CN108982630A - A kind of preparation method and application of the electrochemical immunosensor of interlayer type detection prostate-specific antigen - Google Patents
A kind of preparation method and application of the electrochemical immunosensor of interlayer type detection prostate-specific antigen Download PDFInfo
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Abstract
The invention belongs to novel nano-material, immunoassay and biosensor technique fields, provide a kind of preparation method and application of the electrochemical immunosensor of interlayer type detection prostate-specific antigen, secondary antibody is specifically marked as analogue enztme using the nanocomposite that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube, a kind of interlayer type immunosensor is constructed using triangle gold-nano-piece as base material, realizes quick, Sensitive Detection to prostate-specific antigen.
Description
Technical field
The invention belongs to novel nano-material, immunoassay and biosensor technique fields, provide a kind of interlayer type inspection
The preparation method and application of the electrochemical immunosensor of prostate-specific antigen is surveyed, specifically using molybdenum disulfide nano flower
The nanocomposite for loading hollow nucleocapsid Au-Ag-Pt nanocube marks secondary antibody as analogue enztme, with triangle gold nano
Piece constructs a kind of interlayer type immunosensor as base material, realizes to the quick, sensitive of prostate-specific antigen
Detection.
Background technique
Prostate cancer (PCa) is the second common non-skin cancer (being only second to lung cancer) in male, accounts for the 14% of all new cancers
Whole world male's case.The detection of prostate-specific antigen concentration can be not only used for the diagnosis to carcinoma of prostate, also
The property and state that prostate cancer can be judged by the variation of the content and amount of detection prostate-specific antigen, because
This is sick in diagnosis, the assessment of prostate cancer medically by carrying out quantitative detection to prostate-specific antigen in serum
Become range, forecast assessment curative effect, monitoring recurrence etc. to play an important role, be marked by the biology of specificity and sensibility
Will object and analysis tool carry out the diagnosis of early stage, can reduce prostate cancer incidence, can effectively improve survival rate.
Electrochemical immunosensor is a kind of analysis method combined based on antigen and antibody specificity, has detection fast
Speed, detection limit is low, high sensitivity, the advantage low with preparation cost easy to operate.In recent years, electrochemical immunosensor is by pass
Note, is widely used in the detection of tumor markers.The present invention utilizes self-assembling technique, utilizes novel molybdenum disulfide nano flower
The nanocomposite for loading hollow nucleocapsid Au-Ag-Pt nanocube marks secondary antibody as signal amplifier as analogue enztme,
Using triangle gold-nano-piece as base material, it is prepared for a kind of electro-chemistry immunity of interlayer type detection prostate-specific antigen
Sensor.The nanocomposite that novel molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube has unique
Structure possesses a large amount of active site, to H2O2Reduction show excellent catalytic, this nanocomposite
The characteristic that molybdenum disulfide nano spends brilliant catalytic performance and large specific surface area can not only be given full play to, and can also effective benefit
With the unique catalytic performance of hollow nucleocapsid Au-Ag-Pt nanocube, brilliant electric conductivity and good biocompatibility, lead to
It crosses and plays both novel nano material synergistic effects, molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube
Nanocomposite mark secondary antibody as signal amplifier and triangle gold-nano-piece as substrate material as analogue enztme
Expect the excellent electric conductivity shown, the sensitivity of immunosensor can be effectively improved.It is constructed based on the above advantage
Interlayer type immunosensor realizes the quantitative detection to prostate-specific antigen, have detection range is wide, Monitoring lower-cut is low,
High sensitivity, it is easy to operate, detection speed it is fast the advantages that, and have good reproducibility, stability and selectivity, be forefront
The early diagnosis of gland cancer provides a kind of reliable detection means.
Summary of the invention
The present invention provides a kind of preparation sides of the electrochemical immunosensor of interlayer type detection prostate-specific antigen
Method and application, the electrochemical immunosensor include: working electrode, to electrode and reference electrode, and the working electrode is
Triangle gold-nano-piece, prostate specific antibody, bovine serum albumin, prostate-specific are successively modified in glass-carbon electrode, surface
Property antigen, the nanocomposite that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube hatches prostate-specific
Property antibody secondary antibody marker, the reference electrode be saturated calomel electrode, it is described to electrode be platinum electrode.
An object of the present invention is to provide a kind of electrochemistry immuno-sensing of interlayer type detection prostate-specific antigen
The preparation method of device.
The second object of the present invention is to prepared interlayer type electrochemical immunosensor is used to prostate specific to resist
Former quantitative detection.
Technical solution of the present invention, comprising the following steps:
(1) triangle gold-nano-piece is prepared；
(2) nanocomposite that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube is prepared；
(3) the nanocomposite hatching prostate that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube is prepared
The secondary antibody marker of specific antibody；
(4) working electrode of the electrochemical immunosensor of interlayer type detection prostate-specific antigen is prepared；
(5) working curve of the electrochemical immunosensor of interlayer type detection prostate-specific antigen is prepared.
Wherein step (1) prepares triangle gold-nano-piece and includes:
1. preparing gold seeds solution
25.0 μ L of μ L ~ 30.0,50.0 mmol/L chlorauric acid solutions are added to 4.0 ~ 5.0 mL, 0.1 mol/L 16
In alkyl trimethyl ammonium chloride solution, then under continuous stirring by 200.0 ~ 300.0 μ L, 10.0 mmol/L sodium borohydrides
It is added drop-wise in above-mentioned solution, reacts 2 h at room temperature, obtain gold seeds solution；
2. preparing triangle gold-nano-piece
First respectively prepare two kinds of solution of a, b: a solution be by 40.0 μ L, 50.0 mmol/L chlorauric acid solutions and 15.0 μ L,
The IodineSodium Solution of 10.0 mmol/L is added in the hexadecyltrimethylammonium chloride solution of 8.0 mL, 0.1 mol/L；B is molten
Liquid is to be added to the IodineSodium Solution of 500.0 μ L, 50.0 mmol/L chlorauric acid solutions and 300.0 μ L, 10.0 mmol/L
40.0 mL, 0.1 mol/L hexadecyltrimethylammonium chloride solution in；Respectively by 40.0 ~ 60.0 μ L and 400.0 ~
600.0 μ L, 0.1 mol/L ascorbic acid solution are added in a, b solution, and 100.0 μ of μ L ~ 200.0 L are then diluted 10
Times gold seeds solution be added to and react 1 s in a solution after, a solution of 3.0 ~ 4.0 mL is added to the stirring of b solution rapidly
More than ten seconds, 1 ~ 3 h is then stood at room temperature, and final centrifugation, cleaning 3 times obtain triangle gold-nano-piece.
Wherein step (2) prepares the nano combined material that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube
Material includes:
1. preparing hollow nucleocapsid Au-Ag-Pt nanocube
The gold chloride of 10.0 μ L, 25.0 mmol/L are added to 10.0 mL, 75.0 mmol/L cetyl trimethyl chlorinations
In ammonium salt solution, 0.6 mL, 10.0 mmol/L sodium borohydride solutions is then added dropwise, reacts 2 h under room temperature, obtains gold seeds
Solution.Then by 0.1 ~ 0.3 mL, 10.0 mmol/L chlorauric acid solutions and 2.0 ~ 4.0 mL, 0.2 mol/L cetyl
Trimethyl ammonia chloride ammonium salt solution is added in 25.0 mL deionized waters, and 1.5 ~ 2.5 mL, 0.1 mol/L ascorbic acid are added
By above-mentioned solution reaction more than ten second, 0.3 ~ 0.5 mL is then added and dilutes 100 times of gold seeds solution, be stored at room temperature 8 h it
Afterwards, 0.5 ~ 1.0 mL, 10.0 mmol/L silver nitrates and 2.0 ~ 4.0 mL, 0.1 mol/L ascorbic acid solution are added to
Above-mentioned solution is placed in 50 DEG C of water-baths and stands 12 h, obtains gold and silver nano cubic colloidal solution.Take 4.0 ~ 6.0 mL golden
Silver nanoparticle cube colloidal solution continues agitating and heating in oil bath pan and flows back into 100 DEG C, by 200.0 ~ 400.0 after 15 min
μ L, 5.0 mmol/L platinum acid chloride solutions are added drop-wise in solution, are persistently stirred to react 15 min, and cooling, centrifugation, cleaning 3 times obtain
Hollow nucleocapsid Au-Ag-Pt nanocube；
2. preparing molybdenum disulfide nano flower
1.2 ~ 1.6 g ammonium molybdates are added in 15.0 mL deionized waters, then 0.8 ~ 1.0 g ethylenediamine is added
It states in solution, after ultrasonic dissolution, the salt acid for adjusting pH of appropriate 1.0 mol/L is added to 4.0 ~ 6.0, under the conditions of 50 DEG C
2 h are reacted, eccentric cleaning 3 times, is dried in vacuo under 50 DEG C of environment, obtains the complex of ammonium molybdate and ethylenediamine；Take 0.1 ~
The complex of 0.2 g ammonium molybdate and ethylenediamine is added in 15.0 mL deionized waters, and 0.2 ~ 0.4 gL-, half Guang is then added
Propylhomoserin, ultrasonic disperse are put into the autoclave of 25.0 mL polytetrafluoroethyllining linings, are heated to 200 DEG C, keep the temperature 14 h, most
Whole eccentric cleaning 3 times, vacuum drying obtain molybdenum disulfide nano flower；
3. preparing the nanocomposite that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube
By 0.1 ~ 0.2 g molybdenum disulfide nano flower ultrasonic disperse into 20.0 mL dehydrated alcohols, it is heated at reflux in oil bath pan
To 100 DEG C, 20.0 ~ 30.0 μ L3- aminopropyl triethoxysilanes are then added, cooling after reacting 2 h, centrifugation, clearly
It washes 3 times, freeze-drying obtains amidized molybdenum disulfide nano flower；By the hollow nucleocapsid gold and silver of 2.0 ~ 4.0 mL, 2.0 mg/mL
Platinum nanocube is added to the amidized molybdenum disulfide nano flower of 2.0 mL, 2.0 mg/mL, 1 h of ultrasonic disperse, in room temperature
Under environment, 6 h are shaken, eccentric cleaning 2 times, molybdenum disulfide nano flower is obtained and loads receiving for hollow nucleocapsid Au-Ag-Pt nanocube
Nano composite material.
Wherein step (3) prepares the nano combined material that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube
Expect that the secondary antibody marker for hatching prostate specific antibody includes:
1. 2.0 ~ 4.0 mL, 10.0 μ g/mL prostate specific antibody are added to 2.0 mL, 2.0 mg/mL molybdenum disulfide
Nano flower loads in the nanocomposite of hollow nucleocapsid Au-Ag-Pt nanocube, and 12 h are shaken under 4 DEG C of environment, centrifugation
Cleaning is dispersed in phosphate buffer solution.
Wherein step (4) prepares the working electrode of the electrochemical immunosensor of interlayer type detection prostate-specific antigen
Include:
1. the glass-carbon electrode Al for being 3.0 ~ 5.0 mm by diameter2O3Polishing powder is polished to mirror surface, respectively successively in anhydrous second
It is cleaned by ultrasonic in alcohol, ultrapure water；
2. the triangle gold-nano-piece dispersant liquid drop of 6.0 μ L, 0.5 ~ 3.0 mg/mL are added on electrode surface, dry in the air at room temperature
It is dry, with ultrapure water electrode surface, dry at room temperature；
3. continuing the prostate specific antibody of 6.0 μ L, 4.0 ~ 8.0 μ g/mL being added drop-wise to electrode surface, in 4 DEG C of refrigerators
Middle drying；
4. the bovine serum albumin solution of 3.0 μ L, 0.7 ~ 1.4 wt% are then added drop-wise to electrode surface, to close non-spy
Specific activities site is rinsed electrode surface with the phosphate buffer that pH is 7.0, is dried in 4 DEG C of refrigerators；
5. a series of prostate-specific antigen solution of various concentrations of 6.0 μ L, 0.00005 ~ 100.0 ng/mL is added dropwise,
Electrode surface is rinsed with the phosphate buffer that pH is 7.0, is dried in 4 DEG C of refrigerators；
6. 6.0 μ L, 2.0 ~ 4.0 mg/mL molybdenum disulfide nanos flower are loaded receiving for hollow nucleocapsid Au-Ag-Pt nanocube
The secondary antibody marker of nano composite material hatching prostate specific antibody is added drop-wise to electrode surface, and the phosphate for being 7.0 with pH is slow
Fliud flushing rinses electrode surface, dries in 4 DEG C of refrigerators, prepares the electro-chemistry immunity of interlayer type detection prostate-specific antigen
The working electrode of sensor.
Wherein step (5) prepares the working curve of the electrochemical immunosensor of interlayer type detection prostate-specific antigen
Include:
1. being tested using electrochemical workstation with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is
To electrode, prepared sensor is working electrode, the phosphate for being 5.3 ~ 8.0 in the pH of 10.0 mL, 50.0 mmol/L
It is tested in buffer solution；
2. the used time, m- current method detected analyte, input voltage is -0.4 V, 0.1 s of sampling interval, runing time
200 s；
3. being infused in the phosphate buffer solution for being 7.4 to the pH of 10.0 mL, 50.0 mmol/L after background current tends towards stability
Enter the hydrogen peroxide solution of 10.0 ~ 15.0 μ L, 5.0 mol/L, records under various concentration corresponding to prostate-specific antigen
Current value draws the working curve of the electrochemical immunosensor of detection prostate-specific antigen；
4. utilizing working curve method, the concentration of prostate-specific antigen in sample to be tested is obtained.
Raw material used in the present invention can be bought in chemical reagents corporation or biopharmaceutical company.
Beneficial achievement of the invention
(1) present invention loads the nanocomposite of hollow nucleocapsid Au-Ag-Pt nanocube by novel molybdenum disulfide nano flower
Mark secondary antibody as signal amplifier as analogue enztme, molybdenum disulfide nano is colored and hollow nucleocapsid Au-Ag-Pt nanocube has
There is unique structure, possesses a large amount of active site, to H2O2Reduction show excellent catalytic, it is prepared
The nanocomposite that novel molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube can give full play to collaboration
Effect shows excellent catalytic performance, electric conductivity and biocompatibility and triangle gold-nano-piece as base material
With excellent electric conductivity, the sensitivity of sensor can be effectively improved, constructed interlayer type immunosensor is realized
To the quantitative detection of prostate-specific antigen, have that detection range is wide, Monitoring lower-cut is low, high sensitivity, easy to operate, inspection
The advantages that degree of testing the speed is fast, and there is good reproducibility, stability and selectivity, for early diagnosis provide it is a kind of reliable
Detection means；
(2) interlayer type electrochemical immunosensor constructed by the present invention realizes accurate quantification detection prostate-specific antigen
Purpose, linear detection range is the ng/mL of 0.00005 ng/mL ~ 100, and lowest detection lower limit is 16.6 fg/mL；
(3) electrochemical immunosensor of method of the invention building, easy to operate, detection rapidly, can be used for actual sample
Quickly detection.
Specific embodiment
Now the present invention is further illustrated by specific embodiment, but not limited to this.
Embodiment 1 prepares triangle gold-nano-piece
1. preparing gold seeds solution
25.0 μ L, 50.0 mmol/L chlorauric acid solutions are added to 4.0 mL, 0.1 mol/L hexadecyltrimethylammonium chloride
In solution, then 200.0 μ L, 10.0 mmol/L sodium borohydrides are added drop-wise under continuous stirring in above-mentioned solution, in room temperature
Under conditions of react 2 h, obtain gold seeds solution；
2. preparing triangle gold-nano-piece
First respectively prepare two kinds of solution of a, b: a solution be by 40.0 μ L, 50.0 mmol/L chlorauric acid solutions and 15.0 μ L,
The IodineSodium Solution of 10.0 mmol/L is added in the hexadecyltrimethylammonium chloride solution of 8.0 mL, 0.1 mol/L；B is molten
Liquid is to be added to the IodineSodium Solution of 500.0 μ L, 50.0 mmol/L chlorauric acid solutions and 300.0 μ L, 10.0 mmol/L
40.0 mL, 0.1 mol/L hexadecyltrimethylammonium chloride solution in；Respectively by 40.0 μ L and 400.0 μ L, 0.1
Mol/L ascorbic acid solution is added in a, b solution, and it is molten that the 100.0 μ L gold seeds solution for diluting 10 times is then added to a
After reacting 1 s in liquid, a solution of 3.0 mL is added to b solution rapidly and is stirred more than ten seconds, then stands 1 at room temperature
H, final centrifugation, cleaning 3 times, obtains triangle gold-nano-piece.
Embodiment 2 prepares triangle gold-nano-piece
1. preparing gold seeds solution
27.5 μ L, 50.0 mmol/L chlorauric acid solutions are added to 4.5 mL, 0.1 mol/L hexadecyltrimethylammonium chloride
In solution, then 250.0 μ L, 10.0 mmol/L sodium borohydrides are added drop-wise under continuous stirring in above-mentioned solution, in room temperature
Under conditions of react 2 h, obtain gold seeds solution；
2. preparing triangle gold-nano-piece
First respectively prepare two kinds of solution of a, b: a solution be by 40.0 μ L, 50.0 mmol/L chlorauric acid solutions and 15.0 μ L,
The IodineSodium Solution of 10.0 mmol/L is added in the hexadecyltrimethylammonium chloride solution of 8.0 mL, 0.1 mol/L；B is molten
Liquid is to be added to the IodineSodium Solution of 500.0 μ L, 50.0 mmol/L chlorauric acid solutions and 300.0 μ L, 10.0 mmol/L
40.0 mL, 0.1 mol/L hexadecyltrimethylammonium chloride solution in；Respectively by 50.0 μ L and 500.0 μ L, 0.1
Mol/L ascorbic acid solution is added in a, b solution, and it is molten that the 250.0 μ L gold seeds solution for diluting 10 times is then added to a
After reacting 1 s in liquid, a solution of 3.5 mL is added to b solution rapidly and is stirred more than ten seconds, then stands 2 at room temperature
H, final centrifugation, cleaning 3 times, obtains triangle gold-nano-piece.
Embodiment 3 prepares triangle gold-nano-piece
1. preparing gold seeds solution
30.0 μ L, 50.0 mmol/L chlorauric acid solutions are added to 5.0 mL, 0.1 mol/L hexadecyltrimethylammonium chloride
In solution, then 300.0 μ L, 10.0 mmol/L sodium borohydrides are added drop-wise under continuous stirring in above-mentioned solution, in room temperature
Under conditions of react 2 h, obtain gold seeds solution；
2. preparing triangle gold-nano-piece
First respectively prepare two kinds of solution of a, b: a solution be by 40.0 μ L, 50.0 mmol/L chlorauric acid solutions and 15.0 μ L,
The IodineSodium Solution of 10.0 mmol/L is added in the hexadecyltrimethylammonium chloride solution of 8.0 mL, 0.1 mol/L；B is molten
Liquid is to be added to the IodineSodium Solution of 500.0 μ L, 50.0 mmol/L chlorauric acid solutions and 300.0 μ L, 10.0 mmol/L
40.0 mL, 0.1 mol/L hexadecyltrimethylammonium chloride solution in；Respectively by 60.0 μ L and 600.0 μ L, 0.1
Mol/L ascorbic acid solution is added in a, b solution, and it is molten that the 200.0 μ L gold seeds solution for diluting 10 times is then added to a
After reacting 1 s in liquid, a solution of 4.0 mL is added to b solution rapidly and is stirred more than ten seconds, then stands 3 at room temperature
H, final centrifugation, cleaning 3 times, obtains triangle gold-nano-piece.
Embodiment 4 prepares the nanocomposite packet that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube
It includes:
1. preparing hollow nucleocapsid Au-Ag-Pt nanocube
The gold chloride of 10.0 μ L, 25.0 mmol/L are added to 10.0 mL, 75.0 mmol/L cetyl trimethyl chlorinations
In ammonium salt solution, 0.6 mL, 10.0 mmol/L sodium borohydride solutions is then added dropwise, reacts 2 h under room temperature, obtains gold seeds
Solution.Then by 0.1 mL, 10.0 mmol/L chlorauric acid solutions and 2.0 mL, 0.2 mol/L hexadecyltrimethylammonium chloride
Solution is added in 25.0 mL deionized waters, and 1.5 mL, 0.1 mol/L ascorbic acid are added to above-mentioned solution reaction more than ten
Second, the gold seeds solution that 0.3 mL dilutes 100 times is then added, is stored at room temperature after 8 h, by 0.5 mL, 10 mmol/L nitric acid
Silver and 2.0 mL, 0.1 mol/L ascorbic acid solution are added to above-mentioned solution, are placed in 50 DEG C of water-baths and stand 12 h, obtain
Gold and silver nano cubic colloidal solution.4.0 mL gold and silver nano cubic colloidal solution are taken, continue agitating and heating reflux in oil bath pan
To 100 DEG C, 200.0 μ L, 5.0 mmol/L platinum acid chloride solutions are added drop-wise in solution after 15 min, are persistently stirred to react 15
Min, cooling, centrifugation, cleaning 3 times, obtains hollow nucleocapsid Au-Ag-Pt nanocube；
2. preparing molybdenum disulfide nano flower
1.2 g ammonium molybdates are added in 15.0 mL deionized waters, then 0.8 g ethylenediamine is added in above-mentioned solution, ultrasound
After dissolution, the salt acid for adjusting pH of appropriate 1.0 mol/L is added to 4.0, reacts 2 h under the conditions of 50 DEG C, eccentric cleaning 3 times,
It is dried in vacuo under 50 DEG C of environment, obtains the complex of ammonium molybdate and ethylenediamine；
It takes the complex of 0.1 g ammonium molybdate and ethylenediamine to be added in 15.0 mL deionized waters, 0.2 gL-, half Guang is then added
Propylhomoserin, ultrasonic disperse are put into the autoclave of 25.0 mL polytetrafluoroethyllining linings, are heated to 200 DEG C, keep the temperature 14 h, most
Whole eccentric cleaning 3 times, vacuum drying obtain molybdenum disulfide nano flower；
3. preparing the nanocomposite that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube
By 0.1 g molybdenum disulfide nano flower ultrasonic disperse into 20.0 mL dehydrated alcohols, it is heated at reflux in oil bath pan to 100
DEG C, 20.0 μ L 3- aminopropyl triethoxysilanes are then added, cooling after reacting 2 h, centrifugation is cleaned 3 times, and freezing is dry
It is dry to obtain amidized molybdenum disulfide nano flower；
The hollow nucleocapsid Au-Ag-Pt nanocube of 2.0 mL, 2.0 mg/mL is added to the amination of 2.0 mL, 2.0 mg/mL
Molybdenum disulfide nano flower, 1 h of ultrasonic disperse, under room temperature environment, shake 6 h, eccentric cleaning 2 times, obtain molybdenum disulfide and receive
Popped rice loads the nanocomposite of hollow nucleocapsid Au-Ag-Pt nanocube.
Embodiment 5 prepares the nanocomposite packet that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube
It includes:
1. preparing hollow nucleocapsid Au-Ag-Pt nanocube
The gold chloride of 10.0 μ L, 25.0 mmol/L are added to 10.0 mL, 75.0 mmol/L cetyl trimethyl chlorinations
In ammonium salt solution, 0.6 mL, 10.0 mmol/L sodium borohydride solutions is then added dropwise, reacts 2 h under room temperature, obtains gold seeds
Solution.Then by 0.2 mL, 10.0 mmol/L chlorauric acid solutions and 3.0 mL, 0.2 mol/L hexadecyltrimethylammonium chloride
Solution is added in 25.0 mL deionized waters, and 2.0 mL, 0.1 mol/L ascorbic acid are added to above-mentioned solution reaction more than ten
Second, the gold seeds solution that 0.4 mL dilutes 100 times is then added, is stored at room temperature after 8 h, by 0.75 mL, 10.0 mmol/L
Silver nitrate and 3.0 mL, 0.1 mol/L ascorbic acid solution are added to above-mentioned solution, are placed in 50 DEG C of water-baths and stand 12 h,
Obtain gold and silver nano cubic colloidal solution.5.0 mL gold and silver nano cubic colloidal solution are taken, continue agitating and heating in oil bath pan
100 DEG C are flowed back into, 300.0 μ L, 5.0 mmol/L platinum acid chloride solutions are added drop-wise in solution after 15 min, are persistently stirred to react
15 min, cooling, centrifugation, cleaning 3 times, obtain hollow nucleocapsid Au-Ag-Pt nanocube；
2. preparing molybdenum disulfide nano flower
1.4 g ammonium molybdates are added in 15.0 mL deionized waters, then 0.9 g ethylenediamine is added in above-mentioned solution, ultrasound
After dissolution, the salt acid for adjusting pH of appropriate 1.0 mol/L is added to 5.0, reacts 2 h under the conditions of 50 DEG C, eccentric cleaning 3 times,
It is dried in vacuo under 50 DEG C of environment, obtains the complex of ammonium molybdate and ethylenediamine；Take the cooperation of 0.15 g ammonium molybdate and ethylenediamine
Object is added in 15.0 mL deionized waters, and 0.3 gL- cysteine is then added, and ultrasonic disperse is put into 25.0 mL polytetrafluoros
The autoclave of ethylene liner is heated to 200 DEG C, keeps the temperature 14 h, final eccentric cleaning 3 times, and vacuum drying obtains curing
Molybdenum nano flower；
3. preparing the nanocomposite that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube
By 0.15 g molybdenum disulfide nano flower ultrasonic disperse into 20.0 mL dehydrated alcohols, it is heated at reflux in oil bath pan to 100
DEG C, 25.0 μ L 3- aminopropyl triethoxysilanes are then added, cooling after reacting 2 h, centrifugation is cleaned 3 times, and freezing is dry
It is dry to obtain amidized molybdenum disulfide nano flower；The hollow nucleocapsid Au-Ag-Pt nanocube of 3.0 mL, 2.0 mg/mL is added to
The amidized molybdenum disulfide nano flower of 2.0 mL, 2.0 mg/mL, 1 h of ultrasonic disperse shake 6 h under room temperature environment, from
The heart cleans 2 times, obtains the nanocomposite that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube.
Embodiment 6 prepares the nanocomposite packet that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube
It includes:
1. preparing hollow nucleocapsid Au-Ag-Pt nanocube
The gold chloride of 10.0 μ L, 25.0 mmol/L are added to 10.0 mL, 75.0 mmol/L cetyl trimethyl chlorinations
In ammonium salt solution, 0.6 mL, 10.0 mmol/L sodium borohydride solutions is then added dropwise, reacts 2 h under room temperature, obtains gold seeds
Solution.Then by 0.3 mL, 10.0 mmol/L chlorauric acid solutions and 4.0 mL, 0.2 mol/L hexadecyltrimethylammonium chloride
Solution is added in 25.0 mL deionized waters, and 2.5 mL, 0.1 mol/L ascorbic acid are added to above-mentioned solution reaction more than ten
Second, the gold seeds solution that 0.5 mL dilutes 100 times is then added, is stored at room temperature after 8 h, by 1.0 mL, 10.0 mmol/L nitre
Sour silver and 4.0 mL, 0.1 mol/L ascorbic acid solution are added to above-mentioned solution, are placed in 50 DEG C of water-baths and stand 12 h, obtain
To gold and silver nano cubic colloidal solution.6.0 mL gold and silver nano cubic colloidal solution are taken, continues agitating and heating in oil bath pan and returns
100 DEG C are flowed to, 400.0 μ L, 5.0 mmol/L platinum acid chloride solutions are added drop-wise in solution after 15 min, are persistently stirred to react 15
Min, cooling, centrifugation, cleaning three times, obtain hollow nucleocapsid Au-Ag-Pt nanocube；
2. preparing molybdenum disulfide nano flower
1.6 g ammonium molybdates are added in 15.0 mL deionized waters, then 1.0 g ethylenediamines are added in above-mentioned solution, ultrasound
After dissolution, the salt acid for adjusting pH of appropriate 1.0 mol/L is added to 6.0, reacts 2 h under the conditions of 50 DEG C, eccentric cleaning 3 times,
It is dried in vacuo under 50 DEG C of environment, obtains the complex of ammonium molybdate and ethylenediamine；Take the cooperation of 0.2 g ammonium molybdate and ethylenediamine
Object is added in 15.0 mL deionized waters, and 0.4 gL- cysteine is then added, and ultrasonic disperse is put into 25.0 mL polytetrafluoros
The autoclave of ethylene liner is heated to 200 DEG C, keeps the temperature 14 h, final eccentric cleaning 3 times, and vacuum drying obtains curing
Molybdenum nano flower；
3. preparing the nanocomposite that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube
By 0.2 g molybdenum disulfide nano flower ultrasonic disperse into 20.0 mL dehydrated alcohols, it is heated at reflux in oil bath pan to 100
DEG C, 30.0 μ L3- aminopropyl triethoxysilanes are then added, cooling after reacting 2 h, centrifugation is cleaned 3 times, freeze-drying
Obtain amidized molybdenum disulfide nano flower；The hollow nucleocapsid Au-Ag-Pt nanocube of 4.0 mL, 2.0 mg/mL is added to
The amidized molybdenum disulfide nano flower of 2.0 mL, 2.0 mg/mL, 1 h of ultrasonic disperse shake 6 h under room temperature environment, from
The heart cleans 2 times, obtains the nanocomposite that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube.
The nanocomposite that embodiment 7 prepares the hollow nucleocapsid Au-Ag-Pt nanocube of molybdenum disulfide nano flower load is incubated
Change prostate specific antibody secondary antibody marker include:
1. 2.0 mL, 10.0 μ g/mL prostate specific antibody are added to 2.0 mL, 2.0 mg/mL molybdenum disulfide nanos flower
In the nanocomposite for loading hollow nucleocapsid Au-Ag-Pt nanocube, 12 h, eccentric cleaning point are shaken under 4 DEG C of environment
It is dispersed in phosphate buffer solution.
Embodiment 8 prepares molybdenum disulfide nano flower and loads hollow nucleocapsid Au-Ag-Pt nanocube nanocomposite hatching
The secondary antibody marker of prostate specific antibody includes:
1. 3.0 mL, 10.0 μ g/mL prostate specific antibody are added to 2.0 mL, 2.0 mg/mL molybdenum disulfide nanos flower
In the nanocomposite for loading hollow nucleocapsid Au-Ag-Pt nanocube, 12 h, eccentric cleaning point are shaken under 4 DEG C of environment
It is dispersed in phosphate buffer solution.
The nanocomposite that embodiment 9 prepares the hollow nucleocapsid Au-Ag-Pt nanocube of molybdenum disulfide nano flower load is incubated
Change prostate specific antibody secondary antibody marker include:
1. 4.0 mL, 10.0 μ g/mL prostate specific antibody are added to 2.0 mL, 2.0 mg/mL molybdenum disulfide nanos
Flower loads in the nanocomposite of hollow nucleocapsid Au-Ag-Pt nanocube, and 12 h, eccentric cleaning are shaken under 4 DEG C of environment
It is dispersed in phosphate buffer solution.
Embodiment 10 prepares the working electrode packet of the electrochemical immunosensor of interlayer type detection prostate-specific antigen
It includes:
1. the glass-carbon electrode Al for being 3.0 mm by diameter2O3Polishing powder is polished to mirror surface, respectively successively in dehydrated alcohol, ultrapure
It is cleaned by ultrasonic in water；
2. the triangle gold-nano-piece dispersant liquid drop of 6.0 μ L, 0.5 mg/mL are added on electrode surface, dry, uses at room temperature
Ultrapure water electrode surface, dries at room temperature；
3. continuing the prostate specific antibody of 6.0 μ L, 4.0 μ g/mL being added drop-wise to electrode surface, done in 4 DEG C of refrigerators
It is dry；
4. the bovine serum albumin solution of 3.0 μ L, 0.7 wt% are then added drop-wise to electrode surface, to close non-specific work
Property site, rinse electrode surface with the phosphate buffer that pH is 7.0, dry in 4 DEG C of refrigerators；
5. a series of prostate-specific antigen solution of various concentrations of 6.0 μ L, 0.00005 ~ 100.0 ng/mL is added dropwise,
Electrode surface is rinsed with the phosphate buffer that pH is 7.0, is dried in 4 DEG C of refrigerators；
6. 6.0 μ L, 2.0 mg/mL molybdenum disulfide nanos flower are loaded the nano combined of hollow nucleocapsid Au-Ag-Pt nanocube
The secondary antibody marker of material hatching prostate specific antibody is added drop-wise to electrode surface, is rushed with the phosphate buffer that pH is 7.0
Electrode surface is washed, is dried in 4 DEG C of refrigerators, the electrochemical immunosensor of interlayer type detection prostate-specific antigen is prepared
Working electrode.
Embodiment 11 prepares the working electrode packet of the electrochemical immunosensor of interlayer type detection prostate-specific antigen
It includes:
1. the glass-carbon electrode Al for being 4.0 mm by diameter2O3Polishing powder is polished to mirror surface, respectively successively in dehydrated alcohol, ultrapure
It is cleaned by ultrasonic in water；
2. the triangle gold-nano-piece dispersant liquid drop of 6.0 μ L, 2.0 mg/mL are added on electrode surface, dry, uses at room temperature
Ultrapure water electrode surface, dries at room temperature；
3. continuing the prostate specific antibody of 6.0 μ L, 6.0 μ g/mL being added drop-wise to electrode surface, done in 4 DEG C of refrigerators
It is dry；
4. the bovine serum albumin solution of 3.0 μ L, 1.0 wt% are then added drop-wise to electrode surface, to close non-specific work
Property site, rinse electrode surface with the phosphate buffer that pH is 7.0, dry in 4 DEG C of refrigerators；
5. a series of prostate-specific antigen solution of various concentrations of 6.0 μ L, 0.00005 ~ 100.0 ng/mL is added dropwise,
Electrode surface is rinsed with the phosphate buffer that pH is 7.0, is dried in 4 DEG C of refrigerators；
6. 6.0 μ L, 3.0 mg/mL molybdenum disulfide nanos flower are loaded the nano combined of hollow nucleocapsid Au-Ag-Pt nanocube
The secondary antibody marker of material hatching prostate specific antibody is added drop-wise to electrode surface, is rushed with the phosphate buffer that pH is 7.0
Electrode surface is washed, is dried in 4 DEG C of refrigerators, the electrochemical immunosensor of interlayer type detection prostate-specific antigen is prepared
Working electrode.
Embodiment 12 prepares the working electrode packet of the electrochemical immunosensor of interlayer type detection prostate-specific antigen
It includes:
1. the glass-carbon electrode Al for being 5.0 mm by diameter2O3Polishing powder is polished to mirror surface, respectively successively in dehydrated alcohol, ultrapure
It is cleaned by ultrasonic in water；
2. the triangle gold-nano-piece dispersant liquid drop of 6.0 μ L, 3.0 mg/mL are added on electrode surface, dry, uses at room temperature
Ultrapure water electrode surface, dries at room temperature；
3. continuing the prostate specific antibody of 6.0 μ L, 8.0 μ g/mL being added drop-wise to electrode surface, done in 4 DEG C of refrigerators
It is dry；
4. the bovine serum albumin solution of 3.0 μ L, 1.4 wt% are then added drop-wise to electrode surface, to close non-specific work
Property site, rinse electrode surface with the phosphate buffer that pH is 7.0, dry in 4 DEG C of refrigerators；
5. a series of prostate-specific antigen solution of various concentrations of 6.0 μ L, 0.00005 ~ 100.0 ng/mL is added dropwise,
Electrode surface is rinsed with the phosphate buffer that pH is 7.0, is dried in 4 DEG C of refrigerators；
6. 6.0 μ L, 4.0 mg/mL molybdenum disulfide nanos flower are loaded the nano combined of hollow nucleocapsid Au-Ag-Pt nanocube
The secondary antibody marker of material hatching prostate specific antibody is added drop-wise to electrode surface, is rushed with the phosphate buffer that pH is 7.0
Electrode surface is washed, is dried in 4 DEG C of refrigerators, the electrochemical immunosensor of interlayer type detection prostate-specific antigen is prepared
Working electrode.
Embodiment 13 prepares the working curve packet of the electrochemical immunosensor of interlayer type detection prostate-specific antigen
It includes:
1. being tested using electrochemical workstation with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is
To electrode, prepared sensor is working electrode, and the phosphate-buffered for being 5.3 in the pH of 10.0 mL, 50.0 mmol/L is molten
It is tested in liquid；
2. the used time, m- current method detected analyte, input voltage is -0.4 V, 0.1 s of sampling interval, runing time
200 s；
3. being infused in the phosphate buffer solution for being 5.3 to the pH of 10.0 mL, 50.0 mmol/L after background current tends towards stability
Enter the hydrogen peroxide solution of 10.0 μ L, 5.0 mol/L, record current value corresponding to prostate-specific antigen under various concentration,
Draw the working curve of the electrochemical immunosensor of detection prostate-specific antigen；
4. utilizing working curve method, the concentration of prostate-specific antigen in sample to be tested is obtained.
Embodiment 14 prepares the working curve packet of the electrochemical immunosensor of interlayer type detection prostate-specific antigen
It includes:
1. being tested using electrochemical workstation with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is
To electrode, prepared sensor is working electrode, and the phosphate-buffered for being 7.4 in the pH of 10.0 mL, 50.0 mmol/L is molten
It is tested in liquid；
2. the used time, m- current method detected analyte, input voltage is -0.4 V, 0.1 s of sampling interval, runing time
200 s；
3. being infused in the phosphate buffer solution for being 6.8 to the pH of 10.0 mL, 50.0 mmol/L after background current tends towards stability
Enter the hydrogen peroxide solution of 13.0 μ L, 5.0 mol/L, record current value corresponding to prostate-specific antigen under various concentration,
Draw the working curve of the electrochemical immunosensor of detection prostate-specific antigen；
4. utilizing working curve method, the concentration of prostate-specific antigen in sample to be tested is obtained.
Embodiment 15 prepares the working curve packet of the electrochemical immunosensor of interlayer type detection prostate-specific antigen
It includes:
1. being tested using electrochemical workstation with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is
To electrode, prepared sensor is working electrode, and the phosphate-buffered for being 8.0 in the pH of 10.0 mL, 50.0 mmol/L is molten
It is tested in liquid；
2. the used time, m- current method detected analyte, input voltage is -0.4 V, 0.1 s of sampling interval, runing time
200 s；
3. being infused in the phosphate buffer solution for being 7.4 to the pH of 10.0 mL, 50.0 mmol/L after background current tends towards stability
Enter the hydrogen peroxide solution of 15.0 μ L, 5.0 mol/L, record current value corresponding to prostate-specific antigen under various concentration,
Draw the working curve of the electrochemical immunosensor of detection prostate-specific antigen；
4. utilizing working curve method, the concentration of prostate-specific antigen in sample to be tested is obtained.
Embodiment 16 prepares the working curve packet of the electrochemical immunosensor of interlayer type detection prostate-specific antigen
It includes:
1. being tested using electrochemical workstation with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is
To electrode, prepared sensor is working electrode, and the phosphate-buffered for being 8.0 in the pH of 10.0 mL, 50.0 mmol/L is molten
It is tested in liquid；
2. the used time, m- current method detected analyte, input voltage is -0.4 V, 0.1 s of sampling interval, runing time
200 s；
3. being infused in the phosphate buffer solution for being 8.0 to the pH of 10.0 mL, 50.0 mmol/L after background current tends towards stability
Enter the hydrogen peroxide solution of 15.0 μ L, 5.0 mol/L, record current value corresponding to prostate-specific antigen under various concentration,
Draw the working curve of the electrochemical immunosensor of detection prostate-specific antigen；
4. utilizing working curve method, the concentration of prostate-specific antigen in sample to be tested is obtained.
Claims (6)
1. a kind of preparation method of the electrochemical immunosensor of interlayer type detection prostate-specific antigen, the interlayer type electricity
Chemo-immunity sensor, method includes: working electrode, reference electrode and to electrode, and the working electrode is its table of glass-carbon electrode
Triangle gold-nano-piece, prostate specific antibody, bovine serum albumin, prostate-specific antigen, curing are successively modified in face
Molybdenum nano flower loads the secondary antibody of the nanocomposite hatching prostate specific antibody of hollow nucleocapsid Au-Ag-Pt nanocube
Marker, the reference electrode are saturated calomel electrode, and described is platinum electrode to electrode；
It is characterized in that, the method for the preparation the following steps are included:
(1) triangle gold-nano-piece is prepared；
(2) nanocomposite that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube is prepared；
(3) the nanocomposite hatching prostate that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube is prepared
The secondary antibody marker of specific antibody；
(4) working electrode of the electrochemical immunosensor of interlayer type detection prostate-specific antigen is prepared；
(5) working curve of the electrochemical immunosensor of interlayer type detection prostate-specific antigen is prepared.
2. a kind of preparation side of the electrochemical immunosensor of interlayer type detection prostate-specific antigen as described in claim 1
Method, which is characterized in that the step (1) prepares triangle gold-nano-piece and includes:
1. preparing gold seeds solution
25.0 μ L of μ L ~ 30.0,50.0 mmol/L chlorauric acid solutions are added to 4.0 ~ 5.0 mL, 0.1 mol/L 16
In alkyl trimethyl ammonium chloride solution, then under continuous stirring by 200.0 ~ 300.0 μ L, 10.0 mmol/L sodium borohydrides
It is added drop-wise in above-mentioned solution, reacts 2 h at room temperature, obtain gold seeds solution；
2. preparing triangle gold-nano-piece
First respectively prepare two kinds of solution of a, b: a solution be by 40.0 μ L, 50.0 mmol/L chlorauric acid solutions and 15.0 μ L,
10.0 the IodineSodium Solution of mmol/L is added in the hexadecyltrimethylammonium chloride solution of 8.0 mL, 0.1 mol/L；B is molten
Liquid is to be added to the IodineSodium Solution of 500.0 μ L, 50.0 mmol/L chlorauric acid solutions and 300.0 μ L, 10.0 mmol/L
40.0 mL, 0.1 mol/L hexadecyltrimethylammonium chloride solution in；Respectively by 40.0 ~ 60.0 μ L and 400.0 ~
600.0 μ L, 0.1 mol/L ascorbic acid solution are added in a, b solution, and 100.0 μ of μ L ~ 200.0 L are then diluted 10
Times gold seeds solution be added to and react 1 s in a solution after, a solution of 3.0 ~ 4.0 mL is added to the stirring of b solution rapidly
More than ten seconds, 1 ~ 3 h is then stood at room temperature, and final centrifugation, cleaning 3 times obtain triangle gold-nano-piece.
3. a kind of preparation side of the electrochemical immunosensor of interlayer type detection prostate-specific antigen as described in claim 1
Method, which is characterized in that the step (2) prepares the nanometer that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube
Composite material includes:
1. preparing hollow nucleocapsid Au-Ag-Pt nanocube
The gold chloride of 10.0 μ L, 25.0 mmol/L are added to 10.0 mL, 75.0 mmol/L cetyl trimethyl chlorinations
In ammonium salt solution, 0.6 mL, 10.0 mmol/L sodium borohydride solutions is then added dropwise, reacts 2 h under room temperature, obtains gold seeds
Solution；Then by 0.1 ~ 0.3 mL, 10.0 mmol/L chlorauric acid solutions and 2.0 ~ 4.0 mL, 0.2 mol/L cetyl
Trimethyl ammonia chloride ammonium salt solution is added in 25.0 mL deionized waters, and 1.5 ~ 2.5 mL, 0.1 mol/L ascorbic acid are added
By above-mentioned solution reaction more than ten second, 0.3 ~ 0.5 mL is then added and dilutes 100 times of gold seeds solution, be stored at room temperature 8 h it
Afterwards, 0.5 ~ 1.0 mL, 10.0 mmol/L silver nitrates and 2.0 ~ 4.0 mL, 0.1 mol/L ascorbic acid solution are added to
Above-mentioned solution is placed in 50 DEG C of water-baths and stands 12 h, obtains gold and silver nanocube colloidal solution；Take 4.0 ~ 6.0 mL
Gold and silver nanocube colloidal solution is persistently stirred at reflux in oil bath pan and is heated to 100 DEG C, after 15 min by 200.0 ~
400.0 μ L, 5.0 mmol/L platinum acid chloride solutions are added drop-wise in solution, are persistently stirred to react 15 min, cooling, centrifugation, cleaning 3
It is secondary, obtain hollow nucleocapsid Au-Ag-Pt nanocube；
2. preparing molybdenum disulfide nano flower
1.2 ~ 1.6 g ammonium molybdates are added in 15.0 mL deionized waters, then 0.8 ~ 1.0 g ethylenediamine is added
It states in solution, after ultrasonic dissolution, the salt acid for adjusting pH of appropriate 1.0 mol/L is added to 4.0 ~ 6.0, under the conditions of 50 DEG C
2 h are reacted, eccentric cleaning 3 times, is dried in vacuo under 50 DEG C of environment, obtains the complex of ammonium molybdate and ethylenediamine；Take 0.1 ~
The complex of 0.2 g ammonium molybdate and ethylenediamine is added in 15.0 mL deionized waters, and 0.2 ~ 0.4 gL-, half Guang is then added
Propylhomoserin, ultrasonic disperse are put into the autoclave of 25.0 mL polytetrafluoroethyllining linings, are heated to 200 DEG C, keep the temperature 14 h, most
Whole eccentric cleaning 3 times, vacuum drying obtain molybdenum disulfide nano flower；
3. preparing the nanocomposite that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube
By 0.1 ~ 0.2 g molybdenum disulfide nano flower ultrasonic disperse into 20.0 mL dehydrated alcohols, it is heated at reflux in oil bath pan
To 100 DEG C, 20.0 ~ 30.0 μ L 3- aminopropyl triethoxysilanes are then added, cooling after reacting 2 h, centrifugation, clearly
It washes 3 times, freeze-drying obtains amidized molybdenum disulfide nano flower；By the hollow nucleocapsid gold and silver of 2.0 ~ 4.0 mL, 2.0 mg/mL
Platinum nanocube is added to the amidized molybdenum disulfide nano flower of 2.0 mL, 2.0 mg/mL, 1 h of ultrasonic disperse, in room temperature
Under environment, 6 h are shaken, eccentric cleaning 2 times, molybdenum disulfide nano flower is obtained and loads receiving for hollow nucleocapsid Au-Ag-Pt nanocube
Nano composite material.
4. a kind of preparation side of the electrochemical immunosensor of interlayer type detection prostate-specific antigen as described in claim 1
Method, which is characterized in that the step (3) prepares the nanometer that molybdenum disulfide nano flower loads hollow nucleocapsid Au-Ag-Pt nanocube
Composite material hatching prostate specific antibody secondary antibody marker include:
1. 2.0 ~ 4.0 mL, 10.0 μ g/mL prostate specific antibody are added to 2.0 mL, 2.0 mg/mL molybdenum disulfide
Nano flower loads in the nanocomposite of hollow nucleocapsid Au-Ag-Pt nanocube, and 12 h are shaken under 4 DEG C of environment, centrifugation
Cleaning is dispersed in phosphate buffer solution.
5. a kind of preparation side of the electrochemical immunosensor of interlayer type detection prostate-specific antigen as described in claim 1
Method, which is characterized in that the step (4) prepares the work of the electrochemical immunosensor of interlayer type detection prostate-specific antigen
Include: as electrode
1. the glass-carbon electrode Al for being 3.0 ~ 5.0 mm by diameter2O3Polishing powder is polished to mirror surface, respectively successively in anhydrous second
It is cleaned by ultrasonic in alcohol, ultrapure water；
2. the triangle gold-nano-piece dispersant liquid drop of 6.0 μ L, 0.5 ~ 3.0 mg/mL are added on electrode surface, dry in the air at room temperature
It is dry, with ultrapure water electrode surface, dry at room temperature；
3. continuing the prostate specific antibody of 6.0 μ L, 4.0 ~ 8.0 μ g/mL being added drop-wise to electrode surface, in 4 DEG C of refrigerators
Middle drying；
4. the bovine serum albumin solution of 3.0 μ L, 0.7 ~ 1.4 wt% are then added drop-wise to electrode surface, to close non-spy
Specific activities site is rinsed electrode surface with the phosphate buffer that pH is 7.0, is dried in 4 DEG C of refrigerators；
5. a series of prostate-specific antigen solution of various concentrations of 6.0 μ L, 0.00005 ~ 100.0 ng/mL is added dropwise,
Electrode surface is rinsed with the phosphate buffer that pH is 7.0, is dried in 4 DEG C of refrigerators；
6. 6.0 μ L, 2.0 ~ 4.0 mg/mL molybdenum disulfide nanos flower are loaded receiving for hollow nucleocapsid Au-Ag-Pt nanocube
The secondary antibody marker of nano composite material hatching prostate specific antibody is added drop-wise to electrode surface, and the phosphate for being 7.0 with pH is slow
Fliud flushing rinses electrode surface, dries in 4 DEG C of refrigerators, prepares the electro-chemistry immunity of interlayer type detection prostate-specific antigen
The working electrode of sensor.
6. a kind of preparation side of the electrochemical immunosensor of interlayer type detection prostate-specific antigen as described in claim 1
Method, which is characterized in that the step (5) prepares the work of the electrochemical immunosensor of interlayer type detection prostate-specific antigen
Include: as curve
1. being tested using electrochemical workstation with three-electrode system, saturated calomel electrode is reference electrode, and platinum electrode is
To electrode, prepared sensor is working electrode, the phosphate for being 5.3 ~ 8.0 in the pH of 10.0 mL, 50.0 mmol/L
It is tested in buffer solution；
2. the used time, m- current method detected analyte, input voltage is -0.4 V, 0.1 s of sampling interval, runing time
200 s；
3. after background current tends towards stability, to 10.0 mL, 50.0 mmol/L phosphate buffer solution in injection 10.0 ~
The hydrogen peroxide solution of 15.0 μ L, 5.0 mol/L record current value corresponding to prostate-specific antigen under various concentration, draw
The working curve of the electrochemical immunosensor of system detection prostate-specific antigen；
4. utilizing working curve method, the concentration of prostate-specific antigen in sample to be tested is obtained.
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| CN110068601A (en) * | 2019-04-27 | 2019-07-30 | 山东理工大学 | A kind of preparation method and application of the electrochemical sensor based on the mesoporous stick label of mulberries shape Au@PtPd |
| CN111273014A (en) * | 2020-03-06 | 2020-06-12 | 安徽大学 | Photoelectrochemical immunosensor for detecting prostate specific antigen and preparation method thereof |
| CN111721820A (en) * | 2020-07-13 | 2020-09-29 | 江西科技师范大学 | Non-labeled electrochemical immunosensor for detecting prostate specific antigen and preparation method thereof |
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| CN111721820B (en) * | 2020-07-13 | 2023-05-09 | 江西科技师范大学 | Non-labeled electrochemical immunosensor for detecting prostate specific antigen |
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| CN113862735B (en) * | 2021-09-24 | 2023-08-08 | 鲍宁 | Coating liquid, coating electrode, and preparation method and application of coating liquid |
| CN114965633A (en) * | 2022-04-12 | 2022-08-30 | 烟台南山学院 | Preparation method and application of ratio sensor constructed based on gold cysteine and gold platinum molybdenum disulfide |
| CN114965633B (en) * | 2022-04-12 | 2023-08-01 | 烟台南山学院 | Preparation method of ratio sensor constructed based on cysteine gold and gold platinum molybdenum disulfide |
| CN114772643A (en) * | 2022-04-27 | 2022-07-22 | 广东省科学院生物与医学工程研究所 | Composite nano material and preparation method and application thereof |
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