CN103645315A - Platinum-based alloy structured nanorod simulation enzyme solution and application thereof in ELISA (Enzyme-Linked Immunosorbent Assay) - Google Patents

Platinum-based alloy structured nanorod simulation enzyme solution and application thereof in ELISA (Enzyme-Linked Immunosorbent Assay) Download PDF

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CN103645315A
CN103645315A CN201310701342.1A CN201310701342A CN103645315A CN 103645315 A CN103645315 A CN 103645315A CN 201310701342 A CN201310701342 A CN 201310701342A CN 103645315 A CN103645315 A CN 103645315A
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吴晓春
阿迪蒂亚萨兰
胡晓娜
张会
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National Center for Nanosccience and Technology China
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Abstract

The invention relates to a platinum-based alloy structured nanorod simulation enzyme solution and application thereof in ELISA (Enzyme-Linked Immunosorbent Assay). The platinum-based alloy structured nanorod simulation enzyme solution contains a gold kernel-platinum-based alloy-antibody or antigen composite structure which consists of a cylindrical gold nanorod kernel, an island-shaped platinum-based alloy structure and an antibody or antigen, wherein the outside surface of the cylindrical gold nanorod kernel is coated with the island-shaped platinum-based alloy structure, and the outside surface of the cylindrical gold nanorod kernel is coated with the antibody or antigen. When applied to the ELISA, the platinum-based alloy structured nanorod simulation enzyme solution disclosed by the invention has the advantages that the response range is wide in detection on the antibody or antigen, the sensitivity is high, and the detection limit can reach the level of pg/mL. Particularly, when a gold kernel/copper-platinum alloy shell nanorod is used, the separation of binding sites and the catalysis sites can be realized, and coexisting protein has not obvious influence on the catalytic activity of nanostructures.

Description

A kind of nano bar analogue enzyme solution of platinum base alloy structure and the application in ELISA
Technical field
The present invention relates to nanometer rods and the application thereof of platinum base alloy structure, relate in particular to a kind of nano bar analogue enzyme solution of platinum base alloy structure and the application in ELISA.
Background technology
Enzyme-linked immuno assay (ELISA) is the method for the strong detection albumen extensively approved of a kind of quilt.The method has the running program of high sensitivity and standard, conventionally uses the immunoreagent generation signal of horseradish peroxidase (HRP) mark to detect target molecule.Although have the various and sensitivity of method compared with advantages of higher, it also has some shortcomings.Such as a large amount of expensive albumen of needs, the reaction needed of diffusion control is much walked hatching and needs a lot of cleaning steps etc.Past these years in, people are mainly devoted to ELISA and how improve sensing range, increase the research of sensitivity and specific recognition etc.
The ELISA that nano particle participates in has following several mode.Nano particle can provide for traditional ELISA more interesting instrument after connecting biomolecule.On the one hand, because the specific surface area of nano particle is larger, can provide more connection site for identification antibody or HRP, thus realize signal amplification (Anal.Chem.2013,85,6228-6232).On the other hand, in phasmon ELISA, nano particle can be used as chromogenic substrate.This is mainly the change that can bring color due to the change in size of phasmon nano particle (comprising reunion), based on this theory, prostate specific antigen (prostate specific antigen, PSA) and the detectability of HIV-1 capsid antigen p24 in whole blood can be low to moderate 1 * 10 -18gmL -1(Nat.Chem.2012,7,821-824).In addition, nano particle self has class peroxidase activity, can directly replace the HRP in traditional E LISA.
In the last few years, Fe 3o 4, CeO 2in succession find to have type oxide enzyme and class peroxidase activity with the nano particle such as Pt, can replace the HRP in traditional E LISA, realize the high-sensitivity detection to target molecule.As utilize Fe 3o 4the panimmunity detection method of class peroxidase activity design, can realize to the detection of hepatitis B virus surface antigen and troponin (Nat.Nanotechnol.2007,2,577-583).In early stage, the research of gold-core/platinum-shell nanostructured (Au@Pt) class peroxidase activity is found, its can replace in traditional E LISA HRP realize to the highly sensitive detection of mouse interleukin (Biomaterials, 2011,32,1139-1147).
Yet, at present the nano bar analogue enzyme solution of platinum base alloy structure is replaced to the detection of HRP in ELISA as class peroxidase and still lacks further investigation and report.
Summary of the invention
The object of the present invention is to provide a kind of nano bar analogue enzyme solution of platinum base alloy structure and the application in ELISA, the nano bar analogue enzyme solution antagonist of described platinum base alloy structure or the detection responding range of antigen are wide, highly sensitive, and detectability can reach the level of pg/mL.
For realizing object of the present invention, the present invention by the following technical solutions:
In first aspect, the invention provides a kind of nano bar analogue enzyme solution of platinum base alloy structure, contain by cylindric gold nanorods kernel, be coated on the island platinum base alloy structure of described cylindric gold nanorods kernel outside surface and be coated in the antibody of described cylindric gold nanorods kernel outside surface or golden core-platinum base alloy-antibody or the antigen composite structure that antigen forms.
Golden core-platinum base alloy-the antibody or the antigen composite structure that in the nano bar analogue enzyme solution of platinum base alloy structure of the present invention, contain contain three part-structures: as the cylindric gold nanorods of kernel; As the island platinum base alloy structure of catalytic center, it is coated on described cylindric gold nanorods kernel outside surface, the head part of especially described cylindric gold nanorods kernel outside surface; With antibody or the antigen as immune binding site, it is coated in described cylindric gold nanorods kernel outside surface.The composite structure of said structure is because the specific surface area of cylindric gold nanorods kernel is large, and for immune response provides more binding site, so the enlarge-effect of signal is obvious; Island platinum base alloy structure has the enzymatic activity that is similar to HRP etc., can realize the ELISA reaction of analogue enztme characteristic.
As of the present invention preferred, the nanometer rods of described platinum base alloy structure is selected from one or more in gold-core/platinum-shell nanometer rods, golden core/copper platinum alloy shell nanometer rods, gold core/platinum palldium alloy shell nanometer rods and gold core/silver-platinum alloy shell nanometer rods; Golden core/copper platinum alloy shell nanometer rods more preferably, wherein copper platinum is mainly island deposition at the two ends of cylindric gold nanorods kernel, and antibody or antigen are mainly coated in the outer surface of rod between described cylindric gold nanorods kernel two ends.
Herein, other parts gold-core/platinum-shell nanometer rods is called again " nanometer rods of gold core/platinum nanometer island shell structure ", gold core/copper platinum alloy shell nanometer rods is called again " nanometer rods of golden core/copper platinum alloy nanometer island shell structure ", and it is identical that those skilled in the art can understand its implication.
When using golden core/copper platinum alloy shell nanometer rods, can realize the separated of binding site and catalytic site, the albumen coexisting is on the not significantly impact of the catalytic activity of nanostructured.
In second aspect, the invention provides a kind of method of preparing the nano bar analogue enzyme solution of the platinum base alloy structure as described in first aspect, comprising:
(1) the nanometer rods solution of platinum base alloy structure is mixed and shaken up with surfactant, place centrifugally after at least 3 hours, obtain the nanometer rods precipitation of surperficial electronegative platinum base alloy structure, abandon supernatant resuspended;
(2) in the solution of the resuspended gained of step (1), add coated damping fluid and antibody or antigen, mix rear constant-temperature incubation, then free antibody or the antigen of centrifugal removal, obtain the nanometer rods of the platinum base alloy structure of antibody or Modified antigen, the resuspended nano bar analogue enzyme solution that obtains described platinum base alloy structure.
Wherein, in step (1), with surfactant, processing is mainly for increasing the stability of nanometer rods in buffer solution, also for reducing bio-toxicity.Described surfactant solution can be one or more in kayexalate (PSS), polyethyleneimine (PEI), polyvinylpyrrolidone (PVP) and diallyl dimethyl ammoniumchloride (PDDAC), is preferably kayexalate (PSS).
Wherein, in described step (1), can, by deionized water or the resuspended precipitation of coated damping fluid, preferably use the resuspended precipitation of deionized water.
Preferably, in described step (1), in the solution of resuspended gained, the nanometer rods concentration of surperficial electronegative platinum base alloy structure is 0.1-5nM, for example 0.2nM, 0.3nM, 0.5nM, 0.8nM, 1nM, 2nM, 3nM, 4nM or 5nM, more preferably 2nM.
Preferably, in described step (2), coated damping fluid is selected from the buffer solution containing bovine serum albumin (BSA); Wherein, buffer solution can be one or more in trishydroxymethylaminomethane (Tris) damping fluid, hydroxyethyl piperazine second thiosulfonic acid (HEPES) damping fluid and phosphoric acid (PBS) damping fluid, more preferably trishydroxymethylaminomethane (Tris) damping fluid.
Preferably, the temperature of constant-temperature incubation is 37 ℃ in described step (2), can certainly be other applicable temperature, such as 25 ℃, 30 ℃, 35 ℃, 40 ℃, 35-39 ℃ or 33-38 ℃ etc.
Preferably, in described step (2), the time of constant-temperature incubation is 15-120min, for example 18min, 20min, 25min, 30min, 45min, 60min, 80min, 100min or 110min, more preferably 30min.
Preferably, in described step (2), antibody is one or more in goat anti-human igg, sheep anti-mouse igg, goat anti-rabbit igg and mouse interleukin antibody, more preferably goat anti-human igg.
Preferably, in described step (2), with respect to the nanometer rods of the electronegative platinum base alloy structure in 0.1-5pmol surface, the addition of antibody is 0.5-10 μ g.Wherein, the nanometer rods of surperficial electronegative platinum base alloy structure can from 0.1-5pmol, for example, be selected in 0.2pmol, 0.5pmol, 0.8pmol, 1pmol, 1.2pmol, 2pmol, 3pmol, 3.2pmol, 3.8pmol, 4.5pmol or 4.8pmol; Antibody can from 0.5-10 μ g, for example, be selected in 0.5 μ g, 0.7 μ g, 0.9 μ g, 1.2 μ g, 1.5 μ g, 2 μ g, 4 μ g, 6 μ g, 8 μ g or 9 μ g.Certainly, those skilled in the art can understand the nanometer rods of surperficial electronegative platinum base alloy structure and the consumption of antibody is not limited only to above-mentioned numerical range, also can on the basis of aforementioned proportion, suitably expand or dwindle.
More preferably, in described step (2), with respect to the nanometer rods of the electronegative platinum base alloy structure in 2pmol surface, the addition of antibody is 5 μ g.
In the third aspect, the invention provides the nano bar analogue enzyme solution detectable antigens of the platinum base alloy structure of a kind of use as described in first aspect or the method for antibody, comprising:
(1) nano bar analogue enzyme solution of antibody or antigen coated platinum base alloy structure is added in the coated microwell plate of antigen or antibody, hatch and with buffer solution, clean afterwards;
(2) then add the phosphoric acid-citric acid solution that contains chromogenic substrate to react;
(3) finally add stop buffer cessation reaction, and use microplate reader to monitor the variation of its absorbance.
Preferably, the microwell plate in described step (1) is the microwell plate in 96 holes; The microwell plate in 96 holes is microwell plates the most frequently used in ELISA, but does not also get rid of other microwell plate for situation of the present invention.
Preferably, the antigen on described microwell plate be selected from can with the nanometer rods of described platinum base alloy structure on human IgG, mouse IgG, rabbit igg and the mouse interleukin antigen of antibody mediated immunity identification in one or more, human IgG more preferably.
Preferably, the temperature of hatching in described step (1) is 4-44 ℃, for example 5 ℃, 7 ℃, 9 ℃, 10 ℃, 12 ℃, 13 ℃, 15 ℃, 20 ℃, 25 ℃, 28 ℃, 32 ℃, 35 ℃, 40 ℃, 42 ℃ or 43 ℃, and more preferably 37 ℃.
Preferably, the time of hatching in described step (1) is 30-360min, for example 40min, 60min, 100min, 150min, 180min, 210min, 240min, 280min, 300min, 320min, 40-120min, 50-300min or 120-280min, more preferably 240min.
Preferably, in described step (1), the addition of analogue enztme solution is 75-200 μ L, for example 80 μ L, 90 μ L, 100 μ L, 120 μ L, 130 μ L, 150 μ L, 170 μ L or 190 μ L, more preferably 100 μ L.
Preferably, in described step (2), chromogenic substrate is 3,3 ', 5,5 '-tetramethyl benzidine (TMB), 2,2 '-hydrazine-bis-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) diamine salts (ABTS) or o-phenylenediamine (OPD), more preferably 3,3 ', 5,5 '-tetramethyl benzidine (TMB) or 2,2 '-hydrazine-bis-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) diamine salts (ABTS).
Preferably, the pH of phosphoric acid-citric acid solution is 3-6 in described step (2), for example 3.2,3.5,3.7,3.9,4.2,4.5,4.8,5.2,5.4,5.6,5.8,3.5-5 or 3.8-5.5, and more preferably 4.2.
Preferably, in described step (2), the time of reaction is 10-30min, for example 12min, 15min, 18min, 21min, 24min, 26min or 28min, more preferably 20min.
Preferably, in described step (2), stop buffer is sulfuric acid solution or sodium dodecylsulphonate (SDS) solution, and wherein the concentration of sulfuric acid solution can be 1.5M, 2M or 3M, and the mass concentration of sodium dodecyl sulfate solution can be 0.1%, 0.5%, 1% or 2%.
In fourth aspect, the application of the nano bar analogue enzyme solution that the invention provides the platinum base alloy structure as described in first aspect in enzyme-linked immuno assay.
As of the present invention preferred, the nanometer rods of described platinum base alloy structure is golden core/copper platinum alloy shell nanometer rods, wherein copper platinum is mainly island deposition at the two ends of cylindric gold nanorods kernel, and antibody or antigen are mainly coated in the outer surface of rod between described cylindric gold nanorods kernel two ends.
Beneficial effect of the present invention is: in the nano bar analogue enzyme solution of platinum base alloy structure of the present invention, contain golden core-platinum base alloy-antibody or antigen composite structure, wherein platinum base alloy is similar to HRP enzyme as catalytic center; The detection responding range of analogue enztme solution antagonist of the present invention or antigen is wide, highly sensitive, and detectability can reach the level of pg/mL.Especially use golden core/copper platinum alloy shell nanometer rods, take ABTS during as substrate, detect and be limited to 90pg/mL, take TMB during as substrate, detectability is lower than 90pg/mL.In addition, while using golden core/copper platinum alloy shell nanometer rods, can realize the separated of binding site and catalytic site, the albumen coexisting is on the not significantly impact of the catalytic activity of nanostructured.Method provided by the present invention is simple to operation, repeatability is high, and the nano particle that uses is with low cost, be easy to preparation.
Accompanying drawing explanation
Fig. 1 is that (a) (b) transmission electron microscope photo and the corresponding UV, visible light of nano particle absorb spectrogram (c) to the prepared gold-core/platinum-shell of the embodiment of the present invention 1 (Au Pt) with golden core/copper platinum alloy shell (Au PtCu), wherein Au NRs represents gold nanorods, curve (I), (II) and (III) represent respectively Au@Pt, Au@PtCu and Au NRs UV, visible light absorption spectra.
Fig. 2 be in the embodiment of the present invention 2 Au@Pt nanometer rods and Au@PtCu nanometer rods as the concentration of substrate of class peroxidase and the relation of catalytic oxidation speed, and the Michaelis constant of utilizing two counting backward techniques to try to achieve.
Fig. 3 is the impact on the TMB oxidation rate of Au@Pt nanometer rods and the catalysis of Au@PtCu nanometer rods of situation that in the embodiment of the present invention 4, albumen exists, wherein (a) shows that the albumen coexisting can obviously reduce the catalytic activity of Au@Pt, Au Pt+0.05%BSA indicates the situation that 0.05%BSA coexists, and Au Pt+ contrast represents the situation that does not have albumen to coexist; (b) impact that the albumen that demonstration coexists brings Au@PtCu almost can be ignored, Au PtCu-contrast represents the situation that does not have albumen to coexist, Au@PtCu@antibody+0.05%BSA indicates the situation that antibody and 0.05%BSA coexist, and Au@PtCu+0.05%BSA indicates the situation that 0.05%BSA coexists.
Fig. 4 is the structural representation of Au@PtCu nanometer rods in the present invention, and the ELISA course of reaction schematic diagram participating in.
Fig. 5 be take TMB(a in the embodiment of the present invention 6 and 7) and during ABTS(b) as substrate, the relation between the absorbance obtaining and testing protein concentration.
Embodiment
Below in conjunction with embodiment, embodiment of the present invention are described in detail.It will be understood to those of skill in the art that following examples are only the preferred embodiments of the present invention, so that understand better the present invention, thereby should not be considered as limiting scope of the present invention.For a person skilled in the art, the present invention can have various modifications and variations, within the spirit and principles in the present invention all, any modification of doing, is equal to and replaces or improvement etc., within all should being included in protection scope of the present invention.Experimental technique in following embodiment, if no special instructions, is conventional method; Experiment material used, if no special instructions, is and is purchased available from routine biochemistry chemical reagent work.
In following examples, described reagent is as follows: cetyl trimethyl ammonium bromide (CTAB, Amresco), potassium chloroplatinate (K 2ptCl 4,, Alfa), cupric chloride (CuCl 2, Alfa), ascorbic acid (AA, Alfa), polystyrene-based sodium sulfonate (PSS, Alfa), goat anti-human igg (ZSGB-Bio), human IgG (ZSGB-Bio), the goat anti-human igg of HRP mark (Bioss), 3,3 ', 5,5 '-tetramethyl benzidine (TMB, Alfa), 2,2 '-hydrazine-bis-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) diamine salts (ABTS, Alfa).
Testing tool has: microplate reader (Infinite tMm200), Varian Cary50, TEM(Tecnai G 220S-TWIN), Delsa Nano C(Beckman Coulter).
Embodiment 1: the nanometer rods of golden core/(copper) platinum alloy nanometer island shell structure prepare purifying
The nanometer rods of golden core described in the present invention/(copper) platinum alloy nanometer island shell structure can obtain by method well-known to those skilled in the art.In the present invention, we use gold nanorods for seed, reduce the codeposition of Pt or Pt and Cu realize the preparation of nanostructured by reductive agent.
The preparation of the nanometer rods of gold core/platinum nanometer island shell structure (Au@Pt): get the gold nanorods solution of 1mL purifying, add successively the K of the 2mM of 75 μ L 2ptCl 4solution, the AA solution of the 0.1M of 22.5 μ L, is placed on after mixing in 30 ℃ of water-baths and reacts half an hour, adds afterwards the CTAB solution of the 0.1M of 0.5mL, and within centrifugal 5 minutes under 12000rpm, once precipitation is dispersed in deionized water standby.
The preparation of the nanometer rods (Au@PtCu) of gold core/copper platinum alloy nanometer island shell structure: get 1mL purifying gold nanorods solution, add successively the K of the 2mM of 75 μ L 2ptCl 4solution, the CuCl of the 10mM of 40 μ L 2solution, the AA solution of the 0.1M of 22.5 μ L, is placed on after mixing in 30 ℃ of water-baths and reacts half an hour, adds afterwards the CTAB solution of the 0.1M of 0.5mL, and within centrifugal 5 minutes under 12000rpm, once precipitation is dispersed in deionized water standby.
The TEM shape appearance figure of Au@Pt and Au@PtCu nanometer rods and corresponding UV, visible light absorb figure as shown in Figure 1.From Fig. 1 (a), can find out, Pt is island growth pattern on gold nanorods surface; Introduce after Cu, the codeposition of Cu and Pt still keeps island growth, different, and these islands are to be mainly deposited on excellent two ends (b).In addition, the codeposition of the deposition of Pt and Pt and Cu all can cause local plasmon resonance peak (LSPR) to produce red shift (c).
Embodiment 2: enzyme kinetics parameter is analyzed
Its reaction kinetics is investigated in variation by monitoring TMB oxidation product absorption peak absorbance.Use Varian Cary50 kinetics model, every 0.1min interval measurement once.Test condition is: the concentration of Au@Pt or Au@PtCu nanometer rods is 15pM, the phosphate buffer solution of the pH4.5 of the 0.1M of 2.4mL, and temperature of reaction is set as 30 ℃.When take TMB during as substrate, H 2o 2concentration be fixed on 2mM; When with H 2o 2during for substrate, the concentration of TMB is fixed on 0.13mM.Apparent kinetics parameter is from Lineweaver-Burk equation: 1/V=(K m/ V max) (1/[C])+1/V maxobtain.Wherein, V is reaction velocity, V maxbe maximum reaction velocity, [C] is concentration of substrate, K mit is Michaelis constant.
The kinetic parameter obtaining is as shown in table 1 and Fig. 2.
Table 1Au@Pt and Au@PtCu nanometer rods are as the apparent kinetics parameter of class peroxidase
Figure BDA0000440808750000091
Wherein, K cat=V max/ [E], [E] is the concentration of enzyme, K catit is catalyzer constant.
Enzymic catalytic reaction dynamics can use equation (1) to describe:
Figure BDA0000440808750000101
K m＝((k -1+k 2)/k 1)
V = k 2 [ E 0 ] × [ S ] K m + [ S ] = V max × [ S ] K m + [ S ]
At this, K mby k -1, k 1and k 2determine.Concerning nano particle analogue enztme, surface is coated and k 2to apparent K mthere is contribution.As can be seen from Table 1, take TMB during as substrate, Au@PtCu nanometer rods has larger K mand V max; With H 2o 2during for substrate, Au@PtCu nanometer rods has less K mlarger V max, K mreduction be mainly derived from k 1increase, illustrate that Pt and Cu form after alloy, its variation that electronic structure is brought can affect the proximities to substrate, to H 2o 2affinity strengthen.
Embodiment 3: the nanorod surfaces of golden core/(copper) platinum alloy nanometer island shell structure is carried out goat anti-human igg antibody's modification
Get coated golden core/(copper) platinum alloy shell nanostructured solution of 1mL purifying original CT AB once, the PSS solution that adds the 20mg/mL of 50 μ L, more than at room temperature placing 3h after mixing, under 12000rpm, centrifugal 5 minutes once afterwards, precipitation is dispersed in 200 μ L deionized waters standby, and after purifying, the concentration of alloy nano rod is about 2nM.
The coated 2nM alloy nano rod solution of PSS of getting 1mL purifying, is dispersed in 1mL containing in the Tris buffer solution (0.05M, pH8) of 0.05%BSA.Add the 1mg/mL goat anti-human igg solution of 5 μ L, after mixing, put into 37 ℃ of constant temperature ovens, after hatching 30 minutes, within centrifugal 5 minutes under 8000rpm rotating speed, remove free goat anti-human igg's molecule.Outwell supernatant, sediment is dispersed in to 1mL containing standby in the Tris damping fluid (50mM, pH8) of 0.05%BSA.
Embodiment 4: the impact research of the albumen coexisting on catalytic activity
(the DMSO solution of 1mg/mL TMB is dissolved in phosphoric acid-citric acid solution of 9mL0.05M pH4.2 1mL tmb substrate solution, and adds 2 μ L30%H 2o 2) in, with after one times of deionized water dilution, add the Au PtCu nanometer rods solution of 20 μ L2nM, monitor the dynamic variation of its oxidation TMB.
In 1mL tmb substrate solution, add the Au@PtCu nanometer rods solution of 20 μ L2nM, 5 μ L1mg/mL goat anti-human igg's solution and 0.05%BSA solution, monitor the dynamic variation that it is oxidized TMB.
In 1mL tmb substrate solution, add the Au@PtCu nanometer rods solution of 20 μ L2nM, 0.05%BSA solution, monitors the dynamic variation that it is oxidized TMB.
As a comparison, we have also carried out the experiment of oxidation TMB to Au@Pt nanometer rods solution (containing or do not contain 0.05%BSA).Result as shown in Figure 3.
As seen from Figure 3, the albumen coexisting can obviously reduce the catalytic activity (Fig. 3 (a)) of Au@Pt; But concerning Au PtCu, the impact that albumen brings almost can be ignored, illustrate that catalytic activity is not suppressed, this is mainly due to albumen, to tend to be adsorbed on the side of nanometer rods, and concerning Au@PtCu, side does not almost have catalytic site, so be not affected.This phenomenon hint Au@PtCu has the catalytic site of realizing potentiality separated with binding site.
Table 2 is to the coated Au@PtCu nanometer rods size of difference and the data of zeta potential measurement.Result shows that the coated nanometer rods (Au@PtCu) of PSS is electronegative; After 0.05%BSA hatching (Au@PtCu@0.05%BSA), the surface charge of nanometer rods increases a little; Further, with goat anti-human igg's hatching (Au@PtCu@0.05%BSA@antibody), electric charge can continue to increase, and the existence of nanorod surfaces albumen is described.
Size and the zeta current potential of the different coated Au@PtCu nanometer rods of table 2
Figure BDA0000440808750000111
Embodiment 5: immune recognition reaction
First transparent, flat tygon 96 orifice plates are cleaned with the PBS of 0.05M pH7.4.Afterwards to the PBS monoclonal antibody human IgG solution that is dispersed in 0.05M pH7.4 that adds respectively 100 μ L variable concentrations in hole.Wherein human IgG concentration range, from 9pg to 0.9 μ g, is hatched and is spent the night at 4 ℃.With the Tris buffer(of 50mM pH8, contain 0.05%Tween-20 afterwards) clean 2 times, finally with the Tris buffer of 50mM pH8, again clean.Then with the Tris buffer of 1%BSA, in 37 ℃, seal 2 hours.Finally use again Tris buffer(0.05M, pH8) clean 3 times.Then to the Au@PtCu nanometer rods solution that adds in each hole 100 μ L goat anti-human iggs to modify, in 37 ℃ of hatchings 4 hours.Afterwards with Tris buffer(50mM, pH8 containing 0.05%Tween-20) clean, the last Tris buffer of 50mM pH8 that uses again cleans once.
Fig. 4 has shown the structural representation of Au@PtCu nanometer rods in the present invention, and the ELISA course of reaction schematic diagram participating in.Can find out, the ELISA reaction that in the present invention, Au@PtCu nanometer rods participates in can realize the separated of binding site and catalytic site.
Embodiment 6: take the detection reaction of TMB during as chromogenic substrate
To adding 100 μ L tmb substrate solution in each hole of the microwell plate of embodiment 5, (the DMSO solution of 1mg/mL TMB is dissolved in phosphoric acid-citric acid solution of 9mL0.05M pH4.2, and adds 2 μ L30%H 2o 2).React and after 20 minutes, add 25 μ L2M H 2sO 4cessation reaction, the absorbance at detection 450nm place.Contrast, positive contrast, negative contrast and blank have been set up in whole experiment.
Result, as shown in Fig. 5 (a), can find out that between determined antigen and absorbance, having good exponential relationship, responding range is 90pg/mL-9 μ g/mL, and lowest detectable limit will be lower than 90pg/mL.
Embodiment 7: take the detection reaction of ABTS during as chromogenic substrate
To the substrate solution that adds 100 μ L ABTS in each hole of the microwell plate of embodiment 5, (0.4mg/mL is dissolved in phosphoric acid-citrate buffer solution of 0.1M pH4.2, containing 2 μ L30%H 2o 2).React after 20 minutes and add 100 μ L1%SDS stop buffers to stop reaction, detect the absorbance of 405nm.Whole experiment is provided with contrast, positive contrast, negative contrast and blank.
Result, as shown in Fig. 5 (b), can find out that between determined antigen and absorbance, having good exponential relationship, responding range is 90pg/mL-9 μ g/mL, and lowest detection is limited to 90pg/mL.
Applicant's statement, the present invention illustrates detailed features of the present invention and detailed method by above-described embodiment, but the present invention is not limited to above-mentioned detailed features and detailed method, do not mean that the present invention must rely on above-mentioned detailed features and detailed method could be implemented.Person of ordinary skill in the field should understand, any improvement in the present invention is selected the selection of the equivalence replacement of component and the interpolation of auxiliary element, concrete mode etc., within all dropping on protection scope of the present invention and open scope to the present invention.

Claims (10)

1. a nano bar analogue enzyme solution for platinum base alloy structure, contains by cylindric gold nanorods kernel, is coated on the island platinum base alloy structure of described cylindric gold nanorods kernel outside surface and is coated in the antibody of described cylindric gold nanorods kernel outside surface or golden core-platinum base alloy-antibody or the antigen composite structure that antigen forms.
2. the nano bar analogue enzyme solution of platinum base alloy structure according to claim 1, it is characterized in that, the nanometer rods of described platinum base alloy structure is selected from one or more in gold-core/platinum-shell nanometer rods, golden core/copper platinum alloy shell nanometer rods, gold core/platinum palldium alloy shell nanometer rods and gold core/silver-platinum alloy shell nanometer rods; Be preferably golden core/copper platinum alloy shell nanometer rods, wherein copper platinum is mainly island deposition at the two ends of cylindric gold nanorods kernel, and antibody or antigen are mainly coated in the outer surface of rod between described cylindric gold nanorods kernel two ends.
3. a method of preparing the nano bar analogue enzyme solution of platinum base alloy structure as claimed in claim 1 or 2, comprising:
(1) the nanometer rods solution of platinum base alloy structure is mixed and shaken up with surfactant, place centrifugally after at least 3 hours, obtain the nanometer rods precipitation of surperficial electronegative platinum base alloy structure, abandon supernatant resuspended;
(2) in the solution of the resuspended gained of step (1), add coated damping fluid and antibody or antigen, mix rear constant-temperature incubation, then free antibody or the antigen of centrifugal removal, obtain the nanometer rods of the platinum base alloy structure of antibody or Modified antigen, the resuspended nano bar analogue enzyme solution that obtains described platinum base alloy structure.
4. method according to claim 3, it is characterized in that, described surfactant solution is selected from one or more in kayexalate, polyethyleneimine, polyvinylpyrrolidone and diallyl dimethyl ammoniumchloride, is preferably kayexalate.
5. according to the method described in claim 3 or 4, it is characterized in that, in described step (1), by deionized water or the resuspended precipitation of coated damping fluid, preferably use the resuspended precipitation of deionized water;
Preferably, in described step (1), in the solution of resuspended gained, the nanometer rods concentration of surperficial electronegative platinum base alloy structure is 0.1-5nM, more preferably 2nM.
6. according to the method described in claim 3-5 any one, it is characterized in that, in described step (2), coated damping fluid is selected from the buffer solution containing BSA;
Preferably, described buffer solution is selected from one or more in TRIS buffer, hydroxyethyl piperazine second thiosulfonic acid damping fluid and phosphate buffer, more preferably TRIS buffer;
Preferably, in described step (2), the temperature of constant-temperature incubation is 37 ℃;
Preferably, in described step (2), the time of constant-temperature incubation is 15-120min, more preferably 30min;
Preferably, in described step (2), antibody is one or more in goat anti-human igg, sheep anti-mouse igg, goat anti-rabbit igg and mouse interleukin antibody, more preferably goat anti-human igg;
Preferably, in described step (2), with respect to the nanometer rods of the electronegative platinum base alloy structure in 0.1-5pmol surface, the addition of antibody is 0.5-10 μ g;
More preferably, in described step (2), with respect to the nanometer rods of the electronegative platinum base alloy structure in 2pmol surface, the addition of antibody is 5 μ g.
7. use the nano bar analogue enzyme solution detectable antigens of platinum base alloy structure as claimed in claim 1 or 2 or a method for antibody, comprising:
(1) nano bar analogue enzyme solution of antibody or antigen coated platinum base alloy structure is added in the coated microwell plate of antigen or antibody, hatch and with buffer solution, clean afterwards;
(2) then add the phosphoric acid-citric acid solution that contains chromogenic substrate to react;
(3) finally add stop buffer cessation reaction, and use microplate reader to monitor the variation of its absorbance.
8. method according to claim 7, is characterized in that, the microwell plate in described step (1) is the microwell plate in 96 holes;
Preferably, the antigen on described microwell plate be selected from can with the nanometer rods of described platinum base alloy structure on human IgG, mouse IgG, rabbit igg and the mouse interleukin antigen of antibody mediated immunity identification in one or more, human IgG more preferably;
Preferably, the temperature of hatching in described step (1) is 4-44 ℃, more preferably 37 ℃;
The time of preferably, hatching in described step (1) is 30-360min, more preferably 240min;
Preferably, in described step (1), the dosage of analogue enztme solution is 75-200 μ L, more preferably 100 μ L;
Preferably, in described step (2), chromogenic substrate is 3,3 ', 5,5 '-tetramethyl benzidine, 2,2 '-hydrazine-bis-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) diamine salts or o-phenylenediamine, more preferably 3,3 ', 5,5 '-tetramethyl benzidine or 2,2 '-hydrazine-bis-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) diamine salts;
Preferably, the pH of phosphoric acid-citric acid solution is 3-6 in described step (2), more preferably 4.2;
Preferably, in described step (2), the time of reaction is 10-30min, more preferably 20min;
Preferably, in described step (2), stop buffer is sulfuric acid solution or sodium dodecyl sulfate solution.
9. the application of the nano bar analogue enzyme solution of platinum base alloy structure as claimed in claim 1 or 2 in enzyme-linked immuno assay.
10. application according to claim 9, it is characterized in that, the nanometer rods of described platinum base alloy structure is golden core/copper platinum alloy shell nanometer rods, wherein copper platinum is mainly island deposition at the two ends of cylindric gold nanorods kernel, and antibody or antigen are mainly coated in the outer surface of rod between described cylindric gold nanorods kernel two ends.
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