CN108896756A - A kind of colloid gold nano-polyaniline-gold nano complex microsphere and its preparation method and application - Google Patents
A kind of colloid gold nano-polyaniline-gold nano complex microsphere and its preparation method and application Download PDFInfo
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Abstract
The present invention relates to technical field of immunoassay, a kind of colloid gold nano-polyaniline-gold nano complex microsphere and its preparation method and application is disclosed.Polyaniline wraps up Au nanoparticle in complex microsphere of the present invention, and is coupled colloid gold nanoparticle on polyaniline surface.The present invention substitutes the colloidal gold cholylglycine monoclonal antibody routinely applied with the colloid gold nano-polyaniline-gold nano complex microsphere with three-decker, monoclonal antibody is set to be multiplied, when carrying out immunochromatography detection, detection sensitivity can be increased, detection limit can directly pass through naked-eye observation down to the preferable degree of 0.2mg/L.
Description
Technical field
The present invention relates to technical field of immunoassay, and in particular to a kind of colloid gold nano-polyaniline-gold nano is compound micro-
Ball and its preparation method and application.
Background technique
Liver cell, which is damaged and accumulates, causes liver diseases, detects the liver cell extent of damage and can be carried out to liver function and comments in real time
Valence is of great significance for early diagnosing, preventing and treating liver diseases.Glycocholic acid is one of main component of bile acid,
It is combined by cholesterol and the glycine in liver, the measurement of content relatively has sensibility to diagnosing hepatism, is evaluating liver function
One critically important index of energy.Correlative study points out, bile acid through kidney excretion it is obvious that and with disease in the liver and gallbladder degree phase
It closes, therefore the glycocholic acid detected in urine has certain guidance value to clinic, and clinically detects the more extracting vein bloods of glycocholic acid, and
For some pediatric patients, be not easy to the patient of extracting vein blood, detect the glycocholic acid in its urine have its convenience with to disease
The directive significance of disease diagnosis.
Existing patent CN105481877A discloses a kind of immune inspection of glycocholic acid based on anti-glycocholic acid specific antibody
Test agent and preparation method thereof, it is special with the glycocholic acid obtained by above-mentioned glycocholic acid specificity hapten conjugation protein immune animal
The time resolution immune chromatography test paper of specific monoclonal antibodies preparation can rapidly measure the content of glycocholic acid in sample, improve
The specificity and accuracy of existing glycocholic acid clinical diagnosis.
But above-mentioned existing scheme needs just to can reach the inspection of 0.5mg/L by time-resolved fluoroimmunoassay chromatography instrument
Survey limit, can not directly by naked eyes carry out fast qualitative judgement, and sensitivity it is bad cause detection limit it is higher.
Summary of the invention
In view of this, making the purpose of the present invention is to provide a kind of colloid gold nano-polyaniline-gold nano complex microsphere
Its cholylglycine monoclonal antibody is used to directly can visually carry out result viewing when immune detection, and detects limit in 0.1-0.2mg/L
Between, have high sensitivity；
Another object of the present invention is that providing the preparation method of above-mentioned complex microsphere；
Another object of the present invention is that providing above-mentioned complex microsphere in preparation qualitative detection glycocholic acid product
Related application.
To achieve the goals above, the present invention provides the following technical solutions：
A kind of colloid gold nano-polyaniline-gold nano complex microsphere, polyaniline wrap up Au nanoparticle, and in polyaniline
Surface is coupled colloid gold nanoparticle.
Wherein, the coupling is coupled by-Au-NH- key and is wrapped up polyaniline, forms emeraldine salt, as follows：
In acid medium, aniline and HAuCl4Between redox reaction can occur.Initially, by aniline reduction
HAuCl4Lead to the formation of metal Au0 atom.As time goes by, it is former that more new gold are produced in this system
Son will generate nucleation when the concentration of gold atom reaches critical hypersaturated state.Atomic nucleus is by further increasing golden original
Son and grow into nano-pore.Finally, Dominant particle can flock together to form biggish gold nano sphere, size distribution is opposite
It is smaller.At the same time, aniline monomer is transferred in polyaniline, and in gold nanosphere surface coated.
The present invention is needed for the immunochromatography technique of existing detection glycocholic acid by time-resolved fluoroimmunoassay chromatograph
Device and the not high defect of sensitivity, the present invention are substituted conventional use of with colloid gold nano-polyaniline-gold nano complex microsphere
Colloidal gold cholylglycine monoclonal antibody carries out immunochromatography detection, directly directly can observe testing result by naked eyes, and sensitive
It spends higher.
The present invention is divided into two stages when preparing the complex microsphere, first by HAuCl4Aqueous solution with contain aniline list
The HCl solution of body is stirred to react to dark green, at this time HAuCl4As oxidant be reduced to Au nanoparticle be wrapped in it is poly-
Inside aniline, it is centrifugally separating to obtain the polyaniline microsphere for being precipitated as package Au nanoparticle；Prepared polyaniline microsphere surface
Can be with the group of coupling protein due to not having to exist, therefore it can not be coupled with glycocholic acid monoclonal antibody, therefore the of preparation method of the present invention
Two-stage is that polyaniline microsphere is resuspended and is stirred to react overnight with colloid gold nanoparticle, and precipitating is resuspended after centrifugation, is wrapped
It wraps up in the polyaniline of Au nanoparticle and (flow diagram is shown in figure in the complex microsphere of polyaniline surface coupling colloid gold nanoparticle
1)。
It is to be connected to polyaniline surface that colloid gold nanoparticle, which mainly acts on, connects glycocholic acid monoclonal antibody, and due to the
The one stage polyaniline microsphere surface of preparation can connect multiple colloid gold nanoparticles, and the glycocholic acid monoclonal antibody of coupling will be at multiplication
Add, therefore its sensitivity is higher, can reach the degree by naked eyes identification.
Preferably, the preparation method is that：
The HAuCl of 1.0mmol/L4Aqueous solution instills the HCl solution that 0.1mol/L contains 0.1mmol/L aniline monomer, stirs
It mixes uniformly；When the color of mixture becomes bottle green, what is be centrifugally separating to obtain is precipitated as the polyaniline of package Au nanoparticle
Microballoon；The polyaniline microsphere is resuspended in deionized water, is slowly added to the colloid gold nanoparticle of concentration=24 Au ± 1mol/L, delays
At room temperature overnight, centrifugation, precipitating is resuspended for slow stirring, obtains the polyaniline of blackish green package Au nanoparticle and in polyaniline
The complex microsphere of surface coupling colloid gold nanoparticle.
Wherein, the HAuCl4The volume ratio of aqueous solution and the HCl solution containing aniline monomer is 3:10；The deionization
The volume ratio of water and colloid gold nanoparticle is 1:3.
In specific implementation method of the present invention, the preparation method is specially：
By the HAuCl of 3mL4Aqueous solution (1.0mmol/L) instills 10mL HCl solution (0.1mol/L), wherein containing
0.1mmol/L aniline monomer, stirs evenly.When the color of mixture becomes bottle green, the precipitating being centrifugally separating to obtain (is wrapped
Wrap up in the polyaniline microsphere of Au nanoparticle)；After being resuspended with the deionized water of 1mL, it is slowly added to the colloid gold nanoparticle of 3mL
(C (Au)=24 ± 1mol/L) is slowly stirred at room temperature overnight, and 12000rpm is centrifuged 20 minutes under the conditions of 4 DEG C, and is added
The deionized water of 1.0mL suspends again, blackish green colloid gold nano-polyaniline-gold nano complex microsphere can be obtained, in 4 DEG C
Under the conditions of save.
According to the complex microsphere can bring beneficial effect, the present invention provides the complex microsphere or the systems
Application of the complex microsphere of Preparation Method preparation in detection glycocholic acid and/or preparation detection glycocholic acid product.Preferably, described
Product is immuno-chromatographic test paper strip.
In the specific embodiment of the invention, the present invention provides a kind of for detecting the immune chromatography test paper of glycocholic acid
Item, including sample pad, gold-labelled pad, nitrocellulose filter and water absorption pad, and successively overlap；It is adsorbed in the gold-labelled pad described multiple
Close the glycocholic acid monoclonal antibody of the complex microsphere label of microballoon or preparation method preparation；The nitrocellulose filter, which is drawn, detection line
And nature controlling line, the detection line are that glycocholic acid is mostly anti-, the nature controlling line is sheep anti-mouse igg, and schematic diagram is referring to fig. 2.
In the specific embodiment of the invention, the complex microsphere label of the complex microsphere or preparation method preparation
Glycocholic acid monoclonal antibody can mark coupling by the following method：
Use Na2CO3The complex microsphere solution ph of 1mL is adjusted to 9.0-9.5 by aqueous solution (2mmol/L).By the sweet of 100uL
Cholic acid monoclonal antibody is slowly dropped in complex microsphere solution.Slowly after concussion 5min, it is transferred to 4 DEG C of refrigerator overnight.It will be molten
Liquid is centrifuged 12000 revolving speeds and is centrifuged 20min, with the Na of 1mL2CO3Aqueous solution (2mmol/L) is resuspended, and the 10% of 20ul is slowly added dropwise
BSA mixes reaction 30 minutes to close the residue of complex microsphere excess surface.9000rpm is centrifuged 15 minutes under the conditions of 4 DEG C,
Supernatant is abandoned, gained is precipitated as the glycocholic acid monoclonal antibody marked.
In the specific embodiment of the invention, the glycocholic acid monoclonal antibody is with the glycocholic acid for being coupled bovine serum albumin with mostly anti-
Immunogene prepares；The glycocholic acid of the coupling bovine serum albumin has 1 structure of formula (n=1)：
Specifically preparation method is：First glycocholic acid and bovine serum albumin are coupled using mixed anhydride method, dialysed pure
Immunizing antigen is obtained after change；New zealand white rabbit is immunized in immunizing antigen, to rabbit ear venous blood sampling, is centrifugated at 4 DEG C
Take supernatant is to resist more, is saved backup at -20 DEG C.Balb/c mouse is immunized in immunizing antigen, extracting spleen cell fusion prepares sweet gallbladder
Acid hybridization tumor cell strain, sensitized mice abdominal cavity prepares ascites, and is purified with caprylic acid-ammonium and resisted up to glycocholic acid monoclonal
Body；
In order to obtain the preferable monoclonal antibody of specificity, the present invention passes through the system of multiple hybridoma cell strain and monoclonal antibody
It is standby, one plant of glycocholic acid hybridoma cell strain that can secrete the higher monoclonal antibody of specificity is filtered out, CG-706 is named as,
Deposit number is CGMCC No.15292.
It is detected using glycocholic acid standard items of the immuno-chromatographic test paper strip of the present invention to various concentration, is as a result shown
Show, it, can be directly by the colour developing of naked eyes resolved detection lines as a result, glycocholic acid contains in normal human in 0.1-0.2mg/L
Amount is 0.4-2.98mg/L, can meet existing testing requirements, specific testing result situation is referring to Fig. 3.
From the above technical scheme, the present invention is compound with colloid gold nano-polyaniline-gold nano with three-decker
Microballoon substitutes the colloidal gold cholylglycine monoclonal antibody routinely applied, and monoclonal antibody is made to be multiplied, can when carrying out immunochromatography detection
Increase detection sensitivity, detection limit can directly pass through naked-eye observation down to the preferable degree of 0.2mg/L.
Biological deposits material information explanation
CG-706, classification naming：Glycocholic acid hybridoma cell strain；China Microbiological bacterium is preserved on January 25th, 2018
Kind preservation administration committee common micro-organisms center, address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, the Chinese Academy of Sciences
Institute of microbiology, deposit number are CGMCC No.15292.
Detailed description of the invention
Fig. 1 show the preparation flow figure of colloid gold nano-polyaniline-gold nano complex microsphere of the present invention；
Fig. 2 show the structural schematic diagram of immuno-chromatographic test paper strip of the present invention；
Fig. 3 show immuno-chromatographic test paper strip testing result of the present invention as positive, negative and invalid schematic diagram；
Fig. 4 show the glycocholic acid standard items testing result of various concentration；Wherein A-D respectively indicate 1,0.5,0.2,
The glycocholic acid standard items test result of 0.1mg/L, E indicate PBS buffer solution test result.
Specific embodiment
The invention discloses a kind of colloid gold nano-polyaniline-gold nano complex microspheres and its preparation method and application, originally
Field technical staff can use for reference present disclosure, be suitably modified realization of process parameters.In particular, it should be pointed out that all similar
Replace and change apparent to those skilled in the art, they are considered as being included in the present invention.Institute of the present invention
It states complex microsphere and its preparation method and application to be described by preferred embodiment, related personnel can obviously not depart from
Complex microsphere as described herein and its preparation method and application is modified in the content of present invention, spirit and scope or is suitably become
More with combine, carry out implementation and application the technology of the present invention.
Just a kind of colloid gold nano-polyaniline-gold nano complex microsphere provided by the present invention and preparation method thereof below
It is described further with application.
Embodiment 1：Prepare colloid gold nano-polyaniline-gold nano complex microsphere
By the HAuCl of 3mL4Aqueous solution (1.0mmol/L) instills 10mL HCl solution (0.1mol/L), wherein containing
0.1mmol/L aniline monomer, stirs evenly.When the color of mixture becomes bottle green, the precipitating being centrifugally separating to obtain is (i.e. poly-
Aniline-protection gold nanoparticle)；After being resuspended with the deionized water of 1mL, it is slowly added to the colloid gold nanoparticle (C of 3mL
(Au) ≈ 24mol/L), it is slowly stirred at room temperature overnight, 12000rpm is centrifuged 20 minutes under the conditions of 4 DEG C, and adds 1.0mL's
Deionized water suspends again, blackish green polyaniline-composite nano-microsphere can be obtained, partial size is in 30-40nm, under the conditions of 4 DEG C
It saves.
Embodiment 2：Prepare the immuno-chromatographic test paper strip
1, the synthesis of immunizing antigen
10-40mg glycocholic acid and the n,N-Dimethylformamide of 10-30mg are weighed, 1-3mL DMF is dissolved in, adds 20-
40mg DCC, 4 DEG C are stirred overnight, and then reactant is centrifuged 10min at 10000rpm, and supernatant is taken to be added to containing 100-
In the phosphate buffered saline solution of 150mg bovine serum albumin, 4 DEG C are stirred to react 12 hours.Centrifuging and taking supernatant is put again after reaction
Enter in bag filter, is dialysed three days under the conditions of 4 DEG C with phosphate buffered saline solution, changed the liquid once every 12 hours, can be obtained and exempt from
Epidemic disease antigen.
2, the mostly anti-generation of glycocholic acid
It is immunized using multiple spot hypodermic injection.It is female using 2 new zealand white rabbits, the monthly age 3 months, weight
1.5-2.5 kilogram.Immunizing antigen with the phosphate buffered saline now matched at 1mg/ml, respectively take 1ml immunizing antigen solution with etc.
The Freund's complete adjuvant of volume, carrying out emulsification with emulsifier mixes it completely for a phase.Using the immune side of subcutaneous multi-point injection
Formula, every rabbit inject 2ml；First immunisation carries out first time booster immunization after two weeks, with isometric incomplete Freund's adjuvant
With the immunizing antigen mixing and emulsifying of 1mg/ml, 2ml every carries out subcutaneous multi-point injection and is immunized, and carries out reinforcing exempting from every two weeks later
Epidemic disease is primary；It is immune after a week to rabbit ear venous blood sampling in last time after booster immunization 4 times, it is centrifugated at 4 DEG C
10min, revolving speed are 10000rpm, and supernatant is taken to save backup at -20 DEG C.
3, the preparation of glycocholic acid monoclonal antibody
It is immunized using multiple spot hypodermic injection.It is female using 3 Bal/c, the monthly age 3 months, weight 25-30g.With
Immunizing antigen is configured to 1mg/ml by the phosphate buffer now matched, and respectively takes 1ml immunizing antigen solution and isometric Freund complete
Full adjuvant, carrying out emulsification with emulsifier mixes it completely for a phase.Using the immunization ways of subcutaneous multi-point injection, every mouse note
Penetrate 0.2ml；First immunisation carries out first time booster immunization after two weeks, with exempting from for isometric incomplete Freund's adjuvant and 1mg/ml
Epidemic disease antigen mixing and emulsifying, 2ml every carries out subcutaneous multi-point injection and is immunized, and it is primary to carry out booster immunization every two weeks later；Reinforce
After 4 times immune, tail portion is carried out in the immune mouse of the immune antigen after a week of last time and takes blood, measures anti-blood with ELISA method
Clear potency takes the splenocyte of the preferable mouse of serum titer, with fusion agent PEG4000 by the marrow of splenocyte and in vitro culture
Oncocyte fusion detects the cell supernatant positive and Competitive assays situation with indirect ELISA, carries out sub- gram using limiting dilution assay
Grand, after all positives of 3 subclones, taking hybridoma concentration is 106-108/ mL injects mouse peritoneal 0.5mL/ only, and 14 days
After take and be centrifugated 10min at 4 DEG C of ascites, revolving speed is 10000rpm, takes supernatant to obtain a large amount of monoclonal antibodies, -20
It DEG C saves backup.
According to the method described above, the list that the hybridoma cell strain that the present invention chooses that deposit number is CGMCC No.15292 is secreted
It is anti-.
3, the preparation of gold-labelled pad
Use Na2CO3The polyaniline of 1mL-nanosphere solution ph is adjusted to 9.0-9.5 by aqueous solution (2mmol/L).It will
The glycocholic acid monoclonal antibody of 100uL is slowly dropped in polyaniline-nanosphere solution.Slowly after concussion 5min, it is transferred to
4 DEG C of refrigerator overnight.Solution is centrifuged 12000 revolving speeds and is centrifuged 20min, with the Na of 1mL2CO3Aqueous solution (2mmol/L) is resuspended, slowly
10% BSA of 20ul is added dropwise to close polyaniline-nanosphere excess surface residue, mixes reaction 30 minutes.In 4 DEG C of items
9000rpm is centrifuged 15 minutes under part, abandons supernatant, and 200ul dilution is added to obtained precipitating, metal spraying draw on film instrument with
15ul/cm is uniformly layered on glass fibre membrane, freeze-drying, spare；
4, the preparation of nitrocellulose filter
The glycocholic acid polyclonal antibody of preparation is diluted to concentration 2mg/mL as detection line, sheep anti-mouse igg is diluted to
Then 2mg/mL is drawn film instrument with metal spraying and is sprayed on nitrocellulose filter with the even concentration of 1ul/cm as nature controlling line, detection line
It is 0.4cm at a distance from before nature controlling line, is dried in 37 DEG C of constant temperature oven, it is spare；
5, the preparation of test strips
Sample pad, gold-labelled pad, nitrocellulose filter and water absorption pad are successively pasted from top to bottom on the floor plastics PVP,
Wherein water absorption pad is pasted in the top of nitrocellulose filter, and mutually overlapping 0.2mm is pasted in the top of nitrocellulose filter, in nitric acid fibre
Glass fibre membrane (gold-labelled pad) and sample pad are successively pasted in the lower section for tieing up plain film, and nitrocellulose filter and gold-labelled pad are mutually overlapping
Test strips, are then cut into the strip of 4mm, finally install test strips by the mutually overlapping 2mm of 0.3mm, gold-labelled pad and sample pad
To get immuno-chromatographic test paper strip in the flat shelly-shaped detection card shell of strip.
Embodiment 3：Sensitivity (detection limit) detection
It is 1,0.5,0.2,0.1mg/L, after mixing, each concentration that the standard items of glycocholic acid, which are diluted to concentration with PBS,
Standard items take 50ul to be added in sample pad and are detected, as a result see Fig. 4.
As seen from Figure 4, under not by instrument, naked eyes visible detection is limited between 0.2mg/L (in Fig. 4 the present invention
C).Therefore the detection line that the present invention can define the immuno-chromatographic test paper strip of detection liver and gallbladder acid is 0.2mg/L, and sweet gallbladder in normal human
The content of acid is 0.4~2.98mg/L, can meet existing testing requirements.
Embodiment 4：Clinical sample detection
28 urina sanguinis samples that 50ul is fetched from hospital are added in sample pad, each sample repeats to survey 3 times, is laid flat waiting
This 28 samples it can be learnt that whether glycocholic acid is exceeded in urine sample, while being used existing ELISA by 3-5min
Method synchronizes detection, the results are shown in Table 1.
Table 1
Note：+ positive (exceeded) ,-negative (not exceeded) ,/invalid
As can be seen from Table 1, the common sense for being 0.4~2.98mg/L according to the content of glycocholic acid in normal human, passes through this
The immuno-chromatographic test paper strip of invention can accurately qualitative detection go out by whether exceeded and quantitative detection the knot of sample product glycocholic acid
Fruit is consistent.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications are also answered
It is considered as protection scope of the present invention.
Claims (10)
1. a kind of colloid gold nano-polyaniline-gold nano complex microsphere, which is characterized in that polyaniline wraps up Au nanoparticle, and
Colloid gold nanoparticle is coupled on polyaniline surface.
2. the preparation method of complex microsphere described in claim 1, which is characterized in that including：
By HAuCl4Aqueous solution and the HCl solution containing aniline monomer are stirred to react to dark green, are centrifugally separating to obtain and are precipitated as
Then the polyaniline microsphere for wrapping up Au nanoparticle is resuspended and is stirred to react overnight, after centrifugation with colloid gold nanoparticle
Precipitating is resuspended, obtains the polyaniline of package Au nanoparticle and in the compound micro- of polyaniline surface coupling colloid gold nanoparticle
Ball.
3. preparation method according to claim 2, which is characterized in that including：
The HAuCl of 1.0mmol/L4Aqueous solution instills the HCl solution that 0.1mol/L contains 0.1mmol/L aniline monomer, and stirring is equal
It is even；When the color of mixture becomes bottle green, what is be centrifugally separating to obtain is precipitated as the polyaniline microsphere of package Au nanoparticle；
The polyaniline microsphere is resuspended in deionized water, is slowly added to the colloid gold nanoparticle of concentration=24 Au ± 1mol/L, slowly stirs
It mixes and stays overnight at room temperature, centrifugation, precipitating is resuspended, and obtains the polyaniline of blackish green package Au nanoparticle and on polyaniline surface
It is coupled the complex microsphere of colloid gold nanoparticle.
4. preparation method according to claim 3, which is characterized in that the HAuCl4Aqueous solution and HCl containing aniline monomer
The volume ratio of solution is 3:10.
5. preparation method according to claim 3, which is characterized in that the volume of the deionized water and colloid gold nanoparticle
Than being 1:3.
6. the complex microsphere of any one of complex microsphere described in claim 1 or claim 2-5 preparation method preparation is sweet in detection
Application in cholic acid and/or preparation detection glycocholic acid product.
7. applying according to claim 6, which is characterized in that the product is immuno-chromatographic test paper strip.
8. a kind of immuno-chromatographic test paper strip, which is characterized in that including sample pad, gold-labelled pad, nitrocellulose filter and water absorption pad, and
Successively overlap；The compound of any one of complex microsphere described in claim 1 or claim 2-5 preparation is adsorbed in the gold-labelled pad
The glycocholic acid monoclonal antibody of microballoon label；The nitrocellulose filter, which is drawn, detection line and nature controlling line, and the detection line is that glycocholic acid is more
Anti-, the nature controlling line is sheep anti-mouse igg.
9. immuno-chromatographic test paper strip according to claim 8, which is characterized in that the glycocholic acid monoclonal antibody resists with to be coupled ox more
The glycocholic acid of haemocyanin prepares for immunogene.
10. immuno-chromatographic test paper strip according to claim 8 or claim 9, which is characterized in that the glycocholic acid monoclonal antibody is by deposit number
Acquisition is secreted for the hybridoma of CGMCC No.15292.
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Cited By (2)
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| CN113413839A (en) * | 2021-07-07 | 2021-09-21 | 西南交通大学 | Salt response polyaniline microcapsule, self-warning coating and preparation method |
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| CN113413839A (en) * | 2021-07-07 | 2021-09-21 | 西南交通大学 | Salt response polyaniline microcapsule, self-warning coating and preparation method |
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| CN108896756B (en) | 2021-05-11 |
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