CN106018819B - A kind of ELISA detection method based on the nano enzyme with peroxidase activity - Google Patents
A kind of ELISA detection method based on the nano enzyme with peroxidase activity Download PDFInfo
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- CN106018819B CN106018819B CN201610347888.5A CN201610347888A CN106018819B CN 106018819 B CN106018819 B CN 106018819B CN 201610347888 A CN201610347888 A CN 201610347888A CN 106018819 B CN106018819 B CN 106018819B
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- 238000001514 detection method Methods 0.000 title claims abstract description 98
- 108040007629 peroxidase activity proteins Proteins 0.000 title claims abstract description 52
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 50
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 50
- 230000000694 effects Effects 0.000 title claims abstract description 43
- 238000002965 ELISA Methods 0.000 title claims abstract description 26
- 102000003992 Peroxidases Human genes 0.000 title claims abstract 16
- 239000010931 gold Substances 0.000 claims abstract description 117
- 229910052737 gold Inorganic materials 0.000 claims abstract description 74
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 claims abstract description 62
- 239000000427 antigen Substances 0.000 claims abstract description 57
- BASFCYQUMIYNBI-UHFFFAOYSA-N platinum Chemical compound [Pt] BASFCYQUMIYNBI-UHFFFAOYSA-N 0.000 claims abstract description 57
- 102000036639 antigens Human genes 0.000 claims abstract description 52
- 108091007433 antigens Proteins 0.000 claims abstract description 52
- 239000002245 particle Substances 0.000 claims abstract description 50
- 238000012360 testing method Methods 0.000 claims abstract description 42
- 229910052697 platinum Inorganic materials 0.000 claims abstract description 27
- 238000000034 method Methods 0.000 claims abstract description 25
- 238000012986 modification Methods 0.000 claims abstract description 19
- 230000004048 modification Effects 0.000 claims abstract description 19
- 230000003197 catalytic effect Effects 0.000 claims abstract description 17
- 239000000758 substrate Substances 0.000 claims abstract description 9
- 150000001875 compounds Chemical class 0.000 claims abstract description 8
- 235000013339 cereals Nutrition 0.000 claims description 64
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 32
- 150000005208 1,4-dihydroxybenzenes Chemical class 0.000 claims description 22
- 239000011668 ascorbic acid Substances 0.000 claims description 21
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 18
- 239000002131 composite material Substances 0.000 claims description 18
- 229960005070 ascorbic acid Drugs 0.000 claims description 16
- 235000010323 ascorbic acid Nutrition 0.000 claims description 15
- 238000005406 washing Methods 0.000 claims description 15
- 102000004169 proteins and genes Human genes 0.000 claims description 12
- 108090000623 proteins and genes Proteins 0.000 claims description 12
- 239000002105 nanoparticle Substances 0.000 claims description 11
- 241000894007 species Species 0.000 claims description 8
- 238000002360 preparation method Methods 0.000 claims description 6
- 239000011248 coating agent Substances 0.000 claims description 5
- 238000000576 coating method Methods 0.000 claims description 5
- 235000007164 Oryza sativa Nutrition 0.000 claims description 4
- 238000002835 absorbance Methods 0.000 claims description 4
- 230000015572 biosynthetic process Effects 0.000 claims description 4
- 235000009566 rice Nutrition 0.000 claims description 4
- 240000007594 Oryza sativa Species 0.000 claims 1
- 230000009977 dual effect Effects 0.000 claims 1
- 239000010420 shell particle Substances 0.000 claims 1
- 230000007423 decrease Effects 0.000 abstract description 4
- 238000003745 diagnosis Methods 0.000 abstract description 3
- 201000010099 disease Diseases 0.000 abstract description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 3
- 238000001179 sorption measurement Methods 0.000 abstract description 3
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 238000012544 monitoring process Methods 0.000 abstract description 2
- 102000013415 peroxidase activity proteins Human genes 0.000 description 36
- 239000000243 solution Substances 0.000 description 26
- 102100031051 Cysteine and glycine-rich protein 1 Human genes 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 238000010586 diagram Methods 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 239000011534 wash buffer Substances 0.000 description 5
- 108091005804 Peptidases Proteins 0.000 description 4
- 239000004365 Protease Substances 0.000 description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 4
- 101710134784 Agnoprotein Proteins 0.000 description 3
- 241000209094 Oryza Species 0.000 description 3
- 239000002253 acid Substances 0.000 description 3
- 238000013461 design Methods 0.000 description 3
- 229910000397 disodium phosphate Inorganic materials 0.000 description 3
- 238000011534 incubation Methods 0.000 description 3
- 238000007689 inspection Methods 0.000 description 3
- 229910002621 H2PtCl6 Inorganic materials 0.000 description 2
- 108010001336 Horseradish Peroxidase Proteins 0.000 description 2
- 229920001213 Polysorbate 20 Polymers 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 238000002389 environmental scanning electron microscopy Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 2
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 2
- 239000001509 sodium citrate Substances 0.000 description 2
- 235000011083 sodium citrates Nutrition 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 230000000007 visual effect Effects 0.000 description 2
- GDSOZVZXVXTJMI-SNAWJCMRSA-N (e)-1-methylbut-1-ene-1,2,4-tricarboxylic acid Chemical compound OC(=O)C(/C)=C(C(O)=O)\CCC(O)=O GDSOZVZXVXTJMI-SNAWJCMRSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 239000002211 L-ascorbic acid Substances 0.000 description 1
- 235000000069 L-ascorbic acid Nutrition 0.000 description 1
- 101100434911 Mus musculus Angpt1 gene Proteins 0.000 description 1
- 206010070834 Sensitisation Diseases 0.000 description 1
- 238000004847 absorption spectroscopy Methods 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- RFXSFVVPCLGHAU-UHFFFAOYSA-N benzene;phenol Chemical compound C1=CC=CC=C1.OC1=CC=CC=C1.OC1=CC=CC=C1 RFXSFVVPCLGHAU-UHFFFAOYSA-N 0.000 description 1
- HFACYLZERDEVSX-UHFFFAOYSA-N benzidine Chemical compound C1=CC(N)=CC=C1C1=CC=C(N)C=C1 HFACYLZERDEVSX-UHFFFAOYSA-N 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 238000011953 bioanalysis Methods 0.000 description 1
- 230000000903 blocking effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 238000005034 decoration Methods 0.000 description 1
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 1
- 238000009585 enzyme analysis Methods 0.000 description 1
- RJHLTVSLYWWTEF-UHFFFAOYSA-K gold trichloride Chemical class Cl[Au](Cl)Cl RJHLTVSLYWWTEF-UHFFFAOYSA-K 0.000 description 1
- 229910021505 gold(III) hydroxide Inorganic materials 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 1
- 238000010992 reflux Methods 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 229910001961 silver nitrate Inorganic materials 0.000 description 1
- SQGYOTSLMSWVJD-UHFFFAOYSA-N silver(1+) nitrate Chemical class [Ag+].[O-]N(=O)=O SQGYOTSLMSWVJD-UHFFFAOYSA-N 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- HRXKRNGNAMMEHJ-UHFFFAOYSA-K trisodium citrate Chemical class [Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HRXKRNGNAMMEHJ-UHFFFAOYSA-K 0.000 description 1
- 238000001429 visible spectrum Methods 0.000 description 1
Classifications
-
- 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
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/581—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates)
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J23/00—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00
- B01J23/38—Catalysts comprising metals or metal oxides or hydroxides, not provided for in group B01J21/00 of noble metals
- B01J23/48—Silver or gold
- B01J23/52—Gold
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J31/00—Catalysts comprising hydrides, coordination complexes or organic compounds
- B01J31/02—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides
- B01J31/06—Catalysts comprising hydrides, coordination complexes or organic compounds containing organic compounds or metal hydrides containing polymers
-
- B01J35/23—
-
- B01J35/50—
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
Abstract
The invention discloses a kind of ELISA detection method based on the nano enzyme with peroxidase activity, and suitable for double antibody sandwich method or dual-antigen sandwich method, first modification detects antibody/detection antigen on gold nano grain；ELISA is carried out again to detect to form capture antibody/capture antigen determined antigen/test antibodies detection antibody/detection antigen gold nano grain compound；Then silver-colored platinum dye is carried out, silver-colored shell and platinum shell are wrapped up on gold nano grain surface, the nano enzyme Au@AgPt particles with peroxidase activity are obtained, recycles it to be catalyzed peroxidase substrate and obtains color products, so as to detect to obtain the amount of determined antigen/test antibodies.The present invention is had the advantages of simple, quick and good stability, nano enzyme particulate catalytic activity decrease caused by can effectively avoiding non-specific adsorption and modification, new platform is provided for environmental monitoring and medical diagnosis on disease using the method after first modifying into enzyme.
Description
Technical field
The invention belongs to technical field of analysis and detection, and in particular to a kind of ELISA detection method based on nano enzyme, more
It is to be related to a kind of ELISA detection method based on the nano enzyme with peroxidase activity body.
Background technology
Peroxidase be it is a kind of in the presence of hydrogen peroxide, the protease of electron donor oxidation can be catalyzed.At present, mistake
Oxide enzyme has been widely used in chemical sensitisation, environment measuring and medical diagnosis on disease, for example, horseradish peroxidase
(HRP) the detection antibody of mark detects available for ELISA.However, protease has easy in inactivation, preparation not easy to maintain and difficult etc. no
Foot, significantly limit the application of peroxidase.
Nano enzyme be with the nano material similar to protease catalytic capability that they have synthesis be simple, cost is cheap and
The advantages of catalytic activity is strong, it can be good at making up the deficiency of protease.In various nano enzymes, Pt nanoparticle is due to extremely strong
Peroxidase activity and be widely used in environmental monitoring and medical diagnosis on disease.But Pt nanoparticle catalytic activity is unstable
Determine, easily reunite, and can decline catalytic activity due to surface modification antibody, limit with peroxidase activity
Property nano enzyme analysis detect in application.
Therefore, develop simple, quick, stable and can avoid, because modification causes the method for catalytic activity decline, preparing
Nano enzyme with strong peroxidase activity has great importance.
The content of the invention
It is an object of the invention in place of overcome the deficiencies in the prior art, there is provided a kind of ELISA inspections based on nano enzyme
Prepared by survey method, the method that this kind of nano enzyme is contaminated by silver-colored platinum, have peroxidase activity, the present invention can solve the problem that existing skill
Nanometer enzymatic activity is unstable in art, easily due to modification cause catalytic activity to reduce the problem of.
One of the technical solution adopted for the present invention to solve the technical problems is：
A kind of ELISA detection method based on the nano enzyme with peroxidase activity, suitable for double antibody sandwich method
Or dual-antigen sandwich method, the detecting step of wherein double antibody sandwich method include：
A) according to the corresponding capture antibody of species selection and detection antibody of determined antigen in testing sample；
B) the detection antibody on 5~30nm of particle diameter gold nano grain described in modification step a), obtain being modified with detection
The gold nano grain of antibody；The detection antibody can also be binding proteins specific, small molecular protein or cell antibody etc.；
C) ELISA detections are carried out：Coating capture antibody → addition testing sample make determined antigen with capture antibody binding →
Add the gold nano grain for being modified with detection antibody that step b) is obtained and make determined antigen and detection antibody binding therein, shape
Into capture antibody-determined antigen-detection antibody-gold nano-particle complex；
D) the capture antibody-determined antigen-detection antibody-gold nano-particle complex obtained to step c) carries out silver-colored platinum
Dye：Add Ag+Solution and hydroquinones simultaneously make its final concentration be respectively 62.5~250 μM and 0.04~5mM, utilize hydroquinones
Reduce Ag+Silver-colored shell is wrapped up with gold nano grain surface in the composite；Washing；Add PtCl6 2-Solution and ascorbic acid are simultaneously
It is respectively 0.25~1mM and 2~50mM to make its final concentration, and PtCl is reduced using ascorbic acid6 2-To wrap up the gold of silver-colored shell
Nano grain surface wraps up platinum shell again, obtains the nano enzyme Au@AgPt particles with peroxidase activity, and it is anti-to form capture
Body-determined antigen-detection antibody-Au@AgPt particle composites；
E) under 0.1~10M hydrogen peroxide existence conditions, capture antibody-determined antigen-detection for being obtained using step d)
There is the nano enzyme Au@AgPt particulate catalytic peroxidase bottom of peroxidase activity in antibody-Au@AgPt particle composites
Thing obtains product, detects the amount of product to obtain the amount of determined antigen.
The detecting step of dual-antigen sandwich method includes：
A ') according to the corresponding capture antigen of species selection and detection antigen of test antibodies in testing sample；
B ') the modification step a ' on 5~30nm of particle diameter gold nano grain) described in detection antigen, obtain being modified with inspection
Survey the gold nano grain of antigen；
C ') carry out ELISA detections：Coating capture antigen → addition testing sample make test antibodies with capture antigen binding →
Add step b ') the obtained gold nano grain for being modified with detection antigen and make test antibodies and detection antigen binding therein,
Form capture antigen-test antibodies-detection antigen-gold nano grain compound；
D ') to step c ') obtained capture antigen-test antibodies-detection antigen-gold nano grain compound carries out silver-colored platinum
Dye：Add Ag+Solution and hydroquinones simultaneously make its final concentration be respectively 62.5~250 μM and 0.04~5mM, utilize hydroquinones
Reduce Ag+Silver-colored shell is wrapped up with gold nano grain surface in the composite；Washing；Add PtCl6 2-Solution and ascorbic acid are simultaneously
It is respectively 0.25~1mM and 2~50mM to make its final concentration, and PtCl is reduced using ascorbic acid6 2-To wrap up the gold of silver-colored shell
Nano grain surface wraps up platinum shell again, obtains the nano enzyme Au@AgPt particles with peroxidase activity, and it is anti-to form capture
Original-test antibodies-detection antigen-Au@AgPt particle composites；
E ') under 0.1~10M hydrogen peroxide existence conditions, utilize step d ') obtained capture antigen-test antibodies-inspection
Surveying in antigen-Au@AgPt particle composites has the nano enzyme Au@AgPt particulate catalytic peroxidase of peroxidase activity
Substrate obtains product, detects the amount of product to obtain the amount of test antibodies.
In one embodiment：The step b) or b ') in, gold nano grain particle diameter is 13~20nm.
In one embodiment：The step d) or d ') in, Ag+Final concentration of 100~150 μM of solution.
In one embodiment：The step d) or d ') in, final concentration of 0.2~1mM of hydroquinones.
In one embodiment：The step d) or d ') in, PtCl6 2-Final concentration of 0.4~0.6mM of solution.
In one embodiment：The step d) or d ') in, final concentration of 10~20mM of ascorbic acid.
In one embodiment：The step e) or e ') in, the concentration of hydrogen peroxide is 1~5M.
In one embodiment：The step e) or e ') in, the peroxidase substrate is TMB (3,3', 5,5'- tetramethyls
Benzidine)；Color products are obtained using the nano enzyme Au@AgPt particulate catalytics TMB with peroxidase activity, in 450nm
The absorbance of color products is detected, so that the amount of determined antigen or test antibodies is calculated.
The two of the technical solution adopted for the present invention to solve the technical problems are：
A kind of nano enzyme preparation method with peroxidase activity, it is characterised in that：Including：
1) modifying protein on 5~30nm of particle diameter gold nano grain, obtain being modified with the gold nano grain of protein；
Or directly select 5~30nm of particle diameter gold nano grain, not modifying protein；
Wherein, the protein can be antibody or antigen, and the gold nano grain after the modification being correspondingly prepared can
Double antibody sandwich method or dual-antigen sandwich method applied to ELISA；Certainly, the protein can also be other species；
2) gold nano grain for being modified with albumen obtained to step 1) carries out silver-colored platinum dye, or directly to not modifying protein
Gold nano grain carry out silver-colored platinum dye：Add Ag+Solution and hydroquinones and make its final concentration be respectively 62.5~250 μM and
0.04~5mM, Ag is reduced using hydroquinones+To wrap up silver-colored shell on gold nano grain surface；Washing；Add PtCl6 2-Solution
With ascorbic acid and make its final concentration be respectively 0.25~1mM and 2~50mM, utilize ascorbic acid reduction PtCl6 2-To wrap up
Platinum shell is wrapped up on the gold nano grain surface of silver-colored shell again, obtains Au@AgPt particles, is described to have peroxidase
The nano enzyme of activity.
Wherein, when the protein is antibody or antigen, obtained nano enzyme can be used in ELISA detections, effect
In peroxidase substrate and for detecting.When the protein is other species, other purposes can be respectively used for.If
Silver-colored platinum dye directly is carried out to the gold nano grain of unmodified protein matter, then the nano enzyme obtained can also merely be used as peroxidating
Thing enzyme uses.
Present invention also offers the nano enzyme with peroxidase activity according to prepared by above-mentioned preparation method.
Compared with background technology, it has the following advantages that the technical program：
First, the present invention utilizes the strong suction-operated of gold nano grain surface and albumen, it is possible to prevente effectively from detection antibody
Or non-specific adsorption occurs for the gold nano grain of detection Modified antigen；Secondly, the present invention first will detection antibody or detection antigen
Modification after carrying out ELISA detection reactions, recycles silver-colored platinum to contaminate to be formed with strong peroxidase activity on gold nano grain
Nano particle, i.e., first modify again into enzyme, it is traditional first into enzymatic caused by modification in enzyme again modification technique so as to avoid
Activity decrease；Then, the nano enzyme with peroxidase activity for preparing of the present invention is with catalytic activity is strong, stable and reproduction
Property it is good the advantages of, its be catalyzed formed color products can carry out Visual retrieval or by ELIASA carry out Visual retrieval,
Easily realize commercialization.In summary, compared with the ELISA method based on Pt nanoparticle, method of the invention is simple, fast
Victory, stability are good, and catalytic activity caused by non-specific adsorption and modification can be avoided to reduce, for peroxidase activity
Property nano enzyme bioanalysis and it is biomedical in application new platform is provided.
Brief description of the drawings
The invention will be further described with reference to the accompanying drawings and examples.
Fig. 1 is the ELISA detection principle diagrams (double antibody sandwich method) of the present invention.
Fig. 2 is the experimental result schematic diagram that silver-colored platinum contaminates condition optimizing in embodiment 2, and (A) and (B) is respectively various concentrations nitre
Background signal (control group for being not added with AuNPs) and sample signal when acid is silver-colored, when (C) and (D) is respectively various concentrations chloroplatinic acid
Background signal (control group for being not added with AuNPs) and sample signal.
Fig. 3 is the photo (A) and purple of Au/BSA particles, Au/BSA@Ag particles and Au/BSA@AgPt particles in embodiment 3
Outer visible spectrum schematic diagram (B).
Fig. 4 is the ESEM signal of Au/BSA particles, Au/BSA@Ag particles and Au/BSA@AgPt particles in embodiment 3
Scheme (A) and transmission electron microscope schematic diagram (B), and the Elemental redistribution (C) of Au/BSA@AgPt particles.
Fig. 5 is the testing result signal for detecting determined antigen CRP in embodiment 5 using the ELISA detection method of the present invention
Scheme (A) and selective schematic diagram (B), and the ELISA detection method detection determined antigen H using the present invention5N1Testing result
Schematic diagram (C) and selective schematic diagram (D).
Embodiment
Present disclosure is illustrated below by embodiment：
Embodiment 1：The preparation of gold nano grain
100mL 0.01wt% gold chlorides are added in round-bottomed flask, boiling reflux, at the uniform velocity adds 1mL with continuous stirring
3wt% sodium citrates, continue stirring and boil 30min, the gold nano grain (AuNPs) that synthesis particle diameter is 16nm.By adjusting chlorine
The ratio of auric acid and sodium citrate, the gold nano grain of different-grain diameter size can be obtained.
Embodiment 2：The contrast test of silver-colored platinum dye condition
Among the present embodiment, gold nano grain is modified using bovine serum albumin(BSA) BSA.It is of course also possible to repaiied without BSA
Decorations, the gold nano grain directly obtained to embodiment 1 carry out silver-colored platinum dye, can equally obtain the nanometer with peroxidase activity
Enzyme.
First, 100 μ L 2wt%BSA/PBS solution are added into 96 orifice plates, 1h, 300 μ L PBS washings are incubated at 37 DEG C
Three times.The 16nm AuNPs (control group be not added with AuNPs) for preparing, 37 DEG C incubations are then added in 100 μ L 2.5nM embodiments 1
1h, 300 μ L PBS are washed three times.Then 300 μ L Blocking Buffer are added and (contain 2wt%BSA and 0.1wt%Tween
20 PBS solution), 37 DEG C are incubated 1h, and 300 μ L PBS are washed three times, obtain the gold nano grain Au/BSA of BSA modifications, and it is wrapped
Absorption is wrapped up in 96 orifice surfaces.
Then, 50 μ L various concentrations silver nitrates (AgNO are added into above-mentioned 96 orifice plate3) solution and 50 μ L 0.4mM are to benzene
Diphenol, make Ag+Final concentration be respectively 0,31.25,62.5,125,250,600 μM and the final concentration of 0.2mM of hydroquinones,
37 DEG C of reaction 20min, 300 μ L water washings three times.Then 50 μ L various concentrations chloroplatinic acids (H are added2PtCl6) solution and 50 μ
L20mM ascorbic acid, makes PtCl6 2-Final concentration be respectively 0,62.5,125,250,500,1000 μM and the end of ascorbic acid is dense
Spend for 10mM, 37 DEG C of reaction 20min, 300 μ L water washings three times, obtain the nano enzyme Au/BSA@with peroxidase activity
AgPt particles.
Finally, the μ L 1mM TMB/2mM H of substrate 100 of peroxidase are added into 96 orifice plates3PO4Solution, utilize tool
The nano enzyme Au/BSA@AgPt particles for having peroxidase activity obtain color products in 37 DEG C of catalytic reaction 20min, utilize enzyme
Mark instrument and determine absorption of the different samples in 450nm.
As a result as shown in Fig. 2 Ag+The final concentration of solution is preferred with 100~150 μM, PtCl6 2-The final concentration of solution is with 0.4
~0.6mM is preferred, and effect is preferable.
Embodiment 3：The sign of the nano enzyme Au/BSA@AgPt particles with peroxidase activity obtained after silver-colored platinum dye
AgNO is added in the gold nano grain Au/BSA of the BSA modifications obtained into 1mL 2.5nM embodiments 23Solution and
Hydroquinones simultaneously makes Ag+Final concentration with hydroquinones is respectively 125 μM and 0.2mM, and 37 DEG C are reacted 20min.300 μ L water washings
Three times, obtain Au/BSA@Ag particles.Then add H2PtCl6Solution and ascorbic acid simultaneously make PtCl6 2-It is dense with the end of ascorbic acid
Degree is respectively 0.5mM and 10mM, 37 DEG C of reaction 20min, 300 μ L water washings three times, obtains having receiving for peroxidase activity
Rice enzyme Au/BSA@AgPt particles.Using camera, ultraviolet-visible absorption spectroscopy instrument, ESEM and high-resolution-ration transmission electric-lens to silver
The front and rear gold nano grain of dye is characterized.
As a result as shown in Figure 3 and Figure 4, in the nano enzyme Au/BSA@AgPt particles with peroxidase activity obtained,
Silver-colored shell and platinum shell is enclosed with successively outside gold nano grain.
Embodiment 4：The modification detection antibody on gold nano grain
The gold nano grain AuNPs that the particle diameter for taking 1mL embodiments 1 to obtain is 16nm adds thereto in 1.5mL centrifuge tubes
Enter 10 μ L 1mg/mL detection antibody and 50 μ L 0.2M Na2HPO4, 20min is incubated at 37 DEG C.Then 50 μ L are added thereto
2%BSA/PBS solution, 20min is incubated at 37 DEG C after mixing, it is then centrifuged into 10min under 14000rpm, uses 1mL
0.1%BSA/10mM Na2HPO4It is resuspended, the centrifugation/resuspension process repeats 2 times, is finally scattered in 250 μ L0.1%BSA/
10mM Na2HPO4In solution, obtain being modified with the gold nano grain of detection antibody；4 DEG C save backup.
Embodiment 5：ELISA detection reactions (double antibody sandwich method)
A) according to the corresponding capture antibody of species selection and detection antibody of determined antigen in testing sample；The present embodiment it
In, determined antigen is CRP and H5N1, respective capture antibody and detection antibody are selected respectively according to two kinds of determined antigens；
B) two kinds are obtained using the method for embodiment 4 and is modified with CRP and H respectively5N1The gold nano of corresponding detection antibody
Grain；
C) ELISA detections are carried out：
The μ g/mL of 100 μ L 2 capture antibody, 4 DEG C of overnight incubations are added in 96 orifice plates, 300 μ L Wash Buffer (contain
The PBS solutions of 0.1wt%Tween 20) wash three times.Then, 300 μ L Block Buffer are added and (contains 2wt%BSA's
Wash Buffer), 37 DEG C are incubated 1h, and 300 μ L Wash Buffer are washed three times；
100 μ L testing samples are added, 25 DEG C are incubated 1h and make determined antigen and capture antibody binding, 300 μ L Wash
Buffer is washed three times；
The gold nano grain for being modified with detection antibody that 100 μ L 2.5nM steps b) are obtained is added, 37 DEG C of incubation 1h, makes to treat
Antigen and detection antibody binding therein are surveyed, 300 μ L Wash Buffer are washed twice, 300 μ L water washings one time, in 96 orifice plates
In obtain capture antibody-determined antigen-detection antibody-gold nano-particle complex；
D) the capture antibody-determined antigen-detection antibody-gold nano-particle complex obtained to step c) carries out silver-colored platinum
Dye：50 μ L AgNO are added in 96 orifice plates3Solution and 50 μ L hydroquinones simultaneously make Ag+Final concentration with hydroquinones is respectively
125 μM and 0.2mM, 37 DEG C of reaction 20min, Ag is reduced using hydroquinones+Wrapped with gold nano grain surface in the composite
Wrap up in silver-colored shell；300 μ L water washings three times；Then add 50 μ L H2PtCl6Solution and 50 μ L ascorbic acid simultaneously make PtCl6 2-With it is anti-
The final concentration of bad hematic acid is respectively 0.5mM and 10mM, 37 DEG C of reaction 20min, PtCl is reduced using ascorbic acid6 2-To wrap up
Platinum shell is wrapped up on the gold nano grain surface of silver-colored shell again, 300 μ L water washings three times, respectively obtains with peroxidase activity
The nano enzyme Au/CRP@AgPt particles and Au/H of property5N1@AgPt particles, i.e., form capture antibody-to be measured respectively in 96 orifice plates
Antigen-detection antibody A u/CRP@AgPt particle composites and capture antibody-determined antigen-detection antibody A u/H5N1@AgPt particles
Compound；
E) under 0.1~10M hydrogen peroxide existence conditions, 100 μ L 1mM TMB/2mM H are added into 96 orifice plates3PO4It is molten
Liquid, 37 DEG C of reaction 20min, the capture antibody-determined antigen-detection antibody A u/CRP@AgPt particles obtained using step d) are answered
Compound and capture antibody-determined antigen-detection antibody A u/H5N1Receiving with peroxidase activity in@AgPt particle composites
Rice enzyme Au/CRP@AgPt particles and Au/H5N1@AgPt particulate catalytics TMB obtains color products, in 450nm detection color products
Absorbance, so that determined antigen CRP and H to be calculated respectively5N1Amount.
As a result as shown in figure 5, using the method for the present invention to antigens c RP and H to be measured5N1Detected respectively, linear and choosing
Selecting property is good.
The ELISA detections of double antibody sandwich method are above mentioned embodiment provided, but the present disclosure applies equally to double antigens sandwich
The ELISA detections of method：
A ') according to the corresponding capture antigen of species selection and detection antigen of test antibodies in testing sample；
B ') with reference to embodiment 4 method, the modification step a ' on 5~30nm of particle diameter gold nano grain) described in detection
Antigen, obtain being modified with the gold nano grain of detection antigen；
C ') with reference to the design parameter progress ELISA detections of embodiment 5：Coating capture antigen → addition testing sample makes to treat
Survey antibody with capture antigen binding → addition step b ') obtain be modified with detection antigen gold nano grain and make test antibodies
With detection antigen binding therein, capture antigen-test antibodies-detection antigen-gold nano grain compound is formed；
D ') with reference to the design parameter of embodiment 5, to step c ') obtained capture antigen-test antibodies-detection antigen-gold
Nano-particle complex carries out silver-colored platinum dye：Add Ag+Solution and hydroquinones and make its final concentration be respectively 62.5~250 μM and
0.04~5mM, Ag is reduced using hydroquinones+Silver-colored shell is wrapped up with gold nano grain surface in the composite；Washing；Add
PtCl6 2-Solution and ascorbic acid simultaneously make its final concentration be respectively 0.25~1mM and 2~50mM, are reduced using ascorbic acid
PtCl6 2-To wrap up platinum shell again on the gold nano grain surface for having wrapped up silver-colored shell, obtain that there is receiving for peroxidase activity
Rice enzyme Au@AgPt particles, form capture antigen-test antibodies-detection antigen-Au@AgPt particle composites；
E ') with reference to the design parameter of embodiment 5, under 0.1~10M hydrogen peroxide existence conditions, utilize step d ') obtain
Capture antigen-test antibodies-detection antigen-Au@AgPt particle composites in have peroxidase activity nano enzyme Au@
AgPt particulate catalytic peroxidase substrates TMB obtains color products, 450nm detection color products absorbance, with
To the amount of test antibodies.
It is described above, only present pre-ferred embodiments, therefore the scope that the present invention is implemented can not be limited according to this, i.e., according to
The equivalent changes and modifications that the scope of the claims of the present invention and description are made, all should still it belong in the range of the present invention covers.
Claims (10)
- A kind of 1. ELISA detection method based on the nano enzyme with peroxidase activity, it is characterised in that：Suitable for dual anti- Body sandwich method or dual-antigen sandwich method, including：A) according to the corresponding capture antibody of species selection and detection antibody of determined antigen in testing sample；B) the detection antibody on 5~30nm of particle diameter gold nano grain described in modification step a), obtain being modified with detection antibody Gold nano grain；C) ELISA detections are carried out：Coating capture antibody → addition testing sample makes determined antigen and capture antibody binding → addition What step b) was obtained is modified with the gold nano grain of detection antibody and makes determined antigen and detection antibody binding therein, and formation is caught Obtain antibody-determined antigen-detection antibody-gold nano-particle complex；D) the capture antibody-determined antigen-detection antibody-gold nano-particle complex obtained to step c) carries out silver-colored platinum dye：Add Enter Ag+Solution and hydroquinones simultaneously make its final concentration be respectively 62.5~250 μM and 0.04~5mM, are reduced using hydroquinones Ag+Silver-colored shell is wrapped up with gold nano grain surface in the composite；Washing；Add PtCl6 2-Solution and ascorbic acid simultaneously make it Final concentration is respectively 0.25~1mM and 2~50mM, and PtCl is reduced using ascorbic acid6 2-To wrap up the gold nano of silver-colored shell Particle surface wraps up platinum shell again, obtains the nano enzyme Au@AgPt particles with peroxidase activity, formed capture antibody- Determined antigen-detection antibody-Au@AgPt particle composites；E) under 0.1~10M hydrogen peroxide existence conditions, the capture antibody-determined antigen-detection obtained using step d) is resisted There is the nano enzyme Au@AgPt particulate catalytic peroxidase substrates of peroxidase activity in body-Au@AgPt particle composites Product is obtained, detects the amount of product to obtain the amount of determined antigen；Or,A ') according to the corresponding capture antigen of species selection and detection antigen of test antibodies in testing sample；B ') the modification step a ' on 5~30nm of particle diameter gold nano grain) described in detection antigen, it is anti-to obtain being modified with detection Former gold nano grain；C ') carry out ELISA detections：Coating capture antigen → addition testing sample makes test antibodies and capture antigen binding → addition Step b ') obtain be modified with detection antigen gold nano grain and make test antibodies and detection antigen binding therein, formation Capture antigen-test antibodies-detection antigen-gold nano grain compound；D ') to step c ') obtained capture antigen-test antibodies-detection antigen-gold nano grain compound carries out silver-colored platinum dye： Add Ag+Solution and hydroquinones simultaneously make its final concentration be respectively 62.5~250 μM and 0.04~5mM, using hydroquinones also Former Ag+Silver-colored shell is wrapped up with gold nano grain surface in the composite；Washing；Add PtCl6 2-Solution and ascorbic acid simultaneously make Its final concentration is respectively 0.25~1mM and 2~50mM, and PtCl is reduced using ascorbic acid6 2-To wrap up the Jenner of silver-colored shell Platinum shell is wrapped up on rice grain surface again, obtains the nano enzyme Au@AgPt particles with peroxidase activity, and it is anti-to form capture Original-test antibodies-detection antigen-Au@AgPt particle composites；E ') under 0.1~10M hydrogen peroxide existence conditions, utilize step d ') obtained capture antigen-test antibodies-detection resists There is the nano enzyme Au@AgPt particulate catalytic peroxidase substrates of peroxidase activity in original-Au@AgPt particle composites Product is obtained, detects the amount of product to obtain the amount of test antibodies.
- 2. detection method according to claim 1, it is characterised in that：The step b) or b ') in, gold nano grain particle diameter For 13~20nm.
- 3. detection method according to claim 1, it is characterised in that：The step d) or d ') in, Ag+The final concentration of solution For 100~150 μM.
- 4. detection method according to claim 1, it is characterised in that：The step d) or d ') in, the end of hydroquinones is dense Spend for 0.2~1mM.
- 5. detection method according to claim 1, it is characterised in that：The step d) or d ') in, PtCl6 2-The end of solution Concentration is 0.4~0.6mM.
- 6. detection method according to claim 1, it is characterised in that：The step d) or d ') in, the end of ascorbic acid is dense Spend for 10~20mM.
- 7. detection method according to claim 1, it is characterised in that：The step e) or e ') in, the concentration of hydrogen peroxide For 1~5M.
- 8. detection method according to claim 1, it is characterised in that：The step e) or e ') in, the peroxidase Substrate is TMB；Color products are obtained using the nano enzyme Au@AgPt particulate catalytics TMB with peroxidase activity, 450nm detects the absorbance of color products, so that the amount of determined antigen or test antibodies is calculated.
- A kind of 9. nano enzyme preparation method with peroxidase activity, it is characterised in that：Including：1) modifying protein on 5~30nm of particle diameter gold nano grain, obtain being modified with the gold nano grain of protein；2) gold nano grain for being modified with albumen obtained to step 1) carries out silver-colored platinum dye：Add Ag+Solution and hydroquinones simultaneously make Its final concentration is respectively 62.5~250 μM and 0.04~5mM, and Ag is reduced using hydroquinones+To be wrapped on gold nano grain surface Wrap up in silver-colored shell；Washing；Add PtCl6 2-Solution and ascorbic acid simultaneously make its final concentration be respectively 0.25~1mM and 2~50mM, profit PtCl is reduced with ascorbic acid6 2-To wrap up platinum shell again on the gold nano grain surface for having wrapped up silver-colored shell, Au@AgPt are obtained Particle, it is the described nano enzyme with peroxidase activity.
- A kind of 10. nano enzyme with peroxidase activity prepared by preparation method according to claim 9.
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