD-Ag, constant temperature 4 DEG C of incubated overnight, rotating speed is centrifugal 8 min under the condition of 8000 rpm, abandoning supernatant, adds 200 μ L, and pH is the PBS buffer solution of 7.4, preserve under the condition of-20 DEG C, obtained Ab ₂-HS-β-CD-Ag-GOD conjugate solution;

(5) preparation of multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene MWCNTs-CD-Fc compound substance

Take 0.4-0.6 g multi-walled carbon nano-tubes MWCNTs, join the H prepared ₂sO ₄and HNO ₃for in the mixed solution of 3:1, ultrasonic 3 h of constant temperature 40 DEG C, are cooled to room temperature, be diluted to 500 mL with ultrapure water, use the filter membrane of 0.22 μm to carry out vacuum filtration, milli-Q water is neutral to pH, constant temperature 60 DEG C of vacuum drying, obtained functionalized multi-wall carbonnanotubes MWCNTs-COOH;

Take 20-30 mg MWCNTs-COOH, be distributed in 50 mL ultrapure waters, then add the beta-schardinger dextrin-CD of 150-250 mg, shake 12 h, centrifugal, wash 4 times, constant temperature 60 DEG C of vacuum drying, 60-100 mg ferrocenecarboxylic acid Fc joins in the methylene chloride of 50 mL, add 20-30 mg MWCNTs-CD, stir 12 h, centrifugal drying, obtain multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene MWCNTs-CD-Fc compound substance;

(6) multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene-capture antibody MWCNTs-CD-Fc-Ab ₁preparation

The MWCNTs-CD-Fc taking 3-5 mg is distributed to 2 mL, pH is in the PBS buffer solution of 7.4, add 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC, 10 mg N-hydroxysuccinimide NHS, constant temperature 4 DEG C stirs 0.5 h, add 200 μ L, 0.5-1.5 mg/mL capture antibody Ab ₁, constant temperature 4 DEG C concussion 12h, obtained multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene-capture antibody MWCNTs-CD-Fc-Ab ₁.

3. the detection method of CSFV antigens c SFV

(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is contrast electrode, platinum electrode is auxiliary electrode, prepared a kind of electrochemical immunosensor based on HS-β-CD-Ag-GOD conjugate is working electrode, in pH6.0 ~ 8.0 phosphate buffered solution of 10 mL, 40 ~ 60 mmol/L, measure its curent change;

(2) according to gained current differential and CSFV antigens c SFV concentration linear, drawing curve;

(3) method for drafting of foundation working curve carries out the detection of CSFV antigens c SFV in sample, and testing result checks in from working curve.

useful achievement of the present invention

(1) the present invention is based on HS-β-CD-Ag-GOD conjugate, construct a kind of sandwich type electrochemical immunosensor, the glucose oxidase ADA-GOD of adamantanecarboxylic acid functionalization can transforming glucose be gluconic acid, and transmit two protons and two electronics to oxygen molecule, produce hydrogen peroxide, HS-β-CD-Ag nano material is as a kind of analogue enztme, and the reduction of catalysis hydrogen peroxide, achieves the dual amplification of electrochemical signals;

(2) the HS-β-CD-Ag nano material that the present invention adopts has good molecule distinguishability, effectively can catch the glucose oxidase ADA-GOD of adamantanecarboxylic acid functionalization and the detection antibody A DA-Ab of adamantanecarboxylic acid functionalization ₂;

(3) the present invention utilizes the specific surface area that MWCNTs-CD-Fc compound substance is large, good biocompatibility and high electric conductivity, carrys out fixed trapped antibody A b effectively ₁, improve the detection sensitivity of immune sensing;

(4) the present invention is by highly sensitive, the specific detection of this sensor application in CSFV antigens c SFV, and the range of linearity is 0.001-5 ng/mL, detects and is limited to 0.33 pg/mL.

Embodiment

The preparation of embodiment 1 HS-β-CD-Ag nano material

By 1 mL, the AgNO of 0.005 mol/L ₃with 1 mL, the trisodium citrate of 0.02 mol/L joins in the high purity water of 97 mL, stirs, concussion dropping 1 mL, the NaBH of 1.5 mg/mL ₄, fully stir 20 min, add the sulfhydrylation beta cyclodextrin HS-β-CD of 0.0238 g, magnetic agitation 24 h, rotating speed is centrifugal 10 min under the condition of 11000 rpm, constant temperature 60 DEG C of vacuum drying;

The preparation of embodiment 2 HS-β-CD-Ag nano material

By 1 mL, the AgNO of 0.01 mol/L ₃with 1 mL, the trisodium citrate of 0.03 mol/L joins in the high purity water of 97 mL, stirs, concussion dropping 1 mL, the NaBH of 1.79 mg/mL ₄, fully stir 20 min, add the sulfhydrylation beta cyclodextrin HS-β-CD of 0.0238 g, magnetic agitation 24 h, rotating speed is centrifugal 10 min under the condition of 11000 rpm, constant temperature 60 DEG C of vacuum drying;

The preparation of embodiment 3 HS-β-CD-Ag nano material

By 1 mL, the AgNO of 0.015 mol/L ₃with 1 mL, the trisodium citrate of 0.04 mol/L joins in the high purity water of 97 mL, stirs, concussion dropping 1 mL, the NaBH of 2.0 mg/mL ₄, fully stir 20 min, add the sulfhydrylation beta cyclodextrin HS-β-CD of 0.0238 g, magnetic agitation 24 h, rotating speed is centrifugal 10 min under the condition of 11000 rpm, constant temperature 60 DEG C of vacuum drying;

The preparation of the glucose oxidase ADA-GOD of embodiment 4 adamantanecarboxylic acid functionalization

Take 25 mg adamantanecarboxylic acid ADA in 40 mL ultrapure waters, add 200 μ L, the NaOH solution of 0.5 mol/mL, 100 mL are diluted to after solution clarification, 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC is added in the adamantanecarboxylic acid ADA solution of 1 mL, 10 mg N-hydroxysuccinimide NHS and 2 mL, pH is the PBS buffer solution of 7.4, stirring at room temperature 0.5 h, add 200 μ L, the glucose oxidase of 0.5 mg/mL, constant temperature 4 DEG C spends the night, rotating speed is centrifugal 8 min under the condition of 8000 rpm, preserve under the condition of-20 DEG C, the glucose oxidase ADA-GOD solution of the adamantanecarboxylic acid functionalization of obtained 100 μ g/mL,

The preparation of the glucose oxidase ADA-GOD of embodiment 5 adamantanecarboxylic acid functionalization

Take 33 mg adamantanecarboxylic acid ADA in 40 mL ultrapure waters, add 200 μ L, the NaOH solution of 1 mol/mL, 100 mL are diluted to after solution clarification, 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC is added in the adamantanecarboxylic acid ADA solution of 2 mL, 10 mg N-hydroxysuccinimide NHS and 2 mL, pH is the PBS buffer solution of 7.4, stirring at room temperature 0.5 h, add 200 μ L, the glucose oxidase of 1 mg/mL, constant temperature 4 DEG C spends the night, rotating speed is centrifugal 8 min under the condition of 8000 rpm, preserve under the condition of-20 DEG C, the glucose oxidase ADA-GOD solution of the adamantanecarboxylic acid functionalization of obtained 200 μ g/mL,

The preparation of the glucose oxidase ADA-GOD of embodiment 6 adamantanecarboxylic acid functionalization

Take 41 mg adamantanecarboxylic acid ADA in 40 mL ultrapure waters, add 200 μ L, the NaOH solution of 1.5 mol/mL, 100 mL are diluted to after solution clarification, 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC is added in the adamantanecarboxylic acid ADA solution of 1-3 mL, 10 mg N-hydroxysuccinimide NHS and 2 mL, pH is the PBS buffer solution of 7.4, stirring at room temperature 0.5 h, add 200 μ L, the glucose oxidase of 1.5 mg/mL, constant temperature 4 DEG C spends the night, rotating speed is centrifugal 8 min under the condition of 8000 rpm, preserve under the condition of-20 DEG C, the glucose oxidase ADA-GOD solution of the adamantanecarboxylic acid functionalization of obtained 300 μ g/mL,

The detection antibody A DA-Ab of embodiment 7 adamantanecarboxylic acid functionalization ₂preparation

Take 25 mg adamantanecarboxylic acid ADA in 40 mL ultrapure waters, add 200 μ L, the NaOH solution of 0.5 mol/mL, 100 mL are diluted to after solution clarification, the PBS buffer solution that 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC, 10 mg N-hydroxysuccinimide NHS and 2 mL, pH are 7.4 is added, stirring at room temperature 0.5 h in the adamantanecarboxylic acid ADA solution of 1-3 mL, add 200 μ L, 0.5 mg/mL detects antibody A b ₂, constant temperature 4 DEG C spends the night, and rotating speed is centrifugal 8 min under the condition of 8000 rpm, adds 1 mL, and pH is the PBS buffer solution of 7.4, preserves under the condition of-20 DEG C, the detection antibody A DA-Ab of the adamantanecarboxylic acid functionalization of obtained 100 μ g/mL ₂solution;

The detection antibody A DA-Ab of embodiment 8 adamantanecarboxylic acid functionalization ₂preparation

Take 33 mg adamantanecarboxylic acid ADA in 40 mL ultrapure waters, add 200 μ L, the NaOH solution of 1 mol/mL, 100 mL are diluted to after solution clarification, the PBS buffer solution that 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC, 10 mg N-hydroxysuccinimide NHS and 2 mL, pH are 7.4 is added, stirring at room temperature 0.5 h in the adamantanecarboxylic acid ADA solution of 2 mL, add 200 μ L, 1 mg/mL detects antibody A b ₂, constant temperature 4 DEG C spends the night, and rotating speed is centrifugal 8 min under the condition of 8000 rpm, adds 1 mL, and pH is the PBS buffer solution of 7.4, preserves under the condition of-20 DEG C, the detection antibody A DA-Ab of the adamantanecarboxylic acid functionalization of obtained 200 μ g/mL ₂solution;

The detection antibody A DA-Ab of embodiment 9 adamantanecarboxylic acid functionalization ₂preparation

Take 41 mg adamantanecarboxylic acid ADA in 40 mL ultrapure waters, add 200 μ L, the NaOH solution of 1.5 mol/mL, 100 mL are diluted to after solution clarification, the PBS buffer solution that 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC, 10 mg N-hydroxysuccinimide NHS and 2 mL, pH are 7.4 is added, stirring at room temperature 0.5 h in the adamantanecarboxylic acid ADA solution of 1-3 mL, add 200 μ L, 1.5 mg/mL detect antibody A b ₂, constant temperature 4 DEG C spends the night, and rotating speed is centrifugal 8 min under the condition of 8000 rpm, adds 1 mL, and pH is the PBS buffer solution of 7.4, preserves under the condition of-20 DEG C, the detection antibody A DA-Ab of the adamantanecarboxylic acid functionalization of obtained 300 μ g/mL ₂solution.

Embodiment 10 Ab ₂the preparation of-HS-β-CD-Ag-GOD conjugate solution

By the detection antibody A DA-Ab of adamantanecarboxylic acid functionalization ₂be diluted to 40 μ g/mL with the glucose oxidase ADA-GOD of adamantanecarboxylic acid functionalization, get 200 μ L, 40 μ g/mL ADA-Ab ₂with 200 μ L, 40 μ g/mL ADA-GOD, add 200 μ L, 3 mg/mL HS-β-CD-Ag, constant temperature 4 DEG C of incubated overnight, rotating speed is centrifugal 8 min under the condition of 8000 rpm, abandoning supernatant, adds 200 μ L, and pH is the PBS buffer solution of 7.4, preserve under the condition of-20 DEG C, obtained Ab ₂-HS-β-CD-Ag-GOD conjugate solution;

Embodiment 11 Ab ₂the preparation of-HS-β-CD-Ag-GOD conjugate solution

By the detection antibody A DA-Ab of adamantanecarboxylic acid functionalization ₂be diluted to 50 μ g/mL with the glucose oxidase ADA-GOD of adamantanecarboxylic acid functionalization, get 200 μ L, 50 μ g/mL ADA-Ab ₂with 200 μ L, 50 μ g/mL ADA-GOD, add 200 μ L, 4 mg/mL HS-β-CD-Ag, constant temperature 4 DEG C of incubated overnight, rotating speed is centrifugal 8 min under the condition of 8000 rpm, abandoning supernatant, adds 200 μ L, and pH is the PBS buffer solution of 7.4, preserve under the condition of-20 DEG C, obtained Ab ₂-HS-β-CD-Ag-GOD conjugate solution;

Embodiment 12 Ab ₂the preparation of-HS-β-CD-Ag-GOD conjugate solution

By the detection antibody A DA-Ab of adamantanecarboxylic acid functionalization ₂be diluted to 60 μ g/mL with the glucose oxidase ADA-GOD of adamantanecarboxylic acid functionalization, get 200 μ L, 60 μ g/mL ADA-Ab ₂with 200 μ L, 60 μ g/mL ADA-GOD, add 200 μ L, 5 mg/mL HS-β-CD-Ag, constant temperature 4 DEG C of incubated overnight, rotating speed is centrifugal 8 min under the condition of 8000 rpm, abandoning supernatant, adds 200 μ L, and pH is the PBS buffer solution of 7.4, preserve under the condition of-20 DEG C, obtained Ab ₂-HS-β-CD-Ag-GOD conjugate solution;

The preparation of embodiment 13 multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene MWCNTs-CD-Fc compound substance

Take 0.4g multi-walled carbon nano-tubes MWCNTs, join the H prepared ₂sO ₄and HNO ₃for in the mixed solution of 3:1, ultrasonic 3 h of constant temperature 40 DEG C, are cooled to room temperature, be diluted to 500 mL with ultrapure water, use the filter membrane of 0.22 μm to carry out vacuum filtration, milli-Q water is neutral to pH, constant temperature 60 DEG C of vacuum drying, obtained functionalized multi-wall carbonnanotubes MWCNTs-COOH;

Take 20mg MWCNTs-COOH, be distributed in 50 mL ultrapure waters, then add the beta-schardinger dextrin-CD of 150 mg, shake 12 h, centrifugal, wash 4 times, constant temperature 60 DEG C of vacuum drying, 60 mg ferrocenecarboxylic acid Fc join in the methylene chloride of 50 mL, add 20 mg MWCNTs-CD, stir 12 h, centrifugal drying, obtain multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene MWCNTs-CD-Fc compound substance;

The preparation of embodiment 14 multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene MWCNTs-CD-Fc compound substance

Take 0.5 g multi-walled carbon nano-tubes MWCNTs, join the H prepared ₂sO ₄and HNO ₃for in the mixed solution of 3:1, ultrasonic 3 h of constant temperature 40 DEG C, are cooled to room temperature, be diluted to 500 mL with ultrapure water, use the filter membrane of 0.22 μm to carry out vacuum filtration, milli-Q water is neutral to pH, constant temperature 60 DEG C of vacuum drying, obtained functionalized multi-wall carbonnanotubes MWCNTs-COOH;

Take 25 mg MWCNTs-COOH, be distributed in 50 mL ultrapure waters, then add the beta-schardinger dextrin-CD of 200 mg, shake 12 h, centrifugal, wash 4 times, constant temperature 60 DEG C of vacuum drying, 80 mg ferrocenecarboxylic acid Fc join in the methylene chloride of 50 mL, add 25 mg MWCNTs-CD, stir 12 h, centrifugal drying, obtain multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene MWCNTs-CD-Fc compound substance;

The preparation of embodiment 15 multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene MWCNTs-CD-Fc compound substance

Take 0.6 g multi-walled carbon nano-tubes MWCNTs, join the H prepared ₂sO ₄and HNO ₃for in the mixed solution of 3:1, ultrasonic 3 h of constant temperature 40 DEG C, are cooled to room temperature, be diluted to 500 mL with ultrapure water, use the filter membrane of 0.22 μm to carry out vacuum filtration, milli-Q water is neutral to pH, constant temperature 60 DEG C of vacuum drying, obtained functionalized multi-wall carbonnanotubes MWCNTs-COOH;

Take 30 mg MWCNTs-COOH, be distributed in 50 mL ultrapure waters, then add the beta-schardinger dextrin-CD of 250 mg, shake 12 h, centrifugal, wash 4 times, constant temperature 60 DEG C of vacuum drying, 100 mg ferrocenecarboxylic acid Fc join in the methylene chloride of 50 mL, add 30 mg MWCNTs-CD, stir 12 h, centrifugal drying, obtain multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene MWCNTs-CD-Fc compound substance;

Embodiment 16 multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene-capture antibody MWCNTs-CD-Fc-Ab ₁preparation

The MWCNTs-CD-Fc taking 3 mg is distributed to 2 mL, pH is in the PBS buffer solution of 7.4, add 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC, 10 mg N-hydroxysuccinimide NHS, constant temperature 4 DEG C stirs 0.5 h, add 200 μ L, 0.5 mg/mL capture antibody Ab ₁, constant temperature 4 DEG C concussion 12h, obtained multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene-capture antibody MWCNTs-CD-Fc-Ab ₁;

Embodiment 17 multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene-capture antibody MWCNTs-CD-Fc-Ab ₁preparation

The MWCNTs-CD-Fc taking 4 mg is distributed to 2 mL, pH is in the PBS buffer solution of 7.4, add 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC, 10 mg N-hydroxysuccinimide NHS, constant temperature 4 DEG C stirs 0.5 h, add 200 μ L, 1 mg/mL capture antibody Ab ₁, constant temperature 4 DEG C concussion 12h, obtained multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene-capture antibody MWCNTs-CD-Fc-Ab ₁;

Embodiment 18 multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene-capture antibody MWCNTs-CD-Fc-Ab ₁preparation

The MWCNTs-CD-Fc taking 5 mg is distributed to 2 mL, pH is in the PBS buffer solution of 7.4, add 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC, 10 mg N-hydroxysuccinimide NHS, constant temperature 4 DEG C stirs 0.5 h, add 200 μ L, 1.5 mg/mL capture antibody Ab ₁, constant temperature 4 DEG C concussion 12h, obtained multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene-capture antibody MWCNTs-CD-Fc-Ab ₁;

The detection method of embodiment 19 CSFV antigens c SFV

Arbitrary described one highly sensitive CSFV antigens c SFV immunosensor in embodiment 1 ~ 18, for the detection of CSFV antigens c SFV, comprises the following steps:

(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is contrast electrode, platinum electrode is auxiliary electrode, prepared a kind of electrochemical immunosensor based on HS-β-CD-Ag-GOD conjugate is working electrode, in the pH6.0 phosphate buffered solution of 10 mL, 40mmol/L, measure its curent change;

(2) according to gained current differential and CSFV antigens c SFV concentration linear, drawing curve;

(3) method for drafting of foundation working curve carries out the detection of CSFV antigens c SFV in sample, and testing result checks in from working curve.

The detection method of embodiment 20 CSFV antigens c SFV

(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is contrast electrode, platinum electrode is auxiliary electrode, prepared a kind of electrochemical immunosensor based on HS-β-CD-Ag-GOD conjugate is working electrode, in the pH7.0 phosphate buffered solution of 10 mL, 50 mmol/L, measure its curent change;

(2) according to gained current differential and CSFV antigens c SFV concentration linear, drawing curve;

(3) method for drafting of foundation working curve carries out the detection of CSFV antigens c SFV in sample, and testing result checks in from working curve.

The detection method of embodiment 21 CSFV antigens c SFV

(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is contrast electrode, platinum electrode is auxiliary electrode, prepared a kind of electrochemical immunosensor based on HS-β-CD-Ag-GOD conjugate is working electrode, in pH 8.0 phosphate buffered solution of 10 mL, 60 mmol/L, measure its curent change;

(2) according to gained current differential and CSFV antigens c SFV concentration linear, drawing curve;

(3) method for drafting of foundation working curve carries out the detection of CSFV antigens c SFV in sample, and testing result checks in from working curve.

Claims (3)

1., based on a preparation method for the electrochemical immunosensor of HS-β-CD-Ag-GOD conjugate, it is characterized in that, step is as follows:

(1) glass-carbon electrode of diameter 4 mm is used successively the alundum (Al2O3) burnishing powder polishing of 1.0,0.3 and 0.05 mm, EtOH Sonicate cleans, cleaner with ultrapure water, then electrode is placed in 5 mmolL ^-1in potassium ferricyanide solution, scan under-0.2 ~ 0.6 V current potential, make spike potential difference be less than 80 mV;

(2) by pH 7.0, the MWCNTs-CD-Fc-Ab of the phosphate buffered solution dispersion of 0.1 mol/L ₁modify in glassy carbon electrode surface, drying at room temperature, described MWCNTs-CD-Fc-Ab ₁concentration be 1.0 ~ 3.0 mgmL ^-1;

(3) painting 3 μ L, 100 μ gmL are dripped ^-1bovine serum albumin solution to electrode surface, dry under wet condition at 4 DEG C;

(4) painting 6 μ L, 0.001 ~ 5.0 ngmL is dripped ^-1cSFV antigens c SFV standard solution to electrode surface, dry under wet condition at 4 DEG C;

(5) Ab of painting 6 μ L is dripped ₂-CD-Ag-GOD solution, to electrode surface, dries under wet condition at 4 DEG C, obtained a kind of electrochemical immunosensor based on HS-β-CD-Ag-GOD conjugate.

2. the preparation method of a kind of electrochemical immunosensor based on HS-β-CD-Ag-GOD conjugate as claimed in claim 1, described Ab ₂the preparation method of-HS-β-CD-Ag-GOD conjugate solution, step is as follows:

(1) preparation of HS-β-CD-Ag nano material

By the AgNO of 1 mL, 0.005-0.015 mol/L ₃join in the high purity water of 97 mL with the trisodium citrate of 1 mL, 0.02-0.04 mol/L, stir, concussion dropping 1 mL, the NaBH of 1.5-2.0 mg/mL ₄, fully stir 20 min, add the sulfhydrylation beta cyclodextrin HS-β-CD of 0.0238 g, magnetic agitation 24 h, rotating speed is centrifugal 10 min under the condition of 11000 rpm, constant temperature 60 DEG C of vacuum drying;

(2) preparation of the glucose oxidase ADA-GOD of adamantanecarboxylic acid functionalization

Take 25-41 mg adamantanecarboxylic acid ADA in 40 mL ultrapure waters, add 200 μ L, the NaOH solution of 0.5-1.5 mol/mL, 100 mL are diluted to after solution clarification, 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC is added in the adamantanecarboxylic acid ADA solution of 1-3 mL, 10 mg N-hydroxysuccinimide NHS and 2 mL, pH is the PBS buffer solution of 7.4, stirring at room temperature 0.5 h, add 200 μ L, the glucose oxidase of 0.5-1.5 mg/mL, constant temperature 4 DEG C spends the night, rotating speed is centrifugal 8 min under the condition of 8000 rpm, preserve under the condition of-20 DEG C, the glucose oxidase ADA-GOD solution of the adamantanecarboxylic acid functionalization of obtained 100-300 μ g/mL,

(3) the detection antibody A DA-Ab of adamantanecarboxylic acid functionalization ₂preparation

Take 25-41 mg adamantanecarboxylic acid ADA in 40 mL ultrapure waters, add 200 μ L, the NaOH solution of 0.5-1.5 mol/mL, 100 mL are diluted to after solution clarification, the PBS buffer solution that 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC, 10 mg N-hydroxysuccinimide NHS and 2 mL, pH are 7.4 is added, stirring at room temperature 0.5 h in the adamantanecarboxylic acid ADA solution of 1-3 mL, add 200 μ L, 0.5-1.5 mg/mL detects antibody A b ₂, constant temperature 4 DEG C spends the night, and rotating speed is centrifugal 8 min under the condition of 8000 rpm, adds 1 mL, and pH is the PBS buffer solution of 7.4, preserves under the condition of-20 DEG C, the detection antibody A DA-Ab of the adamantanecarboxylic acid functionalization of obtained 100-300 μ g/mL ₂solution;

(4) Ab ₂the preparation of-HS-β-CD-Ag-GOD conjugate solution

By the detection antibody A DA-Ab of adamantanecarboxylic acid functionalization ₂be diluted to 40-60 μ g/mL with the glucose oxidase ADA-GOD of adamantanecarboxylic acid functionalization, get 200 μ L, 40-60 μ g/mL ADA-Ab ₂with 200 μ L, 40-60 μ g/mL ADA-GOD, add 200 μ L, 3-5 mg/mL HS-β-CD-Ag, constant temperature 4 DEG C of incubated overnight, rotating speed is centrifugal 8 min under the condition of 8000 rpm, abandoning supernatant, adds 200 μ L, and pH is the PBS buffer solution of 7.4, preserve under the condition of-20 DEG C, obtained Ab ₂-HS-β-CD-Ag-GOD conjugate solution;

(5) preparation of multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene MWCNTs-CD-Fc compound substance

Take 0.4-0.6 g multi-walled carbon nano-tubes MWCNTs, join the H prepared ₂sO ₄and HNO ₃for in the mixed solution of 3:1, ultrasonic 3 h of constant temperature 40 DEG C, are cooled to room temperature, be diluted to 500 mL with ultrapure water, use the filter membrane of 0.22 μm to carry out vacuum filtration, milli-Q water is neutral to pH, constant temperature 60 DEG C of vacuum drying, obtained functionalized multi-wall carbonnanotubes MWCNTs-COOH;

Take 20-30 mg MWCNTs-COOH, be distributed in 50 mL ultrapure waters, then add the beta-schardinger dextrin-CD of 150-250 mg, shake 12 h, centrifugal, wash 4 times, constant temperature 60 DEG C of vacuum drying, 60-100 mg ferrocenecarboxylic acid Fc joins in the methylene chloride of 50 mL, add 20-30 mg MWCNTs-CD, stir 12 h, centrifugal drying, obtain multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene MWCNTs-CD-Fc compound substance;

(6) multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene-capture antibody MWCNTs-CD-Fc-Ab ₁preparation

The MWCNTs-CD-Fc taking 3-5 mg is distributed to 2 mL, pH is in the PBS buffer solution of 7.4, add 10 mg 1-ethyls-(3-dimethylaminopropyl) phosphinylidyne diimmonium salt hydrochlorate EDC, 10 mg N-hydroxysuccinimide NHS, constant temperature 4 DEG C stirs 0.5 h, add 200 μ L, 0.5-1.5 mg/mL capture antibody Ab ₁, constant temperature 4 DEG C concussion 12h, obtained multi-walled carbon nano-tubes-beta-schardinger dextrin--ferrocene-capture antibody MWCNTs-CD-Fc-Ab ₁.

3. a kind of electrochemical immunosensor based on HS-β-CD-Ag-GOD conjugate of preparing of preparation method as claimed in claim 1, detects for CSFV antigens c SFV, it is characterized in that, comprise the steps:

(1) electrochemical workstation is used to test with three-electrode system, saturated calomel electrode is contrast electrode, platinum electrode is auxiliary electrode, prepared a kind of electrochemical immunosensor based on HS-β-CD-Ag-GOD conjugate is working electrode, in pH6.0 ~ 8.0 phosphate buffered solution of 10 mL, 40 ~ 60 mmol/L, measure its curent change;

(2) according to gained current differential and CSFV antigens c SFV concentration linear, drawing curve;

(3) method for drafting of foundation working curve carries out the detection of CSFV antigens c SFV in sample, and testing result checks in from working curve.