CN101491835B - Preparation method of heparin modified gold nano-particles - Google Patents
Preparation method of heparin modified gold nano-particles Download PDFInfo
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- CN101491835B CN101491835B CN 200910024843 CN200910024843A CN101491835B CN 101491835 B CN101491835 B CN 101491835B CN 200910024843 CN200910024843 CN 200910024843 CN 200910024843 A CN200910024843 A CN 200910024843A CN 101491835 B CN101491835 B CN 101491835B
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Abstract
The invention belongs to the field of nano diagnosis, in particular to a method for preparing heparin-modified gold nano particles. The method for preparing the heparin-modified gold nano particles is to add low molecular weight heparin into a gold nano particle solution which is reduced by a reducing agent, and obtain the gold nano particles which are modified by the low molecular weight heparin, wherein the low molecular weight heparin is hydrosulphonyl-containing low molecular weight heparin, and the reducing agent is sodium borohydride or sodium citrate. The method adopts the hydrosulphonyl-containing low molecular weight heparin as a modifier to stabilize gold nano particles which are prepared by taking the sodium borohydride or the sodium citrate as the reducing agent, and obtains the gold nano particles with small particle diameter and uniform distribution by controlling the proportion of the hydrosulphonyl-containing low molecular weight heparin and the sodium borohydride or the sodium citrate. Moreover, the method can be applied to detection of proteins or viruses, and has the advantages of quickness and convenience compared with other virus detection methods such as an immunological method and PCR polymerase chain reaction. The invention provides a novel method for industrialized production for quick virus detection.
Description
Technical field
The invention belongs to field of nano diagnosis, relate to a kind of method for preparing heparin-modified gold nano particles, be particularly related to a kind of low molecular weight heparin of band sulfydryl that utilizes and be trim, sodium borohydride or natrium citricum are the method that reducing agent prepares heparin-modified gold nano particles.
Background technology
In recent years, along with the rise of nanosecond science and technology, golden nanometer particle surface self assembly has been subjected to paying close attention to widely, and the gold particle of nanoscale shows potential using value in a lot of fields with its unique character, has caused the research interest that people are dense.Golden nanometer particle is the most stable metal nanoparticle, the gold particle of nanoscale has the character of unique, relevant with size aspects such as optics, calorifics, electricity, magnetics and chemistry: absorb (SPR) as surface plasmon resonance, Raman scattering (RS), and high catalytic activity, biocompatibility and stability etc.These characteristics make golden nanometer particle (GNPs) have important use at aspects such as analysis, catalysis, photoelectric device, biology sensor, biomedicines.
Mammiferous cell surface has the carbohydrate of the intensive glycocalix of one deck, and these compounds and physiology and pathological research have very big contacting, as: metabolism, inflammation infects etc.
Heparin (heparin) then is the Sulfated Heparan sulfate of a class height.Heparan sulfate is owing to have a large amount of negative electrical charges, be easy to and the protein of virus surface positively charged between produce nonspecific electrostatic interaction, thereby make virus be attached on cell surface, play an important role in the initial period of multiple poisoning intrusion cell.Comprise multiple flaviviridae in these viruses, as dengue fever virus (Dengue), typical case's hog cholera virus (classical swinefever, CSF), mite passes encephalitis viruses (tick-borne encephalitis viruses, TBE), herpes simplex virus 1 (herpes simplex virus 1), 8-type nerpes vinrus hominis (human herpesvirus 8) and papillomavirus (papilloma virus).Generally believe the motion of the interaction partners virus of virus surface molecule and Heparan sulfate and pathogenic considerable influence arranged.In addition, HS also can produce specificity with some virus surface proteins matter and interact, and participates in entering and course of infection of regulation and control virus directly.
The optical effect relevant with size based on the golden nanometer particle uniqueness, we can be connected to heparin the gold particle surface, the mechanism of action on analog cell surface, be combined the characteristics of virus with protein bound or specificity by heparin, thereby cause that the optical change that the gathering of golden nanometer particle causes detects virus.
At present, how modify golden nanometer particles with chemistry or biomolecule such as DNA, protein, polymer, dendrimer, dendritic polymer or functionalization sulfydryl etc., but the research of carbohydrate or sugar-modified golden nanometer particle is deep not enough.Existing research is modified golden nanometer particle with micromolecular monose or disaccharide unit mostly, and structure is single; The polysaccharide that perhaps adopts the method for positive and negative Electrostatic Absorption will have electric charge is adsorbed on nanoparticle surface.This static adhesion does not have the adhesion of chemical bond strong, is subjected to the influence of effects of ion easily, makes the nano particle instability of the polyose modification of formation.
Therefore, the present invention has prepared the low molecular weight heparin gold nano-particles modified of band sulfydryl, and the step of synthesizing nano-particle is simple to operation, and cost is low, and good stability, has remedied the deficiency of other sugar-modified golden nanometer particle methods.And can be used for the detection of virus and protein, the present invention has expanded new direction in field of nano diagnosis
Summary of the invention
The purpose of this invention is to provide a kind of method for preparing heparin-modified gold nano particles.
The method for preparing heparin-modified gold nano particles provided by the present invention is to be starting material with the heparin, at first uses HNO
2A certain proportion of Na is used in solution degradation then
2CO
3, H
2O, NaHCO
3Mixed solution be neutralized to neutrality, at NH
4HCO
3After the dialysis, freeze-drying obtains the low molecular weight heparin that reduction end is aldehyde radical in the solution; Reduction end is the low molecular weight heparin of aldehyde radical and mercaptoethylmaine or p-Mercaptoaniline reaction, makees catalyst with sodium cyanoborohydride, adds the freeze-drying of dialysing after the thermal response, obtains the low molecular weight heparin with sulfydryl; Be the low molecular weight heparin that adds the band sulfydryl in the solution of gold nanoparticles of reducing agent reduction to sodium borohydride or natrium citricum, obtain low molecular weight heparin as the golden nanometer particle of trim.
In the said method, described HNO for the degraded heparin
2Solution was with 1: 1～1: 20 0.5M H
2SO
4With 5.5M NaNO
2The solution preparation, the reaction time is 0.5～4 hour, and reaction temperature is 0～10 ℃, and the solution that is used for neutralization is 1: 1: 1～1: 10: 10 1M Na
2CO
3, H
2O, 1M NaHCO
3Mixed solution is used for the NH that dialyses
4HCO
3Solution concentration is 50mM.
In the said method, when reduction end is that the consumption of the low molecular weight heparin of aldehyde radical is when being 1g, the solvent that is used for the terminal low molecular weight heparin for aldehyde radical of dissolving and reducing is the phosphate buffer (pH7.0) of the 0.2M of 1～100ml, the consumption of mercaptoethylmaine or p-Mercaptoaniline is 0.1～1M, the consumption of described reaction system catalyst sodium cyanoborohydride is 1～2g, reaction time is 2～10 hours, and reaction temperature is 55～65 ℃.
In the said method, the concentration of the amount of substance of chlorauric acid solution is 1～5mM, the consumption of described borane reducing agent sodium hydride or natrium citricum is 0.01～0.1mM, the initial concentration of the low molecular weight heparin of described band sulfydryl is 0.1～10g, reaction time is 0.5～3 hour, and reaction temperature is 65～100 ℃.
The concrete steps of the preparation of a kind of heparin-modified gold nano particles provided by the invention are as follows: the heparin of 1g, and with 1: 1～1: 20 0.5MH
2SO
4With 5.5M NaNO
2Solution is mixed with 5mlHNO
2Solution degradation was reacted 0.5～4 hour down at 0～10 ℃, took normal jolting solution in the reaction, after reaction finishes, with 1: 1: 1～1: 10: 10 1M Na of 3ml
2CO
3, H
2O, 1M NaHCO
3Mixed solution neutralization, regulate pH to 7, with the 50mM NH of gained solution with 500ml～1000ml
4HCO
3Three freeze-drying of solution dialysis obtain the low molecular weight heparin that reduction end is aldehyde radical; The above-mentioned reduction end of 1g is the low molecular weight heparin of aldehyde radical, be dissolved in the 0.2M phosphate buffer (pH7.0) of 10～100ml, the mercaptoethylmaine or the p-Mercaptoaniline that add 10ml 0.1～1M again, sodium cyanoborohydride catalyst with 1～2g, under 55～65 ℃ of temperature, reacted 2～10 hours, the solution that obtains dialysis freeze-drying obtains the low molecular weight heparin with sulfydryl; The golden nanometer particle of preparation fast detecting virus, the sodium borohydride or the natrium citricum reducing agent that add 5ml 0.01～0.1mM earlier in to 20ml 1～5mM chlorauric acid solution, after reacting 0.5～3 hour under 65～100 ℃ the temperature, the low molecular weight heparin that adds the above-mentioned band sulfydryl of 0.1～10g, stirring at normal temperature 0.5～2 hour, after the solution of gold nanoparticles that obtains is centrifugal with the centrifuge of 10000～30000r/min, Tris-HCl buffer solution (pH7.6) with the 10mM of 10～100ml dissolves again, the above-mentioned steps triplicate obtains heparin-modified gold nano particles solution.
Compare with other detection methods, the present invention has following advantage: at first prepared the low molecular weight heparin gold nano-particles modified of band sulfydryl, the step of synthesizing nano-particle is simple to operation, and cost is low, and good stability.And can be used for the detection of virus and protein, the present invention has expanded new direction in field of nano diagnosis.
Description of drawings
Fig. 1 is the ultraviolet absorpting spectrum of the low molecular weight heparin gold nano-particles modified of band sulfydryl
Fig. 2 is transmission electron microscope (TEM) collection of illustrative plates of the low molecular weight heparin gold nano-particles modified of band sulfydryl
The specific embodiment
Embodiment 1: reduction end is the preparation of the low molecular weight heparin of aldehyde radical
0.5M H with 1: 1～1: 20
2SO
4With 5.5M NaNO
2Solution is mixed with 5mlHNO
2Solution adds the heparin of 1g then, fully mix, 0～10 ℃ of reaction 0.5～4 hour down, in the course of reaction every solution of jolting in 5～10 seconds, after reaction finishes, with 1: 1: 1～1: 10: 10 1M Na of 3ml
2CO
3, H
2O, 1M NaHCO
3Mixed solution neutralization, regulate pH to 7, with the 50mM NH of gained solution with 500ml～1000ml
4HCO
3Three freeze-drying of solution dialysis obtain the low molecular weight heparin that reduction end is aldehyde radical.
Embodiment 2: the preparation of the low molecular weight heparin of band sulfydryl
The reduction end of 1g is the low molecular weight heparin of aldehyde radical, be dissolved in the 0.2M phosphate buffer (pH7.0) of 10～100ml, the mercaptoethylmaine solution that adds 10ml 0.1～1M again after stirring, adds the sodium cyanoborohydride catalyst of 1～2g, under 55～65 ℃ of temperature, reacted 2～10 hours, after reaction finishes, with solution centrifugal, get supernatant dialysis back freeze-drying, namely get the low molecular weight heparin sample of band sulfydryl.
Embodiment 3: the preparation of the low molecular weight heparin of band sulfydryl
The reduction end of 1g is the low molecular weight heparin of aldehyde radical, be dissolved in the 0.2M phosphate buffer (pH7.0) of 10～100ml, the p-Mercaptoaniline solution that adds 10ml 0.1～1M again after stirring, adds the sodium cyanoborohydride catalyst of 1～2g, under 55～65 ℃ of temperature, reacted 2～10 hours, after reaction finishes, with solution centrifugal, get supernatant dialysis back freeze-drying, namely get the low molecular weight heparin sample of band sulfydryl.
Embodiment 4: the preparation of heparin-modified gold nano particles
The sodium borohydride reduction agent that adds earlier 5ml 0.01～0.1mM in 20ml 1～5mM chlorauric acid solution, after reacting 0.5～3 hour under 65～100 ℃ the temperature, obtain inornate naked golden nanometer particle, in solution, add the low molecular weight heparin that 0.1～10g is with sulfydryl again, stirring at normal temperature 0.5～2 hour, after gained solution is centrifugal with the centrifuge of 10000～30000r/min, Tris-HCl buffer solution (pH7.6) with the 10mM of 10～100ml is resuspended with nano particle, more than operate triplicate, remove the heparin of not participating in reaction in the solution, obtain stable, the heparin-modified gold nano particles of good water solubility.
Embodiment 5: the preparation of heparin-modified gold nano particles
The natrium citricum reducing agent that adds earlier 5ml 0.01～0.1mM in 20ml 1～5mM chlorauric acid solution, after reacting 0.5～3 hour under 65～100 ℃ the temperature, obtain inornate naked golden nanometer particle, in solution, add the low molecular weight heparin that 0.1～10g is with sulfydryl again, stirring at normal temperature 0.5～2 hour, after gained solution is centrifugal with the centrifuge of 10000～30000r/min, Tris-HCl buffer solution (pH7.6) with the 10mM of 10～100ml is resuspended with nano particle, more than operate triplicate, remove the heparin of not participating in reaction in the solution, obtain stable, the heparin-modified gold nano particles of good water solubility.
Claims (4)
1. a method for preparing heparin-modified gold nano particles is characterized in that it being to be prepared by following process: be starting material with the heparin, at first use HNO
2A certain proportion of Na is used in solution degradation then
2CO
3, H
2O, NaHCO
3Mixed solution be neutralized to neutrality, at NH
4HCO
3After the dialysis, freeze-drying obtains the low molecular weight heparin that reduction end is aldehyde radical in the solution; Reduction end is the low molecular weight heparin of aldehyde radical and mercaptoethylmaine or p-Mercaptoaniline reaction, makees catalyst with sodium cyanoborohydride, adds the freeze-drying of dialysing after the thermal response, obtains the low molecular weight heparin with sulfydryl; Be the low molecular weight heparin that adds the band sulfydryl in the solution of gold nanoparticles of reducing agent reduction to sodium borohydride or natrium citricum, obtain low molecular weight heparin as the golden nanometer particle of trim.
2. a kind of method for preparing heparin-modified gold nano particles according to claim 1 is characterized in that: described degraded solutions was with 1: 1～1: 20 0.5M H
2SO
4With 5.5M NaNO
2Solution is mixed with HNO
2Solution, the reaction time is 0.5～4 hour, and reaction temperature is 0～10 ℃, and the solution that is used for neutralization is 1: 1: 1～1: 10: 10 1M Na
2CO
3, H
2O, 1M NaHCO
3Mixed solution is used for the NH that dialyses
4HCO
3Solution concentration is 50mM.
3. a kind of method for preparing heparin-modified gold nano particles according to claim 1, it is characterized in that: when described reduction end is that the consumption of the low molecular weight heparin of aldehyde radical is when being 1g, the solvent that is used for the terminal low molecular weight heparin for aldehyde radical of dissolving and reducing is the phosphate buffer of the 0.2M of 1～100ml, pH7.0, the consumption of mercaptoethylmaine or p-Mercaptoaniline is 0.1～1M, the consumption of described reaction system catalyst sodium cyanoborohydride is 1～2g, reaction time is 2～10 hours, and reaction temperature is 55～65 ℃.
4. method for preparing heparin-modified gold nano particles is characterized in that concrete steps are as follows: the liver rope of 1g, and with 1: 1～1: 20 0.5M H
2SO
4With 5.5M NaNO
2Solution is mixed with 5mlHNO
2Solution degradation was reacted 0.5～4 hour down at 0～10 ℃, took normal jolting solution in the reaction, and reaction is used 3ml1: 1: 1～1: 10: 10 1M Na after finishing
2CO
3, H
2O, 1M NaHCO
3Mixed solution neutralization, regulate pH to 7, with the 50mMNH of gained solution with 500ml～1000ml
4HCO
3Three freeze-drying of solution dialysis obtain the low molecular weight heparin that reduction end is aldehyde radical; The above-mentioned reduction end of 1g is the low molecular weight heparin of aldehyde radical, be dissolved in the 0.2M phosphate buffer of 10～100ml, pH7.0, the mercaptoethylmaine or the p-Mercaptoaniline that add 10ml0.1～1M again, with the sodium cyanoborohydride catalyst of 1～2g, under 55～65 ℃ of temperature, reacted 2～10 hours, the solution dialysis freeze-drying that obtains obtains the low molecular weight heparin with sulfydryl; The golden nanometer particle of preparation fast detecting virus, the sodium borohydride or the natrium citricum reducing agent that add earlier 5ml0.01～0.1mM in 20ml1～5mM chlorauric acid solution, after reacting 0.5～3 hour under 65～100 ℃ the temperature, the low molecular weight heparin that adds the above-mentioned band sulfydryl of 0.1～10g, stirring at normal temperature 0.5～2 hour, after the solution of gold nanoparticles that obtains is centrifugal with the centrifuge of 10000～30000r/min, Tris-HCl buffer solution with the 10mM of 10～100ml, pH7.6, again dissolving, the above-mentioned steps triplicate obtains heparin-modified gold nano particles solution.
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| CN102049226B (en) * | 2010-11-16 | 2013-05-08 | 中国烟草总公司郑州烟草研究院 | Method for preparing TMV template-based nano-gold material |
| CN102435571B (en) * | 2011-09-16 | 2014-07-23 | 东华大学 | Method for detecting heparin content with polyethyleneimine-stabilized gold nanoparticle |
| TWI429460B (en) | 2011-12-19 | 2014-03-11 | Ind Tech Res Inst | Anticoagulant-conjugated carbon nanocapsule, anti-thrombotic drug containing thereof and preparation thereof |
| CN108403704A (en) * | 2018-05-31 | 2018-08-17 | 江南大学 | Heparin disaccharides is grafted the purposes of sulphation polymethyl acyl ethanol amine |
| CN111975010A (en) * | 2019-05-21 | 2020-11-24 | 中国药科大学 | Preparation of gold nanoparticles based on D-arabinose reduction |
| CN110596065B (en) * | 2019-09-25 | 2021-10-15 | 福建医科大学 | Acid phosphatase detection method based on cysteamine-N-acetyl-L-cysteine-gold nanocluster fluorescent material |
| CN110672567B (en) * | 2019-09-26 | 2022-01-11 | 南通大学 | Low molecular weight heparin gold nano material and application thereof in heparanase detection |
| CN112692297A (en) * | 2019-10-07 | 2021-04-23 | 福建医科大学 | Osmium nano particle with anticoagulation effect and oxidase activity |
| CN116376832A (en) * | 2019-11-11 | 2023-07-04 | 百脉迪生物科技(苏州)有限公司 | Method for selecting optimal effect of promoting neural differentiation of stem cells |
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| CN101245387A (en) * | 2008-01-22 | 2008-08-20 | 中国科学院上海应用物理研究所 | Nano-gold signal probe for DNA detection, production method and method for detecting DNA |
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| CN101245387A (en) * | 2008-01-22 | 2008-08-20 | 中国科学院上海应用物理研究所 | Nano-gold signal probe for DNA detection, production method and method for detecting DNA |
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| "肝素稳定的金纳米微粒自组装薄膜的光谱研究";郭艳丽等;《光谱学与光谱分析》;20081031;第28卷(第10期);全文 * |
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