CN109596697A - A kind of electrochemical immunosensor and its preparation method and application - Google Patents
A kind of electrochemical immunosensor and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of electrochemical immunosensor and preparation method thereof and the application in PSA detection kit is being prepared, is belonging to electrochemical immunosensor technical field.The electrochemical immunosensor, including working electrode, the working electrode includes that platinum electrode surface is coated with graphene oxide/poly ion liquid composite layer modified electrode, and antibody 1 and antibody 2 with object to be analyzed specific binding, the antibody 1 is fixed on graphene oxide/poly ion liquid composite layer, and the antibody 2 is marked with ferrocenecarboxylic acid.The present invention provides a kind of novel graphene oxide/poly ion liquid compounds, then it modifies on naked platinum electrode, by the way of sandwich immunoassay, make electrochemical immunosensor, ferrocene labelled antibody 2 has the effect of signal amplification, and minimum detection limit of the sensor for the detection of PSA has reached 1.0pg/mL.
Description
Technical field
The present invention relates to electrochemical immunosensor technical fields, and in particular to a kind of electrochemical immunosensor and its system
Preparation Method and preparing the application in PSA detection kit.
Background technique
PSA (prostate specific antigen, prostate specific antigen) is directly divided by prostate epithelial cell
Secrete one of prostate duct system glycoprotein.PSA content is usually not higher than 4ng/mL in the serum of normal person, and
When canceration occurs for prostate, the PSA content in serum can then be steeply risen.Therefore, PSA at prostate cancer biological marker
Object, while being also considered as the strongest marker of organ specificity in tumor markers.In recent years, PSA is as prostate cancer
Unique designation object carry out preliminary screening international common approval have been obtained, and clinically using for many years.In addition, serum
The content of middle PSA can also embody the stage of patient disease development, and in the serum amount of PSA increase 0.2ng/mL or more then can be by
Suspect cancer return.For prostate cancer, the unique identification object of alternative PSA is not yet found at present, it, can by detecting PSA
Screening as early as possible goes out patients with prostate cancer, then gives and timely and effectively treats, and is the generation for reducing related neoplasms metastatic disease,
Improve the effective way of the quality of life even survival rate of patient.
At present measurement prostate-specific antigen PSA method it is more, as radio immunoassay, enzyme immunoassay (EIA),
Chemiluminescence immunoassay, electrochemical immunoanalytical method and NMR spectrum inspection technique.Some methods need expensive instrument
Device, some methods need the operator of professional training, and some method sensitivity is to be improved.And electrochemical immunoanalytical method
Required instrument and equipment is simple, easy to operate, low in cost, high specificity, is the prefered method of current detection PSA.
CN 104267184A discloses a kind of immune-electrochemistry sensor based on AuNPS@AgNCs nanocomposite
And its building and application method.Ab1 is fixed with graphene, PSA is as target analytes, the SA- of marked by streptavidin
AuNPs@AgNCs is easy in conjunction with the monoclonal antibody Ab2 of biotin modification, by the idiosyncrasy of antigen-antibody, with " Sanming City
Control " sandwich method preparation PSA immunosensor, the sensor electro-catalysis is restored into deposition silver and H respectively2O2, can be used for quick, clever
The PSA concentration and prostate cancer of super low loading and hyperplasia of prostate are suffered from the detection blood serum of patients with human breast carcinoma quick, specificity is good
The value of f-PSA and t-PSA in person's serum, by in diagnosis of prostate cancer gray area identification and diagnosis have important meaning
Justice.
Core of the electrode as electrochemical sensor can prepare modification electricity using nano material either composite material etc.
Pole, so that electrode more functionalization, is applicable to diversified experiment demand.These materials mainly include: graphene, stone
Black alkene-gold nano grain, graphene-magnetism gold nanosphere composite material, graphene-single stranded DNA, carbon nanotube-ionic liquid
Etc..PSA electrochemical immunosensor based on the building of these materials has many advantages, such as high sensitivity, rapidly and efficiently, but equally
There is also certain deficiencies, such as have to be hoisted or monoclonal antibody-antigen-reactive specificity to have as the stability of electrode modified material
It is to be reinforced.
Therefore, prepare that sensitivity is higher, the better electrode composite material of stability and double antibody-antigen model are electrifications
Learn the developing direction of immunoassay.
Summary of the invention
A kind of the purpose of the present invention is to provide sensitivity higher, the better electrode composite material of stability, and answered
It is used to prepare in the electrochemical immunosensor of detection PSA, provides effective detection hand for the identification and diagnosis of prostate cancer
Section.
To achieve the above object, the present invention adopts the following technical scheme:
A kind of electrochemical immunosensor, including working electrode, the working electrode include that platinum electrode surface cladding is aerobic
Graphite alkene/poly ion liquid composite layer (GO-PIL) modified electrode, and specifically bound with object to be analyzed
Antibody 1 and antibody 2, the antibody 1 is fixed on graphene oxide/poly ion liquid composite layer, and the antibody 2 is marked with
Ferrocenecarboxylic acid.
Graphene has unique two-dimensional structure and excellent physicochemical properties, and derivative graphene oxide exists
The oxygen-containing function such as hydroxyl (- OH), epoxy group [- C (O) C-], carbonyl (- C=O), carboxyl (- COOH) and ester group (- COO-)
Group, due to the presence of these oxygen-containing functional groups, the thickness of graphene oxide can reach (1.1 ± 0.2) nm, significantly larger than graphene
0.3354nm.The presence of these oxygen-containing functional groups impart graphene oxide it is good dispersibility and reactivity, be easy with
Compound containing groups such as amino, carboxyl and isocyanate group reacts, to realize the covalent knot to graphene oxide
It closes and is modified；In conjunction with graphene oxide can also realize non-covalent bond with other compounds using hydrogen bond or pi-pi bond etc..
Ionic liquid is a kind of room temperature or the salt that near room temperature is in a liquid state, is made of zwitterion, it there is steam to force down,
Liquid temperature range is wide, stability is good, does not allow easy firing, good conductivity, electrochemical window mouth width, good biocompatibility and hypotoxicity
Equal dramatic benefits, can not only substitute some traditional solvents, and in material synthesis processes, and ionic liquid can be real simultaneously
The multiple actions such as template, dispersing agent and stabilizer are now played, the performance of composite material can be improved.
It is equal that intermediate ion liquid of the present invention carries out compound obtained stabilization with graphene oxide while free radical polymerization occurs
Even, with good conductivity, the electrode modified material having no toxic side effect solves common decorative material coated in easily de- on platinum electrode
The problem of falling, while electric conductivity is improved, there is signal amplification.
Electrochemical sensor provided by the invention utilizes the idiosyncrasy of antigen-antibody, prepared by " sandwich " sandwich method,
Middle secondary antibody marks ferrocenecarboxylic acid, realizes that signal amplifies using this active material of ferrocene, makes up general sandwich immunoassay reaction
This defect of middle steric hindrance.
The present invention also provides the preparation methods of the electrochemical immunosensor, comprising the following steps:
(1) it prepares modified electrode: graphene oxide and the ionic liquid with ethylene linkage is carried out freely in the presence of initiator
Graphene oxide/poly ion liquid composite material is made in base polymerization reaction, then is mixed to prepare oxidation stone with chitosan-acetic acid solution
Black alkene/poly ion liquid solution, is applied on platinum electrode, dry, and modified electrode is made；
(2) after the activation of modified electrode made from step (1), the electrode after activation sessile antibody 1: is added dropwise in antibody 1
Surface, standing make antibody 1 be fixed on graphene oxide/poly ion liquid composite layer；
(3) object to be analyzed combines: by step (2) electrode obtained for being fixed with antibody 1 after BSA is closed, being added dropwise
Object to be analyzed is incubated for；
(4) it binding antibody 2: is added dropwise on step (3) electrode obtained for combining object to be analyzed and is marked with ferrocene
The working electrode is made in the antibody 2 of formic acid, stationary incubation.
Preferably, the graphene oxide can also be redox graphene, reducing agent is added in graphene oxide
As DMF and and ion liquid polymerization, obtain the polymer of redox graphene and ionic liquid.Redox graphene polymerization
Electric conductivity afterwards will greatly enhance.
Preferably, the ionic liquid with ethylene linkage is chlorination -1- allyl -3- methylimidazole, 1- in step (1)
- 3 butyl tetrafluoroborate of vinyl, 1- ethyl -3- vinyl imidazole tetrafluoroborate or chlorination N- trimethyl-(4- vinyl
Benzyl) ammonium.Comprehensively consider viscosity, dissolubility, operability, the toxic side effect of ionic liquid, selective chlorination -1- allyl -3-
Methylimidazole is original material.
Preferably, the mass ratio of graphene oxide and chlorination -1- allyl -3- methylimidazole is 1:1-3.It is more excellent
Choosing, the mass ratio of the two are that 1:2. is prepared by the ratio of test discovery graphene oxide and the ionic liquid in 1:2 or so
Composite conductivity is most strong.
Preferably, in step (1), the initiator is azodiisobutyronitrile, the condition of Raolical polymerizable be
Under inert gas shielding, 70-80 DEG C of reaction 14-15h.
The mass percent concentration of the chitosan-acetic acid solution is 1.5%, is uniformly mixed with compound material ultrasound.It is made
GO-PIL solution drop coating on platinum electrode, it is dry under infra-red drying lamp.
Preferably, in step (1), before drop coating GO-PIL solution, platinum electrode first polished processing to redox peaks electricity
Gesture separating degree is close to 64mV.Platinum electrode aluminum oxide powder sanding and polishing to mirror surface, cleans electricity with secondary distilled water repeatedly
Pole surface, it is spare after spontaneously drying in air.Naked platinum electrode is immersed and contains 5mM Fe (CN)6 3-/4-With the electrolysis of 0.1M KCl
Cyclic voltammetry scan is carried out in matter solution, when redox peaks potential separating degree is close to 64mV, indicates that electrode sanding and polishing closes
Lattice.
Preferably, the activation is obtained in step (1) for the EDC-NHS dropwise addition for being 10mM by concentration in step (2)
Modified electrode surface activates 30~50min at room temperature.
Preferably, the electrode surface after activation is added dropwise in antibody 1,12~15h is stored under 4 DEG C of environment, is used after taking-up
0.1mol/L PBS cleaning is dried.
In step (4), the preparation method of markup antibody 2 is monoclonal antibody, the EDC- that will resist object to be analyzed
NHS, PBS buffer solution and ferrocenecarboxylic acid are made through ultrasonic mixing, wherein resisting the monoclonal antibody of object to be analyzed and two cyclopentadienyls
The mass ratio of iron formate is 1:10~100.
Preferably, antibody 2 is added dropwise on the working electrode (s, 1h is incubated at 37 DEG C.
The present invention also provides the electrochemical immunosensors in preparing prostate specific antigen detection kit
Application.
Prostate-specific antigen (PSA) is a kind of Specific marker of carcinoma of prostate, and measuring to it facilitates reality
The early screening of existing prostate cancer, reduces aspiration biopsy false positive bring wound and infection, reduces cancer mortality, can be effective
Easily assess after cancer development stage and predicted treatment recurrence may and rehabilitation situation, the control of prostate cancer can be played
Positive effect.Using PSA as the object to be analyzed of electrochemical immunosensor in the present invention, preparation is specifically bound therewith
Antibody 1 and antibody 2.
Specifically, the antibody 1 is mouse anti-psa monoclonal antibody, is coated antibody, and the antibody 2 is the anti-prostate of mouse
Specific antigen (PSA) monoclonal antibody, is the antibody for label.The preparation method of markup antibody 2 is the anti-prostate specific of mouse
Antigen (PSA) monoclonal antibody (abbreviation Ab2), EDC-NHS, PBS buffer solution and ferrocenecarboxylic acid through ultrasonic mixing be made, wherein Ab2 with
The mass ratio of ferrocenecarboxylic acid is 1:50.
Electrochemical immunosensor in the prostate specific antigen detection kit can pass for three-electrode electro Chemical
Sensor, including working electrode, reference electrode and to electrode, Ag/AgCl electrode or saturated calomel electrode can be selected in reference electrode, right
Platinum electrode can be selected in electrode, and working electrode is the platinum electrode of above-mentioned " sandwich " sandwich method preparation.
It is that the present invention has the utility model has the advantages that
The present invention provides a kind of novel graphene oxide/poly ion liquid compounds (GO-PIL), and then modification is arrived
On naked platinum electrode, the electric conductivity of decorative material is detected.Show that the compound has well by multiple electrochemistry experiment
Electric conductivity and electro-catalysis effect.
By the way of sandwich immunoassay, ferrocene labelled antibody 2 is used to prepare detection prostate-specific antigen PSA's
Electrochemical immunosensor working electrode.Pass through multiple electrochemistry experiment such as cyclic voltammetry, differential pulse voltammetry, electrification
It learns impedance method and shows that the sensor has the effect of signal amplification.
The sensor of building is used for the detection of prostate-specific antigen (PSA), obtained its 0.002~
In the concentration range of 5500ng/mL PSA, the peak current of PSA differential pulse voltammetry shows good with the logarithm of its concentration
Linear relationship, coefficient R2Reach 0.9785, minimum detection limit has reached 1.0pg/mL (K=3).Meanwhile to the electrification
Learn detection architecture selectivity, reproducibility, stability be tested, it was demonstrated that the detection architecture selectivity preferably, reproducibility and
Have good stability, sensitivity it is higher.
Detailed description of the invention
Fig. 1 is bare electrode (a:Pt), modified electrode (b:Pt/GO-PIL), fixes Ab1 (c:Pt/GO-PIL/Ab1), electrode
(e:Pt/GO-PIL/Ab1/BSA/PSA) and unlabelled antibody 2 after closing (d:Pt/GO-PIL/Ab1/BSA), being reacted with PSA
The cyclic voltammogram CV of (f:Pt/GO-PIL/Ab1/BSA/PSA/Ab2) after progress sandwich immunoassay reaction.
Fig. 2 is the differential pulse voltammogram DPV, same Fig. 1 of electrode that a-f is represented of each electrode.
Fig. 3 is AC impedance the figure EIS, same Fig. 1 of electrode that a-f is represented of each electrode.
Fig. 4 is bare electrode (a:Pt), modified electrode (b:Pt/GO-PIL), fixes Ab1 (c:Pt/GO-PIL/Ab1), electrode
(e:Pt/GO-PIL/Ab1/BSA/PSA) and resisting using label after closing (d:Pt/GO-PIL/Ab1/BSA), being reacted with PSA
Body 2 carries out the cyclic voltammogram CV of (f:Pt/GC-IL/Ab1/BSA/PSA/Ab2-Fc) after sandwich immunoassay reaction.
Fig. 5 is the differential pulse voltammogram DPV, same Fig. 4 of electrode that a-f is represented of each electrode.
Fig. 6 is AC impedance the figure EIS, same Fig. 4 of electrode that a-f is represented of each electrode.
Fig. 7 is that the electrochemical sensor constructed using the antibody 2 of label carries out sandwich immunoassay to the PSA of serial various concentration
After reaction DPV figure, the PSA concentration that curve represents from top to bottom be followed successively by 0ng/mL, 0.002ng/mL, 0.02ng/mL,
0.2ng/mL, 2ng/mL, 20ng/mL, 0.2 μ g/mL, 1.1 μ g/mL and 5.5 μ g/mL.
Fig. 8 is the linear relationship curve of DPV peak current and PSA concentration, and wherein illustration is the line of peak current and log concentration
Sexual intercourse curve.
Fig. 9 is that interference test result (has been separately added into 5 μ of 0.2ng/mL BSA, IgG, IgM and myoglobins
The DPV peak current of the PSA of L0.002ng/mL).
Specific embodiment
The present invention is further explained in the light of specific embodiments, but the present invention is not limited thereto.
Reagent involved in embodiment is as follows: graphene oxide (Aladdin company)；Ionic liquid (chlorination -1- allyl -
3- methylimidazole, Aladdin company)；(0.03g chitosan is dissolved in the acetic acid of 2g 2mol/L to 1.5% chitosan-acetic acid solution
In solution, it is made into 1.5% chitosan-acetic acid solution 2mL)；Electrolyte solution: 5mM Fe (CN)6 3-/4-With 0.1mM KCl conduct
Electrolyte solution；The phosphate buffer solution (PBS) of 0.1mol/L；Azodiisobutyronitrile AIBN (Aladdin company)；10mM's
EDC-NHS (1- (3- dimethyl propyl) -3- ethyl-carbodiimide hydrochloride and N- hydroxysuccinimide)；Methanol CH3OH (Ah
Latin company)；1% BSA；IgM；IgG；Myoglobins, ferrocenecarboxylic acid.
PSA (prostate-specific antigen), Ab1 (mouse anti-psa monoclonal antibody), Ab2 (the anti-prostate-specific antigen of mouse
(PSA) monoclonal antibody) it is purchased from people from Zhengzhou Fu Bosai biotechnology Co., Ltd).
Other reagents are that analysis is pure, and experimental water is secondary distilled water.
Embodiment 1
1, graphene oxide/poly ion liquid compound (GO-PIL) preparation
0.011g graphene oxide is weighed, is placed in a small beaker with 20mL methanol, room temperature ultrasound 30min, so that oxidation
Graphene uniform is dispersed in methanol solution.Then 0.058g AIBN and 0.022g ionic liquid is weighed, it is good to be added to ultrasound
In the methanol solution of graphene oxide, N2Under environment keep 40min after seal, in 70 DEG C of oil bath pan heating stirring 14~
15h, cooling centrifugation, washing and drying to obtain the final product.
2, the preparation of modified electrode
The 2mg GO-PIL being dried for standby in step 1 is taken to be dispersed in 1mL secondary distilled water, and the chitosan with 1.5%
Acetum 1:1 ultrasonic mixing is uniform, obtain can modified electrode 1mg/mL GO-PIL solution.
By platinum electrode aluminum oxide powder sanding and polishing to mirror surface, with secondary distilled water cleaning electrode surface repeatedly,
It is spare after being spontaneously dried in air.Naked platinum electrode is immersed and contains 5mM Fe (CN)6 3-/4-In the electrolyte solution of 0.1M KCl
Cyclic voltammetry scan is carried out, makes redox peaks potential separating degree close to 64mV.Pipette 5 μ L GO-PIL, drop coating is in having polished
On platinum electrode, then it is placed under infra-red drying lamp and dries to get modified electrode.
3, the activation of electrode and the fixation of Ab1
It takes 5 μ L 10mM EDC-NHS to be added dropwise on the platinum electrode for modifying one layer of GO-PIL, then activates 30 at room temperature
It is rinsed after~50min with the PBS of 0.1mol/L, drying for standby.
The Ab1 of 5 μ L, 1.1 μ g/mL is added dropwise in activated electrode surface, is placed in 12~15h of storage under 4 DEG C of environment, takes
It is cleaned and is dried with 0.1mol/L PBS after out.
4, the label of Ab2
Take the μ L 0.1mol/L of the Ab2 of 20 μ L 1.1mg/mL, the EDC-NHS of 20 μ L 10mM, 160 PBS and 1.0mg
Fc-COOH ultrasound obtains the Ab2 that 0.11mg/mL is marked by Fc-COOH, 4 DEG C of preservations after uniformly mixing 30min, then is diluted
The Ab2 marked by Fc-COOH for obtaining 0.2 μ g/mL is spare.
5, electro-chemical test
Electricity is carried out using electrochemical AC impedance method (EIS), differential pulse voltammetry (DPV) and cyclic voltammetry (CV)
Test chemical, the above experiment are containing 5mM Fe (CN)6 3-/4-It carries out in 0.1M KCl electrolyte solution, is repaired to investigate
Adorn the electrochemical behavior of electrode and some chemical properties of GO-PIL compound.
5.1 respectively characterize modification front and back electrode with cyclic voltammetry, differential pulse voltammetry and AC impedence method.Table
It is rinsed again with 0.1mol/L PBS after having levied, closes 2h in the environment of 4 DEG C in the BSA solution that 5 μ L 1% are added dropwise on electrode,
And carry out electrochemical Characterization.After being dried after having characterized again with 0.1mol/L PBS flushing, then it is added dropwise 5 μ L, 0.2 μ g/mL's
PSA is incubated for 1h under conditions of 37 DEG C, carries out electrochemical Characterization again.It is rinsed again with 1%PBS later, 5 μ L0.2 μ is added dropwise
The unmarked Ab2 of g/mL is characterized after storing 1h at 37 DEG C.
As shown in Figs. 1-2, a of a and Fig. 2 of Fig. 1 are the electrochemical behavior of naked platinum electrode, and the b of the b and Fig. 2 of Fig. 1 are
Electrochemical behavior after having modified the GO-PIL of one layer of experiment preparation on naked platinum electrode., it is apparent that when in bare electrode
On modified after GO-PIL compound, peak current obviously rises, this is because poly ion liquid have good electric conductivity with
And the bigger serface of graphene oxide and good electron transmission ability illustrate so that electron transport rate significantly improves
Effect after modification is very ideal.And when toward on modified electrode plus after Ab1,1%BSA, PSA and Ab2, peak current gradually under
Drop, this is because the above substance forms biggish steric hindrance on the electrode so that electron transport rate is gradually reduced, and
So that peak current is gradually reduced.
As shown in figure 3, electrochemical AC impedance is made of a semicircle and straight line, electrode modification mistake can be indicated
The variation of surface impedance in journey, half diameter of a circle are equivalent to the resistance of electronics transfer, so the variation of electrochemical AC impedance
Journey is just opposite with the behavior of CV and DPV.After having modified GO-PIL compound toward on naked platinum electrode, resistance is decreased obviously,
Illustrate to test the ability that prepared GO-PIL compound has enhancing electron transmission.And when toward adding Ab1,1% on modified electrode
After BSA, PSA and Ab2, due to the effect of steric hindrance, resistance is significantly increased.
5.2 couples of Ab2 be marked after modified electrode characterization
The Ab1 of 5 μ L, 1.1 μ g/mL is added dropwise in activated electrode surface, is placed in 12~15h of storage under 4 DEG C of environment, takes
It is cleaned and is dried with PBS after out, close 2h in the environment of 4 DEG C in the BSA that 5 μ L 1% are added dropwise on electrode later, then 5 μ L are added dropwise
The PSA of 0.2 μ g/mL is incubated for 1h under conditions of 37 DEG C, 5 μ L, the 0.2 μ g/mL Ab2 labeled with Fc-COOH is added dropwise, in 37
1h is incubated at DEG C.Each step is both needed to be cleaned with PBS, is characterized (as Figure 4-Figure 6) with CV, DPV, EIS.
It is compared to the Ab2 of no ferrocene label, after having added ferrocene to mark Ab2, the peak current of either CV or DPV
Than not having the peak current of markd Ab2 big, and the peak current that EIS ratio does not have markd Ab2 to measure is small, this illustrates to mark
The Fc-COOH of note Ab2 increases electron transport rate, to affect peak current and AC impedance.
6, the measurement of series of concentrations PSA
Using label antibody 2 construct electrochemical sensor to serial various concentration (0.2pg/mL, 2pg/mL,
0.02ng/mL, 0.2ng/mL, 2ng/mL, 20ng/mL, 2 μ g/mL, 1.1 μ g/mL and 5.5 μ g/mL) PSA sandwich exempt from
Epidemic disease reaction, and (Fig. 7) is characterized with DPV, obtain linear relationship (Fig. 8, the ip of the logarithm of peak current and PSA concentration and concentration
=-0.0459lgC (pg/mL)+2.4813, R2=0.9785).
It is obtained by the above results, the electrochemistry immuno-sensing constructed with the antibody 2 that GO-PIL compound and ferrocene mark
Device lowest detection is limited to 1.0pg/mL (K=3).
7, interference is tested
Under similarity condition, to the 5 μ L0.002ng/ for being separately added into 0.2ng/mL BSA, IgG, IgM and myoglobins
The PSA of mL is tested, even if concentration is higher than 100 times of test substance PSA as the result is shown, chaff interferent influences not experimental result
Greatly.Illustrate that constructed sensor has very strong anti-interference ability (Fig. 9) to PSA.
8, reproducibility is tested
Nine platinum electrodes are tested under identical condition, the sensor constructed using the antibody 2 that ferrocene is marked
The PSA of 5 μ L, 0.2 μ g/mL is tested, the results are shown in Table 1.
Table 1
| Electrode | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 |
| Peak current * 10-4A | 5.320 | 5.344 | 5.354 | 5.389 | 5.252 | 5.227 | 5.315 | 5.363 | 3.345 |
Table 1 as the result is shown the peak current of DPV relative standard deviation be 1.5%, illustrate the modified electrode have it is good
Reproducibility.
9, stability test
Under similarity condition, the electrode for securing Ab1 is placed under 4 DEG C of environment and is stored 12 hours, 7 days and 15 days, used
The sensor that markup antibody 2 constructs is measured PSA, and the results are shown in Table 2.
Table 2
| Time | 12h | 7d | 15d |
| Peak current * 10-4A | 1.538 | 1.530 | 1.527 |
As seen from Table 2, as storage time extends, DPV peak current successively has dropped 0.52%, 0.2%, this means that
The constructed electrochemical sensor of this experiment has good stability.
Claims (9)
1. a kind of electrochemical immunosensor, including working electrode, which is characterized in that the working electrode includes platinum electrode surface
It is coated with graphene oxide/poly ion liquid composite layer modified electrode, and is specifically bound with object to be analyzed
Antibody 1 and antibody 2, the antibody 1 is fixed on graphene oxide/poly ion liquid composite layer, and the antibody 2 is marked with
Ferrocenecarboxylic acid.
2. the preparation method of electrochemical immunosensor as described in claim 1, which comprises the following steps:
(1) it prepares modified electrode: graphene oxide and the ionic liquid with ethylene linkage is subjected to radical polymerization in the presence of initiator
Close react be made graphene oxide/poly ion liquid composite material, then with chitosan-acetic acid solution be mixed to prepare graphene oxide/
Poly ion liquid solution, is applied on platinum electrode, dry, and modified electrode is made；
(2) sessile antibody 1: after the activation of modified electrode made from step (1), being added dropwise the electrode surface after activation for antibody 1,
Standing makes antibody 1 be fixed on graphene oxide/poly ion liquid composite layer；
(3) object to be analyzed combines: by step (2) electrode obtained for being fixed with antibody 1 after BSA is closed, being added dropwise wait divide
Analysis object is incubated for；
(4) it binding antibody 2: is added dropwise on step (3) electrode obtained for combining object to be analyzed and is marked with ferrocenecarboxylic acid
Antibody 2, the working electrode is made in stationary incubation.
3. preparation method as claimed in claim 2, which is characterized in that in step (1), the ionic liquid with ethylene linkage is chlorine
Change -1- allyl -3- methylimidazole.
4. preparation method as claimed in claim 3, which is characterized in that graphene oxide and chlorination -1- allyl -3- methyl miaow
The mass ratio of azoles is 1:1-3.
5. preparation method as claimed in claim 2, which is characterized in that in step (1), the initiator is two isobutyl of azo
Nitrile, the condition of Raolical polymerizable are 70-80 DEG C of reaction 14-15h under inert gas protection.
6. preparation method as claimed in claim 2, which is characterized in that in step (1), drop coating graphene oxide/polyion liquid
Before liquid solution, first polished processing to redox peaks potential separating degree is 64mV to platinum electrode.
7. preparation method as claimed in claim 2, which is characterized in that in step (2), it is 10mM's that the activation, which is by concentration,
The modified electrode surface made from step (1) is added dropwise in EDC-NHS, activates 30~50min at room temperature.
8. application as claimed in claim 2, which is characterized in that the preparation method of the antibody 2 is to resist object to be analyzed
Monoclonal antibody, EDC-NHS, PBS buffer solution and ferrocenecarboxylic acid are made through ultrasonic mixing, wherein resisting the list of object to be analyzed
The mass ratio of clonal antibody and ferrocenecarboxylic acid is 1:10~100.
9. electrochemical immunosensor as described in claim 1 is preparing answering in prostate specific antigen detection kit
With.
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Cited By (4)
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|---|---|---|---|---|
| CN112213482A (en) * | 2020-09-30 | 2021-01-12 | 同济大学 | Method for electrochemically detecting antigen content in sample based on immunohistochemical space |
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| WO2022125501A1 (en) * | 2020-12-07 | 2022-06-16 | Graphene-Dx, Inc. | Graphene-based sensor for detection of prostate biomarkers |
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