CN102556959A - Preparation method of metal nanoparticle dimer - Google Patents
Preparation method of metal nanoparticle dimer Download PDFInfo
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- CN102556959A CN102556959A CN2011104550505A CN201110455050A CN102556959A CN 102556959 A CN102556959 A CN 102556959A CN 2011104550505 A CN2011104550505 A CN 2011104550505A CN 201110455050 A CN201110455050 A CN 201110455050A CN 102556959 A CN102556959 A CN 102556959A
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Images
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, be limited in light in the nanoscale on metal nanoparticle surface, promptly produce local surface plasma resonance.Noble metal nano particles is because the local surface plasma resonance effect causes it to have unique photoelectric property; Strengthen phenomenon as produce significant electromagnetic field at the particle surface near-field region; This makes the Raman scattering of molecule signal at this place strengthen several to tens one magnitude; Thereby realize that monomolecular Raman spectrum detects, this electromagnetic field enhancement effect also can improve the fluorescence generation signal of molecule, the higher hamonic wave of atom produces efficient etc.Because metal nanoparticle surface plasma body resonant vibration effect receives concern 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, field of materials puts in the research of this new branch of science.Along with the deepening continuously and expanding of correlative study, the phasmon nanostructured demonstrates huge potential using value in various fields such as chemical biosensor, near field imaging technique, negative refractive index material, the waveguides of optic communication nanometer.
In the research of metal Nano structure, the metal nanoparticle dimer is because it is simple in structure, be prone to characteristics such as modeling and by broad research, comprises that the optical property observation etc. of Theoretical Calculation and individual particle is reported in media successively.Yet the research to the plasmon resonance coupling of metal nanoparticle still rests between the nano particle of constant spacing, and the spacing of nano particle is difficult to regulation and control in these researchs.Produced in high yields to the controllable metal nano structure is follow-up further investigation of influence and key in application link, also is one at present and has challenging work.
Summary of the invention
The objective of the invention is to propose the dimeric preparation method of a kind of high purity metal nano particle, not high with the assembling productive rate that overcomes existing scheme, the loaded down with trivial details deficiency that waits of purification step.
Above-mentioned purpose of the present invention, can realize through following technical scheme: the dimeric preparation method of a kind of metal nanoparticle, said method prepare the metal nanoparticle dimer that gained is the size symmetry, it is characterized in that comprising step.
S1: to the end modified lipoic acid of single stranded oligonucleotide ssDNA, wherein the dimeric spacing Modulatory character of the length of single stranded oligonucleotide and metal nanoparticle is mated; S2: be paired into dsDNA through single stranded oligonucleotide ssDNA hybridization end modified and that have a complementary series with two; S3: be assembled into the dimer of discrete type through the combination of modifying sulfydryl that lipoic acid had and metal nanoparticle, and processing sealed on the metal nanoparticle surface with excess of sulfur is sad; S4: adopt electrophoresis that the metal nanoparticle dimer of size symmetry is separated.
Further; Among the step S1; In the aqueous solution that contains 70% acetonitrile, add reactive intermediate DHLA-NHS and terminal, and add triethylamine, reacted 12 hours as a small amount of acid binding agent through amido modified ssDNA; And the single stranded oligonucleotide ssDNA that utilizes vacuum evaporation apparatus will pass through the lipoic acid modification concentrates evaporative removal acetonitrile composition wherein;
Among the step S2, with two through end modified and single stranded oligonucleotide ssDNA with complementary series under equimolar condition, in 1 * PBS buffer solution, slowly reduce to room temperature and match and hybridize into dsDNA from 95 ℃;
Among the step S3; In the metal nanoparticle aqueous solution of the NaCl that contains 50mM, add the equimolar dsDNA of relative particle; Reacted 6 hours, and added excessive N aCl subsequently and make metal nanoparticle reunion sedimentation, quicken the metal nanoparticle coupling and become dimer; Precipitum is resuspended in after centrifugal in the NaCl solution of 50mM, adds sad all the other positions of excess of sulfur and seal processing the metal nanoparticle surface;
Among the step S4, electrophoretic separation goes out the metal nanoparticle dimer of size symmetry in 3% agarose.
Further, said metal nanoparticle is at least the nano particle or the nanoshell of one of gold, silver, platinum, four kinds of metals of palladium, or is semiconductor-quantum-point.
Further, before step S1, but can also specific modification fluorescent dye or the detection thing of specificity combining target to single stranded oligonucleotide ssDNA.
Above-mentioned purpose of the present invention, can also realize through following technical scheme: it is the asymmetrical metal nanoparticle dimer of size that the dimeric preparation method of a kind of metal nanoparticle, said method prepare 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, the wherein dimeric spacing Modulatory character coupling of the length of single stranded oligonucleotide and metal nanoparticle; S2 ': single stranded oligonucleotide ssDNA1 that lipoic acid is modified and the relatively large metal nanoparticle of size are to mix, modify under 1 the condition in mol ratio; S3 ': the less relatively metal nanoparticle of single stranded oligonucleotide ssDNA2 that lipoic acid is modified and size mixes, modifies under greater than 1 condition in mol ratio; S4 ': two groups of metal nanoparticles are mixed assembling, and make between the relatively large metal nanoparticle of the less relatively metal nanoparticle of size and size mol ratio greater than 1; S5 ': adopt electrophoresis that the asymmetrical metal nanoparticle dimer of size is separated.
Further; Among the step S1 '; The single stranded oligonucleotide ssDNA1 of preparatory sequence complementary pairing and single stranded oligonucleotide ssDNA2 add reactive intermediate DHLA-NHS and terminal through amido modified ssDNA1, and add the triethylamine as a small amount of acid binding agent in the aqueous solution that contains 70% acetonitrile; Reacted 12 hours; And utilize vacuum evaporation apparatus will pass through the single stranded oligonucleotide ssDNA1 that lipoic acid modifies to concentrate, evaporative removal acetonitrile composition wherein divides subsynchronous or asynchronous modification single stranded oligonucleotide ssDNA2 with identical method.
Among step S2 ' and the 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; In the less relatively metal nanoparticle aqueous solution of the size of the NaCl that contains 50mM, add simultaneously relative particle mol ratio greater than 1 ssDNA2, reacted 12 hours.
Among the step S5 ', electrophoretic separation goes out the asymmetrical metal nanoparticle dimer of size in 3% agarose.
Further, said metal nanoparticle is at least the nano particle or the nanoshell of one of gold, silver, platinum, four kinds of metals of palladium, or is semiconductor-quantum-point.
Further, at step S1 ' before, but to single stranded oligonucleotide ssDNA1 and/or single stranded oligonucleotide ssDNA2 simultaneously or select the detection thing of a specific modification fluorescent dye or specificity combining 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 prone to control, good reproducibility.In addition; Method of the present invention also can be used to conveniently, between the preparation gold nano grain of high yield and silver nano-grain, semiconductor-quantum-point, the gold nanoshell optional both as the size of constructing excimer with form symmetry and asymmetric dimeric structure, and the distance between the nano particle can be regulated and control through the length that changes double-stranded DNA.
Description of drawings
Fig. 1 is the symmetrical dimer transmission electron microscope photo that constitutes through the 13nm gold grain that the present invention makes;
Fig. 2 is the symmetrical dimer low temperature electromicroscopic photograph that constitutes through the 13nm gold grain that the present invention makes;
Fig. 3 is the asymmetric dimer transmission electron microscope photo that the 13nm that makes through the present invention and 5nm gold grain constitute;
Fig. 4 is the symmetrical dimer agarose electrophoresis photo that constitutes through the 13nm gold grain that the present invention makes.
The specific embodiment
In recent years, utilizing large biological molecule DNA is forward position and hot research content in the research of phasmon nanostructured as constructing the controlled assembling of primitive guide wire nano particle.DNA has the high special recognition capability, follows simple A-T, C-G base pairing rules, therefore has the designability of height; The dna double spiral has structural rigidity; And rigid length can reach 50nm; Functionalized modification technology to DNA is also progressive and perfect constantly at present; These advantages make DNA can be used to modify and the guide wire nano particle carries out complicacy, controlled assembling, construct rich and varied metal Nano structure, comprise gold nano grain dimer, tripolymer, gold nano grain nano wire etc.
In depth study the local surface plasmon resonance coupling of metal nanoparticle for system; And based on the basis and the application problem of nanophotonics such as the enhancing molecular spectrum of 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 defective of purity, the especially high-purity simple and convenient process for preparing of gold nano grain dimer nanostructured of new preparation gold nano grain nanostructured is proposed.Design is as a whole: utilize different length double-stranded DNA that two ends modify two thiol molecule respectively as attaching parts; Form chemical bond through sulfydryl and gold nano grain surface groups of grains is dressed up discrete dimer; And obtain target product with high purity through the one-step method electrophoretic separation, comprise symmetry and asymmetrical gold nano grain dimer.
Summarize step by step: the dimeric concrete grammar of preparation size symmetry gold nano grain is: to the end modified lipoic acid of single stranded oligonucleotide ssDNA (being called for short DHLA); To pass through end modified ssDNA hybridization pairing and form dsDNA with complementary series; Subsequently through 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 on the gold nano grain surface, utilize one step of electrophoresis means to isolate highly purified symmetrical product at last.
Moreover the dimeric concrete grammar of the asymmetrical gold nano grain of preparation size is: under ssDNA1 that DHLA modifies and gold nano grain mol ratio are 1 condition, the large scale gold nano grain is modified earlier; Simultaneously in ssDNA2 (ssDNA2 and ssDNA1 complementary pairing) and gold nano grain mol ratio that DHLA modifies>under 1 the condition; The small size gold nano grain is modified; Subsequently above two groups of particles are mixed assembling; And make the mol ratio of small sized particles and large-size particle>1, separate obtaining highly purified asymmetric product at last through one step of electrophoresis.
Among the above-mentioned preparation method, the dimeric spacing Modulatory character coupling of the length of single stranded oligonucleotide and metal nanoparticle.Metal nanoparticle is at least the nano particle or the nanoshell of one of gold, silver, platinum, four kinds of metals of palladium, or is semiconductor-quantum-point.But can also be simultaneously or select the detection thing of specific modification fluorescent dye in a ground or specificity combining target to single stranded oligonucleotide ssDNA1 and/or single stranded oligonucleotide ssDNA2.
Following constipation closes Fig. 1 to Fig. 4 two embodiment of technical scheme of the present invention is done further detailed description, but following description and explanation about embodiment do not constitute any limitation protection domain of the present invention.
Embodiment 1: in the aqueous solution that contains 70% acetonitrile, add reactive intermediate DHLA-NHS and terminal (P30 and P30C are the oligonucleotides that contains 30 bases through amido modified strand primer P30 and P30C; And sequence is complementary); And add a small amount of triethylamine and make acid binding agent, reacted 12 hours.The primer P30 and the P30C that utilize vacuum evaporation apparatus will pass through the DHLA modification concentrate, and evaporate acetonitrile composition wherein.
The 13nm symmetry gold nano grain dimer that preparation is connected by 30bp dsDNA; With DHLA-P30 and DHLA-P30C under equimolar condition; In 1 * PBS buffer solution, slowly reduce to room temperature and match and hybridize into double-stranded dsP30, in the 13nm of the NaCl that the contains 50mM gold nano grain aqueous solution, add equimolar dsP30 from 95 ℃.Reacted 6 hours.Add excessive N aCl subsequently and make gold nano grain reunion sedimentation; Promote that further the gold nano grain coupling becomes dimer; Precipitum is resuspended in after centrifugal in the NaCl solution of 50 mM, adds excessive DHLA all the other positions, 13nm gold nano grain surface are sealed.Electrophoretic separation goes out target product in 3% agarose at last.
Preparation is formed 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; Simultaneously in the 13nm of the NaCl that the contains 50mM gold nano grain aqueous solution, add P30C; And the mol ratio that makes P30C and 13nm gold nano grain is 5, reacts 12 hours.With above two groups of gold nano grain mixing assemblings through modifying, 13nm gold nano grain and 20nm gold nano grain mol ratio are 3, react 12 hours, and electrophoretic separation goes out target product in 3% agarose at last.
Embodiment 2: in the aqueous solution that contains 70% acetonitrile, add reactive intermediate DHLA-NHS and terminal (P15 and P15C are the oligonucleotides that contains 15 bases through amido modified strand primer P15 and P15C; And sequence is complementary); And add a small amount of triethylamine and make acid binding agent, reacted 12 hours.The primer P15 and the P15C that utilize vacuum evaporation apparatus will pass through the DHLA modification concentrate, and evaporate acetonitrile composition wherein.
The 13nm symmetry gold nano grain dimer that preparation is connected by 15bp dsDNA; With DHLA-P15 and DHLA-P15C under equimolar condition; In 1 * PBS buffer solution, slowly reduce to room temperature and match and hybridize into double-stranded dsP15, in the 13nm of the NaCl that the contains 50mM gold nano grain aqueous solution, add equimolar dsP15 from 95 ℃.Reacted 6 hours.Add excessive N aCl subsequently and make gold nano grain reunion sedimentation; Promote that further the gold nano grain coupling becomes dimer; Precipitum is resuspended in after centrifugal in the NaCl solution of 50mM, adds excessive DHLA all the other positions, 13nm gold nano grain surface are sealed.Electrophoretic separation goes out target product in 3% agarose at last.
Preparation is formed asymmetric dimeric structure by 13nm and 5nm gold nano grain; In containing the 13nm gold nano grain aqueous solution of 50 mM NaCl, add equimolar DHLA-P15; Simultaneously in the 5nm of the NaCl that the contains 50 mM gold nano grain aqueous solution, add P15C; And the mol ratio that makes P15C and 5nm gold nano grain is 5, reacts 12 hours.With above two groups of gold nano grain mixing assemblings through modifying, 5nm gold nano grain and 13nm gold nano grain mol ratio are 3, react 12 hours, and electrophoretic separation goes out target product in 3% agarose at last.
The dimeric dynamic light scattering average grain diameter statistics of the gold nano grain of the different spacing that makes through the inventive method (nm of unit) is as shown in the table.
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The above-mentioned nano particle dimer that these make can be used as the foundation structure and the platform of nanophotonics research, is used for studying that the surface plasmon resonance effect strengthens molecular spectrum, fluorescence energy transfer and based on the high sensor of plasmon resonance effect.
More than, be intended to should be readily appreciated that technical scheme characteristic of the present invention, protection scope of the present invention is not constituted any limitation through the description of practical implementation example.All employing equivalents or equivalence are replaced and the technical scheme of formation, all drop within the patent application right protection domain of the present invention.
Claims (8)
1. dimeric preparation method of metal nanoparticle, said method prepare the metal nanoparticle dimer that gained is the size symmetry, it is characterized in that comprising step:
S1: to the end modified lipoic acid of single stranded oligonucleotide ssDNA, wherein the dimeric spacing Modulatory character of the length of single stranded oligonucleotide and metal nanoparticle is mated;
S2: be paired into dsDNA through single stranded oligonucleotide ssDNA hybridization end modified and that have a complementary series with two;
S3: be assembled into the dimer of discrete type through the combination of modifying sulfydryl that lipoic acid had and metal nanoparticle, and processing sealed on the metal nanoparticle surface with excess of sulfur is sad;
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:
Among the step S1; In the aqueous solution that contains 70% acetonitrile, add reactive intermediate DHLA-NHS and terminal through amido modified ssDNA; And adding is as the triethylamine of a small amount of acid binding agent; Reacted 12 hours, and the single stranded oligonucleotide ssDNA that utilizes vacuum evaporation apparatus will pass through the lipoic acid modification concentrates evaporative removal acetonitrile composition wherein;
Among the step S2, with two through end modified and single stranded oligonucleotide ssDNA with complementary series under equimolar condition, in 1 * PBS buffer solution, slowly reduce to room temperature and match and hybridize into dsDNA from 95 ℃;
Among the step S3; In the metal nanoparticle aqueous solution of the NaCl that contains 50mM, add the equimolar dsDNA of relative particle; Reacted 6 hours, and added excessive N aCl subsequently and make metal nanoparticle reunion sedimentation, quicken the metal nanoparticle coupling and become dimer; Precipitum is resuspended in after centrifugal in the NaCl solution of 50mM, adds sad all the other positions of excess of sulfur and seal processing the metal nanoparticle surface;
Among the step S4, electrophoretic separation goes out the metal nanoparticle dimer of size symmetry in 3% agarose.
3. the dimeric preparation method of a kind of metal nanoparticle according to claim 1 and 2 is characterized in that: said metal nanoparticle is at least the nano particle or the nanoshell of one of gold, silver, platinum, four kinds of metals of palladium, 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 the said step S1, but to the detection thing of specific modification fluorescent dye of single stranded oligonucleotide ssDNA or specificity combining target.
5. dimeric preparation method of metal nanoparticle, it is the asymmetrical metal nanoparticle dimer of size that said method prepares 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, the wherein dimeric spacing Modulatory character coupling of the length of single stranded oligonucleotide and metal nanoparticle;
S2 ': single stranded oligonucleotide ssDNA1 that lipoic acid is modified and the relatively large metal nanoparticle of size are to mix, modify under 1 the condition in mol ratio;
S3 ': the less relatively metal nanoparticle of single stranded oligonucleotide ssDNA2 that lipoic acid is modified and size mixes, modifies under greater than 1 condition in mol ratio;
S4 ': two groups of metal nanoparticles are mixed assembling, and make between the relatively large metal nanoparticle of the less relatively metal nanoparticle of size and size mol ratio 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:
Among the 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 terminal through amido modified ssDNA1; And add triethylamine as a small amount of acid binding agent, reacted 12 hours, and the single stranded oligonucleotide ssDNA1 that utilizes vacuum evaporation apparatus will pass through the lipoic acid modification concentrates; Evaporative removal acetonitrile composition wherein divides subsynchronous or asynchronous modification single stranded oligonucleotide ssDNA2 with identical method;
Among step S2 ' and the 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; In the less relatively metal nanoparticle aqueous solution of the size of the NaCl that contains 50mM, add simultaneously relative particle mol ratio greater than 1 ssDNA2, reacted 12 hours;
Among the step S5 ', electrophoretic separation goes out the asymmetrical metal nanoparticle dimer of size in 3% agarose.
7. according to claim 5 or the dimeric preparation method of 6 described a kind of metal nanoparticles, it is characterized in that: said metal nanoparticle is at least the nano particle or the nanoshell of one of gold, silver, platinum, four kinds of metals of palladium, or is semiconductor-quantum-point.
8. according to claim 5 or the dimeric preparation method of 6 described a kind of high purity metal nano particles; It is characterized in that: said step S1 ' before, but to single stranded oligonucleotide ssDNA1 and/or single stranded oligonucleotide ssDNA2 simultaneously or select the detection thing of a specific modification fluorescent dye or specificity combining target.
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