CN105181953A - 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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- CN105181953A CN105181953A CN201510363249.3A CN201510363249A CN105181953A CN 105181953 A CN105181953 A CN 105181953A CN 201510363249 A CN201510363249 A CN 201510363249A CN 105181953 A CN105181953 A CN 105181953A
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- 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
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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, be specifically related to a kind of preparation method and application of grape cluster sample nano particle (GCNPs)-enzyme-immunological probe.
Background technology
Enzyme-linked immuno assay technology is a kind of very ripe solid-phase immunoassay pattern, has obtained application for many years in clinical analysis of diagnosis, meanwhile, nearly ten years, also obtains a wide range of applications in the fields such as environmental monitoring, the survey of food safety hazard quality testing.The major issue that clinical diagnosis and field of detection of food safety face is the immune detection of low concentration antigen, and its accuracy detected and reliability.Improving one of method of sensitivity of immune detection is exactly prepare high-sensitive immunological probe.
Noble metal nanometer material is a kind of important nano material, not only there is the macrofeature of nano material, the physicochemical property of himself have also organically combined with the performance of nano material simultaneously, there is uniform particle diameter, good dispersion, the characteristics such as specific surface area is large, 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, there is urgent demand, therefore, improve the detection perform of traditional enzyme-linked immune analytic method, there is important using value.Much research adopts new detecting pattern, as chemiluminescence, electrochemiluminescence, compared with traditional enzyme-linked immune analytic method, they have sensitiveer detectability, but these immune analysis methods need equipment costly and reagent, therefore, testing cost significantly improves, and this greatly hinders its application in many fields such as environmental monitoring, the analyses of food hazardous material.
Summary 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 nano particle (GCNPs) and preparation method thereof.
Two of object of the present invention is the purposes providing described grape cluster sample nano particle, and such as, it is preparing the purposes in immunological probe.
Three of object of the present invention is to provide a kind of immunological probe based on described grape cluster sample nano particle (grape cluster sample nanometer-enzyme-immunological probe, or claim GCNPs-enzyme-immunological probe, or signal amplifies GCNPs-enzyme-immunological probe, be called for short immunological probe) and preparation method thereof.
Four of object of the present invention is to provide the application of described immunological probe in enzyme linked immunosorbent detection, such as, based on the enzyme-linked immune detection method of described immunological probe.
For realizing aforementioned invention object, the technical solution used in the present invention comprises:
A kind of preparation method of grape cluster sample nano particle (GCNPs), it comprises: using protein modified Nano Silver triangular plate as template, and using gold chloride as oxygenant, prepares grape cluster sample nano particle by Jia Fanni substitution reaction.
Described GCNPs has the characteristics such as specific surface area is large, favorable dispersibility.
Wherein, described Nano Silver triangle can adopt conventional method to prepare, such as can with reference to Zhang, Q.; Li, N.; Goebl, J.; Lu, Z.; Yin, Y.J.Am.Chem.Soc.2011, the document preparations such as 133,18931-18939.
Among a better embodiment, the preparation method of described grape cluster sample nano particle comprises:
(1) bovine serum albumin is added in the solution of the Nano Silver triangle being of a size of 15nm ~ 30nm and mix, the concentration of bovine serum albumin in the mixed solution of formation is made to be 0.01mg/mL ~ 5mg/mL, and the mol ratio of bovine serum albumin and Nano Silver triangle is 5:1 ~ 20:1, and hold over night;
(2) obtain in mixed reaction solution to step (1) and add ascorbic acid, make the concentration of ascorbic acid in the potpourri of formation be 0.08mM ~ 5mM after the HAuCl that is 0.01mM ~ 0.5mM with the speed implantation concentration of 0.1mL/min ~ 2mL/min
4solution, obtains grape cluster sample nano particle.
The grape cluster sample nano particle prepared by preceding method, its particle diameter is 20nm ~ 50nm.
A kind of immunological probe, it comprises described grape cluster sample nano particle, described grape cluster sample nano particle is modified with the antibody (IgG-HRP) of horseradish peroxidase-labeled.
Wherein, IgG-HRP is adsorbed on GCNPs surface.
A kind of preparation method of immunological probe, it comprises: by described grape cluster sample nanoparticle dispersion in the antibody-solutions of the 0.01 ~ 0.2mg/mL horseradish peroxidase-labeled containing 0.1mM ~ 10mM sal tartari or sodium carbonate, 0 DEG C ~ 8 DEG C concussion 1h ~ 5h, afterwards in the centrifugal 10min ~ 20min of 8000rpm ~ 10000rpm, and then obtain described immunological probe in precipitation.
Further, the preparation method of described immunological probe also can comprise: the precipitation of centrifugal acquisition is resuspended with the solution containing 0.01mg/mL ~ 0.1mg/mL horseradish peroxidase, for subsequent use.
Described IgG-HRP is the monoclonal antibody for a domain of different testing sample or the horseradish peroxidase-labeled of effective group, or polyclonal antibody.
In an exemplary embodiments, the preparation method of described immunological probe can comprise: by the centrifugal 10min of 0.5-2mLGCNPs compound substance 8000rpm, resuspended with 0.01-0.2mg/mLIgG-HRP (antibody of the horseradish peroxidase-labeled) solution containing 0.001M sal tartari, 4 DEG C of concussion 3h, the centrifugal 15min of 9000rpm, the enzyme mark dilution containing 0.01-0.1mg/mL horseradish peroxidase with 0.5-1mL is resuspended.
Among one more specifically case study on implementation, the preparation method of described immunological probe comprises following concrete steps:
I. utilizing based on protein modified Nano Silver triangular plate is template, and gold chloride is oxygenant, prepares GCNPs by Jia Fanni substitution reaction:
A. 200 μ L bovine serum albumen solution (10mg/mL) are joined in 40mL Nano Silver triangle (0.1 μM) solution, mix, hold over night.
B. get the above-mentioned mixed solution of 5-10mL, add 825 μ L, 0.01-0.6M ascorbic acid (AA), then by the HAuCl of 5-20mL, 0.08mM
4enter with the speed injection of 1mL/min, obtain GCNPs;
The preparation of II.GCNPs-enzyme-immunological probe
A. the centrifugal 10min of GCNPs8000rpm 0.5-2mL step Ib obtained, resuspended with the 0.01-0.2mg/mLIgG-HRP solution containing 0.001M sal tartari, 4 DEG C of concussion 3h, the centrifugal 15min of 9000rpm, the enzyme mark dilution containing 0.01-0.1mg/mL horseradish peroxidase with 0.5-1mL is resuspended, namely obtains signal and amplifies GCNPs-enzyme-immunological probe.
Because GCNPs has high specific surface area in the present invention, single nano material can the simultaneously multiple enzymic-labelled antibody molecule of load, and then greatly improve the ratio of enzyme and antibody in immunological probe, therefore, this immunological probe is used to carry out immunoassay, when particularly immune detection being carried out to the analysans of trace, the signal of immune detection can be amplified, improve detection sensitivity.
The application of described immunological probe in enzyme linked immunosorbent detection.
A kind of enzyme-linked immune detection method, it comprises:
The target antigen standard model of described immunological probe and a series of variable concentrations is being suitable for making carrying out enzyme linked immunoassay in the liquid phase environment that in immunological probe, contained antibody is combined with described antigentic specificity, and record detects model, sets up the standard corresponding relation between immunological probe detection signal and antigen concentration thus;
And, the testing sample of described immunological probe and the target antigen containing unknown concentration is being suitable for making carrying out enzyme linked immunoassay in the liquid phase environment that in immunological probe, contained antibody is combined with described antigentic specificity, and foundation detection model and described standard corresponding relation and determine the target antigen concentration in testing sample.
Based on an indirect competitive enzyme-linked immunosorbent analytical approach for described immunological probe, comprise the following steps:
A. be dissolved in carbonate buffer solution using as the small haptens of envelope antigen and the conjugate of ovalbumin or bovine serum albumin and form coating buffer, hatch 1 ~ 4h for 25 DEG C ~ 37 DEG C, again with after phosphate buffer washing, add using as confining liquid, molecular weight that concentration is 0.001wt% be 2000 ~ 10000 polyglycol solution to close in 0 DEG C ~ 8 DEG C and spend the night;
B. the envelope antigen solution obtained by step a mixes with the phosphate buffer of the Small molecular standard items of a series of variable concentrations, add the enzyme mark dilution containing small molecular antibody again, 25 DEG C ~ 37 DEG C hatch after, with phosphate buffer washing, described enzyme mark dilution adopts that the pH value comprising 0.1wt% gelatin and 0.05v/v% Tween-20 is 7.0 ~ 8.0, concentration is the phosphate buffer of 0.01M ~ 0.05M;
C. the antibody diluent containing described immunological probe is added in each competitive reaction mixed system obtained to step b, described antibody diluent adopts that the pH value comprising 0.1wt% gelatin and 0.05v/v% Tween-20 is 7.0 ~ 8.0, concentration is the phosphate buffer of 0.01M ~ 0.05M, 25 DEG C ~ 37 DEG C hatch 0.5h ~ 2h after, wash with phosphate buffer;
D. nitrite ion is added respectively in each hybrid reaction system obtained to step c, 25 DEG C ~ 37 DEG C colour developing 10min ~ 30min, add again as stop buffer, concentration is the sulfuric acid solution of 2M ~ 4M, measure the light absorption value A of each hybrid reaction system when wavelength is 450nm ~ 650nm afterwards, set up the standard corresponding relation of A and antigen amount.
Among one more specifically embodiment, described indirect competitive enzyme-linked immunosorbent analytical approach comprises the following steps:
A. using the conjugate of small haptens and ovalbumin or bovine serum albumin as envelope antigen, and be 0.01-0.1M with concentration, the pH value carbonate buffer solution that is 9-10 adds in the different holes of ELISA Plate as coating buffer after diluting by the volume ratio of 1:2000 ~ 1:16000, hatch 1-4h for 25-37 DEG C, PBST cleansing solution washing 3-5 time, then add as confining liquid, concentration be the molecular weight of 0.001wt-% is the polyglycol solution of 2000-10000, close for 0-8 DEG C and spend the night;
B. the phosphate buffer of the Small molecular standard items containing a series of variable concentrations is added respectively at the different Kong Zhongzai of aforementioned ELISA Plate, add the enzyme mark dilution containing small molecular antibody again, hatch with PBST cleansing solution washing 3 ~ 5 times after 0.5-2h for 25-37 DEG C, described enzyme mark dilution adopts that the pH value comprising 0.1wt% gelatin and 0.05% (v/v) Tween-20 is 7.0-8.0, concentration is the phosphate buffer of 0.01-0.05M;
C. the antibody diluent containing described immunological probe is added respectively at the different Kong Zhongzai of aforementioned ELISA Plate, described antibody diluent adopts that the pH value comprising 0.1wt-% gelatin and 0.05% (v/v) Tween-20 is 7.0-8.0, concentration is the phosphate buffer of 0.01-0.05M, 25-37 DEG C hatch 0.5-2h after, with PBST cleansing solution washing 3 ~ 5 times;
D. nitrite ion is added respectively at the different Kong Zhongzai of aforementioned ELISA Plate, 25-37 DEG C of colour developing 10-30min, last every hole add as stop buffer, concentration is the sulfuric acid solution of 2-4M, measure the light absorption value A of each hole hybrid reaction system when wavelength is 450-650nm again, set up the standard corresponding relation of A and antigen amount.
Among some case study on implementation, described indirect competitive enzyme-linked immunosorbent analytical approach can comprise the following steps:
A. the bag quilt of antigen: using the conjugate of small haptens and ovalbumin or bovine serum albumin as envelope antigen, with the carbonate buffer solution of 0.05M, pH9.6, envelope antigen is diluted with 1:2000 ~ 1:16000, as coating buffer, 100 μ L are added in every hole of ELISA Plate, hatch 2h for 37 DEG C, PBST cleansing solution washs 3 times, is then that the polyglycol PEG of 6000 is as confining liquid with 0.001% molecular weight, every hole 200 μ L, closes for 4 DEG C and spends the night;
B. competitive reaction: every hole adds with the Small molecular standard items 50 μ L of one of the series concentration of standard dilutions phosphate buffer PBS dilution respectively in respective enzyme mark hole, by small molecular antibody with enzyme mark dilution: containing 0.1% gelatin, the phosphate buffer PBST of 0.05% Tween-20, pH7.4,0.01M, after 1:8000 ~ 1:64000 dilution proportion, add in ELISA Plate, every hole 50 μ L, hatches after 30min with PBST cleansing solution washing 3 ~ 5 times for 37 DEG C;
C. add GCNPs-enzyme-immunological probe: with antibody diluent by GCNPs-enzyme-immunological probe with 1:1 ~ 1:8 dilution, every hole adds 100 μ L, hatches after 1h with PBST cleansing solution washing 3 ~ 5 times for 37 DEG C;
D. develop the color: every hole adds nitrite ion 100 μ L, 37 DEG C of colour developing 15min; Last every hole adds stop buffer 2M sulfuric acid solution 50 μ L; Light absorption value A when being 450nm with microplate reader measurement wavelength
450, set up A
450with the relation of antigen amount.
Afterwards, then the uv absorption intensity obtained when detecting with actual sample therewith standard corresponding relation contrast the antigen concentration can determined in actual sample.
Based on a sandwich ELISA method for described immunological probe, comprising:
A. antibody is dissolved in the carbonate bag that concentration is 0.01M ~ 0.1M, pH value is 9 ~ 10 and is buffered liquid, form the solution that protein content is 1 μ g/ml ~ 20 μ g/ml, 1h ~ 4h is hatched again in 25 DEG C ~ 37 DEG C, wash with phosphate buffer afterwards, add afterwards as confining liquid, concentration is the bovine serum albumen solution of 0.1wt% ~ 5wt%, and close in 0 DEG C ~ 8 DEG C and spend the night;
B. the envelope antigen solution obtained by step a mixes with the phosphate buffer of the large molecular criteria product of a series of variable concentrations, hatches 0.5h ~ 2h, washs afterwards with phosphate buffer for 25 DEG C ~ 37 DEG C;
C. the antibody diluent containing described immunological probe is added in each association reaction mixed system obtained to step b, described antibody diluent adopts that the pH value comprising 0.1wt gelatin and 0.05v/v% Tween-20 is 7.0 ~ 8.0, concentration is the phosphate buffer of 0.01M ~ 0.05M, hatch 0.5h ~ 3h, wash with phosphate buffer for 25 DEG C ~ 37 DEG C;
D. nitrite ion is added respectively in each hybrid reaction system obtained to step c, 25 DEG C ~ 37 DEG C colour developing 10min ~ 30min, add again as stop buffer, concentration is the sulfuric acid solution of 2M ~ 4M, measure the light absorption value A of each hybrid reaction system when wavelength is 450nm ~ 650nm afterwards, set up the standard corresponding relation of A and antigen amount.
Among one more specifically embodiment, described sandwich ELISA method comprises the following steps:
A. be 0.01-0.1M with concentration, the pH value carbonate bag that is 9-10 is buffered liquid and antibody dilution formed the solution that protein content is 1 ~ 20 μ g/ml, add respectively again in the different holes of ELISA Plate, hatch 1-4h for 25-37 DEG C, afterwards with PBST cleansing solution washing 3-5 time, add again as confining liquid, concentration is the bovine serum albumen solution of 0.1-5wt-%, close for 0-8 DEG C and spend the night;
B. add the phosphate buffer of the large molecular criteria product of a series of variable concentrations at the different Kong Zhongzai of aforementioned ELISA Plate respectively, hatch 0.5-2h for 25-37 DEG C, afterwards with PBST cleansing solution washing 3-5 time.；
C. add the antibody diluent containing described immunological probe respectively at the different Kong Zhongzai of aforementioned ELISA Plate, described antibody diluent adopts that the pH value comprising 0.1wt% gelatin and 0.05% (v/v) Tween-20 is 7.0-8.0, concentration is the phosphate buffer of 0.01-0.05M.25-37 DEG C hatch 0.5-2h after, with PBST cleansing solution washing 3 ~ 5 times;
D. nitrite ion is added respectively at the different Kong Zhongzai of aforementioned ELISA Plate, 25-37 DEG C of colour developing 10-30min, add again as stop buffer, concentration is the sulfuric acid solution of 2-4M, measure the light absorption value A of each hybrid reaction system when wavelength is 450-650nm afterwards, set up the standard corresponding relation of A and antigen amount.
Among some case study on implementation, described sandwich ELISA detection method can comprise the following steps:
A. quilt is wrapped: being buffered liquid by antibody dilution to protein content with 0.05MPH9.6 carbonate bag is 1 ~ 20 μ g/ml, and in every hole of ELISA Plate, add 100 μ L, hatch 2h for 37 DEG C, PBST cleansing solution washs 3 times.With the bovine serum albumin of 0.1-5% as confining liquid, every hole 200 μ L, closes for 4 DEG C and spends the night;
B. association reaction: the large molecular criteria product that every hole adds one of the series concentration of diluting with standard dilutions phosphate buffer PBS respectively or the sample 100 μ L that handles well, in respective enzyme mark hole, put 37 DEG C and hatch 30min.PBST cleansing solution washs 3 times.(doing blank well, negative control hole and Positive control wells) simultaneously;
C. add GCNPs-enzyme-immunological probe: with antibody diluent by GCNPs-enzyme-immunological probe with 1:1 ~ 1:8 dilution, every hole adds 100 μ L, hatches after 1h with PBST cleansing solution washing 3 ~ 5 times for 37 DEG C;
D. develop the color: every hole adds nitrite ion 100 μ L, 37 DEG C of colour developing 15min; Last every hole adds stop buffer 2M sulfuric acid solution 50 μ L; Light absorption value A when being 450nm with microplate reader measurement wavelength
450, set up A
450with the relation of antigen amount.
Afterwards, then the uv absorption intensity obtained when detecting with actual sample therewith standard corresponding relation contrast the antigen concentration can determined in actual sample.
The suitable type that aforementioned nitrite ion can select industry known.
Preferably, described nitrite ion can comprise:
A liquid: 0.933g citric acid, 3.68g disodium hydrogen phosphate dodecahydrate, 18 μ L massfraction 30% hydrogen peroxide ultrapure waters are settled to 100mL;
B liquid: 60mg3,3 ', 5,5 '-tetramethyl benzidine is dissolved in 100mL ethylene glycol;
Before using, A liquid is mixed with 5:1 volume ratio with B liquid.
Compared with prior art comparatively, the present invention has the following advantages:
1, the present invention adopt based on protein modified Nano Silver triangular plate be template, gold chloride is oxygenant, prepares GCNPs by Jia Fanni substitution reaction, equipment and process is simple, good dispersion, and specific surface area is large;
2, the present invention adopts enzyme linked immunoassay to demonstrate the activity of GCNPs-enzyme-immunological probe, and the method is fast easy, and repeatability is high;
3, GCNPs-enzyme-immunological probe of the present invention significantly promotes than traditional enzymic-labelled antibody immunological probe signal and detection sensitivity;
4, based on the enzyme-linked immunoassay analysis of GCNPs-enzyme-immunological probe of the present invention under the prerequisite of the cost of less change traditional detection method, detection perform improves significantly.
Accompanying drawing explanation
Fig. 1 is the TEM figure of Nano Silver triangular plate in the embodiment of the present invention 1;
Fig. 2 is the TEM figure of GCNPs in the embodiment of the present invention 1;
Fig. 2 a-Fig. 2 b is the enzyme linked immunoassay typical curve of OTA in traditional enzyme linked immunoassay typical curve of ochratoxin A (OTA) and embodiment 2 respectively;
Fig. 3 a-Fig. 3 b is the enzyme linked immunoassay typical curve of ochratoxin A (OTA) in traditional enzyme linked immunoassay typical curve of ochratoxin A (OTA) and the embodiment of the present invention 2 respectively;
Fig. 4 is the enzyme linked immunoassay typical curve of prostate specific antigen (PSA) in traditional enzyme linked immunoassay typical curve of prostate specific antigen (PSA) and the embodiment of the present invention 3 respectively.
Embodiment
Now in conjunction with some embodiments and accompanying drawing, technical scheme of the present invention is further described, but embodiments of the present invention are not limited in this.
In following embodiment, method therefor is conventional method if no special instructions, agents useful for same such as phosphate buffer, carbonate buffer solution, nitrite ion etc. also can be all the known application types of industry, and if not the % special instruction occurred following then all refers to wt%.
Embodiment 1: the preparation of grape cluster sample nano particle (GCNPs)-enzyme-immunological probe
A. the preparation of Nano Silver triangle: 40mL water solution system, comprises 400 μ L, 0.01M liquor argenti nitratis ophthalmicus, 600 μ L, 0.1M citric acid three sodium solution, 96 μ L, 30wt%H
2o
2strong stirring 10min under room temperature.Then, 400 μ L are injected fast, 0.1M sodium borohydride solution.
B. 200 μ L bovine serum albumen solution (10mg/mL) are joined in Nano Silver triangle rubber liquid solution, mix, hold over night.
C. get the above-mentioned mixed solution of 8.2mL, add 825 μ L, 0.015MAA, then by the HAuCl of 10mL, 0.08mM
4enter with the speed injection of 1mL/min, obtain GCNPs particle.
D. by the centrifugal 10min of 1mLGCNPs particle 8000rpm, resuspended with the antibody-solutions of the 0.1mg/mL horseradish peroxidase-labeled containing 0.001M sal tartari, 4 DEG C of concussion 3h, the centrifugal 15min of 9000rpm, the enzyme mark dilution containing 0.05mg/mL horseradish peroxidase with 0.5mL is resuspended, namely obtains GCNPs-enzyme-immunological probe.
Embodiment 2: the detection of ochratoxin A (OTA)
The synthesis of a.OTA envelope antigen adopts carbodiimides (CDI) method.Carboxyl on OTA molecule, after can being activated, is coupled on the lysine amino on protein molecular by CDI.The key step of synthesis, the OTA of 1mg is dissolved in 0.2mL anhydrous dimethyl sulphoxide, adds 2mgCDI, and at room temperature, after magnetic agitation is dissolved, lucifuge continues reaction 10min; The ovalbumin (OVA) of 5mg, is dissolved in the carbonate buffer solution of 1mLpH9.6, and magnetic agitation is dissolved.By the OTA solution activated, dropwise join in OVA solution.After adding, continue magnetic agitation, lucifuge room temperature reaction 4h.After reaction terminates, reaction solution is dialysed 3 times in CBS solution, to remove unreacted OTA and other Small molecular; Last dislysate changes PB (phosphate buffer) into.After the envelope antigen packing of synthesis, be placed in-20 DEG C of preservations.
B. the bag quilt of antigen: using the conjugate of OTA and OVA as envelope antigen, with the carbonate buffer solution of 0.05M, pH9.6, envelope antigen is diluted with 1:2000 ~ 1:16000, as coating buffer, 100 μ L are added in every hole of ELISA Plate, hatch 2h for 37 DEG C, then with 0.001% molecular weight be the polyglycol PEG of 6000 as confining liquid, 4 DEG C close spend the night;
C. competitive reaction: every hole adds the 0.01ng/mL diluted with OTA standard dilutions phosphate buffer PBS respectively, 0.02ng/mL, 0.05ng/mL, 0.1ng/mL, 0.2ng/mL, 0.5ng/mL, the Small molecular standard items of one of 1ng/mL series concentration or the sample 50 μ L that handles well are in respective enzyme mark hole, by small molecular antibody with enzyme mark dilution: containing 0.1% gelatin, 0.05% (v/v) Tween-20, pH7.4, the phosphate buffer PBST of 0.01M, after 1:8000 ~ 1:64000 dilution proportion, add in ELISA Plate, every hole 50 μ L, hatch after 30min with PBST cleansing solution washing 3 ~ 5 times for 37 DEG C,
D. add the ELIAS secondary antibody of having adsorbed gold and silver nano composite material: will add the ELIAS secondary antibody of having adsorbed gold and silver nano composite material with 1:1 ~ 1:8 dilution with antibody diluent, every hole adds 100 μ L, hatch after 1h with PBST cleansing solution washing 3 ~ 5 times for 37 DEG C;
E. develop the color: every hole adds nitrite ion 100 μ L, 37 DEG C of colour developing 15min; Last every hole adds stop buffer 2M sulfuric acid solution 50 μ L; Light absorption value A when being 450nm with microplate reader measurement wavelength
450, contrast with done typical curve the concentration calculating OTA in testing sample.Consult Fig. 3 a-Fig. 3 b, in the present embodiment, obtain the IC that OTA detects
50for 0.05ng/mL, than the IC that traditional ELISA method detects
500.27ng/mL improves 5 times.
Embodiment 3: the detection of prostate specific antigen (PSA)
A. quilt is wrapped: being buffered liquid by PSA antibody dilution to protein content with 0.05MPH9.6 carbonate bag is 1 ~ 20 μ g/ml, and in every hole of ELISA Plate, add 100 μ L, hatch 2h for 37 DEG C, PBST cleansing solution washs 3 times.With the bovine serum albumin of 0.1-5% as confining liquid, every hole 200 μ L, closes for 4 DEG C and spends the night;
B. association reaction: the PSA standard items that every hole adds one of the series concentration of diluting with standard dilutions phosphate buffer PBS respectively or the sample 100 μ L that handles well, in respective enzyme mark hole, put 37 DEG C and hatch 30min.PBST cleansing solution washs 3 times.(doing blank well, negative control hole and Positive control wells) simultaneously;
The combination of c.GCNPs-enzyme-immunological probe: with antibody diluent by GCNPs-enzyme-immunological probe with 1:1 ~ 1:8 dilution, every hole adds 100 μ L, hatches after 1h with PBST cleansing solution washing 3 ~ 5 times for 37 DEG C;
D. develop the color: every hole adds nitrite ion 100 μ L, 37 DEG C of colour developing 15min; Last every hole adds stop buffer 2M sulfuric acid solution 50 μ L; Light absorption value A when being 450nm with microplate reader measurement wavelength
450, set up A
450with the relation of antigen amount.The concentration calculating PSA in testing sample is contrasted with done typical curve.Consult Fig. 4 a-Fig. 4 b, in the present embodiment, obtain the IC that PSA detects
50for 2ng/mL, than the IC that traditional ELISA method detects
500.1ng/mL improves 20 times.
It should be noted that, in this article, term " comprises ", " comprising " or its any other variant are intended to contain comprising of nonexcludability, thus make to comprise the process of a series of key element, method, article or equipment and not only comprise those key elements, but also comprise other key elements clearly do not listed, or also comprise by the intrinsic key element of this process, method, article or equipment.When not more restrictions, the key element limited by statement " comprising ... ", and be not precluded within process, method, article or the equipment comprising described key element and also there is other identical element.
The above is only the specific embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a preparation method for grape cluster sample nano particle, is characterized in that comprising: using protein modified Nano Silver triangular plate as template, and using gold chloride as oxygenant, prepares grape cluster sample nano particle by Jia Fanni substitution reaction.
2. the preparation method of grape cluster sample nano particle according to claim 1, is characterized in that comprising:
(1) bovine serum albumin is added in the solution of the Nano Silver triangle being of a size of 15nm ~ 30nm and mix, the concentration of bovine serum albumin in the mixed solution of formation is made to be 0.01mg/mL ~ 5mg/mL, and the mol ratio of bovine serum albumin and Nano Silver triangle is 5:1 ~ 20:1, and hold over night;
(2) obtain in mixed reaction solution to step (1) and add ascorbic acid, make the concentration of ascorbic acid in the potpourri of formation be 0.08mM ~ 5mM after the HAuCl that is 0.01mM ~ 0.5mM with the speed implantation concentration of 0.1mL/min ~ 2mL/min
4solution, obtains grape cluster sample nano particle.
3. the grape cluster sample nano particle prepared by method described in claim 1 or 2, its particle diameter is 20nm ~ 50nm.
4. an immunological probe, it is characterized in that comprising grape cluster sample nano particle according to claim 3, described grape cluster sample nano particle is modified with the antibody of horseradish peroxidase-labeled, the antibody of described horseradish peroxidase-labeled comprises horseradish peroxidase-labeled monoclonal antibody or polyclonal antibody.
5. the preparation method of an immunological probe, it is characterized in that comprising: by grape cluster sample nanoparticle dispersion according to claim 3 in the antibody-solutions of the 0.01 ~ 0.2mg/mL horseradish peroxidase-labeled containing 0.1mM ~ 10mM sal tartari or sodium carbonate, 0 DEG C ~ 8 DEG C concussion 1h ~ 5h, afterwards in the centrifugal 10min ~ 20min of 8000rpm ~ 10000rpm, and then obtain described immunological probe in precipitation.
6. the preparation method of immunological probe according to claim 5, characterized by further comprising: the precipitation of centrifugal acquisition is resuspended with the solution containing 0.01mg/mL ~ 0.1mg/mL horseradish peroxidase, for subsequent use.
7. immunological probe according to claim 4 or the application of immunological probe in enzyme linked immunosorbent detection that any one of claim 5-6 prepared by method.
8. an enzyme-linked immune detection method, is characterized in that comprising:
The target antigen standard model of immunological probe according to claim 4 or immunological probe that any one of claim 5-6 prepared by method and a series of variable concentrations is being suitable for making carrying out enzyme linked immunoassay in the liquid phase environment that in immunological probe, contained antibody is combined with described antigentic specificity, and record detects model, sets up the standard corresponding relation between immunological probe detection signal and antigen concentration thus;
And, the testing sample of described immunological probe and the target antigen containing unknown concentration is being suitable for making carrying out enzyme linked immunoassay in the liquid phase environment that in immunological probe, contained antibody is combined with described antigentic specificity, and foundation detection model and described standard corresponding relation and determine the target antigen concentration in testing sample.
9., based on an indirect competitive enzyme-linked immunosorbent analytical approach for immunological probe according to claim 4 or immunological probe that any one of claim 5-6 prepared by method, it is characterized in that comprising the following steps:
A. be dissolved in carbonate buffer solution using as the small haptens of envelope antigen and the conjugate of ovalbumin or bovine serum albumin and form coating buffer, hatch 1 ~ 4h for 25 DEG C ~ 37 DEG C, again with after phosphate buffer washing, add using as confining liquid, molecular weight that concentration is 0.001wt% be 2000 ~ 10000 polyglycol solution to close in 0 DEG C ~ 8 DEG C and spend the night;
B. the envelope antigen solution obtained by step a mixes with the phosphate buffer of the Small molecular standard items of a series of variable concentrations, add the enzyme mark dilution containing small molecular antibody again, 25 DEG C ~ 37 DEG C hatch after, with phosphate buffer washing, described enzyme mark dilution adopts that the pH value comprising 0.1wt% gelatin and 0.05v/v% Tween-20 is 7.0 ~ 8.0, concentration is the phosphate buffer of 0.01M ~ 0.05M;
C. the antibody diluent containing described immunological probe is added in each competitive reaction mixed system obtained to step b, described antibody diluent adopts that the pH value comprising 0.1wt% gelatin and 0.05v/v% Tween-20 is 7.0 ~ 8.0, concentration is the phosphate buffer of 0.01M ~ 0.05M, 25 DEG C ~ 37 DEG C hatch 0.5h ~ 2h after, wash with phosphate buffer;
D. nitrite ion is added respectively in each hybrid reaction system obtained to step c, 25 DEG C ~ 37 DEG C colour developing 10min ~ 30min, add again as stop buffer, concentration is the sulfuric acid solution of 2M ~ 4M, measure the light absorption value A of each hybrid reaction system when wavelength is 450nm ~ 650nm afterwards, set up the standard corresponding relation of A and antigen amount.
10., based on a sandwich ELISA method for immunological probe according to claim 4 or immunological probe that any one of claim 5-6 prepared by method, it is characterized in that comprising the following steps:
A. antibody is dissolved in the carbonate bag that concentration is 0.01M ~ 0.1M, pH value is 9 ~ 10 and is buffered liquid, form the solution that protein content is 1 μ g/ml ~ 20 μ g/ml, 1h ~ 4h is hatched again in 25 DEG C ~ 37 DEG C, wash with phosphate buffer afterwards, add afterwards as confining liquid, concentration is the bovine serum albumen solution of 0.1wt% ~ 5wt%, and close in 0 DEG C ~ 8 DEG C and spend the night;
B. the envelope antigen solution obtained by step a mixes with the phosphate buffer of the large molecular criteria product of a series of variable concentrations, hatches 0.5h ~ 2h, washs afterwards with phosphate buffer for 25 DEG C ~ 37 DEG C;
C. the antibody diluent containing described immunological probe is added in each association reaction mixed system obtained to step b, described antibody diluent adopts that the pH value comprising 0.1wt gelatin and 0.05v/v% Tween-20 is 7.0 ~ 8.0, concentration is the phosphate buffer of 0.01M ~ 0.05M, hatch 0.5h ~ 3h, wash with phosphate buffer for 25 DEG C ~ 37 DEG C;
D. nitrite ion is added respectively in each hybrid reaction system obtained to step c, 25 DEG C ~ 37 DEG C colour developing 10min ~ 30min, add again as stop buffer, concentration is the sulfuric acid solution of 2M ~ 4M, measure the light absorption value A of each hybrid reaction system when wavelength is 450nm ~ 650nm afterwards, set up the standard corresponding relation of A and antigen amount.
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