CN107966560A - A kind of preparation method of the immunosensor based on chitosan-gold hybrid particle - Google Patents
A kind of preparation method of the immunosensor based on chitosan-gold hybrid particle Download PDFInfo
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- CN107966560A CN107966560A CN201711170225.1A CN201711170225A CN107966560A CN 107966560 A CN107966560 A CN 107966560A CN 201711170225 A CN201711170225 A CN 201711170225A CN 107966560 A CN107966560 A CN 107966560A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/28—Electrolytic cell components
- G01N27/30—Electrodes, e.g. test electrodes; Half-cells
- G01N27/327—Biochemical electrodes, e.g. electrical or mechanical details for in vitro measurements
- 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 kind of preparation method of the immunosensor based on chitosan gold hybrid particle, the preparation method include the synthesis of catechol group modification of chitosan, the preparation of modification of chitosan gold hybridized nanometer particle, immunosensor structure three big steps.Hybridized nanometer particle prepared by the present invention has good biocompatibility, adhesiveness and electric conductivity, application of the polymer inorganic conduction hybridized nanometer particle in immunosensor, not only effectively improve the fixed amount of antibody and keep the activity of large biological molecule well, the adhesive force and electric conductivity of sensitive coating can effectively be strengthened at the same time, so that constructed immunosensor has the advantages that high specific, long-time stability are good, detection range is wide, Monitoring lower-cut is low etc.；The combination of nanocomposite and electrochemical sensor, can be widely applied to immunoassay and expands be applied to the fields such as food security, biological medicine and environment monitoring.
Description
Technical field
The present invention relates to composite nano materials, immunoassay and electrochemical sensor field, is used more particularly, to one kind
The electrode of hybridized nanometer particle-catechol group modification of chitosan@gold compound nano-particles modification, and it is prepared into electrification
The method for learning immunosensor.
Background technology
It is well known that cancer is a big killer of human health.Clinically for alpha-fetoprotein (AFP), sugar antigen
The detection of the tumor markerses such as (CA125, CA19-9), carcinomebryonic antigen (CEA), to colon cancer, lung cancer, cancer of pancreas, stomach cancer and its
The judgement of the effect of cancers such as its galandular epithelium malignant tumour, progression of the disease, monitoring and prognosis estimation have important references value.Mesh
It is preceding detection serum tumor marker immunoassay method have it is a variety of, such as chemiluminescence immunoassay, fluoroimmunoassay
Method, enzyme-linked immunosorbent assay, radio immunoassay and liposome immunization analytic approach etc., however, there is these methods radiation to endanger
Evil, background noise is big, analysis time is long, the cleaning of very complicated, process instruments are valuable and need professional operator etc.
Shortcoming.
Electrochemical immunosensor is to combine highly sensitive sensing technology with specific immune response, to monitor
A kind of Novel Biosensor of Ag-Ab (Antigen-Antibody) reaction, it had both had the height of electrochemical sensor
The features such as sensitivity and economic simplicity, and there are high specific, strong specificity and the low detection limits of immunoassay.
The key of structure immunosensor is the technique for fixing (fixed amount) of antibody, therefore immunosensor decorative material
Should be with specific surface area is big, avtive spot is more, non-specific adsorption ability is small, can keep the property such as large biological molecule activity well
Matter, traditional decorative material have the natural macromoleculars such as chitosan, nano carbon-base material, nano mesoporous inorganic nano-particle, quantum dot
Deng.However, these materials are difficult to keep the chronobiological of antibody or antigen activity while antibody high load amount is kept, and this
A little materials and electrode surface adhesive force are weaker, so that the long-time service of immunosensor is restricted.Self-assembled nanometer grain
Son has the advantages that preparation is simple, is easy to functionalization, and especially its nano-scale to have in antibody is fixed more unique
Advantage.Based on this, our seminars utilize self-assembled nanometer particle sessile antibody early period, successfully prepare unmarked type and are immunized
Sensor, this sensor take full advantage of the advantage of bio-based polymers nano-particle, realize to the low of tumor markers
Concentration Testing (CN201410721208.2).However, being currently based on polymerinorganic hybridized nanometer particle prepares immunosensor
Method there is not been reported.This combined nanocomposite with bioassay technique builds new, diversified electrochemistry biography
Sensor, is expected to be widely applied to the fields such as food security, biological medicine and environment monitoring.
On the other hand, the adhesion protein that mussel is secreted by byssus can be attached to various base material tables in a wetted condition
Face, this superpower Adhering capacity possessed by mussel adhesion protein, mainly catechol molecular structure distinctive with more Palestine and China and
It is related to the interaction mode of base material etc..Catechol structure is incorporated into the host material of electrode modification, can be had
Effect improves long-time stability of the electrode material in electrode surface, so as to ensure the long-term measuring stability of sensor.
The content of the invention
In view of the above-mentioned problems existing in the prior art, the present invention provides one kind based on catechol group modification of chitosan-
The preparation method of the immunosensor of golden hybridized nanometer particle.The hybridized nanometer particle that inventive sensor uses has excellent
Stability and biocompatibility, the introducing of inorganic gold nanoparticle can significantly improve the electric conductivity of nano-particle, to make up polymerization
The deficiency of poorly conductive of thing nano-particle itself；Meanwhile the introducing of catechol group can effectively improve the length of electrode modified material
Phase adhesiveness.Detection of the inventive sensor for tumor markers has high sensitivity, test limit is low, detection range is wide, steady
The advantages that qualitative good.
Technical scheme is as follows：
A kind of preparation method of the immunosensor based on chitosan-gold hybrid particle, catechol group modification of chitosan
Synthesis, the preparation of modification of chitosan-gold hybridized nanometer particle, the structure of immunosensor comprise the following steps that：
(1) synthesis of catechol group modification of chitosan
Chitosan (CS) is dispersed in the aqueous solution that pH is 4~5.5, it is water-soluble to form chitosan to being completely dissolved for stirring
Liquid；Catechol derivatives are taken to be dissolved in second with 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate (EDCHCl)
The in the mixed solvent of alcohol and water, forms catechol derivatives solution, is then added dropwise to catechol derivatives solution described
In chitosan aqueous solution；The pH for adjusting mixed reaction solution is 4~5.5, reacts 12h under the conditions of ice-water bath；It will finally react molten
Liquid obtains catechol group modification of chitosan every acid water dialysis 12h~48h, freeze-drying, and lucifuge stores for future use；
(2) preparation of modification of chitosan-gold hybridized nanometer particle
Catechol group modification of chitosan in step (1) is soluble in water, aqueous solution of chloraurate, lucifuge is then added dropwise
Stirring removes free gold nano to stable catechol group modification of chitosan-gold hybridized nanometer particle solution, dialysis is formed
Particle, obtains modification of chitosan-gold hybridized nanometer particle solution；
(3) structure of immunosensor
Sensor electrode is immersed in the modification of chitosan-gold hybridized nanometer particle solution, will by electrophoretic deposition
Modification of chitosan-gold hybridized nanometer particle deposition is in electrode surface；The electrode that hybridized nanometer particle is modified then is immersed in chlorine
Gold chloride is reduced in one layer of gold nanoparticle of electrode face finish by constant potential in auric acid solution；Gold nanoparticle is modified again
Electrode immerse the phosphate buffer solution containing antibody in antibody binding；The electrode of binding antibody is finally immersed into cow's serum egg
To close nonspecific binding site in white solution, that is, immunosensor is made.
Concentration 0.5mg/mL~1.0mg/mL of chitosan solution in the step (1)；The catechol derivatives include
Caffeic acid, Dihydrocaffeic acid, dihydroxyphenylalanine；Catechol degree of modification is 10% in the catechol group modification of chitosan
~60%.
Catechol group modification of chitosan concentration is 0.1mg/mL~2.0mg/mL in the step (2), gold chloride concentration
For 0.01~0.5mg/mL；Lucifuge mixing time is 2h~24h；
Electrode needs preprocessed before use in the step (3), and the method for pretreatment is：
1. polishing treatment：Electrode is polished to minute surface with the alumina powder of 1.0 μm, 0.05 μm on chamois leather successively, successively
With absolute ethyl alcohol, ultra-pure water, absolute ethyl alcohol, ultrasound 3min cleaning electrodes surface, nitrogen dry up electrode respectively；
2. the electrode of drying is placed in the potassium ferricyanide solution of 5mol/L, followed in the range of -200mV~600mV
Ring volt-ampere is tested, and scanning spike potential difference is less than 100mV, electrode otherwise is re-started polishing treatment.
Electrophoretic deposition method is to immerse electrode in hybridized nanometer particle solution in the step (3), and application is received with compound
The charged opposite constant potential of rice corpuscles, makes composite nanoparticle form compound particle film in electrode surface；Electrodeposition Conditions
For：Constant potential 0.1V~10V, electrodeposition time 10s~600s.
Structure immunosensor concretely comprises the following steps in the step (3)：
1. the micelle modified electrode of macromolecular is immersed in the aqueous solution of chloraurate of 0.02mg/mL~0.2mg/mL, pass through
Constant potential reduces gold chloride, and dispersed nano Au particle is generated in the micelle modified electrode surface of macromolecular；Constant potential -0.1V
~-2.0V, recovery time 10s~300s；
2. by step 1. gained electrode immerse 6.0~pH of pH 7.5 containing antibody phosphate buffer in, at 4 DEG C
Under the conditions of soak 12h~24h after take out, obtained adsorption has the modified electrode of antibody；
3. the step modified electrode that 2. gained adsorption has antibody is immersed to the cow's serum egg of 5mg/mL~10mg/mL again
In white solution, 0.5h~2h is incubated at 37 DEG C to close non-specific adsorption sites, the immunosensor is made；
4. prepared immunosensor is immersed in the phosphate buffer of 6.0~pH of pH 7.5, less than 4 DEG C preservations
It is spare.
The step 2. in antibody be alpha-fetoprotein antibody, cancer embryo antibody, vascular endothelial growth factor, human primary gastrointestinal cancers it is related
Antibody, oophoroma associated antibodies, breast cancer correlation antigen, micro-capsule seaweeds antibody, human chorionic gonadotropin or forefront
Gland cancer antibody.
The present invention is beneficial to be had technical effect that：
1st, the present invention prepares hybridized nanometer by catechol group modification of chitosan to gold chloride in-situ reducing self assembly
Particle, prepared nano-particle have good biocompatibility, make macromolecular micella in electrode table by electrophoretic deposition technique
Face forms uniform film of nanoparticles, not only realizes the big requirement of electrode modified material specific surface area, and is easy to fixed big point
Sub- protein (such as antibody), effectively improves the fixed amount of antibody, and can keep the activity of antibody well.Importantly,
The introducing of gold nanoparticle can effectively improve the electric conductivity of host material, and the introducing of catechol group then substantially increases electrode
The long-time stability of material.
2nd, inventive sensor has the advantages that high specific, high sensitivity, detection range are wide, test limit is low etc..
3rd, the present invention nano material technology, electrochemical sensing technology are combined with bioassay technique can build it is new, more
Sample electrochemical sensor, is expected to be widely applied to the fields such as food security, biological medicine and environment monitoring.
4th, immunosensor of the present invention can be used for the detection to tumor markers, have high specific, in use, by this
Immunosensor is connected with computer, its electrode contact correspondent probe molecular solution, anti-to measure according to the power of detection signal
Former content.
Brief description of the drawings
Fig. 1：The preparation method schematic diagram of the electrochemical immunosensor of the present invention；
Fig. 2：Catechol group structure diagram；
Fig. 3：Caffeic acid modification of chitosan-gold hybridized nanometer particle prepares schematic diagram in the embodiment of the present invention 1；
Fig. 4：The digital photograph figure of caffeic acid modification of chitosan-gold hybridized nanometer particle in the embodiment of the present invention 1；
Fig. 5：The transmission electron microscope picture of caffeic acid modification of chitosan-gold hybridized nanometer particle in the embodiment of the present invention 1；
Fig. 6：The DPSV curves that immunosensor detects different alpha-fetoproteins are made in the embodiment of the present invention 1.
Embodiment
With reference to the accompanying drawings and examples, the present invention is specifically described.
As shown in Figure 1, the preparation method schematic diagram of the electrochemical immunosensor of the present invention, as can be seen from Figure, first
One layer of polymeric-gold hybridized nanometer particle membrane is first modified on the naked sensing electrode pre-processed, then gold nanoparticle is modified
On micella particle membrane, then the antibody such as alpha-fetoprotein antibody (AFP-Ab) are adsorbed in micella particle film surface and use cow's serum
Protein blocking non-specific adsorption sites, are made the immunosensor, and for detecting the antigens such as alpha-fetoprotein.Pass through test
The change of electrochemical source of current calculates detection result.
Embodiment 1
A kind of preparation method of the immunosensor based on chitosan-gold hybrid particle, comprises the following specific steps that：
(1) synthesis of catechol group modification of chitosan
1.0g chitosans (CS) are dispersed in the aqueous solution of 100mL pH 4.0, stirring 12h forms shell to being completely dissolved
Water solution；Take 2.183g caffeic acids (CAA) and 1.162g 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt
Hydrochlorate (EDCHCl) is dissolved in the in the mixed solvent of ethanol and water, coffee acid solution is formed, then by catechol derivatives solution
It is added dropwise in the chitosan aqueous solution；The pH for adjusting mixed reaction solution is 5.5, reacts 12h under the conditions of ice-water bath；Most
Reaction solution is obtained into caffeic acid modification of chitosan CS-CAA every acid water dialysis 12h, freeze-drying afterwards, lucifuge stores for future use；
(2) preparation of modification of chitosan-gold hybridized nanometer particle
Caffeic acid modification of chitosan-gold hybridized nanometer particle to prepare schematic diagram as shown in Figure 3：By 2mg in step (1)
Caffeic acid modification of chitosan CS-CAA is dissolved in 10mL water, and the aqueous solution of chloraurate 1mL of 0.1mg/mL is then added dropwise, keeps away
Light is stirred to stable caffeic acid modification of chitosan-gold hybridized nanometer particle solution is formed, and dialysis removes free Jenner's grain of rice
Son, obtains modification of chitosan-gold hybridized nanometer particle CS-CAA/Au solution；Fig. 2 is the digital photograph of gained CS-CAA/Au solution
Piece figure, can be using its solution as pink, this is because the characteristic color gone out shown by the formation of Au nano-particles；Fig. 3 is gained
The transmission electron microscope picture of CS-CAA/Au hybridized nanometer particles, it can be seen that form the hybridized nanometer particle that particle diameter is about 80nm, and gold
Nano-particle is dispersed in hybridized nanometer inside particles very well, and this structure helps to improve the electric conductivity of nano-particle.
(3) structure of immunosensor
The structure of immunosensor comprises the following steps that：
1. bare electrode is immersed in the modification of chitosan-gold hybridized nanometer particle solution, will be changed by electrophoretic deposition
For property chitosan-gold hybridized nanometer particle deposition in electrode surface, deposition voltage 0.5V, sedimentation time 300s, obtain hydridization
The electrode of nano-particle CS-CAA/Au modifications；
2. 1. electrode that step is obtained is immersed in 0.1mg/mL chlorauric acid solutions, reducing gold chloride by constant potential exists
One layer of gold nanoparticle of electrode face finish, recovery voltage are -0.2V, recovery time 30s；
3. the phosphate that the electrode of gained is immersed to the alpha-fetoprotein antibody (AFP-Ab) that 0.5mL concentration is 10 μ g/mL delays
In fliud flushing (pH 7.4), taken out after soaking 12h under the conditions of 4 DEG C, obtained adsorption has the modified electrode of antibody；
4. the step modified electrode that 2. gained adsorption has antibody is immersed in the bovine serum albumen solution of 5mg/mL,
2h is incubated under the conditions of 37 DEG C to close non-specific adsorption sites, the alpha-fetoprotein immunosensor is made；
5. prepared immunosensor is immersed in the phosphate buffer that pH is 7.4, less than 4 DEG C save backup.
Embodiment 2
A kind of preparation method of the immunosensor based on chitosan-gold hybrid particle, comprises the following specific steps that：
(1) synthesis of catechol group modification of chitosan
0.5g chitosans (CS) are dispersed in the aqueous solution of 100mL pH 5.5, stirring 12h forms shell to being completely dissolved
Water solution；Take 1.092g Dihydrocaffeic acids (HCA) sub- with 0.581g1- ethyls-(3- dimethylaminopropyls) phosphinylidyne two
Amine hydrochlorate (EDCHCl) is dissolved in the in the mixed solvent of ethanol and water, forms Dihydrocaffeic acid's HCA solution, then that HCA is molten
Liquid is added dropwise in the chitosan aqueous solution；The pH for adjusting mixed reaction solution is 5.5, reacts 24h under the conditions of ice-water bath；
Finally by reaction solution every acid water dialysis 24h, freeze-drying obtains Dihydrocaffeic acid modification of chitosan CS-HCA, lucifuge storage
It is spare；
(2) preparation of modification of chitosan-gold hybridized nanometer particle
1mg caffeic acid modification of chitosan CS-HCA in step (1) is dissolved in 10mL water, 0.05mg/ is then added dropwise
The aqueous solution of chloraurate 1mL of mL, lucifuge stir molten to stable Dihydrocaffeic acid's modification of chitosan-gold hybridized nanometer particle is formed
Liquid, dialysis remove free gold nanoparticle, obtain modification of chitosan-gold hybridized nanometer particle CS-HCA solution；
(3) structure of immunosensor
The structure of immunosensor comprises the following steps that：
1. bare electrode is immersed in the modification of chitosan-gold hybridized nanometer particle solution, will be changed by electrophoretic deposition
For property chitosan-gold hybridized nanometer particle deposition in electrode surface, deposition voltage 0.5V, sedimentation time 300s, obtain hydridization
The electrode of nano-particle CS-HCA/Au modifications；
2. 1. electrode that step is obtained is immersed in 0.1mg/mL chlorauric acid solutions, reducing gold chloride by constant potential exists
One layer of gold nanoparticle of electrode face finish, recovery voltage are -0.2V, recovery time 30s；
3. the electrode of gained is immersed to the phosphate buffer for the cancer embryo antibody (CEA-Ab) that 0.5mL concentration is 10 μ g/mL
In (pH 7.4), taken out after soaking 12h under the conditions of 4 DEG C, obtained adsorption has the modified electrode of antibody；
4. the step modified electrode that 2. gained adsorption has antibody is immersed in the bovine serum albumen solution of 5mg/mL,
2h is incubated under the conditions of 37 DEG C to close non-specific adsorption sites, the carcinomebryonic antigen immunosensor is made；
5. prepared immunosensor is immersed in the phosphate buffer that pH is 7.4, less than 4 DEG C save backup.
Embodiment 3
A kind of preparation method of the immunosensor based on chitosan-gold hybrid particle, comprises the following specific steps that：
(1) synthesis of catechol group modification of chitosan
1.0g chitosans (CS) are dispersed in the aqueous solution of 100mL pH 4.5, stirring 12h forms shell to being completely dissolved
Water solution；Take 4.368g caffeic acids (CAA) and 1.162g1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimine hydrochloric acid
Salt (EDCHCl) is dissolved in the in the mixed solvent of ethanol and water, forms caffeic acid CAA solution, then CAA solution is added dropwise
Into the chitosan aqueous solution；The pH for adjusting mixed reaction solution is 5.5, reacts 24h under the conditions of ice-water bath；Finally will reaction
Solution obtains caffeic acid modification of chitosan CS-CAA every acid water dialysis 48h, freeze-drying, and lucifuge stores for future use；
(2) preparation of modification of chitosan-gold hybridized nanometer particle
2mg caffeic acid modification of chitosan CS-HCA in step (1) is dissolved in 10mL water, 0.2mg/mL is then added dropwise
Aqueous solution of chloraurate 1mL, lucifuge stirs to stable caffeic acid modification of chitosan-gold hybridized nanometer particle solution is formed, thoroughly
Analysis removes free gold nanoparticle, obtains modification of chitosan-gold hybridized nanometer particle CS-CAA liquid；
(3) structure of immunosensor
The structure of immunosensor comprises the following steps that：
1. bare electrode is immersed in the modification of chitosan-gold hybridized nanometer particle solution, will be changed by electrophoretic deposition
For property chitosan-gold hybridized nanometer particle deposition in electrode surface, deposition voltage 1.0V, sedimentation time 180s, obtain hydridization
The electrode of nano-particle CS-CAA/Au modifications；
2. 1. electrode that step is obtained is immersed in 0.1mg/mL chlorauric acid solutions, reducing gold chloride by constant potential exists
One layer of gold nanoparticle of electrode face finish, recovery voltage are -0.2V, recovery time 30s；
3. the electrode of gained is immersed to the phosphate for the prostate-specific antibody (PSA-Ab) that 0.5mL concentration is 10 μ g/mL
In buffer solution (pH 7.4), taken out after soaking 12h under the conditions of 4 DEG C, obtained adsorption has the modified electrode of antibody；
4. the step modified electrode that 2. gained adsorption has antibody is immersed in the bovine serum albumen solution of 5mg/mL,
2h is incubated under the conditions of 37 DEG C to close non-specific adsorption sites, the prostate specific antigen immunosensor is made；
5. prepared immunosensor is immersed in the phosphate buffer that pH is 7.4, less than 4 DEG C save backup.
Test case：
Electrochemical Detection of the immunosensor to alpha-fetoprotein (AFP)
The immunosensor that embodiment 1 is prepared immerses concentration and is followed successively by (a) 0gmL respectively-1, (b) 1.0 × 10- 14g·mL-1, (c) 1.0 × 10-13g·mL-1, (d) 1.0 × 10-12g·mL-1, (e) 1.0 × 10-11g·mL-1, (f) 1.0 ×
10-10g·mL-1, (g) 1.0 × 10-9g·mL-1, (h) 1.0 × 10-8g·mL-1, (i) 1.0 × 10-7g·mL-1, (j) 1.0 ×
10-6g·mL-1Alpha-fetoprotein phosphate delay in solution (pH 7.4), be incubated 2h under the conditions of 37 DEG C；Alpha-fetoprotein will be adsorbed
(AFP) immunosensor takes out, after being eluted with phosphate buffer, using the immunosensor as working electrode, and saturation
Calomel electrode is reference electrode, and platinum electrode is to electrode, on Shanghai Chen Hua CHI660A electrochemical workstations, using electrification
AC impedence method is learned to be measured.
By the electricity for testing immunosensor of the immunosensor of uncombined antigen with combining various concentrations alpha-fetoprotein
Flow valuve change, and calculate before and after electric current change rate (Δ I)) can obtain corresponding linear relationship curve, so as to fulfill to first
The detection of fetoprotein.
Testing result is as shown in fig. 6, as seen from Figure 6, with the immunosensor pair prepared by alpha-fetoprotein concentration
1.0×10-14g·mL-1~1.0 × 10-6g·mL-1The alpha-fetoprotein of concentration range has good linear response, minimum inspection
Survey limit and reach 3.3fgmL-1, linear response equation is：
ΔRct(%)=95.430+6.570lgCCEA, (S/N=3), R2=0.997.
Claims (8)
1. a kind of preparation method of the immunosensor based on catechol group modification of chitosan-gold hybridized nanometer particle, it is special
Sign is the synthesis of catechol group modification of chitosan, the preparation of modification of chitosan-gold hybridized nanometer particle, immunosensor
Structure comprises the following steps that：
(1) synthesis of catechol group modification of chitosan
Chitosan is dispersed in the aqueous solution that pH is 4~5.5, stirring forms chitosan aqueous solution to being completely dissolved；Take catechu
Amphyl is dissolved in the in the mixed solvent of ethanol and water with 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate,
Catechol derivatives solution is formed, then catechol derivatives solution is added dropwise in the chitosan aqueous solution；Adjust
The pH of mixed reaction solution is 4~5.5, reacts 12h under the conditions of ice-water bath；Finally by reaction solution every acid water dialyse 12h~
48h, freeze-drying obtain catechol group modification of chitosan, and lucifuge stores for future use；
(2) preparation of modification of chitosan-gold hybridized nanometer particle
Catechol group modification of chitosan in step (1) is soluble in water, aqueous solution of chloraurate, lucifuge stirring is then added dropwise
Reaction removes free gold nano to stable catechol group modification of chitosan-gold hybridized nanometer particle solution, dialysis is formed
Particle, obtains modification of chitosan-gold hybridized nanometer particle solution；
(3) structure of immunosensor
Sensor electrode is immersed in the modification of chitosan-gold hybridized nanometer particle solution, will be modified by electrophoretic deposition
Chitosan-gold hybridized nanometer particle deposition is in electrode surface；The electrode that hybridized nanometer particle is modified then is immersed in gold chloride
Gold chloride is reduced in one layer of gold nanoparticle of electrode face finish by constant potential in solution；The electricity that gold nanoparticle is modified again
Pole immerse the phosphate buffer solution containing antibody in antibody binding；It is molten that the electrode of binding antibody is finally immersed into bovine serum albumin
To close nonspecific binding site in liquid, that is, immunosensor is made.
2. the preparation method of immunosensor according to claim 1, it is characterised in that catechol is spread out in the step (1)
Biology includes caffeic acid, Dihydrocaffeic acid, dihydroxyphenylalanine；Catechol is modified in the catechol group modification of chitosan
Spend for 10%~60%.
3. the preparation method of immunosensor according to claim 1, it is characterised in that catechu phenolic group in the step (2)
Group's modification of chitosan concentration is 0.1mg/mL~2.0mg/mL, and gold chloride concentration is 0.01mg/mL~0.5mg/mL.
4. the preparation method of immunosensor according to claim 1, its feature is in modification of chitosan-gold hybridized nanometer grain
The preparation of son needs to carry out under the conditions of lucifuge, and the reaction time is 2h~24h.
5. the preparation method of immunosensor according to claim 1, it is characterised in that sensor electricity in the step (3)
Extremely glass-carbon electrode, gold electrode, ito glass electrode, carbon fiber electrode, paper electrode, Graphene electrodes, carbon nanotube electrode, carbon
One kind in paste electrode, screen printing electrode.
6. the preparation method of immunosensor according to claim 1, it is characterised in that electrophoretic deposition in the step (3)
Method is to immerse electrode in hybridized nanometer particle solution, applies the constant potential opposite with hybridized nanometer particle, makes hybridized nanometer
Particle deposition forms particle membrane in electrode surface；Electrophoretic deposition process condition is：Constant potential 0.1V~10V, electrophoretic deposition time
10s~600s.
7. chlorauric acid solution is 0.01mg/mL~1.0mg/mL, the condition of constant potential reduction gold chloride for constant potential -0.1V~-
5.0V, recovery time are 0s~300s.
8. the preparation method of immunosensor according to claim 1, it is characterised in that the antibody in the step (3) is
Alpha-fetoprotein antibody, cancer embryo antibody, vascular endothelial growth factor, human primary gastrointestinal cancers associated antibodies, oophoroma associated antibodies, breast cancer phase
Close antigen, micro-capsule seaweeds antibody, human chorionic gonadotropin or prostate cancer antibody.
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| CN109270138A (en) * | 2018-09-03 | 2019-01-25 | 喀什金慧通实业开发有限责任公司 | Detect the preparation method and applications of the electrochemical immunosensor of brucellosis |
| CN109374705A (en) * | 2018-11-08 | 2019-02-22 | 浙江省农业科学院 | Chitosan/poly- caffeic acid-tyrosinase-carbon nano tube modified glass-carbon electrode and preparation method, application |
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| CN111337555A (en) * | 2020-03-24 | 2020-06-26 | 泉州师范学院 | Method for preparing reduced graphene oxide-nanogold composite material by using perilla extract and application of reduced graphene oxide-nanogold composite material in electrochemical sensor |
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| CN114524952A (en) * | 2022-03-11 | 2022-05-24 | 南通大学 | Preparation method of high-adhesion natural eggshell membrane chitosan hydrogel |
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| CN109270138A (en) * | 2018-09-03 | 2019-01-25 | 喀什金慧通实业开发有限责任公司 | Detect the preparation method and applications of the electrochemical immunosensor of brucellosis |
| CN109270138B (en) * | 2018-09-03 | 2020-08-25 | 青岛科技大学 | Preparation method and application of electrochemical immunosensor for detecting brucellosis |
| CN109374705A (en) * | 2018-11-08 | 2019-02-22 | 浙江省农业科学院 | Chitosan/poly- caffeic acid-tyrosinase-carbon nano tube modified glass-carbon electrode and preparation method, application |
| CN110470721A (en) * | 2019-07-12 | 2019-11-19 | 佛山职业技术学院 | A kind of acrylamide electrochemical fast detecting method |
| CN111337555A (en) * | 2020-03-24 | 2020-06-26 | 泉州师范学院 | Method for preparing reduced graphene oxide-nanogold composite material by using perilla extract and application of reduced graphene oxide-nanogold composite material in electrochemical sensor |
| CN114324540A (en) * | 2021-11-09 | 2022-04-12 | 天津商业大学 | Guanosine tetraphosphate electrochemical type nano enzyme sensor and preparation method thereof |
| CN114324540B (en) * | 2021-11-09 | 2023-12-26 | 天津商业大学 | Guanosine tetraphosphoric acid electrochemical nano-enzyme sensor and preparation method thereof |
| CN114524952A (en) * | 2022-03-11 | 2022-05-24 | 南通大学 | Preparation method of high-adhesion natural eggshell membrane chitosan hydrogel |
| CN114524952B (en) * | 2022-03-11 | 2023-09-15 | 南通大学 | Preparation method of high-adhesion natural eggshell membrane chitosan hydrogel |
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