CN102169119A - Preparation method for nano -gold immune probe - Google Patents
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Abstract
The invention discloses a preparation method for a nano-gold immune probe. In the method, an antibody is covalently fixed on the surface of nano-gold by adopting chemical reaction to form an antibody-nano-gold composite immune probe. The immune probe consists of three parts including the nano-gold, the antibody, and a coupling agent 16- mercaptoundecanoic acid (16-MHDA); the 16-MHDA replaces a citric acid radical on the surface of the nano-gold and forms an Au-S key with high stability with the nano-gold; then epsilon-NH2 of lysine residues at the C terminal of the antibody and carboxyl at the 16-MHDA terminal form a covalent bond similar to a peptide bond through EDC/NHS reaction. Corresponding antibodies can be selected by the preparation method to satisfy the immune receptors of different targets according to detailed application situations. The immune probe for the antibody covalent coupling nano-gold prepared by the preparation method has high specificity, stability and selection, and has very good application prospects in the field such as biochemical immune analysis, biological tissue marking, etc.
Description
Technical field
The present invention relates to the preparation of nm of gold immunological probe, specially refer to a kind of antibody and the nm of gold covalent bonding prepares immunological probe.
Background technology
Nanogold particle is with its unique physical chemical property, and excellent biological compatibility, especially surface plasmon resonance (Surface Plasmon Resonance, SPR) the adjustable optical characteristics that causes, in recent years at biochemical analysis, bio-imaging, catalysis, carrier system, fields such as medical diagnosis obtain widespread use, particularly with the nm of gold conjugated biological molecules as probe at biochemical analysis, aspects such as imaging of tissue demonstrate great potential, be successfully applied to protein, DNA, detection of biomacromolecules such as liposome and analysis, and discern at cancer cell, fields such as nucleus mark have obtained preliminary success.
At present, it is varied to make up the Nano-Au probe method be applied to immunoassay, according to becoming key type, the be divided into covalent approach of coupling and non-covalent method between nm of gold and the antibody.Under non-covalent coupling method, combine by electrostatic interaction or hydrophobic effect (Van der Waals force) between nanogold particle and antibody, the stability of the immunological probe that forms and the specificity of coupling are poor, but simple because of its preparation process, save time, still more commonly used in some immunoassays detect, as a-protein.Compare with non-covalent coupling, the nm of gold of preparation covalent coupling and antibody complex want complicated and time-consuming, but covalent coupling has non-covalent coupling incomparable advantage, the specificity of stability and coupling greatly improves, detectability when the compound of what is more important application covalent coupling preparation detects can improve greatly, even reaches single molecules level.This detects with the disease early diagnosis significant for medical science.
The mode of nm of gold covalent coupling antibody mainly contains following two kinds: the one, and the sulfydryl in the labelled antibody, sulfydryl in the antibody exists with the form of disulfide bond, reduce with reductive agent (mercaptoethanol or dithiothreitol (DTT)), sulfydryl discharges, antibody is cracked into 2 molecule Fac fragments, be modified at the maleimide on nanogold particle surface and the sulfydryl of release and react, the Fac antibody fragment is coupled at the nanogold particle surface.Second kind is the amino by the lysine residue side chain in the antibody.This method mainly contained for two steps, and at first the two ends alkyl sulfhydryl that has sulfydryl and carboxyl respectively is coupled at the surface of nanogold particle by sulfydryl, and carboxyl is exposed to the outside; Then under the condition of NHS and EDC existence, lysine residue side chain amino forms peptide bond, nanogold particle on the antibody covalent coupling in the carboxyl of exposure and the antibody.
It is many that the nm of gold of current preparation covalent coupling and antibody complex are used first method, the crosslinking chemical that adopts has mercaptoacetic acid, 3-mercaptopropionic acid, halfcystine, 11-MUA etc., since these crosslinking chemicals well self assembly on the surface of nm of gold, cause the lining rate of nm of gold surface antibody low, cause the nm of gold immunological probe not reach the set goal in actual applications.In actual applications, the antibody amount that is coupled at the nm of gold surface is many more, and the sensitivity of immunological probe is high more, and the antibody fixed amount is how much closely related with used coupling agent.Therefore, selecting a kind of coupling agent that can improve nm of gold surface antibody lining rate, is to solve the in-problem in actual applications key of nm of gold immunological probe.Therefore, developing a kind of immunological probe preparation method who improves nm of gold surface antibody lining rate, is to solve the in-problem in actual applications key of nm of gold immunological probe.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of nm of gold immunological probe, by selecting long-chain 16-MHDA as coupling agent, be covalent coupling between antibody and the nm of gold in Zhi Bei the immunological probe not only, and the lining rate of nm of gold surface antibody improves greatly, has than common nm of gold immunological probe more advantages of excellent stability and specificity.
Technical scheme of the present invention is achieved in that
Nm of gold immunological probe preparation method, step is as follows:
Form immunological probe by the covalent bond coupling between nm of gold and the antibody, the preparation method of this immunological probe comprises following steps:
(1) preparation of nm of gold: prepare nanogold particle about 30nm with trisodium citrate reduction gold chloride; Be heated to boiling and carry out magnetic agitation with the 80-120ml ultrapure water, getting 0.3-0.5mL 1.0% citric acid three sodium solution adds rapidly in the water of boiling, reaction is carried out adding the 1.0mL0.50-0.70% chlorauric acid solution behind the 2-5min, close thermal source behind the 35-45min, 15-20min stops to stir again afterwards, naturally cool to room temperature, last constant volume 50.0mL refrigerator is preserved;
(2) 16-sulfydryl alkanoic acid 16-MHDA self assembly: under the condition that non-ionic surfactant Tween-20 exists, 16-MHDA replaces the citric acid group by the surface of Au-S key self assembly in nm of gold, get the 2.0-2.5ml nano gold sol and add 1.0-1.2mL Tween-20, room temperature vibration mixing 40-60min, add 0.8-1.2mL 16-MHDA ethanolic solution mixing afterwards again, room temperature vibration 3.5-4.5h.Excessive Tween-20 and 16-MHDA remove for three times by repeated centrifugation.The oily precipitation is aided with ultrasonic dispersion 3-5min with the phosphate buffer dissolving that 1.5-2.0mL pH is 7.0;
(3) the 16-MHDA terminal carboxyl group activates: the mixed solution that earlier the N-hydroxyl amber diacyl amine NHS aqueous solution of 1-(3-the dimethylamino-propyl)-3-ethyl carbodiimide EDC aqueous solution of 0.2-0.4mol/L and 0.03-0.06mol/L is obtained EDC/NHS, get the nm of gold 0.3-0.6mL that 16-MHDA modifies again, add in the new EDC/NHS mixed liquor that disposes of 80-120 μ L and react 10-15min, centrifugal afterwards, topple over supernatant and remove excessive EDC/NHS, precipitation is resuspended with the phosphate buffer of 0.3-0.5mL pH 7.0, with ultrasonic dispersion 3-5min;
(4) antibody coupling: the ε-NH of the terminal lysine residue of the C of antibody
2Combine formation covalent bond, nm of gold and antibody covalent coupling with the succinimide ester that activates.80-100 μ L, 2 μ g/mL antibody join in the solution that 0.3-0.6mL step (3) obtains centrifugal 15-20min behind the reaction 2.5-3h, the supernatant that inclines, and lower sediment is with the phosphate buffer dissolving of pH 7.4, ultrasonic dispersion 3-5min, 4 ℃ of preservations.1-(3-dimethylamino-propyl)-3-ethyl carbodiimide [N-Ethyl-N '-(3-dimethylaminopropyl)-and carbodiimide, EDC] N-hydroxyl amber diacyl amine [N-hydroxysuccinimide succinimide, NHS]
Effect of the present invention is embodied in:
(1) preparation method of the nm of gold immunological probe of the present invention's proposition is a coupling agent system with 16-MHDA, the reaction conditions gentleness, and technology maturation, equipment requirements is low, and preparation technology is simple, and adaptability is easy to realize industrialization production widely.
(2) adopt the prepared nm of gold immunological probe of the technology of the present invention, antibody activity is well kept, and the recognition capability of target recipient is improved greatly.
(3) the prepared nm of gold immunological probe of the present invention has good antijamming capability, and the variation of pH in the environment and ionic strength is had very strong tolerance.
(4) preparation method of this nm of gold immunological probe has good adaptability, can be applied to different target recipient, and discerning simultaneously for the multiple goal acceptor provides possibility, is suitable for preparing the immunological probe of polycomponent, multi-functional step detection.
Embodiment
Nm of gold immunological probe preparation method, step is as follows:
(1) preparation of nm of gold: prepare nanogold particle about 30nm with trisodium citrate reduction gold chloride; Be heated to boiling and carry out magnetic agitation with the 80-120ml ultrapure water, getting 0.3-0.5mL 1.0% citric acid three sodium solution adds rapidly in the water of boiling, reaction is carried out adding the 1.0mL0.50-0.70% chlorauric acid solution behind the 2-5min, close thermal source behind the 40-45min, 15-20min stops to stir again afterwards, naturally cool to room temperature, last constant volume 50.0mL refrigerator is preserved.
(2) the long-chain mercapto alkanoic acid formed by ten six carbon atoms of 16-MHDA (sulfydryl hexadecylic acid) self assembly: 16-MHDA, end is respectively sulfydryl and carboxyl, and sulfydryl is connected on the nm of gold, and carboxyl connects antibody, 16-MHDA connects nm of gold and antibody, plays the function of similar " bridge ".The self assembly of 16-MHDA has realized the connection of the mercapto functional group of 16-MHDA molecule on the nm of gold surface.Under the condition that non-ionic surfactant Tween-20 exists, 16-MHDA replaces the citric acid group by the surface of Au-S key self assembly in nm of gold.The existence of Tween-20 can stop the spontaneous gathering of nm of gold in the 16-MHDA self assembling process.Get the 2.0-2.5ml nano gold sol and add 1.0-1.2mL Tween-20, room temperature vibration mixing 40-60min adds 0.8-1.2mL 16-MHDA ethanolic solution mixing afterwards again, room temperature vibration 3.5-4.5h.Excessive Tween-20 and 16-MHDA remove for three times by repeated centrifugation.The oily precipitation is aided with the ultrasonic dispersion of 25-30kHz 3-5min with the phosphate buffer dissolving that 1.5-2.0mL pH is 7.0.
(3) the 16-MHDA terminal carboxyl group activates: the mixed solution that earlier the NHS aqueous solution of the EDC aqueous solution of 0.2-0.4mol/L and 0.03-0.06mol/L is obtained EDC/NHS.The EDC/NHS mixed solution is a crosslinking chemical commonly used in the cross-linking chemistry, realizes carboxyl and amino bonding.Get the nm of gold 0.3-0.6mL that 16-MHDA modifies, add in the new mixed liquor that disposes of 80-120 μ L and react 10-15min, centrifugal afterwards, topple over supernatant and remove excessive EDC/NHS, precipitation is resuspended with the phosphate buffer of 0.3-0.5mL pH 7.0, with ultrasonic dispersion 3-5min.
(4) antibody coupling: the ε-NH of the terminal lysine residue of antibody C
2Combine formation covalent bond, nm of gold and antibody covalent coupling with the succinimide ester that activates.80-100 μ L, 2 μ g/mL antibody join in the solution that 0.3-0.6mL step (3) obtains centrifugal 15-20min behind the reaction 2.5-3h, the supernatant that inclines, and lower sediment is with the phosphate buffer dissolving of pH 7.4, ultrasonic dispersion 3-5min, 4 ℃ of preservations.
It is that coupling agent prepares the nm of gold immunological probe that the key problem in technology that the present invention solves is to select 16-MHDA, make prepared immunological probe antibody cover rate improve greatly, thereby guaranteed also have high selectivity and specificity when resulting immunological probe has high stable.
Claims (1)
1. the preparation method of a nm of gold immunological probe is characterized in that, forms immunological probe by the covalent bond coupling between nm of gold and the antibody, and the preparation method of this immunological probe comprises following steps:
(1) preparation of nm of gold: the nanogold particle for preparing 30nm with trisodium citrate reduction gold chloride; Be heated to boiling and carry out magnetic agitation with the 80-120ml ultrapure water, getting 0.3-0.5mL 1.0% citric acid three sodium solution adds rapidly in the water of boiling, reaction is carried out adding the 1.0mL0.50-0.70% chlorauric acid solution behind the 2-5min, close thermal source behind the 35-45min, 15-20min stops to stir again afterwards, naturally cool to room temperature, last constant volume 50.0mL refrigerator is preserved;
(2) 16-sulfydryl alkanoic acid 16-MHDA self assembly: under the condition that non-ionic surfactant Tween-20 exists, 16-MHDA replaces the citric acid group by the surface of Au-S key self assembly in nm of gold, get the 2.0-2.5ml nano gold sol and add 1.0-1.2mL Tween-20, room temperature vibration mixing 40-60min, add 0.8-1.2mL 16-MHDA ethanolic solution mixing afterwards again, room temperature vibration 3.5-4.5h.Excessive Tween-20 and 16-MHDA remove for three times by repeated centrifugation, and the oily precipitation is aided with ultrasonic dispersion 3-5min with the phosphate buffer dissolving that 1.5-2.0mL pH is 7.0;
(3) the 16-MHDA terminal carboxyl group activates: the mixed solution that earlier the N-hydroxyl amber diacyl amine NHS aqueous solution of 1-(3-the dimethylamino-propyl)-3-ethyl carbodiimide EDC aqueous solution of 0.2-0.4mol/L and 0.03-0.06mol/L is obtained EDC/NHS, get the nm of gold 0.3-0.6mL that 16-MHDA modifies again, add in the new EDC/NHS mixed liquor that disposes of 80-120 μ L and react 10-15min, centrifugal afterwards, topple over supernatant and remove excessive EDC/NHS, precipitation is resuspended with the phosphate buffer of 0.3-0.5mL pH 7.0, with ultrasonic dispersion 3-5min;
(4) antibody coupling: the ε-NH of the terminal lysine residue of the C of antibody
2Combine the formation covalent bond with the succinimide ester that activates, nm of gold and antibody covalent coupling, 80-100 μ L, 2 μ g/mL antibody join in the solution that 0.3-0.6mL step (3) obtains centrifugal 15-20min behind the reaction 2.5-3h, supernatant inclines, lower sediment is dissolved ultrasonic dispersion 3-5min, 4 ℃ of preservations with the phosphate buffer of pH 7.4.
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| CN104878078A (en) * | 2014-02-28 | 2015-09-02 | 香港理工大学 | Hypersensitive closed tube-type colorimetric loop-mediated isothermal amplification method utilizing carboxyl-modified gold nanometer particles |
| CN105364066A (en) * | 2015-12-02 | 2016-03-02 | 首都师范大学 | Method for modifying surfaces of gold nanoparticles through ionic liquid |
| CN109705660A (en) * | 2017-10-25 | 2019-05-03 | Tcl集团股份有限公司 | A kind of compound ink and preparation method thereof, device |
| CN113945713A (en) * | 2021-09-08 | 2022-01-18 | 安徽医科大学 | Biochip for joint detection of multiple tumor markers and preparation and application thereof |
| CN114107435A (en) * | 2021-11-30 | 2022-03-01 | 广东省人民医院 | Activatable photoacoustic-fluorescence dual-mode probe for real-time monitoring of immunotherapy and application |
| WO2022153898A1 (en) * | 2021-01-13 | 2022-07-21 | デンカ株式会社 | Method for measuring target antigen, and insoluble particles and target antigen measurement kit used therein |
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| CN104878078A (en) * | 2014-02-28 | 2015-09-02 | 香港理工大学 | Hypersensitive closed tube-type colorimetric loop-mediated isothermal amplification method utilizing carboxyl-modified gold nanometer particles |
| CN104878078B (en) * | 2014-02-28 | 2021-01-12 | 香港理工大学 | Ultrasensitive closed-tube colorimetric loop-mediated isothermal amplification method using carboxyl-modified gold nanoparticles |
| CN104614518A (en) * | 2015-01-26 | 2015-05-13 | 珠海丽珠试剂股份有限公司 | Covalent labeling method for quickly detecting colloidal gold |
| CN104614518B (en) * | 2015-01-26 | 2016-06-15 | 珠海丽珠试剂股份有限公司 | A kind of gold colloidal covalent labeling method for quickly detecting |
| CN105364066A (en) * | 2015-12-02 | 2016-03-02 | 首都师范大学 | Method for modifying surfaces of gold nanoparticles through ionic liquid |
| CN109705660A (en) * | 2017-10-25 | 2019-05-03 | Tcl集团股份有限公司 | A kind of compound ink and preparation method thereof, device |
| WO2022153898A1 (en) * | 2021-01-13 | 2022-07-21 | デンカ株式会社 | Method for measuring target antigen, and insoluble particles and target antigen measurement kit used therein |
| CN113945713A (en) * | 2021-09-08 | 2022-01-18 | 安徽医科大学 | Biochip for joint detection of multiple tumor markers and preparation and application thereof |
| CN114107435A (en) * | 2021-11-30 | 2022-03-01 | 广东省人民医院 | Activatable photoacoustic-fluorescence dual-mode probe for real-time monitoring of immunotherapy and application |
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