CN101696972A - Antigen-antibody reaction-based controllable gold nanorod assembling method - Google Patents
Antigen-antibody reaction-based controllable gold nanorod assembling method Download PDFInfo
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- CN101696972A CN101696972A CN200910035791A CN200910035791A CN101696972A CN 101696972 A CN101696972 A CN 101696972A CN 200910035791 A CN200910035791 A CN 200910035791A CN 200910035791 A CN200910035791 A CN 200910035791A CN 101696972 A CN101696972 A CN 101696972A
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Abstract
The invention discloses an antigen-antibody reaction-based controllable gold nanorod assembling method, which belongs to the technical field of nano immunity. In the invention, the antigen-antibody is directionally coupled to the end face or the side face of the gold nanorods by different methods; and the assembling of the gold nanorods is realized by the identification reaction between the antigen and the antibody. The assembling process is reacted in a liquid environment; the operating method is simple; the controllable composting of the gold nanorod can be realized in a mild condition quickly, so large-scale assembled products can be generated within a certain time.
Description
Technical field
The present invention relates to a kind of method of assembling, belong to the nano immune technical field based on the controlled gold nanorods of antigen-antibody reaction.
Background technology
Gold nanorods is owing to have many character that abundant surface plasma resonance derives out, and the current gold nanorods that disperses is widely used in many fields.For example be used for optical storage of data, nano-sensor, bio-imaging, controlling gene conveying and photo-thermal therapy tumour etc.
The Ag-Ab immune response is one of the most common bio-identification reaction, usually is applied to immune response.Now, the demand for development of Science and Technology is used the gold nanorods of assembling, one dimension, two dimension, even 3 d function structure.Because the gold nanorods of assembling not only has the character of single gold nanorods, more derive many colonies character.The character of these optics and electronics be single gold nanorods do not have, the generation of these character be since the assembling after gold nanorods the act on generation adjacent of each monomer of gold nanorods with it.
Gold nanorods is assembled with 2 kinds of modes: (1) end face is assembled end face, the assembling of (2) side-by-side.The method of assembling mainly is by electrostatic interaction or covalent coupling; Hybridization by nucleic acid molecules; Modify superpolymer oneself's effect under selective solvent on the gold nanorods; Be exactly method at last by bio-identification, as biotin-avidin, the Ag-Ab effect.
Summary of the invention
The purpose of this invention is to provide a kind of method of operating simple and fast gold nanorods realize the method for controlled assembling being used for end face to end face assembling and side-by-side assembling.
By the end face that be coupled to gold nanorods or the side of distinct methods, in the assembling that realizes gold nanorods by the identification negative side between antigen-antibody with the antigen-antibody orientation.Experiment is set forth with gentamicin antigen and antibody.Comprise following content:
(1) the side coupling of gentamicin antibody and coating antigen and gold nanorods;
(2) the side-by-side assembling of gold nanorods under the antigen-antibody effect;
(3) the top coupling of antibody and gold nanorods is modified;
(4) the top coupling of coating antigen and gold nanorods is modified;
(5) end face of gold nanorods under the antigen-antibody effect is to the assembling of end face.
Technical scheme of the present invention: a kind of method of assembling based on the controlled gold nanorods of antigen-antibody reaction comprises assembling of gold nanorods side or the assembling of gold nanorods end face;
(1) gold nanorods side assembling:
A antibody modification gold nanorods: the sal tartari that the gentamicin antibody 0.5mL of 100 μ g/mL dropwise adds with 0.1mol/L transfers in pH 8.5-8.8,0.5mL, the 2n mol/L gold nanorods solution in advance, room temperature concussion mixing, 37 ℃ of reaction 1h, reaction finishes the centrifugal 20min of 6000rpm, remove unconjugated antibody, be resuspended in pH 7.4, contain among the 0.01mol/L PBS of 0.5% polyglycol PEG20000, make the gold nanorods probe that gentamicin antibody is modified;
B. coating antigen is modified gold nanorods: method and the coupling of antibody modification gold nanorods are similar, the hydrochloric acid that the gentamicin coating antigen 0.5mL of 100 μ g/mL dropwise adds with 0.1mol/L transfers in pH5,0.5mL, the 2n mol/L gold nanorods solution in advance, room temperature concussion mixing, 37 ℃ of reaction 1h, reaction finishes the centrifugal 20min of 6000rpm, remove unconjugated coating antigen, be resuspended in pH 7.4, contain among the 0.01mol/L PBS of 0.5%PEG20000, make the gold nanorods probe that the gentamicin coating antigen is modified;
C. the assembling of the gold nanorods side-by-side under the Ag-Ab effect:
10 μ L modify in the gold nanorods solution of gentamicin coating antigen and add the gold nanorods solution that 10 μ L modify gentamicin antibody, and the whirlpool mixing is hatched the 0.5h afterreaction for 37 ℃ and finished, get 5 times of 10 μ L reactant liquors dilutions after transmission electron microscope characterize product;
Or (2) gold nanorods end face assembling:
A. the modification of antibody:
The modification of antibody adopts carbodlimide method to carry out the combination of lipoic acid and gentamicin antibody, and operation steps is as follows:
1. the ethanolic solution 0.3mL that contains the 1.625mg lipoic acid mixes with the gentamicin antibody of 0.4mL;
2. the carbodiimide of 75 μ L is dropwise splashed into 1. back room temperature reaction 4h in the solution of above-mentioned steps under the condition of magnetic agitation;
3. dialysed 3 days with the phosphate buffer of pH 7.4, change dislysate every day 3 times;
4. the gentamicin antibody that lipoic acid is modified be placed on 4 ℃ standby;
B. antibody modification gold nanorods end face
Adopt the synthetic gold nanorods of seeded growth method at first will remove unnecessary Vc, AgNO in the solution through the centrifugal 15min of 7500rpm
3Behind little spheroidal particle gold nanorods, the 0.005mol/L cetyl trimethyl ammonium bromide CTAB solution that utilize pH 3.8, contains 0.1% polyglycol PEG20000 carries out resuspended, resuspended liquid is adjusted to pH 3.8 with 1mol/L HCl, CTAB plays a part very crucial in the preparation process of gold nanorods, thereby CTAB mainly is the side that is distributed in the side gold nanorods of gold nanorods have positive charge and the distribution of the end face of gold nanorods seldom, therefore the antibody that adopts lipoic acid to modify can modify gold nanorods end face but not on the side.0.5mL, the gold nanorods solution GNRs of 2n mol/L after resuspended dropwise be added drop-wise to by 50 μ L and be diluted in the gentamicin antibody dilution that the lipoic acid of 1mL modifies, stirring at room reaction 4h, reaction finishes the centrifugal 10min of back 6000rpm, utilization pH 3.8, contain among the 0.005mol/LCTAB solution 1mL of 0.1%PEG20000 and heavily disperse, with twice of this solution washing, heavily disperse with pH3.8, the 0.005mol/L CTAB solution 50 μ L that contain 0.1%PEG20000 at last, this conjugate places 4 ℃ of preservations;
C. the modification of coating antigen:
The modification of coating antigen adopts carbodlimide method to carry out the combination of lipoic acid and coating antigen, and step is as follows:
1. the ethanolic solution 0.3mL that contains the lipoic acid of 1.625mg mixes with the gentamicin coating antigen solution of 0.2mL;
2. the carbodiimide of 75 μ L is dropwise splashed into 1. back room temperature reaction 4h in the solution of above-mentioned steps under the condition of magnetic agitation;
3. dialysed 3 days with the phosphate buffer of pH 7.4, change liquid every day 3 times;
4. the gentamicin coating antigen after lipoic acid being modified be placed on 4 ℃ standby;
D. coating antigen is modified the gold nanorods end face:
The coupling of coating antigen and gold nanorods is similar to the coupling of (b) antibody and gold nanorods, just have any different on the amount of coating antigen, the gentamicin coating antigen 60 μ L that lipoic acid is modified dilute with the pH 3.80 of 940 μ L, the 005mol/L CTAB solution that contains 0.1%PEG20000;
Gold nanorods solution GNRs with pH 3.8,0.5mL, 2n mol/L after resuspended dropwise is added drop-wise in the dilution of the gentamicin coating antigen that lipoic acid modifies, stirring at room reaction 4h, reaction finishes the centrifugal 10min of back 6000rpm, utilization pH 3.8, contain among the 0.005mol/L CTAB solution 1mL of 0.1%PEG20000 and heavily disperse, with twice of this solution washing, heavily disperse with pH 3.8, the 0.005mol/L CTAB solution 50 μ L that contain 0.1%PEG20000 at last, this conjugate places 4 ℃ of preservations;
E. the gold nanorods end face under the Ag-Ab effect is to the assembling of end face:
Add the gold nanorods solution that 10 μ L lipoic acids are modified gentamicin antibody in the gold nanorods solution of the gentamicin coating antigen that 10 μ L lipoic acids are modified, the whirlpool mixing, hatch the 0.5h afterreaction for 37 ℃ and finish, get 5 times of 10 μ L reactant liquors dilutions after transmission electron microscope characterize product.
Beneficial effect of the present invention: the invention provides the method that a kind of new gold nanorods is assembled, by the end face that is coupled to gold nanorods or the side of distinct methods, in the assembling that realizes gold nanorods by the identification negative side between antigen-antibody with the antigen-antibody orientation.Assembling process reacts in liquid environment, and method of operating is simple, and mild condition also realizes the controlled assembling of gold nanorods fast, just can generate large-scale assembling product within a certain period of time.
Description of drawings
Fig. 1 is based on the transmission electron microscope picture of the assembling gold nanorods of antigen-antibody reaction.Assemble the side side of A, gold nanorods, and the end face of B, gold nanorods is assembled end face.
Fig. 2 is based on the UV, visible light figure of the assembling gold nanorods of antigen-antibody reaction.
Embodiment
The side coupling of embodiment 1 gentamicin antibody and coating antigen and gold nanorods:
A. the gentamicin antibody of antibody and gold nanorods coupling: 0.5mL, 100 μ g/mL dropwise adds with the sal tartari of 0.1mol/L and transfers in advance among the 0.5mL, 2n mol/L gold nanorods solution of pH 8.5-8.8, room temperature concussion mixing, 37 ℃ of reaction 1h, reaction finishes the centrifugal 20min of 6000rpm, remove unconjugated antibody, be resuspended among the PBS of 0.01mol/L, pH 7.4 (containing 0.5%PEG2000), make the gold nanorods probe that gentamicin antibody is modified;
B. coating antigen and gold nanorods coupling: method and antibody and gold nanorods coupling are similar, 0.5mL, the hydrochloric acid that dropwise adds with 0.1mol/L of the gentamicin coating antigen of 100 μ g/mL transfers in the gold nanorods solution of pH 5,0.5mL, 2n mol/L in advance, room temperature concussion mixing, 37 ℃ of reaction 1h, reaction finishes the centrifugal 20min of 6000rpm, remove unconjugated coating antigen, be resuspended among the PBS of 0.01mol/L, pH 7.4 (containing 0.5%PEG20000), make the gold nanorods probe that the gentamicin coating antigen is modified;
The gold nanorods side-by-side assembling of embodiment 2 under the antigen-antibody effect:
10 μ L modify in the gold nanorods solution of gentamicin coating antigen and add the gold nanorods solution that 10 μ L modify gentamicin antibody, and the whirlpool mixing is hatched the 0.5h afterreaction for 37 ℃ and finished, get 5 times of 10 μ L reactant liquors dilutions after transmission electron microscope characterize product.
The top coupling of embodiment 3 antibody and gold nanorods is modified:
A. the modification of antibody
The modification of antibody adopts carbodlimide method to carry out the combination of lipoic acid and antibody, and the concrete operations step is as follows:
1. the ethanolic solution of the lipoic acid of 0.3mL (lipoic acid 1.625mg) mixes with the antibody of 0.4mL.
2. the carbodiimide with 75 μ L dropwise splashes into back room temperature reaction 4h in the above-mentioned solution under the condition of magnetic agitation.
3. dialysed 3 days with the phosphate buffer of pH 7.4, change dislysate every day 3 times.
4. the antibody after will modifying be placed on 4 ℃ standby.
B. antibody and the coupling of gold nanorods end face
Adopt the synthetic gold nanorods of seeded growth method at first will remove unnecessary Vc, AgNO in the solution through the centrifugal 15min of 7500rpm
3With utilize the solution that pH 3.8,0.005M cetyl trimethyl ammonium bromide (CTAB) contain 0.1% polyglycol (PEG) to carry out resuspended behind the little spheroidal particle gold nanorods.The pH value of resuspended liquid is adjusted with 1mol/L HCl.CTAB plays a part very crucial in the preparation process of gold nanorods.Thereby CTAB mainly is the side that is distributed in the side gold nanorods of gold nanorods have positive charge and the distribution of the end face of gold nanorods seldom, therefore the antibody that adopts lipoic acid to modify can modify the gold rod end face but not on the side.0.5mL heavily the 2n mol/L GNRs of Fen Saning dropwise is added drop-wise to by 50 μ L and modifies the back antibody dilution in the dilution of the antibody of 1mL.Stirring at room reaction 4h.Reaction finishes the centrifugal 10min of back 6000rpm, and the solution that the 0.005M CTAB (pH 3.8) of utilization 1mL contains 0.1%PEG20000 heavily disperses.With twice of this solution washing.The solution of using the 0.005M CTAB (pH 3.8) of 50 μ L to contain 0.1%PEG20000 at last heavily disperses.This conjugate places 4 ℃ of preservations.
The top coupling of embodiment 4 coating antigens and gold nanorods is modified
A. coating antigen is modified:
The modification of coating antigen adopts carbodlimide method to carry out the combination of lipoic acid and coating antigen, and the concrete operations step is as follows:
1. the ethanolic solution of the lipoic acid of 0.3mL (lipoic acid 1.625mg) mixes with the gentamicin coating antigen solution of 0.2mL.
2. the carbodiimide with 75 μ L dropwise splashes into back room temperature reaction 4h in the above-mentioned solution under the condition of magnetic agitation.
3. dialysed 3 days with the phosphate buffer of pH 7.4, change liquid every day 3 times.
4. the coating antigen after will modifying be placed on 4 ℃ standby.
B. coating antigen and the coupling of gold nanorods end face
The coupling of coating antigen and gold nanorods is similar to the coupling of antibody and gold nanorods, just has any different on the amount of coating antigen, and promptly the solution that contains 0.1%PEG20000 with the 0.005M CTAB (pH 3.8) of 940 μ L behind the coating antigen 60 μ L of Jia Ruing dilutes.
Gold nanorods solution GNRs with pH 3.8,0.5mL, 2n mol/L after resuspended dropwise is added drop-wise in the dilution of the gentamicin coating antigen that lipoic acid modifies, stirring at room reaction 4h, reaction finishes the centrifugal 10min of back 6000rpm, utilization pH 3.8, contain among the 0.005mol/L CTAB solution 1mL of 0.1%PEG20000 and heavily disperse, with twice of this solution washing, heavily disperse with pH 3.8, the 0.005mol/L CTAB solution 50 μ L that contain 0.1%PEG20000 at last, this conjugate places 4 ℃ of preservations.
The gold nanorods end face of embodiment 5 under the antigen-antibody effect is to the assembling of end face
10 μ L modify in the gold nanorods solution of gentamicin coating antigen and add the gold nanorods solution that 10 μ L modify gentamicin antibody, and the whirlpool mixing is hatched the 0.5h afterreaction for 37 ℃ and finished, get 5 times of 10 μ L reactant liquors dilutions after transmission electron microscope characterize product.
Claims (1)
1. the method based on the controlled gold nanorods assembling of antigen-antibody reaction is characterized in that comprising assembling of gold nanorods side or the assembling of gold nanorods end face;
(1) gold nanorods side assembling:
A. antibody modification gold nanorods: the sal tartari that the gentamicin antibody 0.5mL of 100 μ g/mL dropwise adds with 0.1mol/L transfers in pH 8.5-8.8,0.5mL, the 2n mol/L gold nanorods solution in advance, room temperature concussion mixing, 37 ℃ of reaction 1h, reaction finishes the centrifugal 20min of 6000rpm, remove unconjugated antibody, be resuspended in pH 7.4, contain among the 0.01mol/L PBS of 0.5% polyglycol PEG20000, make the gold nanorods probe that gentamicin antibody is modified;
B. coating antigen is modified gold nanorods: the hydrochloric acid that the gentamicin coating antigen 0.5mL of 100 μ g/mL dropwise adds with 0.1mol/L transfers in pH 5,0.5mL, the 2n mol/L gold nanorods solution in advance, room temperature concussion mixing, 37 ℃ of reaction 1h, reaction finishes the centrifugal 20min of 6000rpm, remove unconjugated coating antigen, be resuspended in pH 7.4, contain among the 0.01mol/L PBS of 0.5%PEG20000, make the gold nanorods probe that the gentamicin coating antigen is modified;
C. the assembling of the gold nanorods side-by-side under the Ag-Ab effect:
10 μ L modify in the gold nanorods solution of gentamicin coating antigen and add the gold nanorods solution that 10 μ L modify gentamicin antibody, and the whirlpool mixing is hatched the 0.5h afterreaction for 37 ℃ and finished, get 5 times of 10 μ L reactant liquors dilutions after transmission electron microscope characterize product;
Or (2) gold nanorods end face assembling:
A. the modification of antibody:
The modification of antibody adopts carbodlimide method to carry out the combination of lipoic acid and gentamicin antibody, and operation steps is as follows:
1. the ethanolic solution 0.3mL that contains the 1.625mg lipoic acid mixes with the gentamicin antibody of 0.4mL;
2. the carbodiimide of 75 μ L is dropwise splashed into 1. back room temperature reaction 4h in the solution of above-mentioned steps under the condition of magnetic agitation;
3. dialysed 3 days with the phosphate buffer of pH 7.4, change dislysate every day 3 times;
4. the gentamicin antibody that lipoic acid is modified be placed on 4 ℃ standby;
B. antibody modification gold nanorods end face
Adopt the synthetic gold nanorods of seeded growth method at first will remove unnecessary Vc, AgNO in the solution through the centrifugal 15min of 7500rpm
3Behind little spheroidal particle gold nanorods, utilize pH 3.8, the 0.005mol/L cetyl trimethyl ammonium bromide CTAB solution that contains 0.1%PEG20000 carries out resuspended, resuspended liquid is adjusted to pH 3.8 with 1mol/L HCl, 0.5mL, gold nanorods solution GNRs after 2n mol/L is resuspended dropwise is added drop-wise to by 50 μ L and is diluted in the gentamicin antibody dilution that the lipoic acid of 1mL modifies, stirring at room reaction 4h, reaction finishes the centrifugal 10min of back 6000rpm, utilization pH 3.8, contain among the 0.005mol/L CTAB solution 1mL of 0.1%PEG20000 and heavily disperse, with twice of this solution washing, use pH3.8 at last, the 0.005mol/L CTAB solution 50 μ L that contain 0.1%PEG20000 heavily disperse, and this conjugate places 4 ℃ of preservations;
C. the modification of coating antigen:
The modification of coating antigen adopts carbodlimide method to carry out the combination of lipoic acid and coating antigen, and step is as follows:
1. the ethanolic solution 0.3mL that contains the lipoic acid of 1.625mg mixes with the gentamicin coating antigen solution of 0.2mL;
2. the carbodiimide of 75 μ L is dropwise splashed into 1. back room temperature reaction 4h in the solution of above-mentioned steps under the condition of magnetic agitation;
3. dialysed 3 days with the phosphate buffer of pH 7.4, change liquid every day 3 times;
4. the gentamicin coating antigen after lipoic acid being modified be placed on 4 ℃ standby;
D. coating antigen is modified the gold nanorods end face:
The gentamicin coating antigen 60 μ L that lipoic acid is modified dilute with the pH 3.80 of 940 μ L, the 005mol/L CTAB solution that contains 0.1%PEG20000;
Gold nanorods solution GNRs with pH 3.8,0.5mL, 2n mol/L after resuspended dropwise is added drop-wise in the dilution of the gentamicin coating antigen that lipoic acid modifies, stirring at room reaction 4h, reaction finishes the centrifugal 10min of back 6000rpm, utilization pH 3.8, contain among the 0.005mol/L CTAB solution 1mL of 0.1%PEG20000 and heavily disperse, with twice of this solution washing, heavily disperse with pH 3.8, the 0.005mol/L CTAB solution 50 μ L that contain 0.1%PEG20000 at last, this conjugate places 4 ℃ of preservations;
E. the gold nanorods end face under the Ag-Ab effect is to the assembling of end face:
Add the gold nanorods solution that 10 μ L lipoic acids are modified gentamicin antibody in the gold nanorods solution of the gentamicin coating antigen after 10 μ L lipoic acids are modified, the whirlpool mixing, hatch the 0.5h afterreaction for 37 ℃ and finish, get 5 times of 10 μ L reactant liquors dilutions after transmission electron microscope characterize product.
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| CN103341623A (en) * | 2013-06-25 | 2013-10-09 | 江南大学 | Method for preparing gold nanorod assemblies induced by static electricity acting force |
| CN103528996A (en) * | 2013-10-11 | 2014-01-22 | 南京邮电大学 | Preparation method of gold nanorod SPR probe and method for detecting drug loading release kinetic process of the probe |
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| CN103575875A (en) * | 2013-11-12 | 2014-02-12 | 镇江市第一人民医院 | Novel functionalized gold nanorod immune probe as well as preparation method and application of gold nanorod biological chip |
| CN106771204A (en) * | 2017-01-18 | 2017-05-31 | 中南大学 | A kind of detection method of carcinomebryonic antigen concentration |
| CN106771204B (en) * | 2017-01-18 | 2018-12-14 | 中南大学 | A kind of detection method of carcinomebryonic antigen concentration |
| CN106882764A (en) * | 2017-02-16 | 2017-06-23 | 华东理工大学 | A kind of preparation method of the water phase gold nanorods assembly based on host-guest interaction |
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