CN105181953B - Grape-cluster-like nanoparticles, immune probe, and preparation method and applications of immune probe - Google Patents
Grape-cluster-like nanoparticles, immune probe, and preparation method and applications of immune probe Download PDFInfo
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- CN105181953B CN105181953B CN201510363249.3A CN201510363249A CN105181953B CN 105181953 B CN105181953 B CN 105181953B CN 201510363249 A CN201510363249 A CN 201510363249A CN 105181953 B CN105181953 B CN 105181953B
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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
- G01N33/531—Production of immunochemical test materials
Abstract
The present invention discloses grape-cluster-like nanoparticles (GCNPs), wherein a nanometer silver triangular sheet with protein adsorbed on the surface is adopted as a template, chloroauric acid is adopted as an oxidant, and a Galvanic substitution reaction is performed to prepare the grape-cluster-like nanoparticles, and the grape-cluster-like nanoparticles have characteristics of large specific surface area, good dispersion and the like. The present invention further discloses a signal amplification GCNPs-enzyme-immune probe based on the GCNPs, wherein an enzyme-labeled antibody is adsorbed on the GCNPs surface so as to obtain the probe. The present invention further provides applications of the GCNPs-enzyme-immune probe in enzyme linked immunosorbent assay methods. Compared with the conventional enzyme-labeled antibody probe, the GCNPs-enzyme-immune probe of the present invention has the following characteristics that: the signal and the detection sensitivity are significantly improved, and under the premise that the enzyme linked immunosorbent assay method based on the immune probe less changes the traditional detection method cost, the detection performance is significantly improved.
Description
Technical field
The invention belongs to technical field of immunoassay, and in particular to a kind of grape cluster sample nano-particle (GCNPs)-enzyme-is exempted from
The preparation method and application of epidemic disease probe.
Background technology
Enzyme immunoassay technology is a kind of highly developed solid-phase immunoassay pattern, in clinical analysis of diagnosis
Application for many years is obtained, meanwhile, nearly ten years, also obtain in the fields such as environmental monitoring, food safety hazard analyte detection
It is widely applied.The major issue that clinical diagnosises and field of detection of food safety are faced is the immune detection of low concentration antigen,
And the accuracy and reliability of its detection.It is exactly to prepare high-sensitive immunity to visit to improve one of method of sensitivity of immune detection
Pin.
Noble metal nanometer material is a kind of important nano material, not only the gross feature with nano material, while its
The physicochemical properties of itself have also been organically combined with the performance of nano material, with uniform particle diameter, good dispersion, compare table
The characteristic such as area is big, therefore can be used for the preparation of nano immune probe.
At present, in many fields such as environmental monitoring, the analyses of food hazardous material, all to more highly sensitive immune analysis method
With urgent demand, therefore, the detection performance of traditional enzyme-linked immune analytic method is improved, with important using value.
Many research adopts new detection pattern, such as chemiluminescence, electrochemiluminescence, compared with traditional enzyme-linked immune analytic method, it
There is more sensitive power of test, but these immune analysis methods need equipment costly and reagent, therefore, detect into
Originally significantly improve, this greatly hinders its application in many fields such as environmental monitoring, the analyses of food hazardous material.
The content of the invention
For the deficiencies in the prior art, an object of the present invention is to provide a kind of grape cluster sample nanoparticle
(GCNPs) and preparation method thereof.
The second object of the present invention is the purposes for providing the grape cluster sample nanoparticle, and for example, it is preparing immunity
Purposes in probe.
The third object of the present invention is to provide a kind of immunological probe (grape cluster based on the grape cluster sample nanoparticle
Sample nanometer-enzyme-immunological probe, or claim GCNPs- enzymes-immunological probe, or signal amplifies GCNPs- enzymes-immunological probe, referred to as exempts from
Epidemic disease probe) and preparation method thereof.
The fourth object of the present invention is the application for providing the immunological probe in enzyme linked immunosorbent detection, for example, is based on
The enzyme-linked immune detection method of the immunological probe.
To realize aforementioned invention purpose, the technical solution used in the present invention includes：
A kind of preparation method of grape cluster sample nanoparticle (GCNPs), it includes：With protein modified nanometer silver triangular plate
As template, and using gold chloride as oxidant, grape cluster sample nanoparticle is prepared by Jia Fanni substitution reactions.
The GCNPs has the characteristics such as the big, favorable dispersibility of specific surface area.
Wherein, the nanometer silver triangular plate can be prepared using conventional method, for example, refer to Zhang, Q.；Li,N.；
Goebl,J.；Lu,Z.；It is prepared by the documents such as Yin, Y.J.Am.Chem.Soc.2011,133,18931-18939.
Among a preferred embodiment, the preparation method of described grape cluster sample nanoparticle includes：
(1) bovine serum albumin is added mix homogeneously in the solution of the nanometer silver triangular plate that size is 15nm～30nm, is made
The concentration of bovine serum albumin is 0.01mg/mL～5mg/mL in the mixed solution of formation, and bovine serum albumin and nanometer silver triangle
The mol ratio of piece is 5:1～20:1, and stand overnight；
(2) obtain to step (1) and add in mixed reaction solution ascorbic acid, make ascorbic acid in the mixture to be formed
Concentration be after 0.08mM～5mM with the speed implantation concentration of 0.1mL/min～2mL/min as 0.01mM～0.5mM
HAuCl4Solution, obtains grape cluster sample nanoparticle.
The grape cluster sample nanoparticle prepared by preceding method, its particle diameter is 20nm～50nm.
A kind of immunological probe, it includes described grape cluster sample nanoparticle, modifies on the grape cluster sample nanoparticle
There is the antibody (IgG-HRP) of horseradish peroxidase-labeled.
Wherein, IgG-HRP is absorption on GCNPs surfaces.
A kind of preparation method of immunological probe, it includes：Described grape cluster sample nanoparticle is scattered in containing 0.1mM
In the antibody-solutions of 0.01～0.2mg/mL horseradish peroxidase-labeled of～10mM potassium carbonate or sodium carbonate, 0 DEG C～8 DEG C shakes
Swing 1h～5h, after 8000rpm～10000rpm be centrifuged 10min～20min, and then obtain in precipitation it is described immunity visit
Pin.
Further, the preparation method of described immunological probe may also include：The precipitation for obtaining will be centrifuged to contain
The solution of 0.01mg/mL～0.1mg/mL horseradish peroxidases is resuspended, standby.
The IgG-HRP is the horseradish peroxidase of a domain for different testing sample or effective group
The monoclonal antibody of labelling, or polyclonal antibody.
In an exemplary embodiments, the preparation method of the immunological probe can include：0.5-2mLGCNPs is combined
Material 8000rpm is centrifuged 10min, with the 0.01-0.2mg/mLIgG-HRP (horseradish peroxidases containing 0.001M potassium carbonate
The antibody of labelling) solution is resuspended, 4 DEG C of concussion 3h, and 9000rpm centrifugation 15min contain 0.01-0.1mg/mL Radix Cochleariae officinalises with 0.5-1mL
The enzyme mark diluent of peroxidase is resuspended.
Among one more specifically case study on implementation, the preparation method of the immunological probe includes step in detail below：
I. using being template based on protein modified nanometer silver triangular plate, gold chloride is oxidant, is replaced by Jia Fanni
Reaction prepares GCNPs：
A. 200 μ L bovine serum albumen solutions (10mg/mL) are added in 40mL nanometer silver triangular plates (0.1 μM) solution,
Mix homogeneously, stands overnight.
B. take the above-mentioned mixed solutions of 5-10mL, add 825 μ L, 0.01-0.6M ascorbic acid (AA), then by 5-20mL,
The HAuCl of 0.08mM4Entered with the speed injection of 1mL/min, obtain GCNPs；
The preparation of II.GCNPs- enzymes-immunological probe
A. the GCNPs8000rpm centrifugation 10min for 0.5-2mL steps Ib being obtained, with containing 0.001M potassium carbonate
0.01-0.2mg/mLIgG-HRP solution is resuspended, 4 DEG C of concussion 3h, and 9000rpm centrifugation 15min contain 0.01- with 0.5-1mL
The enzyme mark diluent of 0.1mg/mL horseradish peroxidases is resuspended, that is, obtain signal and amplify GCNPs- enzymes-immunological probe.
Because GCNPs has high specific surface area in the present invention, single nano material can simultaneously load multiple enzyme marks
Note antibody molecule, and then the ratio of enzyme and antibody in immunological probe is greatly improved, therefore, carry out immunity point using the immunological probe
Analysis, when particularly carrying out immune detection to micro analysans, can amplify the signal of immune detection, improve detection sensitivity.
Application of the described immunological probe in enzyme linked immunosorbent detection.
A kind of enzyme-linked immune detection method, it includes：
By a series of target antigen standard sample of described immunological probe and variable concentrations in being suitable to make immunological probe
Enzyme linked immunoassay is carried out in the liquid phase environment that contained antibody is combined with the antigenic specificity, and records detection model, thus
The standard corresponding relation set up between immunological probe detection signal and antigen concentration；
And, the testing sample of the immunological probe and the target antigen containing unknown concentration is being suitable to make immunological probe
In carry out enzyme linked immunoassay in the liquid phase environment that combined with the antigenic specificity of contained antibody, and according to detection model and institute
The target antigen concentration stated standard corresponding relation and determine in testing sample.
A kind of indirect competitive enzyme-linked immunosorbent analysis method based on the immunological probe, comprises the following steps：
A. carbonic acid will be dissolved in as the conjugate of the small haptens of envelope antigen and ovalbumin or bovine serum albumin
Salt buffer and form coating buffer, 25 DEG C～37 DEG C incubation 1～4h, then with phosphate buffer washing after, add using as envelope
Molecular weight that close liquid, that concentration is 0.001wt% is that 2000～10000 polyglycol solution is closed overnight in 0 DEG C～8 DEG C；
The phosphate-buffered of the coated antibody solution that b. step a is obtained and a series of small molecule standard substance of variable concentrations
Liquor mixes, and adds the enzyme mark diluent containing small molecular antibody, after 25 DEG C～37 DEG C incubations, is washed with phosphate buffer
Wash, the enzyme mark diluent adopts the pH value comprising 0.1wt% gelatin and 0.05v/v% tween 20s to be for 7.0～8.0, concentration
The phosphate buffer of 0.01M～0.05M；
C. the antibody diluent containing the immunological probe is added in each competitive reaction mixed system for being obtained to step b,
The antibody diluent adopts the pH value comprising 0.1wt% gelatin and 0.05v/v% tween 20s to be for 7.0～8.0, concentration
The phosphate buffer of 0.01M～0.05M, after 25 DEG C～37 DEG C incubation 0.5h～2h, with phosphate buffer washing；
D. it is separately added into nitrite ion in each hybrid reaction system for being obtained to step c, 25 DEG C～37 DEG C colour developing 10min～
30min, adds as terminate liquid, the sulfuric acid solution that concentration is 2M～4M, and each hybrid reaction system is measured afterwards in wavelength
For 450nm～650nm when light absorption value A, set up the standard corresponding relation of A and amount of antigen.
Among one more specifically embodiment, the indirect competitive enzyme-linked immunosorbent analysis method is comprised the following steps：
A. the conjugate using small haptens and ovalbumin or bovine serum albumin is used as envelope antigen, and is with concentration
0.01-0.1M, pH value press 1 for the carbonate buffer solution of 9-10:2000～1:Conduct after 16000 volume ratio is diluted is coated with
Liquid is added in the different holes of ELISA Plate, and 25-37 DEG C of incubation 1-4h, PBST cleaning mixture is washed 3-5 time, is subsequently adding as confining liquid
, concentration for 0.001wt-% molecular weight for 2000-10000 polyglycol solution, 0-8 DEG C closing overnight；
B. the different Kong Zhongzai in aforementioned ELISA Plate are separately added into a series of small molecule standard substance containing variable concentrations
Phosphate buffer, adds the enzyme mark diluent containing small molecular antibody, with PBST washings after 25-37 DEG C of incubation 0.5-2h
Liquid is washed 3～5 times, the enzyme mark diluent adopt the pH value that includes 0.1wt% gelatin and 0.05% (v/v) tween 20 for
7.0-8.0, concentration are the phosphate buffer of 0.01-0.05M；
C. the different Kong Zhongzai in aforementioned ELISA Plate are separately added into the antibody diluent containing the immunological probe, described anti-
It is 0.01- that body diluent adopts the pH value comprising 0.1wt-% gelatin and 0.05% (v/v) tween 20 for 7.0-8.0, concentration
The phosphate buffer of 0.05M, after 25-37 DEG C of incubation 0.5-2h, is washed 3～5 times with PBST cleaning mixture；
D. the different Kong Zhongzai in aforementioned ELISA Plate are separately added into nitrite ion, and 25-37 DEG C of colour developing 10-30min is last per hole
Add as terminate liquid, concentration for 2-4M sulfuric acid solution, then measure each hole hybrid reaction system in wavelength be 450-650nm
When light absorption value A, set up the standard corresponding relation of A and amount of antigen.
Among some case study on implementation, the indirect competitive enzyme-linked immunosorbent analysis method may comprise steps of：
A. the coating of antigen：It is anti-as coating using the conjugate of small haptens and ovalbumin or bovine serum albumin
Original, with the carbonate buffer solution of 0.05M, pH 9.6 by envelope antigen with 1:2000～1:16000 are diluted, used as coating
Liquid, adds 100 μ L, 37 DEG C of incubation 2h, PBST cleaning mixture to wash 3 times in every hole of ELISA Plate, then uses 0.001% molecular weight
For 6000 Polyethylene Glycol PEG as confining liquid, per the μ L of hole 200,4 DEG C of closings are overnight；
B. competitive reaction：Per hole be separately added into standard dilutions phosphate buffer PBS dilute series concentration it
The one μ L of small molecule standard substance 50 in respective enzyme mark hole, by small molecular antibody with enzyme mark diluent：Containing 0.1% gelatin,
The phosphate buffer PBST of 0.05% tween 20, pH 7.4,0.01M, by 1:8000～1:After 64000 dilution proportions, add
In ELISA Plate, per the μ L of hole 50, washed 3～5 times with PBST cleaning mixture after 37 DEG C of incubation 30min；
Plus GCNPs- enzymes-immunological probe c.：With antibody diluent by GCNPs- enzymes-immunological probe with 1:1～1:8 dilutions,
Add 100 μ L per hole, washed 3～5 times with PBST cleaning mixture after 37 DEG C of incubation 1h；
D. develop the color：The μ L of nitrite ion 100,37 DEG C of colour developing 15min are added per hole；It is last that terminate liquid 2M sulfuric acid solution is added per hole
50μL；Light absorption value A when wavelength is 450nm is measured with microplate reader450, set up A450With the relation of amount of antigen.
The uv absorption intensity obtained when afterwards, then with actual sample detecting is true by contrasting with this standard corresponding relation
Determine the antigen concentration in actual sample.
A kind of sandwich ELISA method based on the immunological probe, including：
A. antibody is dissolved in into concentration for 0.01M～0.1M, the carbonate coating buffer that pH value is 9～10, formation albumen
Matter content is the solution of 1 μ g/ml～20 μ g/ml, then at 25 DEG C～37 DEG C incubation 1h～4h, is washed with phosphate buffer afterwards
Wash, add afterwards as confining liquid, the bovine serum albumen solution that concentration is 0.1wt%～5wt%, and in 0 DEG C～8 DEG C closings
Overnight；
The phosphate-buffered of the envelope antigen solution that b. step a is obtained and a series of macromole standard substance of variable concentrations
Liquor mixes, 25 DEG C～37 DEG C incubation 0.5h～2h, afterwards with phosphate buffer washing；
C. the antibody diluent containing the immunological probe is added in each association reaction mixed system for being obtained to step b,
It is 0.01M that the antibody diluent adopts the pH value comprising 0.1wt gelatin and 0.05v/v% tween 20s for 7.0～8.0, concentration
The phosphate buffer of～0.05M, 25 DEG C～37 DEG C incubation 0.5h～3h, with phosphate buffer washing；
D. it is separately added into nitrite ion in each hybrid reaction system for being obtained to step c, 25 DEG C～37 DEG C colour developing 10min～
30min, adds as terminate liquid, the sulfuric acid solution that concentration is 2M～4M, and each hybrid reaction system is measured afterwards in wavelength
For 450nm～650nm when light absorption value A, set up the standard corresponding relation of A and amount of antigen.
Among one more specifically embodiment, the sandwich ELISA method is comprised the following steps：
A. with concentration as 0.01-0.1M, pH value as 9-10 carbonate coating buffer antibody is diluted to form protein
Content is the solution of 1～20 μ g/ml, then is separately added in the different holes of ELISA Plate, 25-37 DEG C of incubation 1-4h, afterwards with PBST
Cleaning mixture is washed 3-5 time, is added as confining liquid, the bovine serum albumen solution that concentration is 0.1-5wt-%, 0-8 DEG C of closing
Overnight；
B. the different Kong Zhongzai in aforementioned ELISA Plate are separately added into a series of phosphoric acid of the macromole standard substance of variable concentrations
Salt buffer, 25-37 DEG C of incubation 0.5-2h, is washed 3-5 time afterwards with PBST cleaning mixture.；
C. the different Kong Zhongzai in aforementioned ELISA Plate are separately added into the antibody diluent containing the immunological probe, described anti-
It is 0.01- that body diluent adopts the pH value comprising 0.1wt% gelatin and 0.05% (v/v) tween 20 for 7.0-8.0, concentration
The phosphate buffer of 0.05M.After 25-37 DEG C of incubation 0.5-2h, washed 3～5 times with PBST cleaning mixture；
D. the different Kong Zhongzai in aforementioned ELISA Plate are separately added into nitrite ion, and 25-37 DEG C of colour developing 10-30min adds work
For terminate liquid, the sulfuric acid solution that concentration is 2-4M, suction of each hybrid reaction system when wavelength is 450-650nm is measured afterwards
Light value A, sets up the standard corresponding relation of A and amount of antigen.
Among some case study on implementation, the sandwich ELISA detection method may comprise steps of：
A. it is coated with：Antibody is diluted to into protein content for 1～20 μ g/ with 0.05M PH9.6 carbonate coating buffer
Ml, adds 100 μ L, 37 DEG C of incubation 2h, PBST cleaning mixture to wash 3 times in every hole of ELISA Plate.With the Ox blood serum egg of 0.1-5%
Bai Zuowei confining liquids, per the μ L of hole 200,4 DEG C of closings are overnight；
B. association reaction：Per hole be separately added into standard dilutions phosphate buffer PBS dilute series concentration it
One macromole standard substance or the μ L of sample 100 that handle well put 37 DEG C of incubation 30min in respective enzyme mark hole.PBST is washed
Wash liquid to wash 3 times.(while do blank well, negative control hole and Positive control wells)；
Plus GCNPs- enzymes-immunological probe c.：With antibody diluent by GCNPs- enzymes-immunological probe with 1:1～1:8 dilutions,
Add 100 μ L per hole, washed 3～5 times with PBST cleaning mixture after 37 DEG C of incubation 1h；
D. develop the color：The μ L of nitrite ion 100,37 DEG C of colour developing 15min are added per hole；It is last that terminate liquid 2M sulfuric acid solution is added per hole
50μL；Light absorption value A when wavelength is 450nm is measured with microplate reader450, set up A450With the relation of amount of antigen.
The uv absorption intensity obtained when afterwards, then with actual sample detecting is true by contrasting with this standard corresponding relation
Determine the antigen concentration in actual sample.
Aforementioned nitrite ion can select suitable type known to industry.
Preferably, the nitrite ion can include：
A liquid：0.933g citric acids, 3.68g disodium hydrogen phosphate dodecahydrates, the hydrogen peroxide of 18 μ L mass fractions 30% is used super
Pure water is settled to 100mL；
B liquid：3,3 ', 5,5 '-tetramethyl benzidines of 60mg are dissolved in 100mL ethylene glycol；
Using it is front by A liquid and B liquid with 5:1 volume ratio mixes.
Compared with prior art compared with the present invention has advantages below：
1st, it is oxidant that the present invention is adopted and is based on protein modified nanometer silver triangular plate for template, gold chloride, all by gal
Buddhist nun's substitution reaction prepares GCNPs, equipment and process is simple, and good dispersion, specific surface area is big；
2nd, the present invention demonstrates the activity of GCNPs- enzymes-immunological probe using enzyme linked immunoassay, and the method is easy to be quick,
It is repeated high；
3rd, GCNPs- enzymes-immunological probe of the invention is sensitive than traditional enzymic-labelled antibody immunological probe signal and detection
Degree is obviously improved；
4th, the enzyme-linked immunoassay analysis based on GCNPs- enzymes-immunological probe of the present invention are in less change traditional detection
On the premise of the cost of method, detection performance is significantly increased.
Description of the drawings
Fig. 1 is the TEM figures of nanometer silver triangular plate in the embodiment of the present invention 1；
Fig. 2 is the TEM figures of GCNPs in the embodiment of the present invention 1；
Fig. 3 a- Fig. 3 b are respectively traditional enzyme linked immunoassay standard curve of ochratoxin A (OTA) and present invention enforcement
The enzyme linked immunoassay standard curve of ochratoxin A (OTA) in example 2；
Fig. 4 a- Fig. 4 b are respectively traditional enzyme linked immunoassay standard curves and the present invention of prostate specific antigen (PSA)
The enzyme linked immunoassay standard curve of prostate specific antigen (PSA) in embodiment 3.
Specific embodiment
Technical scheme is further described in conjunction with some embodiments and accompanying drawing, but the enforcement of the present invention
Mode is not limited to that.
In following embodiments, if no special instructions method therefor is conventional method, agents useful for same such as phosphate-buffered
Liquid, carbonate buffer solution, nitrite ion etc. also may each be the known application type of industry, and, if not the following % for occurring is special
Do not mentionlet alone bright, refer both to wt%.
Embodiment 1：The preparation of grape cluster sample nanoparticle (GCNPs)-enzyme-immunological probe
A. the preparation of nanometer silver triangular plate：40mL water solution systems, including 400 μ L, 0.01M silver nitrate solutions, 600 μ L,
0.1M citric acid three sodium solutions, 96 μ L, 30wt%H2O2Strong stirring 10min under room temperature.Then, 400 μ L, 0.1M are rapidly injected
Sodium borohydride solution.
B. 200 μ L bovine serum albumen solutions (10mg/mL) are added in nanometer silver triangular plate colloid solution, mixing is equal
It is even, stand overnight.
C. take the above-mentioned mixed solutions of 8.2mL, add 825 μ L, 0.015M AA, then by 10mL, the HAuCl of 0.08mM4With
The speed injection of 1mL/min enters, and obtains GCNPs particles.
D. 1mL GCNPs particles 8000rpm is centrifuged into 10min, with the 0.1mg/mL Radix Cochleariae officinalises mistakes containing 0.001M potassium carbonate
The antibody-solutions of oxide enzyme labelling are resuspended, 4 DEG C of concussion 3h, and 9000rpm centrifugation 15min contain 0.05mg/mL peppery with 0.5mL
The enzyme mark diluent of root peroxidase is resuspended, that is, obtain GCNPs- enzymes-immunological probe.
Embodiment 2：The detection of ochratoxin A (OTA)
The synthesis of a.OTA envelope antigens adopts carbodiimides (CDI) method.Carboxyl on OTA molecules, can be lived by CDI
After change, it is coupled on the lysine amino on protein molecular.The key step of synthesis, the OTA of 1mg is dissolved in 0.2mL anhydrous two
In methyl sulfoxide, 2mg CDI are added, at room temperature, after magnetic agitation dissolving, lucifuge continues to react 10min；The egg white egg of 5mg
In vain (OVA), in being dissolved into the carbonate buffer solution of 1mL pH9.6, magnetic agitation dissolving.By the OTA solution for having activated, dropwise add
Enter in OVA solution.After adding, continue magnetic agitation, lucifuge room temperature reaction 4h.After reaction terminates, reaction solution is in CBS solution
Middle dialysis 3 times, to remove unreacted OTA and other small molecules；Last time dialysis solution changes PB (phosphate buffer) into.Close
Into envelope antigen subpackage after, be placed in -20 DEG C of preservations.
B. the coating of antigen：It is slow with the carbonate of 0.05M, pH 9.6 using the conjugate of OTA and OVA as envelope antigen
Liquid is rushed by envelope antigen with 1:2000～1:16000 are diluted, and as coating buffer, 100 μ L are added in every hole of ELISA Plate,
37 DEG C of incubation 2h, then with the Polyethylene Glycol PEG that 0.001% molecular weight is 6000 as confining liquid, 4 DEG C of closings are overnight；
C. competitive reaction：The 0.01ng/ diluted with OTA standard dilutions phosphate buffers PBS is separately added into per hole
The small molecule of one of mL, 0.02ng/mL, 0.05ng/mL, 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, 1ng/mL series concentration
Standard substance or the μ L of sample 50 that handle well in respective enzyme mark hole, by small molecular antibody with enzyme mark diluent：It is bright containing 0.1%
Glue, 0.05% (v/v) tween 20, the phosphate buffer PBST of pH 7.4,0.01M, by 1:8000～1:64000 ratios are dilute
After releasing, in adding ELISA Plate, per the μ L of hole 50, washed 3～5 times with PBST cleaning mixture after 37 DEG C of incubation 30min；
D. plus the ELIAS secondary antibody of gold silver nano composite material has been adsorbed：Plus gold silver nanometer will be adsorbed with antibody diluent to answer
The ELIAS secondary antibody of condensation material is with 1:1～1:8 dilutions, 100 μ L are added per hole, and 3～5 are washed with PBST cleaning mixture after 37 DEG C of incubation 1h
It is secondary；
E. develop the color：The μ L of nitrite ion 100,37 DEG C of colour developing 15min are added per hole；It is last that terminate liquid 2M sulfuric acid solution is added per hole
50μL；Light absorption value A when wavelength is 450nm is measured with microplate reader450, with the contrast of made standard curve OTA in testing sample is calculated
Concentration.Refering to Fig. 3 a- Fig. 3 b, the IC of OTA detections is obtained in the present embodiment50For 0.05ng/mL, than traditional ELISA method
The IC of detection500.27ng/mL improves 5 times.
Embodiment 3：The detection of prostate specific antigen (PSA)
A. it is coated with：PSA antibody is diluted to into protein content for 1～20 μ with 0.05M PH9.6 carbonate coating buffer
G/ml, adds 100 μ L, 37 DEG C of incubation 2h, PBST cleaning mixture to wash 3 times in every hole of ELISA Plate.With the Ox blood serum of 0.1-5%
, used as confining liquid, per the μ L of hole 200,4 DEG C of closings are overnight for albumen；
B. association reaction：Per hole be separately added into standard dilutions phosphate buffer PBS dilute series concentration it
One PSA standard substance or the μ L of sample 100 that handle well put 37 DEG C of incubation 30min in respective enzyme mark hole.PBST cleaning mixture is washed
Wash 3 times.(while do blank well, negative control hole and Positive control wells)；
The combination of c.GCNPs- enzymes-immunological probe：With antibody diluent by GCNPs- enzymes-immunological probe with 1:1～1:8 is dilute
Release, 100 μ L are added per hole, washed 3～5 times with PBST cleaning mixture after 37 DEG C of incubation 1h；
D. develop the color：The μ L of nitrite ion 100,37 DEG C of colour developing 15min are added per hole；It is last that terminate liquid 2M sulfuric acid solution is added per hole
50μL；Light absorption value A when wavelength is 450nm is measured with microplate reader450, set up A450With the relation of amount of antigen.It is bent with made standard
Line contrast calculates the concentration of PSA in testing sample.Refering to Fig. 4 a- Fig. 4 b, the IC of PSA detections is obtained in the present embodiment50For 2ng/
ML, than the IC that traditional ELISA method is detected500.1ng/mL improves 20 times.
It should be noted that herein, term " including ", "comprising" or its any other variant are intended to non-row
His property is included, so that a series of process, method, article or equipment including key elements not only include those key elements, and
And also include other key elements being not expressly set out, or also include for this process, method, article or equipment institute inherently
Key element.In the absence of more restrictions, the key element for being limited by sentence "including a ...", it is not excluded that including institute
Also there is other identical element in process, method, article or the equipment of stating key element.
The above is only the specific embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
1. a kind of preparation method of grape cluster sample nanoparticle, it is characterised in that include：
(1) bovine serum albumin is added mix homogeneously in the solution of the nanometer silver triangular plate that size is 15nm～30nm, makes to be formed
Mixed solution in the concentration of bovine serum albumin be 0.01mg/mL～5mg/mL, and bovine serum albumin and nanometer silver triangular plate
Mol ratio is 5:1～20:1, and stand overnight；
(2) obtain to step (1) and add in mixed reaction solution ascorbic acid, make the concentration of ascorbic acid in the mixture to be formed
For 0.08mM～5mM, afterwards with the speed implantation concentration of 0.1mL/min～2mL/min as 0.01mM～HAuCl of 0.5mM4It is molten
Liquid, obtains grape cluster sample nanoparticle.
2. the grape cluster sample nanoparticle for being prepared by claim 1 methods described, its particle diameter is 20nm～50nm.
3. a kind of immunological probe, it is characterised in that comprising the grape cluster sample nanoparticle described in claim 2, the grape cluster sample
The antibody of horseradish peroxidase-labeled is modified with nanoparticle, the antibody of the horseradish peroxidase-labeled includes Radix Cochleariae officinalises
Peroxidase labelling monoclonal antibody or polyclonal antibody.
4. a kind of preparation method of immunological probe, it is characterised in that include：By the grape cluster sample nanoparticle described in claim 2
The antibody for being scattered in the 0.01～0.2mg/mL horseradish peroxidase-labeled containing 0.1mM～10mM potassium carbonate or sodium carbonate is molten
In liquid, 0 DEG C～8 DEG C concussion 1h～5h, 10min～20min is centrifuged after 8000rpm～10000rpm, and then in precipitation
Obtain the immunological probe.
5. the preparation method of immunological probe according to claim 4, it is characterised in that also include：The precipitation that centrifugation is obtained
It is resuspended with the solution containing 0.01mg/mL～0.1mg/mL horseradish peroxidases, it is standby.
6. immunological probe described in claim 3 or the immunological probe that prepared by any one of claim 4-5 method are enzyme-linked
Application in immune detection.
7. a kind of enzyme-linked immune detection method, it is characterised in that include：
It is with one by the immunological probe described in claim 3 or by the immunological probe of any one of claim 4-5 method preparation
The target antigen standard sample of row variable concentrations contained antibody in being suitable to make immunological probe is combined with the antigenic specificity
Enzyme linked immunoassay is carried out in liquid phase environment, and records detection model, thus set up immunological probe detection signal and antigen concentration
Between standard corresponding relation；
And, by the testing sample of the immunological probe and the target antigen containing unknown concentration in being suitable to make immunological probe institute
Enzyme linked immunoassay is carried out in the liquid phase environment combined with the antigenic specificity containing antibody, and according to detection model and the mark
Quasi- corresponding relation and determine the target antigen concentration in testing sample.
8. a kind of immunological probe based on described in claim 3 or the immunity spy by the preparation of any one of claim 4-5 method
The indirect competitive enzyme-linked immunosorbent analysis method of pin, it is characterised in that comprise the following steps：
A. carbonate will be dissolved in the conjugate of ovalbumin or bovine serum albumin as the small haptens of envelope antigen to delay
Rush liquid and form coating buffer, and add in the different holes of ELISA Plate, 25 DEG C～37 DEG C 1～4h of incubation, then with phosphate buffer
After washing, add with polyglycol solution that the molecular weight as confining liquid, concentration as 0.001wt% is 2000～10000
In 0 DEG C～8 DEG C closings overnight；
B. it is a series of phosphate buffer of the small molecule standard substance of variable concentrations and step a being obtained, be distributed in before
The envelope antigen solution mixing in the different holes of ELISA Plate is stated, the enzyme mark diluent containing small molecular antibody is added, 25 DEG C～37
After DEG C incubation, with phosphate buffer washing, the enzyme mark diluent using comprising 0.1wt% gelatin and 0.05v/v% tweens-
20 pH value is the phosphate buffer that 7.0～8.0, concentration is 0.01M～0.05M；
C. it is being obtained to step b, be distributed in each competitive reaction mixed system in the different holes of aforementioned ELISA Plate to add and contain
The antibody diluent of the immunological probe, the antibody diluent is using comprising 0.1wt% gelatin and 0.05v/v% tween 20s
PH value be 7.0～8.0, concentration be 0.01M～0.05M phosphate buffer, 25 DEG C～37 DEG C incubation 0.5h～2h after, with
Phosphate buffer is washed；
D. it is being obtained to step c, be distributed in each hybrid reaction system in the different holes of aforementioned ELISA Plate and be separately added into colour developing
Liquid, 25 DEG C～37 DEG C colour developing 10min～30min, add as terminate liquid, concentration for 2M～4M sulfuric acid solution, afterwards
Light absorption value A of each hybrid reaction system when wavelength is 450nm～650nm is measured, A passes corresponding with the standard of amount of antigen are set up
System.
9. a kind of immunological probe based on described in claim 3 or the immunity spy by the preparation of any one of claim 4-5 method
The sandwich ELISA method of pin, it is characterised in that comprise the following steps：
A., antibody is dissolved in the different holes for being distributed in ELISA Plate, concentration for 0.01M～0.1M, the carbonic acid that pH value is 9～10
Salt is coated with buffer, forms the solution that protein content is 1 μ g/ml～20 μ g/ml, then at 25 DEG C～37 DEG C incubation 1h～4h,
Afterwards with phosphate buffer washing, add afterwards as confining liquid, the bovine serum albumin that concentration is 0.1wt%～5wt%
Solution, and in 0 DEG C～8 DEG C closings overnight；
B. it is a series of phosphate buffered solution of the macromole standard substance of variable concentrations and step a being obtained, be distributed in enzyme mark
Coated antibody solution mixing in the different holes of plate, 25 DEG C～37 DEG C incubation 0.5h～2h, afterwards with phosphate buffer washing；
C. it is being obtained to step b, be distributed in each association reaction mixed system in the different holes of aforementioned ELISA Plate to add and contain
The antibody diluent of the immunological probe, the antibody diluent is using comprising 0.1wt% gelatin and 0.05v/v% tween 20s
PH value be 7.0～8.0, concentration be 0.01M～0.05M phosphate buffer, 25 DEG C～37 DEG C incubation 0.5h～3h, with phosphorus
Phthalate buffer is washed；
D. it is being obtained to step c, be distributed in each hybrid reaction system in the different holes of aforementioned ELISA Plate and be separately added into colour developing
Liquid, 25 DEG C～37 DEG C colour developing 10min～30min, add as terminate liquid, concentration for 2M～4M sulfuric acid solution, afterwards
Light absorption value A of each hybrid reaction system when wavelength is 450nm～650nm is measured, A passes corresponding with the standard of amount of antigen are set up
System.
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