CN107490682A - One kind visualization enzyme-linked immune analytic method - Google Patents
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- CN107490682A CN107490682A CN201710913569.0A CN201710913569A CN107490682A CN 107490682 A CN107490682 A CN 107490682A CN 201710913569 A CN201710913569 A CN 201710913569A CN 107490682 A CN107490682 A CN 107490682A
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- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
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Abstract
A kind of visualization enzyme-linked immune analytic method of disclosure of the invention, comprises the following steps：（1）The preparation of Au NBP@Ag nanometer rods；（2）Enzyme linked immunoassay.Primary antibody has onboard been completed, has added antigen, has added the secondary antibody compound of hydrogen peroxide enzyme modification, after then adding the hydrogen peroxide of equivalent, has added Fe2+And hydrochloric acid；Then silver-colored cone nanometer rods covered with gold leaf are added to be reacted, a series of chromatic colours are observed in etching process, visualization semi-quantitative analysis is carried out to target antigen.Visualization immunoassay of the present invention can produce multiple color change, solve the shortcomings that color change is single in conventional colorimetric method, can realize the quantitative detection to object, and the reaction time is fast, and effect is good；Considerably increase this method be used for naked eyes detection the degree of accuracy, detection limit is lower.
Description
Technical field
The invention belongs to analytical chemistry field, and in particular to one kind visualization enzyme-linked immune analytic method.
Background technology
In recent years, due to its simplicity in terms of unique advantage, people give great pass to various colorimetric sensors
Note.Under normal circumstances, colorimetric sensor can be detected with common UV-vis spectrometers, can also be detected by an unaided eye.Due to it
Simplicity, these sensors be used to detecting various biologies and biomedical objects.The colorimetric of most common of which is anti-
It should be horseradish peroxidase（HRP）With 3,3 ', 5,5 '-tetramethyl benzidine (TMB) immune system, this is already used to
It is anti-based on efficient color between the specific recognition to antibody-antigene and HRP and TMB to detect many disease markers
Should.Except TMB, also some other chromogenic substrate, such as o-phenylenediamine (OPD) and 2'- hydrazines-bis- -3- ethyl-benzothiazoles
Quinoline -6- sulfonic acid (ABTS) is also widely used for HRP colorimetric enzyme linked immunosorbent assay（ELISA）.It should be noted, however, that
It is above-mentioned it is all illustrate only a kind of color than colour induction strategy, different target concentrations has corresponded to color intensity
Change.It is known that change of the human eye to color is more sensitive, but depth change for same color and unwise
Sense, therefore, colorimetric sensor above-mentioned is generally detected with spectrometer, rather than is observed with the naked eye.
In modern society, cancer has become one of cause of death maximum in the world.In various cancers, lung cancer it is dead
Die rate highest.It is reported that squamous cell carcinoma antigen (SCCA) is a kind of important cancer markers.In clinical diagnostics,
SCCA detection is significant in terms of early detection and treating cancer.It is used as diagnosing, melanoma and liver
The mark of cell cancer.So far, many methods for being used to detect SCCA have been developed, as Electrochemical Detection, fluorescence are exempted from
Epidemic disease measure, radiommunoassay, enzyme-linked immunoassay（ELISA）Deng.These methods can be used for detecting SCCA sensitivity.So
And some shortcomings are still suffered from, such as using the high and cumbersome operating procedure of complicated instrument, cost.So look for one it is simple and quick,
The low method of cost is come to detect disease marker be necessary.Noble metal nanometer material because of its excellent chemical and physical features and
Get more and more people's extensive concerning.And nanogold color change therein only has two to three kinds, semi-quantitative analysis can only be carried out, can not
It is truly realized the quantitative analysis detection to object.We have found a kind of chromogenic substrate that can produce multiple color change（It is high
Spend uniform Au NBP@Ag nanometer rods）, and apply to the quantitative analysis detection of object.
Therefore, in the present invention, we systematically have studied the optical change of the etching of AuNBP@Ag nanometer rods, its conduct
A kind of application of the thing in disease biomarkers detection that develop the color.By taking SCCA detection as an example, the results showed that, target molecule induction
The etching result of AuNBP@Ag nanometer rods generates a kind of interesting color change:LSPR peaks are initially blue shifts, followed by red shift,
It is finally blue shift.Remove the concentration of quantitative objective thing, solution simultaneously with the change of absorbance and two variables of change of peak position
It is the SCCA of various concentrations corresponding to the change of color.It is proposed that colorimetric sensor can with the naked eye be examined in actual sample
Survey SCCA.Because this method is established based on traditional ELISA, so can be used for the inspection of other diseases label
Survey.
The content of the invention
It is an object of the invention in view of the shortcomings of the prior art, providing a kind of visualization enzyme-linked immune analytic method.This hair
Bright visualization immunoassay can produce multiple color change, solve color change in conventional colorimetric method it is single lack
Point, the quantitative detection to object can be realized, and the reaction time is fast, and effect is good；Considerably increase this method be used for naked eyes
The degree of accuracy of detection, detection limit is lower, is expected to be used widely in clinical medicine detection, has significant commercial value.
To achieve the above object, the present invention adopts the following technical scheme that：
One kind visualization enzyme-linked immune analytic method, specifically includes following steps：
（1）The preparation of silver-colored cone nanometer rods covered with gold leaf：Gold nano bipyramid solution, its longitudinal plasma are synthesized according to seed mediated growth method first
Formant（LSPR）For 800 nm；Then silver nitrate is reduced according to ascorbic acid, makes silver-colored uniform deposition in golden poppet surface, synthesis silver
Gold filled cone nanometer rods solution, LSPR is 800 nm；
（2）By step（1）The silver cone nanometer rods solution covered with gold leaf of middle synthesis centrifuges 15 min under 10000 rpm rotating speed, discards
Supernatant, precipitation are disperseed again with the CTAB solution of 0.16 isometric M；
（3）The Visual retrieval of target antigen：First according to classic ELISA method, primary antibody has onboard been completed, has added antigen,
The secondary antibody compound of hydrogen peroxide enzyme modification is added, then specific reaction occurs for antibody-antigen-antibody, forms sandwich
Sandwich-type structure；After adding isometric hydrogen peroxide, catalase can catalyzing hydrogen peroxide produce water and oxygen, add
Fe2+And hydrochloric acid, while generate the high OH of activity；Now add step（2）Again scattered silver cone nanometer rods covered with gold leaf, OH is just
It can be reacted with silver-colored cone nanometer rods covered with gold leaf, the reaction time is 10 min, and reaction temperature is 37 DEG C, can be seen in etching process
Observe a series of chromatic colours, highly sensitive detection antigen protein.
Step（1）Described in seed mediated growth method synthesis gold nano bipyramid solution, specifically include following steps：
（A）The synthesis of gold kind solution：Under conditions of being stirred vigorously at 30 DEG C, 25 mM that 0.25 mL is prepared with frozen water
NaBH4Solution is added in 10 mL mixed solutions, forms brown yellow solution after the 2 minutes, and the mixed solution includes 0.25 mM
HAuCl4, 50 mM CTAC and 5mM citric acids；Gained brown yellow solution is placed on oil bath at 80 DEG C to be kept for 90 minutes, gold is made
Kind solution, is saved backup at room temperature；
（B）The synthesis of gold nano bipyramid：Then growth-promoting media is prepared, i.e., the M HAuCl of 10 mL 0.014Solution, 2 mL 0.01
M AgNO3Solution, 4 mL 1M HCl solutions and the M ascorbic acid solutions of 1.6 mL 0.1（AA solution）It is added to 200 mL0.1 M
In CTAB solution, after mixing, 8 mL steps are added（A）The gold kind solution of synthesis, stands 3 hours in 30 DEG C of water-baths, synthesizes
Gold nano bipyramid solution（Au NBPs）.
Step（1）Described in ascorbic acid reduction synthesis silver cone nanometer rods solution covered with gold leaf comprise the following steps that：Take 80
ML gold nano bipyramid solution（Au NBPs）15 min are centrifuged in the case where rotating speed is 8000 rpm；Supernatant liquor is removed, is precipitated with 240
ML0.08 mM CTAC solution disperses, and adds 5 mL 0.01mM AgNO3Solution and 2.5mL 0.1mM ascorbic acid solutions
（AA solution）；Gained mixed solution is placed in 60 DEG C of water-baths, stirs 4.5 h, rotating speed is 120 turns/min, in the meantime Ag
It is uniform to be deposited on Au NBPs synthesis silver cone nanometer rods solution covered with gold leaf（Au NBP@Ag）.
Step（3）Described in the preparation method of secondary antibody compound of hydrogen peroxide enzyme modification be：By 0.001 g SCCA
Antibody is dissolved in the phosphate buffer solution of 1.0 mL 0.1 M, pH=7.4, then adds 0.87mg SM (PEG)12Crosslinking agent,
Gained mixed solution is incubated 30 min at room temperature；Then unnecessary crosslinking is removed by desalting column with phosphate buffer solution
Agent；The mM of 1.0 mL 0.5 dissolved with phosphate buffer solution Catalase solution is added, 2h is reacted at 4 DEG C, is made
The secondary antibody compound of hydrogen peroxide enzyme modification, in 4 DEG C of preservations.
Beneficial effects of the present invention：
（1）Conventional ELISA method only produces a kind of color change（Yellow is from shallow to deep）, it is necessary to by instrument, visually can not
Differentiate, and visualization immunoassay of the present invention can produce multiple color（Pink, grey, purple are blue, light green
Color, pink colour, bottle green, brown color etc.）Change, solves the shortcomings that color change is single in conventional colorimetric method, can realize
Quantitative detection to object, and the reaction time is fast, and effect is good；So as to considerably increase this method be used for naked eyes detection standard
Exactness；
（2）Visualization sxemiquantitative immunoassay of the present invention is due to being the change two with the change of absorbance and peak position
Individual variable goes the concentration of quantitative objective thing simultaneously, goes quantitative, colour developing spirit with single absorbance compared to conventional ELISA method
Sensitivity is higher, and the degree of accuracy is more preferable, and detection limit is lower；
（3）The nanometer rods of colour developing silver bipyramid covered with gold leaf are prepared simply, and the LSPR peaks of material are in 650 ~ 900 nm continuously adjustabes, system
Standby cost is low, and reappearance is high, and stability is good, holding time length；
（4）Visualization enzyme-linked immune analytic method of the present invention is by based on traditional ELISA kits and silver cone nanometer rods covered with gold leaf
Composition, raw material are easy to get, can also apply to the detection of other diseases mark, have universal applicability, it is easy to accomplish commodity
Metaplasia is produced.
Brief description of the drawings
Fig. 1 is the schematic diagram of plasma ELISA of the present invention, and wherein left-half is biography
The ELISA schematic diagrames of system, right half part are that plasma senses schematic diagram；
Fig. 2 is the transmission electron microscope picture of gold nano bipyramid (A) of the present invention and silver cone nanometer rods (B) covered with gold leaf；
Fig. 3 is the detection that antigen is carried out using plasma enzyme linked immunosorbent assay（Squamous cell carcinoma antigen, Squamous Cell
Carcinoma Antigen）Colorimetric photo and ultraviolet spectrogram；This than chromatic graph from left to right color it is respectively pink, it is dark red,
Purplish red, royal purple, purple, pale purple, brown, dark red, dark green and yellow, corresponding PSA concentration are followed successively by 0,2.5,5,15,25,
35,45,65,85,105 ng/mL.
Embodiment
Embodiment 1
Following examples combination accompanying drawing come illustrate using the present invention carry out actual sample analysis operating process：
（1）The required glassware used all is soaked with chloroazotic acid before the synthesis, is then rinsed with substantial amounts of water, finally used
Ultra-pure water rinse is clean；
（2）Synthesize the gold nano bipyramid that LSPR peaks are 800nm：Under conditions of being stirred vigorously at 30 DEG C, by 0.25 mL ice
The 25 mM NaBH that water is prepared4Solution is added in 10 mL mixed solutions, forms brown yellow solution after the 2 minutes, and the mixing is molten
Liquid includes 0.25 mM HAuCl4, 50 mM CTAC and 5mM citric acids；Gained brown yellow solution is placed on oil bath at 80 DEG C to keep
90 minutes, gold kind solution is made, saves backup at room temperature；
Then growth-promoting media is prepared, i.e., the M HAuCl of 10 mL 0.014Solution, the M AgNO of 2 mL 0.013Solution, 4 mL 1M
HCl solution and the M AA solution of 1.6 mL 0.1 are added in 200 mL0.1 M CTAB solution, after mixing, add 8 mL gold kinds
Solution, 3 hours are stood in 30 DEG C of water-baths, synthesize gold nano bipyramid solution（Au NBPs）；The Au NBPs' being synthesized is vertical
It is about 800 nm to plasma wavelength；
（3）The nanometer rods of synthesis silver cone covered with gold leaf：The above-mentioned AuNBPs of 80 mL are taken to centrifuge 15 min in the case where rotating speed is 8000 rpm；
Supernatant liquor is removed, precipitation is disperseed with the mM CTAC solution of 240 mL 0.08, adds 5 mL 0.01mM AgNO3Solution and
2.5mL 0.1mM AA solution；Gained mixed solution is placed in 60 DEG C of water-baths, 120 turns of stirrings per minute, 4.5 h, in this phase
Between Ag be uniformly deposited on Au NBPs formed Au NBP@Ag nanometer rods structure；
（4）The synthesis of the compound of the secondary antibody of hydrogen peroxide enzyme modification：0.001 g SCCA antibody is dissolved in 1.0 mL's
In the phosphate buffer solution of 0.1 M, pH=7.4,0.87mg SM (PEG) are then added12Crosslinking agent, gained mixed solution is in room temperature
30 min of lower incubation；Then unnecessary crosslinking agent is removed by desalting column with phosphate buffer solution；Add and delayed with phosphate
The mM of 1.0 mL 0.5 of solution dissolving Catalase solution is rushed, 2h is reacted at 4 DEG C, is made the two of hydrogen peroxide enzyme modification
Anti- compound, in 4 DEG C of preservations；
（5）By step（3）Obtained silver cone nanometer rods solution covered with gold leaf centrifuges 15 min under 10000 rpm rotating speed, discards
Clear liquid, 0.16 M of precipitation equivalent CTAB solution disperse again.
Prepare the gold that LSPR is 800 nm or so first to bore, the A in Fig. 2 is gold nano bipyramid（Au NBPs）Transmission electricity
Mirror figure.And the nanometer rods of the silver cone covered with gold leaf by silver-colored uniform deposition on gold cone, disperseed again with 0.16 M CTAB, the B in Fig. 2
For the nanometer rods of silver-colored cone covered with gold leaf（Au NBP@Ag）Transmission electron microscope picture.
In the present embodiment, selected object is squamous cell carcinoma antigen, it is therefore desirable to by corresponding primary antibody, antigen,
The secondary antibody of enzyme modification connects, and specific implementation method is as follows：The ELISA Plate for being loaded with capture antibody is fixed, in each Kong Zhongjia
Enter the SCCA antigens of 50 μ L various concentrations, then 100 μ L SCCA- hydrogen peroxide multienzyme complexs are added in each hole, mix
Close solution and 1 h is incubated at 37 DEG C.Washed three times with cleaning fluid, finally make all antibody, antigen according to Fig. 1 left-halfs
Mode connects.
The mM H of 100 μ L 6 are added in each hole2O2, after mixing, hatch 10 min at 37 DEG C；Next, by 100
μ LAu NBPs@Ag nanometer rods, the M HCl of 30 μ L 2 and 20 μ L 4mM FeSO4Solution is added in above-mentioned hole, reaction 10
min。
Fig. 3 is as using this plasma enzyme-linked immunosorbent assay SCCA colorimetric photo and ultraviolet spectrogram, the ratio
Color is respectively pink from left to right for chromatic graph, dark red, purplish red, royal purple, purple, pale purple, brown, dark red, dark green and yellow, right
The PSA concentration answered is followed successively by 0,2.5,5,15,25,35,45,65,85,105 ng/mL, and the detection of this method visual colorimetric determination is limited to
2.5 ng/mL。
The foregoing is only presently preferred embodiments of the present invention, all equivalent changes done according to scope of the present invention patent with
Modification, it should all belong to the covering scope of the present invention.
Claims (5)
1. one kind visualization enzyme-linked immune analytic method, it is characterised in that：Specifically include following steps：
（1）The preparation of silver-colored cone nanometer rods covered with gold leaf：Gold nano bipyramid solution, its longitudinal plasma are synthesized according to seed mediated growth method first
Formant is 800 nm；Then according to ascorbic acid reduction, silver-colored uniform deposition is made in golden poppet surface, synthesis silver cone nanometer covered with gold leaf
Rod solution, longitudinal plasma resonance peak are 800 nm；
（2）By step（1）The silver cone nanometer rods solution covered with gold leaf of middle synthesis centrifuges 15 min under 10000 rpm rotating speed, discards
Supernatant, precipitation are disperseed again with the CTAB solution of 0.16 isometric M；
（3）The Visual retrieval of target antigen：First according to classic ELISA method, primary antibody has onboard been completed, has added antigen,
The secondary antibody compound of hydrogen peroxide enzyme modification is added, then specific reaction occurs for antibody-antigen-antibody, forms sandwich
Sandwich-type structure；After adding isometric hydrogen peroxide, Fe is added2+And hydrochloric acid；Then step is added（2）Again scattered silver
Gold filled cone nanometer rods are reacted, and a series of chromatic colours are observed in etching process, target antigen is visualized
Semi-quantitative analysis.
2. enzyme-linked immune analytic method is visualized according to claim 1, it is characterised in that：Step（1）Described in seed
Growth method synthesizes gold nano bipyramid solution, specifically includes following steps：
（A）The synthesis of gold kind solution：Under conditions of being stirred vigorously at 30 DEG C, 25 mM that 0.25 mL is prepared with frozen water
NaBH4Solution is added in 10 mL mixed solutions, forms brown yellow solution after the 2 minutes, and the mixed solution includes 0.25 mM
HAuCl4, 50 mM CTAC and 5mM citric acids；Gained brown yellow solution is placed on oil bath at 80 DEG C to be kept for 90 minutes, gold is made
Kind solution, is saved backup at room temperature；
（B）The synthesis of gold nano bipyramid：Then growth-promoting media is prepared, i.e., the M HAuCl of 10 mL 0.014Solution, the M of 2 mL 0.01
AgNO3Solution, 4 mL 1M HCl solutions and the M ascorbic acid solutions of 1.6 mL 0.1 are added to 200 mL0.1 M CTAB solution
In, after mixing, add 8 mL steps（A）The gold kind solution of synthesis, stands 3 hours, synthesis gold nano is double in 30 DEG C of water-baths
Bore solution.
3. enzyme-linked immune analytic method is visualized according to claim 1, it is characterised in that：Step（1）Described in Vitamin C
Sour reducing process synthesis silver cone nanometer rods solution covered with gold leaf comprises the following steps that：The 80 mL gold nano bipyramid solution are taken to be in rotating speed
15 min are centrifuged under 8000 rpm；Supernatant liquor is removed, precipitation is disperseed with 240 mL0.08 mM CTAC solution, adds 5 mL
0.01mM AgNO3Solution and 2.5mL 0.1mM ascorbic acid solutions；Gained mixed solution is placed in 60 DEG C of water-baths, stirred
4.5 h, rotating speed are 120 turns/min, synthesize silver cone nanometer rods solution covered with gold leaf.
4. enzyme-linked immune analytic method is visualized according to claim 1, it is characterised in that：Step（3）Described in peroxide
The preparation method of secondary antibody compound for changing hydrogen enzyme modification is：By 0.001 g SCCA antibody be dissolved in 1.0 mL 0.1 M, pH=
In 7.4 phosphate buffer solutions, 0.87mg SM (PEG) are then added12Crosslinking agent, gained mixed solution are incubated 30 at room temperature
min；Then unnecessary crosslinking agent is removed by desalting column with phosphate buffer solution；Add and dissolved with phosphate buffer solution
The mM of 1.0 mL 0.5 Catalase solution, react 2h at 4 DEG C, the secondary antibody compound of hydrogen peroxide enzyme modification be made,
4 DEG C of preservations.
5. enzyme-linked immune analytic method is visualized according to claim 1, it is characterised in that：Step（3）Described in reaction
Time is 10 min, and reaction temperature is 37 DEG C.
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| CN109030472A (en) * | 2018-06-12 | 2018-12-18 | 福州大学 | A kind of method of Visual retrieval dibutyl phthalate content |
| CN110632061A (en) * | 2019-01-18 | 2019-12-31 | 长沙理工大学 | Visual colorimetric detection method of aminotriazole |
| CN111519225A (en) * | 2018-11-27 | 2020-08-11 | 广州三孚新材料科技股份有限公司 | Chromium-free manganese-free roughening solution for ABS (acrylonitrile butadiene styrene) plastics and using method thereof |
| CN113030079A (en) * | 2021-04-23 | 2021-06-25 | 中南民族大学 | Method for detecting pesticide carbaryl based on nanogold etching |
| CN113218939A (en) * | 2021-04-23 | 2021-08-06 | 江苏科技大学 | Method for detecting antibiotics based on 'peak exposure' of gold and silver nano-star |
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109030472A (en) * | 2018-06-12 | 2018-12-18 | 福州大学 | A kind of method of Visual retrieval dibutyl phthalate content |
| CN111519225A (en) * | 2018-11-27 | 2020-08-11 | 广州三孚新材料科技股份有限公司 | Chromium-free manganese-free roughening solution for ABS (acrylonitrile butadiene styrene) plastics and using method thereof |
| CN110632061A (en) * | 2019-01-18 | 2019-12-31 | 长沙理工大学 | Visual colorimetric detection method of aminotriazole |
| CN110632061B (en) * | 2019-01-18 | 2022-01-14 | 长沙理工大学 | Visual colorimetric detection method of aminotriazole |
| CN113030079A (en) * | 2021-04-23 | 2021-06-25 | 中南民族大学 | Method for detecting pesticide carbaryl based on nanogold etching |
| CN113218939A (en) * | 2021-04-23 | 2021-08-06 | 江苏科技大学 | Method for detecting antibiotics based on 'peak exposure' of gold and silver nano-star |
| CN113030079B (en) * | 2021-04-23 | 2022-05-20 | 中南民族大学 | Method for detecting pesticide carbaryl based on nanogold etching |
| CN113218939B (en) * | 2021-04-23 | 2022-11-29 | 江苏科技大学 | Method for detecting antibiotics based on 'peak exposure' of gold and silver nano-star |
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