CN105436511B - Platinum nanocage immune probe and preparation method thereof and application thereof in preparing electrochemical immunosensor - Google Patents
Platinum nanocage immune probe and preparation method thereof and application thereof in preparing electrochemical immunosensor Download PDFInfo
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Abstract
The invention discloses a platinum nanocage immune probe and a preparation method thereof and an application thereof in preparing electrochemical immunosensor. According to the invention, cubic platinum nanocage-shaped particles are obtained by substituting platinum atoms for palladium atoms in palladium nano cubic particles. The beneficial effects of the invention are: the method is simple; the obtained platinum nanocage-shaped particles are cubic in shape and have uniform sizes. Experimental results prove that, a cubic nanocage structure has a better catalysis effect than a globular platinum particle does so that the former has a more obvious signal amplification effect. An electrochemical immunosensor prepared by the immune probe which is prepared by platinum nanocage-shaped particles has strong and stable signals and good repeatability; simultaneous detection of double target is realized through marking electric signal material which can be distinguished by peak potential. The platinum nanocage immune probe provided in the invention and electrochemical immunosensors prepared with the same have a large application prospect in the field of immunosense.
Description
Technical field
The present invention relates to a kind of noble metal nano particles and preparation method thereof and the application in immunological probe is prepared, especially
It is related to a kind of platinum nanocages and preparation method thereof and the application in immunological probe and electrochemical immunosensor is prepared.
Background technology
Cancer is a kind of fatal disease, and every year, the number of global range endogenous cause of ill cancer mortality is up to 7,000,000 people, China
There are 1,000,000 people therefore lose life.The probability of cancer patient's survival, is largely dependent upon the discovery time of cancer.Cancer
The early discovery and treatment of disease, is the key of cancer patient's survival.Cancer markers are the marks that cancer occurs, exists and develop
(Magdalena Swierczewska,Gang Liu,Seulki Lee,Xiaoyuan Chen,High-sensitivity
nanosensors for biomarker detection,Chem.Soc.Rev.,2011,41,2641-2655.).Therefore, cancer
The Sensitive Detection of disease mark is the key factor of the early discovery of cancer and treatment.At present, the detection to cancer markers has
The methods such as euzymelinked immunosorbent assay (ELISA), colorimetry, fluorescence method and surface plasma resonance, but often there is time-consuming, equipment and hold high in these methods
Expensive, process is loaded down with trivial details and the low problem of sensitivity.Consider above-mentioned factor, electrochemical method is due to simple to operate, cost
It is low, the features such as sensitivity is high, and become one of optimal method of detection cancer markers (Aicheng Chen,
Sanghamitra Chatterjee,Chem.Soc.Rev.,2013,42,5425-5438.).Electrochemistry experiment is by detection
It is fixed on the electrochemical substance on electrode to provide signal.Generally use the work that sandwich type electrochemical method detects cancer markers
Make principle as follows：(1) using electroactive material come labelling tracer antibody；(2) by tracer antibody discriminatory analysiss thing；(3) utilize
The signal that electroactive material is provided determines the concentration of the target biomarker for being fixed on electrode surface.However, current electrification
The detection that detection is concentrated mainly on single tumor markerses is learned, but due between a kind of single cancer markers and cancer
Susceptiveness and specificity are very poor, therefore can become the screening for cancer instrument of " ideal " without a kind of cancer markers.With it is traditional
Single target immunoassay is compared, many target immunoassays have that analysis time is short, sample volume reduces, detection efficiency is improved, into
The advantages of this reduction, therefore detection has very important significance for the Precise Diagnosis of cancer also have while many targets.
For many target immunoassays, matter of utmost importance is how to make a distinction the different signals of telecommunication and on this basis
Obtain larger electric current.Nano-particle amplification electrochemical signals strengthening electric current during, noble metal Pt nanoparticle due to
With good electric conductivity, it is easy to which it is excellent that load antibodies and the catalytic property to hydrogen peroxide can further enhance electric current etc.
Point.Therefore can be used as good carrier in the application of electrochemical immunosensor.
The present invention is carrier by platinum nanocages, and by first load antibodies, the mode that semiochemicalses are connected afterwards prepares platinum
Nanocages immunological probe.It is expensive due to platinum, therefore nanometer cagelike structure can reduce cost, and the catalytic of platinum
Can be affected than larger by its pattern, and cube shaped nanometer basket structure has more preferably than spherical platinum grain to hydrogen peroxide
Catalytic effect, therefore the effect of its amplification of signal becomes apparent from.Because noble metal nano particles are to work(such as amino, sulfydryls
The signal of telecommunication material of energy base has outstanding binding ability, therefore, the signal of telecommunication thing distinguished using platinum nanocages connecting peak electrical potential energy
Matter can be realized being detected while two kinds of cancer markers or even kinds cancer label.
The content of the invention
An object of the present invention is to provide a kind of cube shaped platinum nanocage granule and preparation method thereof；
The second object of the present invention is to provide a kind of platinum nanometer obtained by the new platinum nanocage particle preparation
Cage immunological probe；
The third object of the present invention is to provide a kind of electro-chemistry immunity prepared by platinum nanocages immunological probe to pass
Sensor.
The above-mentioned purpose of the present invention is realized by following technological means：
A kind of cube shaped platinum nanocage granule of the present invention, it is characterised in that be prepared by the following method and obtain：
(1) after the aqueous solution of cetyl trimethylammonium bromide (CTAB) being heated to into 80-120 DEG C, chlorine palladium sour water is added
Solution, is subsequently adding ascorbic acid, and after reaction 10-60min, centrifugation, precipitate with deionized water cleaning is then dispersed in deionization
In water；
(2) solution for obtaining step (1) and the aqueous solution of CTAB, are heated to 80-100 DEG C, by potassium chloroplatinate water
Solution is added thereto, and stirs 10-20 hours, and product centrifugation, precipitation deionized water cleaning is obtained final product.
In the present invention, it is preferred to, the aqueous solution of the cetyl trimethylammonium bromide (CTAB) described in step (1) it is dense
Spend for 10-25mM, the concentration of the chlorine palladium acid solution for being added is 1-10mM, and volume is cetyl trimethylammonium bromide (CTAB)
Aqueous solution volume 1/20-1/30, the mol ratio of the ascorbic acid for being added and chlorine palladium acid is 1:0.1-1.
In the present invention, it is preferred to, the concentration of the CTAB aqueous solutions that step (2) is added is 6-25mM, the amount for being added
2-5 times of the liquor capacity obtained for step (1), the concentration of the potassium chloroplatinate aqueous solution for being added is 0.1-10mM, is added
Amount be the 1/5-1/2 of liquor capacity that step (1) is obtained.
In the present invention, first palladium nanocube granule has been obtained by step (1), then by step (2) by palladium
Palladium atomic substitutions in nanocube granule are pt atom, so as to obtain a kind of cube shaped nanometer basket structure.This
Bright method is simple, and the platinum nanocage particle size for preparing is homogeneous, it is demonstrated experimentally that cube shaped nanometer basket structure phase
There is more preferable catalytic effect to hydrogen peroxide compared with spherical platinum grain, therefore the effect of its amplification of signal becomes apparent from.Cause
This prepares electrochemical immunosensor signal by force and stable, favorable reproducibility using immunological probe prepared by the platinum nanocages, and logical
The signal of telecommunication material that crossing marker peak current potential can distinguish is detected while realizing dual-target.
Therefore, further, the invention allows for described platinum nanocage granule is preparing the immunity spy of platinum nanocages
Application in pin.
A kind of platinum nanocages immunological probe of the present invention, it is characterised in that be prepared by the following method and obtain：
(1) the platinum nanocage granule described in any of the above item is washed with phosphate buffer, is disperseed；
(2) under room temperature, platinum nanocage granule is mixed with antibody, is centrifuged off supernatant；
(3) precipitation phosphate buffer wash, disperse after with signal of telecommunication material mixing, be centrifuged off supernatant；
(4) precipitation is washed with phosphate buffer, and the platinum nanocages immunological probe for obtaining is distributed in phosphate buffer, in 4
DEG C storage.
In the present invention, it is preferred to, phosphate buffer used is 0.1M in step (1), (2) or (4), and pH value is 5.0-
8.0, and the phosphate buffer containing 0.1MKCl.
In the present invention, it is preferred to, described signal of telecommunication material includes thionine and sulfydryl ferrocene.
Further, the invention allows for described platinum nanocages immunological probe is in electrochemical immunosensor is prepared
Application, it is preferred that prepare respectively be coupled what two or more antibody and spike potential can be distinguished according to the method described in the present invention
Obtained two or more platinum nanocages probes are fixed on electricity by the platinum nanocages immunological probe of two or more signal of telecommunication materials
The surface of pole, obtaining can be while the electrochemical immunosensor detected to two or more labels.
In a particular embodiment of the present invention, as example, giving one kind can be while realizes to tumor markerses CEA
The electrochemical immunosensor detected with AFP, it is to be prepared by the following method to obtain：
(1) heretofore described platinum nanocage granule is washed with phosphate buffer, is disperseed；
(2) under room temperature, platinum nanocage granule is mixed respectively with CEA and AFP antibody, is centrifuged off supernatant；
(3) mix after precipitation is washed, disperseed with PBS with respectively with thionine and sulfydryl ferrocene, be centrifuged off supernatant；
(4) precipitation is washed with PBS, and the platinum nanocages immunological probe for obtaining is distributed in phosphate buffer, and obtained platinum is received
Rice cage immunological probe is in 4 DEG C of storages；
(5) obtained two kinds of platinum nanocages probe is fixed on the surface of electrode by immunoreation, is obtained final product.
The present invention by cube shaped platinum nanocage granule by loading respectively alpha-fetoprotein (AFP) and carcinoembryonic antigen
(CEA) after antibody, by labeling SH groups ferrocene and thionine respectively, two kinds of platinum nanocages immunological probes are prepared for, by exempting from
Obtained two kinds of platinum nanocages probe is fixed on the surface of electrode so as to obtain a kind of electrochemical immunosensor by epidemic disease reaction,
Can be realized to detecting while carcinoembryonic antigen and alpha-fetoprotein, and with relatively low inspection using the electrochemical immunosensor
Rising limit, wider detection range and good repeatability.
Experiment is proved：Using platinum nanocages electro-chemistry immunity probe detect simultaneously variable concentrations carcinoembryonic antigen (CEA) and
Alpha-fetoprotein (AFP), its detection range of linearity is respectively 0.05-200ng/mL and 0.03-100ng/mL.Detection limit is respectively
0.014ng/mL and 0.010ng/mL.
Therefore, further, the invention allows for described electrochemical immunosensor is being prepared to tumor markerses
CEA and AFP carries out the application in the reagent of detection simultaneously.
Description of the drawings
The transmission electron microscope picture of (A) palladium nanocube granule that Fig. 1 is prepared for one embodiment of the invention, (B) platinum nanometer
The transmission electron microscope picture (illustration is the high resolution TEM picture of single platinum nanocages) of caged granule, (C) palladium nanometer is vertical
The scanning electron microscopic picture of cube, (D) scanning electron microscopic picture of platinum nanocages.
Fig. 2 is while detecting square wave voltammogram (A), the electric current of different CEA and AFP using the platinum nanocages probe for preparing
With the linear relationship chart of CEA log concentrations (B), electric current and AFP log concentrations (C).As can be seen from the figure the immunosensor
0.05-200ng/mL and 0.03-100ng/mL is respectively to the detection range of linearity of CEA and AFP.Its detection limit is respectively
0.014ng/mL and 0.010ng/mL.
Specific embodiment
Further describe the present invention with reference to specific embodiment, advantages of the present invention and feature will be with description and
It is apparent.But embodiment is only exemplary, does not constitute any restriction to the scope of the present invention.Those skilled in the art should
It should be appreciated that, the details of technical solution of the present invention and form can be repaiied without departing from the spirit and scope of the invention
Change or replace, but these modifications and replacement are each fallen within protection scope of the present invention.
Embodiment 1：
1st, the preparation of platinum nanocage granule
(1) the CTAB aqueous solutions of 25mL12.5mM are stirred, are heated to 95 DEG C, after 5 minutes, add 6.25mM, the chlorine palladium of 1mL
Aqueous acid, after 5 minutes, rapidly joins 0.1M, the ascorbic acid of 0.1mL, after reaction 20 minutes, centrifugation, precipitation spend from
It is dispersed in 25mL deionized waters after sub- water cleaning, obtains the solution containing palladium nano-particles；The palladium that transmission electron microscope observing is obtained
The pattern of nano-particle, as figs. ia and 1 c show, it can be clearly seen that palladium nano-particles are presented the cube that size is about 20nm
Pattern；
(2) CTAB of the solution of 6mL steps (1) and 21mL10mM is mixed, 90 DEG C is heated to, then with 1mL/min's
Speed is by 5mM, the K of 3mL2PtCl4It is added thereto, reacts 15 hours, product centrifugation, precipitate with deionized water cleaning is contained
The solution of platinum nanocage granule；The pattern of the platinum nanocage granule that transmission electron microscope observing is obtained, as shown in Figure 1B and 1D, can
The cube pattern that size is about 25nm is presented will become apparent from platinum nanocage granule, the illustration in wherein Figure 1B is platinum nanometer
, there is breach on the face of platinum nanocage granule as can be seen from the figure in the high resolution TEM figure of caged granule.
2nd, using platinum nanocage particle preparation immunological probe and its immune performance example：
2.1 are detected sample：Human serum
2.2 method
(1) platinum nanocage granule is washed with phosphate buffer (PBS) (pH7.0, KCl0.1mol/L), is dispersed in
In 6mLPBS；
(2) under room temperature, by 1mL contain the solution of platinum nanocage granule respectively with 100 μ L, the CEA and AFP of 1mg/mL resist
Body mixes, and is centrifuged off supernatant；
(3) precipitation with PBS (pH7.0, KCl0.1mol/L) washing, be dispersed into after 1mL solution with respectively with 0.5mM, 1mL
Thionine and 0.25Mm, 1mL sulfydryl ferrocene stirring at normal temperature 6h, be centrifuged off supernatant；
(4) precipitation is washed with PBS (pH7.0, KCl0.1mol/L), and the platinum nanocages immunological probe for obtaining is distributed to 1mL
In PBS, obtained platinum nanocages immunological probe is in 4 DEG C of storages.
(5)20μL200μg·mL-1CEA and AFP capture antibody mixed solution under the conditions of 4 DEG C porous platinum modify
Glass-carbon electrode on incubated overnight.The unnecessary antibody 0.1M of electrode surface, the PBS of pH=7.3 falls.Then, using 50 μ
L, 10% bovine serum albumin solution is closed 1 hour, to eliminate non-specific adsorption.The PBS of electrode 0.1M, pH=7.3
It is incubated more than 40min at 37 DEG C with human serum after excessive bovine serum albumin is rinsed.Unreacted human serum uses 0.1M,
The PBS of pH=7.3 falls, at 37 DEG C, and with 1：Two kinds of platinum nanocages immunological probes of 1 ratio mixing are incubated more than 40 points
Clock.
Then, the PBS of immune sensing electrode 0.1M, pH=7.3 is to excessive immunological probe.Immunoreation is completed
Afterwards, with 0.1M, the PBS of pH=6.5 is detection liquid, and with square wave voltammetry current responsing signal is detected.
Simultaneously Parallel testing test is done using conventional ELISA method, to illustrate using the inventive method and traditional ELISA side
The matching degree of method measurement result.
3rd, result
It is as shown in table 1 below to the testing result of blood serum sample,：
Table 1
Embodiment 2：
1st, the preparation of platinum nanocage granule
(1) the CTAB aqueous solutions stirring of 25mL10mM, is heated to 80 DEG C, after 5 minutes, adds 1mM, the chlorine palladium sour water of 1mL
Solution, after 5 minutes, rapidly joins 0.1M, the ascorbic acid of 0.1mL, after reaction 60 minutes, centrifugation, precipitate with deionized water
Cleaning, in being then dispersed in 25mL deionized waters, obtain the solution containing palladium nano-particles；
(2) the CTAB mixing of 10mL steps (1) are obtained solution and 21mL6mM, is heated to 80 DEG C, then with 1mL/min
Speed by 0.1mM, the K of 3mL2PtCl4It is added thereto, reacts 20 hours, product centrifugation, precipitation deionized water cleaning, to containing
The solution of platinum nanocage granule.
2nd, using platinum nanocage particle preparation immunological probe and its immune performance example：
2.1 are detected sample：Human serum
2.2 method
(1) platinum nanocage granule is washed with PBS (pH5.0, KCl0.1mol/L), is dispersed in 10mL PBS；
(2) under room temperature, by 1mL contain the solution of platinum nanocage granule respectively with 100 μ L, the CEA and AFP of 0.5mg/mL
Antibody mixes, and is centrifuged off supernatant；
(3) precipitation with PBS (pH5.0, KCl0.1mol/L) washing, be dispersed in after 1mL PBS with respectively with 1mM, 1mL's
Sulfydryl ferrocene stirring at normal temperature 6h of thionine and 0.5mM, 1mL, is centrifuged off supernatant；
(4) precipitation is washed with PBS (pH5.0, KCl0.1mol/L), and the platinum nanocages immunological probe for obtaining is distributed to PBS
In, obtained platinum nanocages immunological probe is in 4 DEG C of storages.
(5)20μL500μg·mL-1CEA and AFP capture antibody mixed solution under the conditions of 4 DEG C porous platinum modify
Glass-carbon electrode on incubated overnight.The unnecessary antibody 0.1M of electrode surface, the PBS of pH=7.3 falls.Then, 100 are used
μ L, 5% bovine serum albumin solution is closed 1 hour, to eliminate non-specific adsorption.The PBS of electrode 0.1M, pH=7.3
It is incubated more than 40min at 37 DEG C with human serum after excessive bovine serum albumin is rinsed.Unreacted human serum uses 0.1M,
The PBS of pH=7.3 falls, at 37 DEG C, and with 1：Two kinds of platinum nanocages immunological probes of 1 ratio mixing are incubated more than 40 points
Clock.Then, the PBS of immune sensing electrode 0.1M, pH=7.3 is to excessive immunological probe.After the completion of immunoreation, with
The PBS of 0.1M, pH=6.5 is detection liquid, and with square wave voltammetry current responsing signal is detected.
Simultaneously Parallel testing test is done using conventional ELISA method, to illustrate using the inventive method and traditional ELISA side
The matching degree of method measurement result.
3rd, result
It is as shown in table 2 below to the testing result of blood serum sample：
Table 2
Embodiment 3：
1st, the preparation of platinum nanocage granule
(1) the CTAB aqueous solutions of 25mL25mM are stirred, are heated to 120 DEG C, after 5 minutes, add 10mM, the chlorine palladium acid of 1mL
Aqueous solution, after 5 minutes, rapidly joins 0.1M, the ascorbic acid of 0.1mL, after reaction 10 minutes, centrifugation, deionized water cleaning,
After be dispersed in 25mL deionized waters, obtain the solution containing palladium nano-particles；
(2) the CTAB mixing of the solution of 10mL steps (1) and 21mL25mM, is heated to 100 DEG C, then with 1mL/min's
Speed is by 10mM, the K of 3mL2PtCl4It is added thereto, reacts 10 hours, product centrifugation, precipitate with deionized water cleaning, to containing
The solution of platinum nanocage granule.
2nd, using platinum nanocage particle preparation immunological probe and its immune performance example：
2.1 are detected sample：Human serum
2.2 method
(1) platinum nanocage granule is washed with PBS (pH8.0, KCl0.1mol/L), is dispersed in 10mL PBS；
(2) under room temperature, by 1mL contain the solution of platinum nanocage granule respectively with 100 μ L, the CEA and AFP of 2mg/mL resist
Body mixes, and is centrifuged off supernatant；
(3) precipitation with PBS (pH8.0, KCl0.1mol/L) washing, dispersion after with respectively with 1mL, the thionine and 1mL of 2mM,
Sulfydryl ferrocene stirring at normal temperature 6h of 1mM, is centrifuged off supernatant；
(4) precipitation is washed with PBS (pH8.0, KCl0.1mol/L), and the platinum nanocages immunological probe for obtaining is distributed to PBS
In, obtained platinum nanocages immunological probe is in 4 DEG C of storages.
(5)20μL1000μg·mL-1CEA and AFP capture antibody mixed solution under the conditions of 4 DEG C porous platinum modify
Glass-carbon electrode on incubated overnight.The unnecessary antibody 0.1M of electrode surface, the PBS of pH=7.3 falls.Then, 200 are used
μ L, 1% bovine serum albumin solution is closed 1 hour, to eliminate non-specific adsorption.The PBS of electrode 0.1M, pH=7.3
Excessive bovine serum albumin is incubated more than 40min with human serum after rinsing at 37 DEG C.Unreacted human serum uses 0.1M, pH
=7.3 PBS falls, at 37 DEG C, and with 1：Two kinds of platinum nanocages immunological probes of 1 ratio mixing are incubated more than 40 points
Clock.Then, the PBS of immune sensing electrode 0.1M, pH=7.3 is to excessive immunological probe.After the completion of immunoreation, with
The PBS of 0.1M, pH=6.5 is detection liquid, and with square wave voltammetry current responsing signal is detected.
Simultaneously Parallel testing test is done using conventional ELISA method, to illustrate using the inventive method and traditional ELISA side
The matching degree of method measurement result.
3rd, result
It is as shown in table 3 below to the testing result of blood serum sample：
Table 3
Embodiment 4：The detection range of linearity and the measure of detection limit of the platinum nanocages probe of the present invention
1st, the preparation of platinum nanocages probe：
Method according to embodiment 1 prepares respectively two kinds of platinum nanocages immunological probes.
2nd, immunosensor building process：
20μL200μg·mL-1CEA and AFP capture antibody mixed solution under the conditions of 4 DEG C porous platinum modification glass
Incubated overnight on carbon electrode.The unnecessary antibody 0.1M of electrode surface, the PBS of pH=7.3 falls.Then, using 150 μ L,
2% bovine serum albumin solution is closed 1 hour, to eliminate non-specific adsorption.The PBS excess of electrode 0.1M, pH=7.3
Bovine serum albumin is respectively 0.05,0.1,0.3,1,2,5,10,30,200ngmL after rinsing with concentration-1CEA and 0.03,
0.1,0.3,1,2,3,10,30,100ng·mL-1AFP antigens mixed solution is incubated more than 40min at 37 DEG C.It is unreacted anti-
Original uses the PBS of 0.1M, pH=7.3 to fall, at 37 DEG C, and with 1：Two kinds of platinum nanocages immunological probes of 1 ratio mixing
Incubation was more than 40 minutes.Then, the PBS of immune sensing electrode 0.1M, pH=7.3 is to excessive immunological probe.Immunity
After the completion of reaction, with 0.1M, the PBS of pH=6.5 is detection liquid, and with square wave voltammetry current responsing signal is detected.
While in order to contrast spherical platinum grain, list of references：Zhongju Song,Ruo Yuan,Yaqin Chai,
Ying Zhou,Wen Jiang,Huilan Su,Xin Che,Jingjing Li,Chem.Commun.,2010,46,6750-
Method in 6752. is prepared for platinum spherical nanoparticle as the electrochemical immunosensor of probe.
3rd, result
The cube shaped platinum nanocages probe prepared using the inventive method detects the square wave of different CEA and AFP simultaneously
The linear relationship chart of voltammogram (A), electric current and CEA log concentrations (B), electric current and AFP log concentrations (C) is as shown in Figure 2.From figure
In it can be seen that the immunosensor 0.05-200ng/mL and 0.03- is respectively to the detection range of linearity of CEA and AFP
100ng/mL.Its detection limit is respectively 0.014ng/mL and 0.010ng/mL.
The detection CEA's and AFP that platinum spherical nanoparticle is prepared respectively with platinum nanocages as electrochemical sensor probe
The detection limit and the comparing result of inspection range that immunosensor is obtained is as shown in table 4.
Table 4
Claims (11)
1. a kind of cube shaped platinum nanocage granule, it is characterised in that be prepared by the following method and obtain：
(1) after the aqueous solution of cetyl trimethylammonium bromide being heated to into 80-120 DEG C, chlorine palladium aqueous acid, Ran Houjia are added
Enter ascorbic acid, after reaction 10-60min, centrifugation, precipitate with deionized water cleaning, in being then dispersed in deionized water；
(2) solution for obtaining step (1) and the aqueous solution of cetyl trimethylammonium bromide, are heated to 80-100 DEG C,
Potassium chloroplatinate aqueous solution is added thereto, 10-20 hours are stirred, product centrifugation, precipitation deionized water cleaning is obtained final product.
2. platinum nanocage granule as claimed in claim 1, it is characterised in that：Cetyl trimethyl described in step (1)
The concentration of the aqueous solution of ammonium bromide is 10-25mM, and the concentration of the chlorine palladium acid solution for being added is 1-10mM, and volume is cetyl
The 1/20-1/30 of the aqueous solution volume of trimethylammonium bromide, the ascorbic acid for being added is 1 with the mol ratio of chlorine palladium acid:0.1-
1。
3. platinum nanocage granule as claimed in claim 1, it is characterised in that：The cetyl front three that step (2) is added
The concentration of the aqueous solution of base ammonium bromide is 6-25mM, and the amount for being added is 2-5 times of the liquor capacity that step (1) is obtained, added
The concentration of the potassium chloroplatinate aqueous solution for entering is 0.1-10mM, and the amount for being added is the 1/5-1/ of the liquor capacity that step (1) is obtained
2。
4. application of the platinum nanocage granule described in any one of claim 1-3 in platinum nanocages immunological probe is prepared.
5. a kind of platinum nanocages immunological probe, it is characterised in that be prepared by the following method and obtain：
(1) the platinum nanocage granule described in any one of claim 1-3 is washed with phosphate buffer, is disperseed；
(2) under room temperature, platinum nanocage granule is mixed with antibody, is centrifuged off supernatant；
(3) precipitation phosphate buffer wash, disperse after with signal of telecommunication material mixing, be centrifuged off supernatant；
(4) precipitation is washed with phosphate buffer, and the platinum nanocages immunological probe for obtaining is distributed in phosphate buffer, in 4 DEG C of storages
Deposit.
6. platinum nanocages immunological probe as claimed in claim 5, it is characterised in that：Phosphoric acid used in step (1), (2) or (4)
Buffer is 0.1M, and pH value is 5.0-8.0, and containing the phosphate buffer of 0.1M KCl.
7. platinum nanocages immunological probe as claimed in claim 5, it is characterised in that：Described signal of telecommunication material include thionine and
Sulfydryl ferrocene.
8. application of the platinum nanocages immunological probe described in any one of claim 5-7 in electrochemical immunosensor is prepared.
9. application as claimed in claim 8, it is characterised in that：Prepare coupling two respectively in accordance with the method for claim 7
The platinum nanocages immunological probe of two or more signal of telecommunication materials that kind or Multiple Antibodies and spike potential can be distinguished, by obtained two
Kind or various platinum nanocages probes are fixed on the surface of electrode, obtain two or more labels can be detected simultaneously
Electrochemical immunosensor.
10. a kind of electrochemical immunosensor that can realize detecting tumor markerses CEA and AFP simultaneously, its feature
It is to be prepared by the following method to obtain：
(1) the platinum nanocage granule described in any one of claim 1-3 is washed with phosphate buffer, is disperseed；
(2) under room temperature, platinum nanocage granule is mixed respectively with CEA and AFP antibody, is centrifuged off supernatant；
(3) mix after precipitation is washed, disperseed with PBS with respectively with thionine and sulfydryl ferrocene, be centrifuged off supernatant；
(4) precipitation is washed with PBS, and the platinum nanocages immunological probe for obtaining is distributed in phosphate buffer, obtained platinum nanocages
Immunological probe is in 4 DEG C of storages；
(5) obtained two kinds of platinum nanocages probe is fixed on the surface of electrode by immunoreation, is obtained final product.
Electrochemical immunosensor described in 11. claim 10 carries out detection simultaneously to tumor markerses CEA and AFP in preparation
Reagent in application.
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| CN103042231A (en) * | 2013-01-11 | 2013-04-17 | 哈尔滨工业大学 | Preparation method of precious metal nano-particles |
| CN103063832A (en) * | 2013-01-05 | 2013-04-24 | 福州大学 | Immunoassay method based on platinum nanoparticle mimic enzyme |
| CN103235021A (en) * | 2013-04-19 | 2013-08-07 | 济南大学 | Manufacturing method and application of sensor for simultaneously detecting three breast-cancer tumor markers |
| CN103575874A (en) * | 2013-10-25 | 2014-02-12 | 济南大学 | Preparation method and application of immunosensor based on dopamine biomemetic modification |
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| CN103063832A (en) * | 2013-01-05 | 2013-04-24 | 福州大学 | Immunoassay method based on platinum nanoparticle mimic enzyme |
| CN103042231A (en) * | 2013-01-11 | 2013-04-17 | 哈尔滨工业大学 | Preparation method of precious metal nano-particles |
| CN103235021A (en) * | 2013-04-19 | 2013-08-07 | 济南大学 | Manufacturing method and application of sensor for simultaneously detecting three breast-cancer tumor markers |
| CN103575874A (en) * | 2013-10-25 | 2014-02-12 | 济南大学 | Preparation method and application of immunosensor based on dopamine biomemetic modification |
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