CN110376380A - A kind of application of electrochemistry Enzyme linked immunosensor and its preparation and detection antigen - Google Patents
A kind of application of electrochemistry Enzyme linked immunosensor and its preparation and detection antigen Download PDFInfo
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- CN110376380A CN110376380A CN201910678544.6A CN201910678544A CN110376380A CN 110376380 A CN110376380 A CN 110376380A CN 201910678544 A CN201910678544 A CN 201910678544A CN 110376380 A CN110376380 A CN 110376380A
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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
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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
- G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
Abstract
The present invention relates to a kind of electrochemistry Enzyme linked immunosensor and its applications of preparation and detection antigen, belong to protein detection technology field.The present invention is prepared for a kind of enzyme-linked immunological probe of electrochemistry, antigen is added in magnetic bead and the bioconjugates of capture antibody and is incubated for, after removing supernatant liquor outside plus under magnetic field condition, the bioconjugates that nanogold, detection antibody and enzyme is added are incubated for, after double-antibody sandwich reacts to get arrive the enzyme-linked immunological probe of electrochemistry.The dispersion liquid of the probe is modified on carbon paste basal electrode surface, electrochemistry Enzyme linked immunosensor, the range of linearity of electrochemistry Enzyme linked immunosensor (MB-eElisa) proposed by the invention are as follows: 0.01-6.0ng mL are obtained‑1, detection limit are as follows: 4.0pg mL‑1(S/N=3)；It is compared with existing electrochemical immunoassay method, this method has lower detection limit and higher sensitivity.
Description
Technical field
The invention belongs to protein detection technology field, more particularly, to a kind of electrochemistry Enzyme linked immunosensor and its
The application of preparation and detection antigen.
Background technique
Antigen is the immune system for referring to stimulation viable organism, and immune Feedback occurs for induction, generates the anti-of humoral immunity
The effector cell of body or cellular immunity, and the substance reacted with it in vivo and in vitro.In addition, antigen is examined in tumor disease
There is highly important theoretical significance in terms of disconnected and clinical treatment.Have now been found that antigen relevant to tumor disease has very
Kind, for example, carcinomebryonic antigen (CEA), alpha-fetoprotein (AFP), prostate-specific albumen (PSA), carbohydrate antigen (CA19-9),
In, CEA is the applicable test object of current tumor disease wide spectrum the most, it is a kind of cell surface that molecular weight is about 200kDa
Glycoprotein, the classifiable tumor marker as diseases such as clinically diagnosing colon, breast cancer, lung cancer, gastric cancer and cancers of pancreas.So
And antigens c EA generally shows low-down content (0-2.5ng mL in the blood of Healthy People-1)；In addition, in infantile tumour
In forming process, small variation can only occur for content of the antigens c EA in blood of human body.Therefore, the clinical assays of antigen are answered
This has the features such as quick, accurate, highly sensitive and high specific, in order to be able to detect antigen in complicated biological sample
The minor change of content.
Electrochemistry immuno-sensing technology has many advantages, such as that portable, response is fast, highly sensitive and inexpensive, therefore it is most often answered
Measurement for antigen.However, the most of antigen electrochemical immunosensors being previously reported are usually at sensing electrode interface
It directly constructs, this usually meets when coping with complex biological sample detection faces the following problem: the detection of redundant and complicated walks
Suddenly, low detection flux and electrode interface are easy to by protein passivation etc..Exempt from studies have shown that magnetic bead (MB) is introduced into electrochemistry
In epidemic disease sensing system, it is possible to prevente effectively from the passivation of electrode interface and also can simplify immune detection process.Relative to fixation
The electrode sensing interface of change, MB have faster kinetics, bigger specific surface area and better stability in the solution；
In addition, can easily be modified on the surface MB upper each only by simple chemical reaction and externally-applied magnetic field cleaning operation
Functional group, DNA, enzyme or the antibody etc. of kind various kinds.It is existing studies have shown that MB can be in conjunction with DNA probe or antibody molecule,
It is widely used in the measurement of DNA and bioprotein.But the signal of current MB based electrochemical immunosensor amplifies plan
Slightly the overwhelming majority is by horseradish peroxidase (HRP) catalyzing hydrogen peroxide (H2O2) reduction reaction.Although however, HRP energy
Enough a variety of redox mediators are catalyzed H2O2Reduction, but H2O2Direct-reduction current potential usually and these media are very close to leading
Cause this enzyme type sensor that there is relatively poor signal-to-noise ratio, this is highly detrimental to the Sensitive Determination of antigen.Therefore, how
Quickly, accurately, in highly sensitive and high specific measurement complex biological sample the content of antigen remains popular class concerned by people
Topic.
Summary of the invention
The present invention solves in the prior art that electrochemical immunosensor detection sensitivity and accuracy be not high, and special
The not strong technical problem of property.The present invention is prepared for a kind of enzyme-linked immunological probe of electrochemistry, and antigen is added to magnetic bead and capture is anti-
Be incubated in the bioconjugates of body, outside plus magnetic field condition under remove supernatant liquor after, be added nanogold, detection antibody and
The bioconjugates of enzyme are incubated for, and arrive the enzyme-linked immunological probe of electrochemistry after double-antibody sandwich reacts.By the probe
Dispersion liquid modify on carbon paste basal electrode surface, obtain electrochemistry Enzyme linked immunosensor, the sensor for antigen detection
When have lower detection limit and higher sensitivity.
According to a first aspect of the present invention, a kind of preparation method of enzyme-linked immunological probe of electrochemistry, including following step are provided
It is rapid:
(1) preparation of the bioconjugates of magnetic bead and capture antibody: magnetic bead and capture antibody are incubated for, described catch is made
It obtains antibody and magnetic bead is covalently attached, add closed protein, the magnetic bead surfaces are at large to be obtained the closed protein for closing
The group of antibody linked obtains magnetic bead and captures the bioconjugates of antibody；
(2) preparation of the bioconjugates of nanogold, detection antibody and enzyme: will test antibody and at least one enzyme is added to
In nano-Au solution, makes to detect antibody and enzyme is covalently attached to the nanometer gold surface, add closed protein, the closing egg
The white site for being used to close the nanogold surface exposure, the bioconjugates for obtaining nanogold, detecting antibody and enzyme；
(3) antigen the preparation of the enzyme-linked immunological probe of electrochemistry: is added to the magnetic bead and capture antibody that step (1) obtains
It is incubated in bioconjugates, the antigen can be combined specifically with the capture antibody and with detection antibody；Outside
The bioconjugates for adding the nanogold for after removal supernatant liquor, adding step (2) under magnetic field condition and obtaining, detecting antibody and enzyme
It is incubated for, arrives the enzyme-linked immunological probe of electrochemistry after double-antibody sandwich reacts.
Preferably, closed protein described in step (1) and step (2) is bovine serum albumin；The enzyme be alkaline phosphatase,
DT- diaphorase, horseradish peroxidase or glucose oxidase.
It is another aspect of this invention to provide that providing the enzyme-linked immunological probe of electrochemistry that any the method is prepared.
It is another aspect of this invention to provide that a kind of preparation method of electrochemistry Enzyme linked immunosensor is provided, it will be described
The dispersant liquid drop of the enzyme-linked immunological probe of electrochemistry be coated in basal electrode surface, the basal electrode is carbon paste electrode, to described
After dispersion liquid dries, make the modification of the electrochemistry enzyme-linked immunological probe on basal electrode surface to get to electrochemistry enzyme linked immunological
Sensor.
Preferably, the basal electrode is the carbon paste electrode of zinc oxide graphene nanometer composite modification.
It is another aspect of this invention to provide that providing the electrochemistry Enzyme linked immunosensor that the method is prepared.
It is another aspect of this invention to provide that providing the electrochemistry Enzyme linked immunosensor for detecting answering for antigen
With.
Preferably, the electrochemistry Enzyme linked immunosensor is placed in the electrolyte containing substrate and is incubated for, make the bottom
Enzyme digestion reaction occurs for the enzyme in the probe of object and sensor surface, detects the electrochemical signals of enzymolysis product, and institute is calculated
State the concentration of antigen.
Preferably, contain surfactant in the electrolyte；
Preferably, the surfactant is cetyl trimethylammonium bromide, lauryl sodium sulfate or dodecyl
Benzene sulfonic acid sodium salt.
Preferably, the antigen is carcinomebryonic antigen, alpha-fetoprotein or prostate-specific albumen；The enzyme is alkaline phosphatase
Enzyme, the substrate are phosphenylic acid salt；
Preferably, the substrate is two based phosphates of 1- naphthyl phosphate or benzene.
In general, through the invention it is contemplated above technical scheme is compared with the prior art, mainly have below
Technological merit:
(1) present invention solves the detection step of redundant and complicated existing for the prior art (the electrode sensing interface of immobilization)
Suddenly, low detection flux and electrode interface are easy to the problems such as being passivated by protein.Electrochemistry enzyme linked immunological proposed by the invention
The range of linearity of sensor (MB-eElisa) are as follows: 0.01-6.0ngmL-1, detection limit are as follows: 4.0pg mL-1(S/N=3)；With it is existing
Some electrochemical immunoassay method comparisons, this method have lower detection limit and higher sensitivity.In addition, the sensor
It is applied to the measurement of antigen (CEA) in Healthy People and affinity antibody to SpA, (commercialized people CEA is tried with standard detecting method
Agent box) comparison after, test result have good accuracy.
(2) the electrochemistry Enzyme linked immunosensor in the present invention, magnetic bead (MB) is introduced into the system, can effectively be kept away
Exempt from the passivation of electrode interface and also can simplify immune detection process.In addition, using nanogold and albumen height binding characteristic,
It may be implemented efficiently to be combined with detection antibody and enzyme.
(3) present invention preferably, utilizes high s/n ratio/reproducible zinc oxide graphene nanometer composite modification carbon paste electricity
Pole (ZnO rGO/CPE) constructs the magnetic bead electrochemistry Enzyme linked immunosensor of alkaline phosphatase (ALP) signal transduction a kind of
(MB-eElisa), specific preparation method is while to introduce magnetic bead (MB), alkalinity when electro-chemistry immunity detects antigen (CEA)
Phosphatase (ALP), surfactant (CTAB) and high s/n ratio/renewable (ZnO@rGO/CPE) etc., significantly improves MB-
The testing process of eElisa and sensitivity.Specifically, CTAB, carbon paste electrode and determinand are provided with hydrophobic property, according to close/thin
Aqueous absorption principle can effectively enhance determinand in the bioaccumulation efficiency on carbon paste electrode surface, and it is sensitive further to promote its sensing
Degree.
(4) enzyme in the electrochemistry Enzyme linked immunosensor in the present invention is preferably alkaline phosphatase (ALP), which can
To be catalyzed the 1- naphthols (1-NP) that enzymatic hydrolysis 1- naphthyl phosphate (1-NPP) is electro-chemical activity, one is constructed close to " zero back
Scape " signal transduction system.Specifically, combining the substrate 1-NPP of zero background using the carbon paste electrode of low background current, it is re-introduced into oxygen
Change zinc graphene nanometer composite and CTAB to the further signal enhanced sensitivity of enzymolysis product 1-NP, to obtain higher noise
Than.
Detailed description of the invention
Fig. 1 is the schematic diagram that ZnO@rGO/CPE sensing interface constructs ALP base MB-eElisa.
Fig. 2 is the XRD of ZnO@rGO nano-complex.
Fig. 3 is the SEM of ZnO@rGO nano-complex.
Fig. 4 is the XPS of ZnO@rGO nano-complex.
Fig. 5 is ZnO@rGO nano-complex Zn2p XPS。
Fig. 6 be ZnO@rGO/GCE (a, b, c) and ZnO@rGO/CPE (d, e, f) without containing (a, d) and containing (b, c, e,
F) the differentiated pulse curve of 1-NP；Wherein curve (c, f) is that CTAB is further added.
Fig. 7 is differentiated pulse figure of 6 ZnO@rGO/CPE modified electrodes in 1-NP.
Fig. 8 is AuNPs and Ab2The ultraviolet-visible absorption spectroscopy figure of-AuNPs-ALP.
Fig. 9 is cyclic voltammogram of the Different electrodes in potassium ferricyanide probe solution, wherein curve (a) CPE；Curve (b)
For ZnO@rGO/CPE；Curve (c) is Ab1-MB-ZnO@rGO/CPE；Curve (d) is ALP-AuNPs-Ab2-CEA-Ab1-MB-
ZnO@rGO/CPE。
Figure 10 is to pass through this in the electrolyte solution containing 1-NPP with CPE (a, b) and ZnO@rGO/CPE (c, d, e)
The differentiated pulse curve of MB-eElisa method measurement CEA；Wherein, (b, d) is that there are CTAB, (e) for there is no under conditions of CEA.
Figure 11 is electrochemistry sensitizative compound figure of the CTAB to 1-NP.
Figure 12 is the Ab of the detection CEA of MB-eElisa2The dosage optimization figure of-AuNPs-ALP.
Figure 13 is the time-optimized figure of enzyme digestion reaction of the detection CEA of MB-eElisa.
Figure 14 is the pH optimization figure of the electrolyte solution of the detection CEA of MB-eElisa.
Figure 15 is ZnO@rGO additional amount optimization figure in the modified electrode of the detection CEA of MB-eElisa.
Figure 16 is the differentiated pulse response curve of various concentration CEA.
Figure 17 is the linear diagram of various concentration CEA respective response signal.
Figure 18 is the stability test figure of MB-eElisa.
Figure 19 is the selectivity test figure of MB-eElisa；Wherein, (a) is CEA, and (b-i) is different to be added in CEA
Chaff interferent: insulin (b), prostate specific antigen (c), human albumin (d), human immunoglobulin(HIg) (e), glucose (f), cream
Acid (g), uric acid (h), dopamine (i).
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to the accompanying drawings and embodiments, right
The present invention is further elaborated.It should be appreciated that described herein, specific examples are only used to explain the present invention, not
For limiting the present invention.As long as in addition, technical characteristic involved in the various embodiments of the present invention described below that
Not constituting conflict between this can be combined with each other.
The construction method of electrochemistry Enzyme linked immunosensor MB-eElisa includes the following steps: in the present invention
(1) magnetic bead MB and capture antibody A b1MB-Ab is obtained after incubation reaction1Bioconjugates.
(2) by alkaline phosphatase and detection antibody A b2It is added in nanogold (AuNPs) solution after incubation reaction
Obtain Ab2- AuNPs-ALP bioconjugates.
(3) bioconjugates obtained by (1) and (2) are added into the solution containing antigens c EA by double-antibody sandwich
MB-Ab is obtained after immune response1-CEA-Ab2- AuNPs-ALP bioprobe.
(4) bioprobe obtained by (3) is modified to the zinc oxide graphene nanometer composite prepared and modifies carbon paste
On ZnO@rGO/CPE electrode, dry at room temperature spare.
(5) the immunological probe electrode that (4) are constructed first is placed on the electrolysis containing 1- naphthyl phosphate (1-NPP) and CTAB
After being incubated for certain time progress enzyme digestion reaction in matter solution, its enzymolysis product 1- naphthols is recorded with Differential Pulse Voltammetry (DPV)
The electrochemical signals of (1-NP), signal magnitude can reflect the content of measured object CEA indirectly.
When the enzyme in step (2) is DT- diaphorase, nitroso can be catalyzed or nitro generates amino；In step (2)
It, can catalyzing hydrogen peroxide reduction when enzyme is horseradish peroxidase；When enzyme in step (2) is glucose oxidase, it can be catalyzed
Grape is glycoxidative.
Embodiment 1
The preparation of electrochemistry Enzyme linked immunosensor and highly sensitive alkali phosphatase enzyme mark magnetic bead electrochemistry are exempted from the present invention
The method that epidemic disease detects antigen has process below and step:
(1) ZnO@rGO/CPE is prepared
Based on improved Kovtyukhova two-step oxidation stripping method, prepare high yield graphene oxide gel (GO,
1.0-100mg mL-1), it takes 0.1-5.0mL GO ultrasonic disperse in the water of 9.0mL, 10-80mg Zn powder and 0.1- is then added
5.0mL NH4Cl aqueous solution (0.2g mL-1), it stands after revolving speed magnetic agitation 1-60min appropriate, the reaction was continued 30min, so
Afterwards by black product with ultrapure water centrifuge washing at least 6 times, the final product that finally 60 DEG C of dry 2-9h are obtained in electric dry oven
(ZnO@rGO)。
0.05g ZnO@rGO and 0.95g graphite powder is accurately weighed in grinding platinum after mixing, 200 μ L paraffin oils are added
Mechanical lapping 30min obtains pasty mixture, by the cavity (CPE, diameter 3mm) of mixture indentation carbon paste electrode bottom, pressure
Electrode surface polishes smooth spare (5%, ZnO@rGO/CPE) on carbon paste paper after reality, in addition, except multiple without rGO nanometers of ZnO@
Other than condensation material, naked CPE is similar with ZnO@rGO/CPE preparation method；As a comparison, ZnO@rGO is modified to glass-carbon electrode (ZnO@
RGO/GCE it) equally be used to detect system enzymolysis product 1-NP, first pass through 50nm Al before GCE modification2O3Powder grinding and polishing
Processing, it is then clean rear spare with ethyl alcohol and secondary water supersound washing.Finally by 5 μ L ZnO@rGO (DMF, 1mg mL-1) dispersion
Liquid is modified to the clean surface GCE, is dried for standby under infrared lamp.
(2) MB-Ab is prepared1
Take 1-40 μ L MBs (10mg mL-1) into 1.5mL PE pipe, under the assistance of externally-applied magnetic field at least with PBST washing lotion
Cleaning 2 times；Then EDC (30 μ L, 50mg mL are added-1) and NHS (30 μ L, 50mg mL-1) room temperature activates on Multifunctional swing bed
After 30min, Ab is further added1(100μL,4μg mL-1) after incubation overnight under room temperature, extra Ab1Adding magnetic outside
PBST is added under field condition and washs 3 removals；Finally, being added on BSA (100 μ L, 1wt%) closing MBs not by Ab1The carboxylic of bonding
Base, extra BSA are removed with PBST washing lotion.Finally obtained MB-Ab1Bioconjugates are easily re-dispersed in 100 μ L 0.01M
PBS (pH 7.4) is placed in (4 DEG C) spare in refrigerator.
(3) Ab is prepared2-AuNPs-ALP
Firstly, with 0.2M K2CO3The pH of 1.0-2.0mL AuNPs (20nm) solution is adjusted to 8.2, is then continuously added to
Ab2(150μL,4μg mL-1) and ALP (150 μ L, 8 μ g mL-1) incubation at room temperature 2h, BSA (200 μ L, 1wt%) further is added
Close other non-specific sites of the surface AuNPs；It is finally mixed object to be centrifugated with high-speed refrigerated centrifuge, what is obtained is heavy
Starch is at least washed 3 times with PBST with above-mentioned low-temperature centrifugation method.Final Ab2- AuNPs-ALP bioconjugates are divided again
200 μ L 1%BSA are dispersed in, (4 DEG C) spare in refrigerator are placed in.
(4) electrochemical immunological detecting of CEA
10 μ L CEA are added to the MB-Ab of the 20 above-mentioned preparations of μ L11h is incubated in bioconjugates under room temperature；Outside
Add after removing supernatant liquor under magnetic field condition, 40 μ L Ab are added2It is incubated for 1h after-AuNPs-ALP again at room temperature；It obtains
MB-Ab1-CEA-Ab2- AuNPs-ALP bioprobe is dispersed to 10 μ L 0.01M PBS (pH again after washing 3 times with PBST
7.4) in, 10 μ L MB-Ab are taken1-CEA-Ab2The dispersion liquid of-AuNPs-ALP modifies the room temperature condition on the surface ZnO rGO/CPE
Under dry it is spare；The immunological probe electrode that this is constructed first is placed on the 0.1M Tris-HCl containing 0.5mM 1-NPP and 6 μM
After being incubated for 15min progress enzyme digestion reaction in electrolyte solution, with the electricity of Differential Pulse Voltammetry (DPV) record hydrolysate 1-NP
Chemical signal.
(5) measurement of actual sample
Experiment practical blood serum sample (Healthy People and cancer patient) used is by Tongji Medical College, Huazhong Science and Technology Univ.-association
It is provided with hospital, it is spare that blood serum sample first collects supernatant liquor after 4000rpm is centrifuged 5min；With MB- set forth above
Before eElisa method measures blood serum sample, blood serum sample PBST is first diluted 100 times, other testing processes are and said determination
Standard sample CEA's is consistent.In addition, using business method-people CEA kit (RAB0411-1KT, Sigma-Aldrich) into
The content of CEA in blood serum sample is determined in one pacing, to verify the accuracy of this method.
Fig. 1 shows that the MB-eElisa system is incubated for using the washing that magnetic bead base strategy greatly simplifies antigen and antibody
Time；In addition, greatly promoting the detection sensitivity of CEA using ALP enzyme digestion reaction and CTAB enhancement effect.
Fig. 2 shows that the ZnO@rGO nano-complex is by success known to the characteristic diffraction peak of the XRD of ZnO and rGO
Ground preparation.
Fig. 3 shows that ZnO rGO nano-complex SEM figure shows typical graphene crimp shape, furthermore on its surface
Many rodlike ZnO nano particles are loaded, this unique appearance structure can provide big ratio for electrochemical sensing application
Surface area and active site abundant.
Fig. 4 shows the characteristic peak that XPS score confirms that ZnO@rGO contains Zn, O, C three.
Fig. 5 shows that XPS high-resolution power spectrum can further confirm that the rodlike substance for being supported on the surface rGO is ZnO nano
Grain.
Fig. 6 is the differentiated pulse curve of the 1-NP of ZnO@rGO/GCE and ZnO@rGO/CPE, and wherein a and d is without containing 1-
The differentiated pulse curve of NP, b and e are the differentiated pulse curves containing 1-NP, and c, f are to travel further into CTAB to contain the micro- of 1-NP
Divide pulse curve.It is compared with the glass carbon modified electrode ZnO@rGO/GCE of business, reproducible ZnO@rGO/ constructed by the system
CPE for this ALP label enzymolysis product 1-NP measurement have better sensitivity and signal-to-noise ratio, by DPV response signal it is found that
The response current of ZnO@rGO/CPE is 12.5 times of ZnO@rGO/GCE, and its background current is only the 0.05 of ZnO@rGO/GCE
Times；In addition, the introducing of CTAB can improve the signal-to-noise ratio of ZnO@rGO/CPE, and unfavorable shadow is caused to ZnO@rGO/GCE
It rings.
Fig. 7 shows that the standard deviation of more ZnO rGO/CPE response signals is 0.8%, reappears performance better than commercialization
Glass carbon modified electrode.
Fig. 8 shows Ab2Red Shift Phenomena has occurred compared with AuNPs in the ultravioletvisible absorption of-AuNPs-ALP, by nanometer gold surface
Ab known to plasma resonance theory2It is successfully adsorbed on AuNPs with ALP, i.e. Ab2The success of-AuNPs-ALP bioconjugates
Building.
Fig. 9 indicates that after ZnO rGO modification, redox peak current and spike potential difference have significantly to be changed CPE
It is kind, illustrate that ZnO@rGO/CPE has better electron transfer capacity；By MB-Ab1Or ALP-AuNPs-Ab2-CEA-Ab1- MB is repaired
After decorations, redox peak current is remarkably decreased phenomenon, and ALP-AuNPs-Ab2-CEA-Ab1Decline the most after-MB modification
Seriously, this illustrates the successful building of various immune bioprobes and MB-Ab1With ALP-AuNPs-Ab2Between have occurred it is effective double
Antibody sandwich immune response.
Figure 10 indicates that sensitivity of the ALP label MB-eElisa of building on ZnO@rGO/CPE is an advantage over CPE,
CTAB is introduced on the basis of this, the sensing sensitivity of ZnO@rGO/CPE has to be promoted significantly, in addition, being immunized in identical other
Under reaction condition, CEA is not contained only, it is known that MB-Ab1With ALP-AuNPs-Ab2Between be immunoreacted and can not occur, on curve e
Do not find any identifiable response signal, i.e. the MB-eElisa of this method building for CEA measurement close to " zero back
Scape ".
Figure 11 is indicated, by the sensitizative compound schematic diagram of Surfactant CTAB it is found that on hydrophobic carbon paste electrode surface
CTAB and hydrophobic 1-NP phenolic substances have synergistic sorption and enrichment effect, so generate transducing signal sensitization.
Figure 12-15 is indicated, from the optimal conditions figure of the detection CEA of MB-eElisa；Ab2The dosage of-AuNPs-ALP
For 40 μ L, the enzyme digestion reaction time is 15min, and the pH value of electrolyte solution is 8.5, the additional amount of ZnO@rGO in modified electrode
5%.
Figure 16-17 is indicated, by the MB-eElisa detection good linear relationship of CEA it is found that its range of linearity are as follows: 0.01-
6.0ng mL-1, detection limit are as follows: 4.0pg mL-1(S/N=3)；It is compared with existing electrochemical immunoassay method, this method tool
There are lower detection limit and higher sensitivity.In addition, sensing electrode ZnO@rGO/CPE passes through simple polishing treatment
It realizes that sensing interface updates regeneration, then is combined with simple operations and high-throughput MB-eElisa, it can be to a large amount of CEA samples
Realize the measurement quickly with low cost.
Figure 18 indicates that the MB-eElisa sensor of building brings test, response letter again after placing respectively 7 days and 14 days
Number 4.66% and 9.68% are only had dropped compared with original state, shows it with good stability.
Figure 19 indicates that this work has selected potential chaff interferent (insulin, prostate specific antigen, people in human serum
Haemocyanin, human immunoglobulin(HIg), glucose, lactic acid, uric acid, dopamine), even if the condition existing for higher concentration chaff interferent
Under, which is substantially unaffected the response signal of CEA, shows that it has selectivity well.
Table 1
aRef. for using the content of CEA in business method-people's CEA kit measurement blood serum sample.
Table 1 is to measure Healthy People and Patients with Various Cancers sample by this MB-eElisa method and commercial ELISA Kit method
The content of middle CEA, wherein Healthy People (A, B), colon cancer (C), breast cancer (D), lung cancer (E), gastric cancer (F), cancer of pancreas (G).By
Table 1 is contained by CEA in Standard Addition Method for Determination Healthy People and Patients with Various Cancers it is found that using the MB-eElisa sensor
Amount, recovery of standard addition illustrate that this method has good accuracy between 96.4% and 109.7%.In addition, this method is also
The measurement of same sample, result height provided by test result and this method are carried out using commercialized people CEA kit
Unanimously, further illustrate this work propose ALP label MB-eElisa sensor in actual human serum sample for
The sensitivity and accuracy that the measurement of CEA has had.
As it will be easily appreciated by one skilled in the art that the foregoing is merely illustrative of the preferred embodiments of the present invention, not to
The limitation present invention, any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should all include
Within protection scope of the present invention.
Claims (10)
1. a kind of preparation method of the enzyme-linked immunological probe of electrochemistry, which comprises the following steps:
(1) preparation of the bioconjugates of magnetic bead and capture antibody: magnetic bead and capture antibody are incubated for, keep the capture anti-
Body and magnetic bead are covalently attached, and add closed protein, the magnetic bead surfaces are at large to obtain antibody to the closed protein for closing
The group of bonding obtains magnetic bead and captures the bioconjugates of antibody；
(2) preparation of the bioconjugates of nanogold, detection antibody and enzyme: will test antibody and at least one enzyme is added to nanometer
In gold solution, make to detect antibody and enzyme is covalently attached to the nanometer gold surface, add closed protein, the closed protein is used
In the site for closing the nanogold surface exposure, the bioconjugates for obtaining nanogold, detecting antibody and enzyme；
(3) antigen the preparation of the enzyme-linked immunological probe of electrochemistry: is added to the biology of magnetic bead and capture antibody that step (1) obtains
It is incubated in conjugate, the antigen can be combined specifically with the capture antibody and with detection antibody；Adding magnetic outside
After removing supernatant liquor under field condition, the bioconjugates for adding nanogold, detection antibody and enzyme that step (2) obtains are carried out
It is incubated for, arrives the enzyme-linked immunological probe of electrochemistry after double-antibody sandwich reacts.
2. the preparation method of the enzyme-linked immunological probe of electrochemistry as described in claim 1, which is characterized in that step (1) and step
(2) closed protein described in is bovine serum albumin；The enzyme be alkaline phosphatase, DT- diaphorase, horseradish peroxidase or
Glucose oxidase.
3. the enzyme-linked immunological probe of electrochemistry being prepared such as any the method for claim 1-2.
4. a kind of preparation method of electrochemistry Enzyme linked immunosensor, which is characterized in that by electrochemical enzymatic as claimed in claim 3
The dispersant liquid drop of connection immunological probe is coated in basal electrode surface, and the basal electrode is carbon paste electrode, dries to the dispersion liquid
Afterwards, make the modification of the electrochemistry enzyme-linked immunological probe on basal electrode surface to get to electrochemistry Enzyme linked immunosensor.
5. the preparation method of electrochemistry Enzyme linked immunosensor as claimed in claim 4, which is characterized in that the basal electrode
For the carbon paste electrode of zinc oxide graphene nanometer composite modification.
6. the electrochemistry Enzyme linked immunosensor being prepared such as claim 4 or 5 the methods.
7. the application that electrochemistry Enzyme linked immunosensor as claimed in claim 6 is used to detect antigen.
8. the use as claimed in claim 7, which is characterized in that being placed in the electrochemistry Enzyme linked immunosensor containing substrate
Electrolyte in be incubated for, make enzyme in the probe of the substrate and sensor surface that enzyme digestion reaction occur, detect enzymolysis product
Electrochemical signals, and the concentration of the antigen is calculated.
9. application as claimed in claim 8, which is characterized in that contain surfactant in the electrolyte；
Preferably, the surfactant is cetyl trimethylammonium bromide, lauryl sodium sulfate or detergent alkylate sulphur
Sour sodium.
10. application as claimed in claim 8, which is characterized in that the antigen is that carcinomebryonic antigen, alpha-fetoprotein or prostate are special
M-band；The enzyme is alkaline phosphatase, and the substrate is phosphenylic acid salt；
Preferably, the substrate is two based phosphates of 1- naphthyl phosphate or benzene.
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