CN102556959B - Preparation method of metal nanoparticle dimer - Google Patents
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Abstract
The invention relates to a method for preparing a high-purity pitch-adjustable metal nanoparticle dimer at high yield. The method is characterized in that two length-adjustable double-stranded DNA molecules respectively modified by a dual-thiol molecule at the tail ends are used as the connection part; Au (gold) nanoparticles are assembled to a discrete dimer through the chemical bond formed between the thiol and the surface of the Au nanoparticles; and the discrete dimer is separated by one-step electrophoresis method to obtain the high-purity target product. The method provided by the invention can be also used for preparing high-purity Au nanoparticle dimer with symmetric and asymmetric compositions. The method has the advantages of less steps, simple operation, easily-controlled conditions, and good repeatability, and solves the problems of single-DNA-modified Au nanoparticles, such as low assembly purity and complicated steps. The method can provide a firm platform for the basic research of plasmon resonance photonics, and can be used for molecular spectrum studies, such as surface-enhanced Raman scattering and metal-enhanced fluorescence. The prepared high-purity pitch-adjustable metal nanoparticle dimer can be used as an ultrahigh-sensitivity sensor based on plasmon resonance.
Description
Technical field
The present invention relates to utilize large biological molecule DNA to assemble the dimeric method of discrete metal nano particle plasmon resonance able to programmely, belong to field of nano material preparation.
Background technology
When the free electron on specific wavelength incident light and metal nanoparticle surface interacts, can excite collective's concussion of free electron, light is limited in the nanoscale on metal nanoparticle surface, produce local surface plasma resonance.Noble metal nano particles has unique photoelectric property because local surface plasma resonance effect causes it, as produced significant Electromagnetic enhancement phenomenon at particle surface near-field region, this makes the Raman scattering of molecule signal at this place strengthen several to tens orders of magnitude, thereby realize monomolecular Raman spectrum, detect, this Electromagnetic enhancement effect also can be put forward the high-order harmonic generation efficiency of high molecular fluorescence generation signal, atom etc.Because metal nanoparticle surface plasmon resonance effect is subject to paying close attention to more and more widely, a large amount of novel metal Nano structures also continue to bring out, this has expedited the emergence of an emerging cross discipline---the birth of phasmon photonic propulsion, and the researcher of more and more physics, chemistry, biology, Material Field puts in the research of this new branch of science.Along with the deepening continuously and expanding of correlative study, phasmon nanostructured demonstrates huge potential using value in various fields such as chemical biosensor, Near-Field Radar Imaging technology, negative refractive index material, the waveguides of optic communication nanometer.
In the research of metal Nano structure, metal nanoparticle dimer is because the features such as it is simple in structure, easy modeling are widely studied, and comprises theoretical calculate and the optical property observation of individual particle etc. is reported in media successively.Yet the research of the plasmon resonance coupling of metal nanoparticle is still rested between the nano particle of constant spacing, and in these researchs, the spacing of nano particle is difficult to regulation and control.High yield preparation on controllable metal nano structure is the key link of the follow-up further investigation of impact and application, is also at present one and has challenging work.
Summary of the invention
The object of the invention is to propose the dimeric preparation method of a kind of high purity metal nano particle, not high to overcome the assembling productive rate of existing scheme, purification step is loaded down with trivial details wait not enough.
Above-mentioned purpose of the present invention, can be achieved through the following technical solutions: the dimeric preparation method of a kind of metal nanoparticle, described method is prepared the metal nanoparticle dimer that gained is size symmetry, it is characterized in that comprising step.
S1: to the end modified lipoic acid of single stranded oligonucleotide ssDNA, wherein the length of single stranded oligonucleotide is mated with the dimeric spacing Modulatory character of metal nanoparticle; S2: be paired into dsDNA through single stranded oligonucleotide ssDNA hybridization end modified and that there is complementary series by two; S3: the sulfydryl having by modification lipoic acid and the combination of metal nanoparticle are assembled into the dimer of discrete type, and with excess of sulfur is sad, processing are sealed in metal nanoparticle surface; S4: adopt electrophoresis that the metal nanoparticle dimer of size symmetry is separated.
Further, in step S1, in the aqueous solution that contains 70% acetonitrile, add reactive intermediate DHLA-NHS and end through amido modified ssDNA, and add the triethylamine as a small amount of acid binding agent, react 12 hours, and utilize vacuum evaporation apparatus that the single stranded oligonucleotide ssDNA modifying through lipoic acid is concentrated, evaporative removal acetonitrile composition wherein;
In step S2, by two through single stranded oligonucleotide ssDNA end modified and that there is complementary series under equimolar condition, in 1 * PBS buffer solution, from 95 ℃, be slowly down to room temperature and match and be hybridized to dsDNA;
In step S3, in the metal nanoparticle aqueous solution of the NaCl that contains 50mM, add the equimolar dsDNA of relative particle, react 6 hours, add subsequently excessive NaCl to make metal nanoparticle reunion sedimentation, accelerate metal nanoparticle coupling and become dimer, precipitum is resuspended in after centrifugal in the NaCl solution of 50mM, adds sad all the other positions to metal nanoparticle surface of excess of sulfur to seal processing;
In step S4, in 3% agarose, electrophoretic separation goes out the metal nanoparticle dimer of size symmetry.
Further, described metal nanoparticle is at least nano particle or the nanoshell of one of four kinds of metals of Au Ag Pt Pd, or is semiconductor-quantum-point.
Further, before step S1, can also specific modification fluorescent dye or detection thing that can specific binding target to single stranded oligonucleotide ssDNA.
Above-mentioned purpose of the present invention, can also be achieved through the following technical solutions: the dimeric preparation method of a kind of metal nanoparticle, it is the asymmetrical metal nanoparticle dimer of size that described method is prepared gained, it is characterized in that comprising step.
S1 ': the single stranded oligonucleotide ssDNA1 of preparatory sequence complementary pairing and single stranded oligonucleotide ssDNA2, and the end modified lipoic acid of difference, wherein the length of single stranded oligonucleotide is mated with the dimeric spacing Modulatory character of metal nanoparticle; S2 ': mix, modify under the condition that the single stranded oligonucleotide ssDNA1 metal nanoparticle relatively large with size that lipoic acid is modified is 1 in mol ratio; S3 ': the single stranded oligonucleotide ssDNA2 metal nanoparticle relatively little with size that lipoic acid is modified is greater than in mol ratio under 1 condition and mixes, modify; S4 ': by two groups of metal nanoparticle Hybrid assemblings, and make mol ratio between metal nanoparticle that size is relatively little and the relatively large metal nanoparticle of size be greater than 1; S5 ': adopt electrophoresis that the asymmetrical metal nanoparticle dimer of size is separated.
Further, in step S1 ', the single stranded oligonucleotide ssDNA1 of preparatory sequence complementary pairing and single stranded oligonucleotide ssDNA2, in the aqueous solution that contains 70% acetonitrile, add reactive intermediate DHLA-NHS and end through amido modified ssDNA1, and add the triethylamine as a small amount of acid binding agent, react 12 hours, and utilize vacuum evaporation apparatus that the single stranded oligonucleotide ssDNA1 modifying through lipoic acid is concentrated, evaporative removal acetonitrile composition wherein, divides subsynchronous or asynchronous modification single stranded oligonucleotide ssDNA2 with identical method.
In step S2 ' and S3 ', in the relatively large metal nanoparticle aqueous solution of the size of the NaCl that contains 50mM, add the equimolar ssDNA1 of relative particle, the ssDNA2 that adds relative particle mol ratio to be greater than 1 in the relatively little metal nanoparticle aqueous solution of the size of the NaCl that contains 50mM, reacts 12 hours simultaneously.
In step S5 ', in 3% agarose, electrophoretic separation goes out the asymmetrical metal nanoparticle dimer of size.
Further, described metal nanoparticle is at least nano particle or the nanoshell of one of four kinds of metals of Au Ag Pt Pd, or is semiconductor-quantum-point.
Further, at step S1 ' before, to single stranded oligonucleotide ssDNA1 and/or single stranded oligonucleotide ssDNA2 simultaneously or select a specific modification fluorescent dye or detection thing that can specific binding target.
Technical scheme of the present invention compares to prior art, its remarkable advantage is embodied in: this preparation method can prepare highly purified size and form symmetric and asymmetric gold nano grain dimer, and related step is few, simple to operate, condition is easily controlled, reproducible.In addition, method of the present invention also can be used to conveniently, high yield prepare between gold nano grain and silver nano-grain, semiconductor-quantum-point, gold nanoshell optional both as constructing the size of excimer and forming symmetrical and asymmetric dimeric structure, and the distance between nano particle can regulate and control by changing the length of double-stranded DNA.
Accompanying drawing explanation
Fig. 1 is the symmetrical dimer transmission electron microscope photo that the 13nm gold grain that makes by the present invention forms;
Fig. 2 is the symmetrical dimer low temperature electromicroscopic photograph that the 13nm gold grain that makes by the present invention forms;
Fig. 3 is the asymmetric dimer transmission electron microscope photo that the 13nm that makes by the present invention and 5nm gold grain form;
Fig. 4 is the symmetrical dimer agarose electrophoresis photo that the 13nm gold grain that makes by the present invention forms.
The specific embodiment
In recent years, utilizing large biological molecule DNA is forward position and the hot research content in the research of phasmon nanostructured as constructing the controlled assembling of primitive guide wire nano particle.DNA has high special recognition capability, follows simple A-T, C-G base pairing rules, therefore has the designability of height; DNA double spiral has structural rigidity, and rigid length can reach 50nm, at present to the functionalized modification technology of DNA also constantly progressive and perfect, these advantages make DNA can be used to modify and guide wire nano particle carries out complexity, controlled assembling, construct rich and varied metal Nano structure, comprise gold nano grain dimer, tripolymer, gold nano grain nano wire etc.
For system is in depth studied the local surface plasmon resonance coupling of metal nanoparticle, and basis and the application problem of the nanophotonics such as the enhancing molecular spectrum based on surface plasmon resonance effect, fluorescence energy transfer, bio-sensing, the present invention is directed to that existing common metal nanoparticle structure assembling preparation process is loaded down with trivial details, productive rate and the low defect of purity, the new especially high-purity simple and convenient process for preparing of gold nano grain dimer nanostructured of gold nano grain nanostructured of preparing is proposed.Design is as a whole: utilize different length double-stranded DNA that two ends modify respectively two thiol molecule as attaching parts, by sulfydryl and gold nano grain surface, form chemical bond groups of grains is dressed up to discrete dimer, and through one-step method electrophoretic separation, obtain highly purified target product, comprise symmetrical and asymmetrical gold nano grain dimer.
Summarize step by step: the dimeric concrete grammar of the symmetrical gold nano grain of preparation size is: to the end modified lipoic acid of single stranded oligonucleotide ssDNA (being called for short DHLA), the ssDNA hybridization pairing with complementary series through end modified is formed to dsDNA, subsequently by controlling the mol ratio of dsDNA and gold nano grain, utilize the combination of sulfydryl and gold nano grain that gold nano grain is assembled into discrete dimer, and with excessive DHLA, processing is sealed in gold nano grain surface, finally utilize electrophoresis means one step to isolate highly purified symmetrical product.
Moreover the dimeric concrete grammar of the asymmetrical gold nano grain of preparation size is: under the condition that the ssDNA1 first modifying at DHLA and gold nano grain mol ratio are 1, large scale gold nano grain is modified; SsDNA2(ssDNA2 and the ssDNA1 complementary pairing at DHLA, modified simultaneously) and the condition of gold nano grain mol ratio >1 under; small size gold nano grain is modified; subsequently by above two groups of particle Hybrid assemblings; and make the mol ratio >1 of small sized particles and large-size particle, finally by electrophoresis one step separation, obtain highly purified asymmetric product.
In above-mentioned preparation method, the length of single stranded oligonucleotide is mated with the dimeric spacing Modulatory character of metal nanoparticle.Metal nanoparticle is at least nano particle or the nanoshell of one of four kinds of metals of Au Ag Pt Pd, or is semiconductor-quantum-point.Can also be simultaneously or alternatively specific modification fluorescent dye or detection thing that can specific binding target to single stranded oligonucleotide ssDNA1 and/or single stranded oligonucleotide ssDNA2.
Constipation closes Fig. 1 to Fig. 4 two of technical solution of the present invention embodiment is described in further detail below, but about description and the explanation of embodiment, protection domain of the present invention is not constituted any limitation below.
Embodiment 1: in the aqueous solution that contains 70% acetonitrile, adding reactive intermediate DHLA-NHS and end is the oligonucleotides that contains 30 bases through amido modified strand primer P30 and P30C(P30 and P30C, and sequence is complementary), and add a small amount of triethylamine to make acid binding agent, react 12 hours.Utilize vacuum evaporation apparatus that the primer P30 modifying through DHLA and P30C is concentrated, and evaporate acetonitrile composition wherein.
The symmetrical gold nano grain dimer of 13nm that preparation is connected by 30bp dsDNA, by DHLA-P30 and DHLA-P30C under equimolar condition, in 1 * PBS buffer solution, from 95 ℃, be slowly down to room temperature and match and be hybridized to double-stranded dsP30, in the 13nm gold nano grain aqueous solution of the NaCl that contains 50mM, add equimolar dsP30.React 6 hours.Add subsequently excessive NaCl to make gold nano grain reunion sedimentation, further promote that gold nano grain coupling becomes dimer, precipitum is resuspended in after centrifugal in the NaCl solution of 50 mM, adds excessive DHLA to seal all the other positions, 13nm gold nano grain surface.Finally in 3% agarose, electrophoretic separation goes out target product.
Preparation forms asymmetric dimeric structure by 20nm and 13nm gold nano grain, in the 20nm of the NaCl that the contains 50mM gold nano grain aqueous solution, add equimolar DHLA-P30, in the 13nm of the NaCl that the contains 50mM gold nano grain aqueous solution, add P30C simultaneously, and the mol ratio that makes P30C and 13nm gold nano grain is 5, react 12 hours.By above two groups of gold nano grain Hybrid assemblings through modification, 13nm gold nano grain and 20nm gold nano grain mol ratio are 3, react 12 hours, and finally in 3% agarose, electrophoretic separation goes out target product.
Embodiment 2: in the aqueous solution that contains 70% acetonitrile, adding reactive intermediate DHLA-NHS and end is the oligonucleotides that contains 15 bases through amido modified strand primer P15 and P15C(P15 and P15C, and sequence is complementary), and add a small amount of triethylamine to make acid binding agent, react 12 hours.Utilize vacuum evaporation apparatus that the primer P15 modifying through DHLA and P15C is concentrated, and evaporate acetonitrile composition wherein.
The symmetrical gold nano grain dimer of 13nm that preparation is connected by 15bp dsDNA, by DHLA-P15 and DHLA-P15C under equimolar condition, in 1 * PBS buffer solution, from 95 ℃, be slowly down to room temperature and match and be hybridized to double-stranded dsP15, in the 13nm gold nano grain aqueous solution of the NaCl that contains 50mM, add equimolar dsP15.React 6 hours.Add subsequently excessive NaCl to make gold nano grain reunion sedimentation, further promote that gold nano grain coupling becomes dimer, precipitum is resuspended in after centrifugal in the NaCl solution of 50mM, adds excessive DHLA to seal all the other positions, 13nm gold nano grain surface.Finally in 3% agarose, electrophoretic separation goes out target product.
Preparation forms asymmetric dimeric structure by 13nm and 5nm gold nano grain, in the 13nm gold nano grain aqueous solution that contains 50 mM NaCl, add equimolar DHLA-P15, in the 5nm of the NaCl that the contains 50 mM gold nano grain aqueous solution, add P15C simultaneously, and the mol ratio that makes P15C and 5nm gold nano grain is 5, react 12 hours.By above two groups of gold nano grain Hybrid assemblings through modification, 5nm gold nano grain and 13nm gold nano grain mol ratio are 3, react 12 hours, and finally in 3% agarose, electrophoretic separation goes out target product.
The dimeric dynamic light scattering average grain diameter statistics of gold nano grain (nm of unit) of the different spacing making by the inventive method is as shown in the table.
。
The above-mentioned nano particle dimer that these make can be used as foundation structure and the platform of nanophotonics research, is used for studying surface plasmon resonance effect and strengthens molecular spectrum, fluorescence energy transfer and the high sensor based on plasmon resonance effect.
More than, by the concrete description of implementing example, be intended to should be readily appreciated that technical scheme feature of the present invention, protection scope of the present invention is not constituted any limitation.All employing equivalents or equivalence are replaced and the technical scheme of formation, within all dropping on patent application right protection domain of the present invention.
Claims (8)
1. the dimeric preparation method of metal nanoparticle, described method is prepared the metal nanoparticle dimer that gained is size symmetry, it is characterized in that comprising step:
S1: to the end modified lipoic acid of single stranded oligonucleotide ssDNA, wherein the length of single stranded oligonucleotide is mated with the dimeric spacing Modulatory character of metal nanoparticle;
S2: be paired into dsDNA through single stranded oligonucleotide ssDNA hybridization end modified and that there is complementary series by two;
S3: controlling dsDNA is 1 with the mol ratio of metal nanoparticle, and the sulfydryl having by modification lipoic acid and the combination of metal nanoparticle be assembled into the dimer of discrete type, and with excess of sulfur is sad, processing is sealed in metal nanoparticle surface;
S4: adopt electrophoresis that the metal nanoparticle dimer of size symmetry is separated.
2. the dimeric preparation method of a kind of metal nanoparticle according to claim 1, is characterized in that:
In step S1, in the aqueous solution that contains 70% acetonitrile, add reactive intermediate DHLA-NHS and end through amido modified ssDNA, and add the triethylamine as a small amount of acid binding agent, react 12 hours, and utilize vacuum evaporation apparatus that the single stranded oligonucleotide ssDNA modifying through lipoic acid is concentrated, evaporative removal acetonitrile composition wherein;
In step S2, by two through single stranded oligonucleotide ssDNA end modified and that there is complementary series under equimolar condition, in 1XPBS buffer solution, from 95 ℃, be slowly down to room temperature and match and be hybridized to dsDNA;
In step S3, in the metal nanoparticle aqueous solution of the NaCl that contains 50mM, add the equimolar dsDNA of relative particle, react 6 hours, add subsequently excessive NaCl to make metal nanoparticle reunion sedimentation, accelerate metal nanoparticle coupling and become dimer, precipitum is resuspended in after centrifugal in the NaCl solution of 50mM, adds sad all the other positions to metal nanoparticle surface of excess of sulfur to seal processing;
In step S4, in 3% agarose, electrophoretic separation goes out the metal nanoparticle dimer of size symmetry.
3. the dimeric preparation method of a kind of metal nanoparticle according to claim 1 and 2, is characterized in that: described metal nanoparticle is at least nano particle or the nanoshell of one of four kinds of metals of Au Ag Pt Pd, or is semiconductor-quantum-point.
4. the dimeric preparation method of a kind of high purity metal nano particle according to claim 1 and 2, is characterized in that: before described step S1, to the specific modification fluorescent dye of single stranded oligonucleotide ssDNA or detection thing that can specific binding target.
5. the dimeric preparation method of metal nanoparticle, it is the asymmetrical metal nanoparticle dimer of size that described method is prepared gained, it is characterized in that comprising step:
S1 ': the single stranded oligonucleotide ssDNAl of preparatory sequence complementary pairing and single stranded oligonucleotide ssDNA2, and the end modified lipoic acid of difference, wherein the length of single stranded oligonucleotide is mated with the dimeric spacing Modulatory character of metal nanoparticle;
S2 ': mix, modify under the condition that the single stranded oligonucleotide ssDNAl metal nanoparticle relatively large with size that lipoic acid is modified is 1 in mol ratio;
S3 ': the single stranded oligonucleotide ssDNA2 metal nanoparticle relatively little with size that lipoic acid is modified is greater than in mol ratio under 1 condition and mixes, modify;
S4 ': by two groups of metal nanoparticle Hybrid assemblings, and make mol ratio between metal nanoparticle that size is relatively little and the relatively large metal nanoparticle of size be greater than 1;
S5 ': adopt electrophoresis that the asymmetrical metal nanoparticle dimer of size is separated.
6. the dimeric preparation method of a kind of metal nanoparticle according to claim 5, is characterized in that:
In step S1 ', the single stranded oligonucleotide ssDNAl of preparatory sequence complementary pairing and single stranded oligonucleotide ssDNA2, in the aqueous solution that contains 70% acetonitrile, add reactive intermediate DHLA-NHS and end through amido modified ssDNAl, and add the triethylamine as a small amount of acid binding agent, react 12 hours, and utilize vacuum evaporation apparatus that the single stranded oligonucleotide ssDNAl modifying through lipoic acid is concentrated, evaporative removal acetonitrile composition wherein, divides subsynchronous or asynchronous modification single stranded oligonucleotide ssDNA2 with identical method;
In step S2 ' and S3 ', in the relatively large metal nanoparticle aqueous solution of the size of the NaCl that contains 50mM, add the equimolar ssDNAl of relative particle, the ssDNA2 that adds relative particle mol ratio to be greater than 1 in the relatively little metal nanoparticle aqueous solution of the size of the NaCl that contains 50mM, reacts 12 hours simultaneously;
In step S5 ', in 3% agarose, electrophoretic separation goes out the asymmetrical metal nanoparticle dimer of size.
7. according to the dimeric preparation method of a kind of metal nanoparticle described in claim 5 or 6, it is characterized in that: described metal nanoparticle is at least nano particle or the nanoshell of one of four kinds of metals of Au Ag Pt Pd, or be semiconductor-quantum-point.
8. according to the dimeric preparation method of a kind of high purity metal nano particle described in claim 5 or 6, it is characterized in that: described step S1 ' before, to single stranded oligonucleotide ssDNAl and/or single stranded oligonucleotide ssDNA2 simultaneously or select a specific modification fluorescent dye or detection thing that can specific binding target.
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| CN110987901B (en) * | 2019-12-27 | 2020-08-28 | 无锡物联网创新中心有限公司 | Au-Au dimer array structure and preparation method and application thereof |
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