CN104568898A - Hollow gold nanoparticle-DNA composite base and preparation method thereof - Google Patents
Hollow gold nanoparticle-DNA composite base and preparation method thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 15
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- 239000000243 solution Substances 0.000 claims description 48
- 108020004414 DNA Proteins 0.000 claims description 36
- 239000000523 sample Substances 0.000 claims description 29
- 239000011780 sodium chloride Substances 0.000 claims description 24
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- 239000010941 cobalt Substances 0.000 description 6
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 description 6
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
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Abstract
The invention discloses a hollow gold nanoparticle-DNA composite base and a preparation method thereof. The composite base disclosed by the invention is composed of a first hollow gold nano layer, a DNA layer and a second hollow gold nano layer. Due to the structure, a coupling effect of surface plasmon between the metal nanoparticles exists in the horizontal direction; coupling of the surface plasmon is also promoted in the direction vertical to the base so as to form relatively many hot spots; and a DNA chain is located in a hot spot coupling area generated between two hollow gold nanoparticle layers, so that the raman scattering intensity of the DNA is significantly enhanced; and compared with a traditional raman active substrate, the composition base prepared by the method has great advantages in enhancement of the raman scattering intensity.
Description
Technical field
The present invention relates to a kind of hollow gold nanometer particle-DNA composite substrate and preparation method thereof: adopt interface self-assembling technique to prepare individual layer hollow gold nanometer particle film, by the base pair complementarity between DNA single chain, upper and lower two-layer hollow gold nanometer particle layer is assembled into together, thus has prepared hollow gold nanometer particle layer-DNA layer-hollow gold nanometer particle layer composite substrate as surface enhanced Raman substrate.
Background technology
Surface enhanced raman spectroscopy (SERS) spectrum is the instrument of a kind of very effective detection interfacial characteristics and intermolecular interaction, characterizing surface molecular absorption behavior and molecular structure, based on the susceptibility of its single molecules level, the feature of molecular specificity, this technology has become one of technology of the highest research interfacial effect of sensitivity, is applied to the orientation of research binding molecule on surface and absorption behavior, absorption interface surface state, the interface orientation of biomacromolecule and configuration, conformation and structure analysis maximum magnitude.SERS spectrum, as a kind of highly sensitive input and the spectral technique with single molecular recognition function, has important application in fields such as life science, analytical chemistry, medical science, food security, environmental healths.
SERS technology overcomes the faint feature of traditional sensing techniques detection signal, effectively compensate for the weakness that Raman signal sensitivity is low, can obtain the information that normal Raman spectrum is difficult to obtain.Especially at surface and interface message context, the Raman rising characteristic of metal and application function are very outstanding, and this technology has been widely used in the physio-chemical study of surface and interface.At life science, utilize this highly sensitive absorption enhancement effect to carry out the detection of biomolecule, as DNA molecular, antibody molecule, protein molecule etc., become one of direction given priority in recent years.
SERS strengthens mechanism and mainly contains Electromagnetic enhancement and Chemical enhancement two kinds.Electromagnetic enhancement mechanism is relevant with the surface phasmon that metal surface produces, the resonance of surface phasmon depends on the pattern, structure, component, coupling condition etc. of nano material, and the Raman signal of the probe molecule being adsorbed onto roughened metal surface can be made to increase by 10
3-10
8doubly.Along with the development of nanosecond science and technology, various technology is utilized to prepare the different metal nano material of pattern and relation one of focus becoming research of research material structure, phasmon characteristic and SERS activity.Prepared the metal nano material come in every shape by chemical synthesis, regulating local electromagnetic intensity around nano particle by controlling the size of nano material, pattern, surfaceness, crystallinity and particle gap etc., being applied to SERS research.But SERS technology also has many limiting factors, and it requires that sample contacts with substrate, loses Raman spectroscopy non-intruding and discontiguous advantage; Its substrate is different to the absorption property of different trace molecules material, and quantitative test is restricted.
At present, on the research of the Chinese scholars preparation that mostly concentrates on solid metal nanoparticle and SERS performance study.Interact with photon because the metal phasmon with hollow-core construction can better be excited by surface plasma body resonant vibration, light is fallen into fixing in hollow-core construction, thus improve the utilization factor to light.And the electromagnetic intensity around after hollow-core construction is excited is higher, and coarse surface can produce more " focus " region, its SERS activity is stronger, and probe molecule detection limit can improve greatly.Therefore, the metal nano material studying hollow-core construction is necessary in SERS performance study field.
Summary of the invention
The object of the present invention is to provide a kind of hollow gold nanometer particle-DNA composite substrate and preparation method thereof.
For realizing above object, the technical solution adopted in the present invention is: a kind of hollow gold nanometer particle-DNA composite substrate, be made up of the first hollow gold nanometer layer, DNA layer, the second hollow gold nanometer layer, described DNA layer is between the first hollow gold nanometer layer and the second hollow gold nanometer layer, and DNA layer is made up of the probeDNA strand described in SEQ ID NO.1 and the reporterDNA strand described in SEQ ID NO.2.
A preparation method for hollow gold nanometer particle-DNA composite substrate, the method comprises the following steps:
A kind of hollow gold nanometer particle-DNA composite substrate, be made up of the first hollow gold nanometer layer, DNA layer, the second hollow gold nanometer layer, described DNA layer is between the first hollow gold nanometer layer and the second hollow gold nanometer layer, and DNA layer is made up of the probeDNA strand described in SEQ ID NO.1 and the reporterDNA strand described in SEQ ID NO.2.
2. a preparation method for hollow gold nanometer particle-DNA composite substrate according to claim 1, it is characterized in that, the method comprises the following steps:
(1) the probeDNA strand as shown in SEQ ID NO.1 and the reporterDNA strand as shown in SEQ ID NO.2 is designed;
(2) at Si substrate assembles first hollow gold nanometer particle layer;
(3) the probeDNA strand that on the first hollow gold nanometer layer prepared in step (2), modification step (1) designs, Product Labeling is Si/HGN-probeDNA;
(4) step (1) design reporterDNA strand is modified on hollow gold nanometer particle, obtain reporterDNA-HGN solution;
(5) the reporterDNA-HGN solution that step (4) prepares is dripped to Si/HGN-probeDNA surface, after incubation at room temperature 24h, with lauryl sodium sulfate aqueous solution and water cleaning that massfraction is 0.01%, on the first hollow gold nanometer layer, assemble the second hollow gold nanometer layer by two complementary single stranded DNAs, obtain hollow gold nanometer particle-DNA composite substrate.
Further, step 2 is specially:
(2.1) be in 1mmol/L hollow gold nanometer particle aqueous solution to 4mL concentration, dropwise add 300 μ L normal butyl alcohols, form water-oil interface, there is one deck hollow gold nanometer particle film at water-oil interface place;
(2.2) the golden nanometer particle film at water-oil interface place is after aging 10 minutes, hollow gold nanometer particle film step 2.1 obtained with Si substrate picks up, fall the residue on surface after 60 DEG C of oven dry with washes of absolute alcohol, obtain the first hollow gold nanometer particle layer on a si substrate.
Further, the external diameter of hollow gold nanometer particle is 30 ~ 40nm, and the wall thickness of hollow gold nanometer particle is 6nm ~ 10nm.
Further, step 3 is specially: probeDNA strand 5 terminal modified-(CH
2)
6-SH, obtains probeDNA-(CH
2)
6-SH; By enough
(40 μ L)concentration be the probeDNA-(CH of 1 μm of ol/L
2)
6the aqueous solution (PH=7.1) of-SH strand is added drop-wise on the first hollow gold nanometer particle layer prepared by step (2), makes probeDNA-(CH
2)
6-SH infiltrates the first hollow gold nanometer particle layer, incubated at room temperature 24h, then washes unnecessary probe DNA by the lauryl sodium sulfate that massfraction is 0.01%, and 60 DEG C of vacuum drying are for subsequent use.
Further, step 4 is specially: reporterDNA strand 5 terminal modified-(CH
2)
3-SH, obtains reporterDNA-(CH
2)
3-SH; By the PBS solution of 500 μ L PH=7.1,100 μ L massfractions be 0.1% lauryl sodium sulfate, 10 μ L concentration be 1 μm of ol/L reporterDNA-(CH
2)
3the aqueous solution (PH=7.1) of-SH strand and 400 μ L concentration are after the golden hollow ball hydrosol of 1mmol/L mixes, leave standstill 6h, then salinization is carried out, salinization is: point to add NaCl solution that isopyknic concentration increases progressively successively for 5 times in mixed solution, NaCl solution adds 100 μ L at every turn; The concentration dripping NaCl solution is first 2mol/L, and the concentration gradient of NaCl solution is 0.05mol/L; 24h, centrifugal 7min under 7000rpm/min is left standstill after salinization; After removing supernatant, add 1mL massfraction be 0.01% lauryl sodium sulfate again centrifuge washing remove supernatant, the PBS buffer solution of precipitation 1mL, obtains the PBS solution of reporterDNA-HGN.
compared with prior art, the invention has the beneficial effects as follows:
1. in the present invention, owing to adopting hollow gold nanometer particle as golden nanometer particle layer, can see from Fig. 2 TEM figure, hollow ball surfaceness is very high, coarse surface more easily produces focus, thus, the electric field excited around hollow ball can be stronger, and the Raman signal intensity of DNA is improved further.
2. the present invention adopts complementary DNA strand to connect upper and lower two-layer hollow gold nanometer particle, and detects the Raman signal of DNA with this structure.Not only there is the coupling effect of the surface phasmon between metal nanoparticle in the horizontal direction in such structure, also the coupling of surface phasmon can be promoted in a direction that is perpendicular to the base, formed more " focus ", DNA is in the focus coupling regime that golden nanometer particle produces, and the Raman scattering intensities of DNA is significantly increased.Compared with traditional Raman active substrate, substrate of the present invention has larger advantage on enhancing Raman scattering intensities.
3. the present invention adopts chemical synthesis to prepare hollow gold nanometer particle, and preparation method simply, easily operates, and cost is low.
4. the present invention adopts the environmentally friendly masking technique of interface self assembly to prepare hollow gold nanometer particle film, and preparation method is simple, efficiency is high, and cost is low.
5. the first gold nano layer of substrate of the present invention and the second gold nano layer are hollow gold nanometer layer, and its chemical stability is good, and biocompatibility is good, the detection of DNA and other molecules also exists and applies widely.
Accompanying drawing explanation
Fig. 1-3 is the sample characterization picture of golden nanometer particle and the XRD curve of preparation, and Fig. 1 is the transmission electron microscope picture of hollow gold nanometer particle, the X-ray diffraction curve of Fig. 2 to be the high-resolution-ration transmission electric-lens figure of hollow gold nanometer particle, Fig. 3 be hollow gold nanometer particle.
Fig. 4 is the SEM phenogram of the hollow gold nanometer particle film of the method assembling adopting interface self assembly.
Fig. 5 is the raman scattering intensity curve of the complementary and incomplementarity single stranded DNA of single layer of gold nano particle sample assembly, corresponds to HGN-probeDNA-probeDNA(incomplementarity respectively) and HGN-probeDNA-reporterDNA(complementation) the raman scattering intensity curve of sample.
Fig. 6 is the SEM figure of Si/HGN-DNA-HGN.
Fig. 7 is the Raman curve of single layer of gold nano particle sample detection DNA raman scattering intensity and double-deck golden nanometer particle sample detection DNA intensity, corresponds to the raman scattering intensity curve of HGN-DNA and HGN-DNA-HGN sample respectively.
Embodiment
A kind of hollow gold nanometer particle-DNA composite substrate, be made up of the first hollow gold nanometer layer, DNA layer, the second hollow gold nanometer layer, described DNA layer is between the first hollow gold nanometer layer and the second hollow gold nanometer layer, and DNA layer is made up of the probeDNA strand described in SEQ ID NO.1 and the reporterDNA strand described in SEQ ID NO.2.
A preparation method for hollow gold nanometer particle-DNA composite substrate, the method comprises the following steps:
(1) the probeDNA strand as shown in SEQ ID NO.1 and the reporterDNA strand as shown in SEQ ID NO.2 is designed.
ProbeDNA is 5 '-acatagcacattcgaggtag-3 ';
ReporterDNA is 5 '-ctacctcgaatgtgctatgt-3 ';
(2) at the first hollow gold nanometer layer of the individual layer of Si substrate assembles one deck arrangement even compact;
First hollow gold nano-sphere is prepared, (the Y. Kobayashi that can be proposed by people such as Kobayashi; M. Horie; M. Konno; B.R. Gonzalez; L. Marzan, J. Phys. Chem. B, 2003 (107), 7420.) (the H.P. Liang that the people such as method, or L.J. Wan propose; L.J. Wan; C.L. Bai; L. Jiang; Gold Hollow Nanospheres:Tunable Surface Plasmon Resonance Controlled by Interior-Cavity Sizes. J. Phys. Chem. B 2005 (109); 7795-7800) method prepare different size and wall thickness hollow gold nanometer particle; and preparation process comparatively comprehensively in detail, and be relatively simply the people such as A.M. Schwartzberg (the A.M. Schwartzberg that disclosed method is carried out on J. Phys. Chem. B; T.Y. Olson; C.E. Talley; J.Z. Zhang; Synthesis, Characterization, and Tunable Optical Properties of Hollow Gold Nanospheres. J. Phys. Chem. B, 2006 (10), 19935-19944).Be specially:
(2.1) hollow gold nano-sphere is that template is synthesized with cobalt nanometer particle, so first synthesize cobalt nanoparticle: synthesis cobalt nanometer particle temperature will control at 35 DEG C, react under oxygen free condition, and require that reaction vessel must be clean, thus all glass apparatus clean up on pretreatment, soak the adsorbent of 1h remover wall surface again through chloroazotic acid, clean up oven dry with ultrapure water subsequently.600 μ L 0.1mol/L trisodium citrates are joined in the there-necked flask filling 75mL ultrapure water.Carry out under reaction is strict controlled in oxygen free condition, first logical argon gas deoxidation 30min in water, drains the oxygen comprised in reactant liquor only, adds the 0.4mol/LCoCl of 100 μ L subsequently
2solution, in there-necked flask, vacuumizes deoxidation and circulates three times, each 3 ~ 5min, under the state causing the space above reaction flask to be also in complete anaerobic, and strong agitation solution; Subsequently by the 1mol/LNaBH of fresh configuration
4solution extraction 100 μ L joins in the mixed solution of vigorous stirring, and greatly after 30s, colourless transparent solution can become taupe, now shows that cobalt nanometer particle is formed.
(2.2) synthesis of hollow gold nanometer particle is sacrifice template with cobalt nanometer particle, uses HAuCl
4carry out displacement reaction and obtain hollow gold nanometer particle: by the cobalt nanometer particle solution prepared in step (2.1), under ar gas environment protection, reaction 1h makes NaBH
4after complete hydrolysis, pipette the HAuCl that 30mL taupe reaction solution adds rapidly the 10mL0.6mmol/L of stirring
4in solution, solution becomes aubergine immediately, namely obtains the hollow gold nanometer particle hydrosol.Then by hollow gold nano-sphere hydrosol centrifuge washing twice, centrifugal rotational speed 8500rpm/min, each centrifugation time 15 minutes, finally this product being concentrated into 4mL(concentration is 1mmol/L).As shown in Figure 1, described hollow gold nanometer particle diameter is 30 ~ 40nm to Electronic Speculum characterization result, and the wall thickness of hollow gold nanometer particle is 6nm ~ 10nm.High-Resolution Map as shown in Figure 2, can see (111), (200) crystal face of hollow gold nanometer particle, and the XRD curve of product as shown in Figure 3, can see (111), (200), (220) of hollow gold nanometer particle, (311), (222) crystal face.
(2.3) silicon substrate is through acetone, absolute ethyl alcohol, ultrapure water successively ultrasonic cleaning, each ultrasonic 5 minutes of often kind of solution, stand-by after drying.
(2.4) hollow gold nanometer particle assembling on a silicon substrate, can with reference to people (N. Chandrasekharan such as P.V. Kamat; P.V. Kamat; Assembling Gold Nanoparticles as Nanostructured Films Using an Electrophoretic Approach. NANO LETTERS. 2001 (1) .67-70) adopt electrophoresis assembling golden nanometer particle to prepare the method for film; Or the people (J.Klug such as Joaqu í n Klug; L.A. Pe rez; E.A. Coronado; G.I. Lacconi; Chemical and Electrochemical Oxidation of Silicon Surfaces Functionalized with APTES:The Role of Surface Roughness in the AuNPs Anchoring Kinetics. J. Phys. Chem. C. 2013 (117), 11,317 11327) adopt the method for Electrostatic Absorption to assemble golden nanometer particle film; And (the Y.K. Park that the people such as Y.K. Park proposes; S. Park; Directing Close-Packing of Midnanosized Gold Nanoparticles at a Water/Hexane Interface) in the method etc. of water-oil interface Self-assembling of gold nanoparticles film.And wherein operate comparatively simple, minimum to the requirement of substrate, quick method is rapidly exactly adopt the method for interface self assembly to prepare golden nanometer particle film, assemble method is as follows: be in 1mmol/L hollow gold nanometer particle aqueous solution to 4mL concentration, dropwise add 300 μ L normal butyl alcohols, one deck hollow gold nanometer particle film will be formed at water-oil interface place, golden nanometer particle film is after aging 10 minutes, hollow gold nanometer particle film step 2.3 obtained with Si substrate picks up, fall the residue on surface with washes of absolute alcohol after oven dry, obtain the first hollow gold nanometer layer on a si substrate.The SEM of sample characterizes as shown in Figure 4, and particle-dense arranges, and is arranged as single layer structure.
(3) adopt the people such as Kang, T disclosed method on NANO LETTERS, probeDNA strand 5 terminal modified-(CH
2)
6-SH, obtains probeDNA-(CH
2)
6-SH; Then be the probeDNA-(CH of 1 μm of ol/L by the concentration of enough (40 μ L)
2)
6the aqueous solution (PH=7.1) of-SH strand is added drop-wise on the first hollow gold nanometer particle layer prepared by step (2), namely on the first hollow gold nanometer particle layer, probeDNA has been modified, incubated at room temperature 24h, unnecessary probe DNA is washed again by the lauryl sodium sulfate (SDS) that massfraction is 0.01%, first hollow gold nanometer layer modifies probeDNA strand, is labeled as Si/HGN-probeDNA;
(4) adopt the people such as Kang, T disclosed method on NANO LETTERS, reporterDNA strand 5 terminal modified-(CH
2)
3-SH, obtains reporterDNA-(CH
2)
3-SH; By the PBS solution of 500 μ L PH=7.1,100 μ L massfractions be 0.1% lauryl sodium sulfate, 10 μ L concentration be 1mmol/L reporterDNA-(CH
2)
3after the aqueous solution (PH=7.1) of-SH strand and the 400 μ L gold hollow ball hydrosol (concentration is 1mmol/L) mix, leave standstill 6h, then salinization is carried out, salinization is: point to add NaCl solution that isopyknic concentration increases progressively successively for 5 times in mixed solution, NaCl solution adds 100 μ L at every turn; The concentration dripping NaCl solution is first 2mol/L, and the concentration gradient of NaCl solution is 0.05mol/L; 24h, centrifugal 7min under 7000rpm/min is left standstill after salinization; Remove supernatant, add 1mL massfraction be 0.01% lauryl sodium sulfate again centrifuge washing remove supernatant, the PBS buffer solution of precipitation 1mL, obtains the PBS solution of reporterDNA-HGN; Disclosed method can referring to document Kang, T on NANO LETTERS for the people such as above-mentioned Kang, T; Yoo, SM; Yoon, I; Lee, SY; Kim, B. Patterned Multiplex Pathogen DNA Detection by Au Particle-on-Wire SERS Sensor. NANO LETTERS. 2010 (10), 1189-1193.
The reporterDNA-HGN solution that (5) step 4 prepared (
40 μ L) be added drop-wise to Si/HGN-probeDNA surface,
make reporterDNA and probeDNA that complementary pairing occur.after incubation at room temperature 24h, with lauryl sodium sulfate aqueous solution and water cleaning that massfraction is 0.01%, on the first hollow gold nanometer layer, by two complementary DNA single chains and then assemble the second hollow gold nanometer layer, obtain hollow gold nanometer particle-DNA composite substrate.
embodiment 1
The present embodiment prepares individual layer hollow gold nanometer particle-DNA Rotating fields, i.e. Si/HGN-DNA structure, and carries out Raman active test.
The DNA single chain of design sample Si/HGN-probeDNA-probeDNA(incomplementarity pairing) and the DNA single chain of Si/HGN-probeDNA-reporterDNA(complementary pairing) structure.
Independent salinization probeDNA and reporterDNA process: be the reporterDNA of 1mmol/L by the PBS solution of 900 μ LPH=7.1,100 μ L0.1% lauryl sodium sulfate and 10 μ L1mmol/LprobeDNA(or 10 μ L concentration) mixing, carry out salinization after this mixed solution being left standstill 6h.Salinization is: point to add NaCl solution that isopyknic concentration increases progressively successively for 5 times in mixed solution, NaCl solution adds 100 μ L at every turn; The concentration dripping NaCl solution is first 2mol/L, and the concentration gradient of NaCl solution is 0.05mol/L; After adding NaCl solution 5 times, in final solution, NaCl concentration is 0.7mol/L.Mixed liquor is centrifugal after leaving standstill 24h, centrifugal rotational speed 7000rpm/min, and centrifugation time is 7min; After being absorbed by supernatant after centrifugal, add 1mL0.01% lauryl sodium sulfate centrifuge washing again, the PBS buffer solution of the lower sediment 1mL after finally being removed by supernatant is for subsequent use.
Complete incomplementarity DNA(probeDNA) and the assembling process of complementary (reporterDNA) DNA on hollow gold nanometer particle film: get probeDNA(or reporterDNA after the salinization of 50 μ L) solution is added drop-wise to Si/HGN-probeDNA surface, after incubation at room temperature 24h, with 0.01% lauryl sodium sulfate and water cleaning, after vacuum drying, identified as samples is designated as Si/HGN-probe/probeDNA(or Si/HGN-probe/reporterDNA).
Obtained Si/HGN-DNA sample is carried out Raman active test, and as shown in Figure 5, result shows the result after test: the Raman signal of the sample Si/HGN-probe/probeDNA of incomplementarity pairing is very faint, and first signal peak is the characteristic peak of Si substrate; In addition, the Raman signal of the sample Si/HGN-probe/reporterDNA of DNA complementary pairing will much larger than the Raman signal of the sample Si/HGN-probe/probeDNA of DNA incomplementarity pairing.
embodiment 2
The present embodiment prepares double-layer hollow golden nanometer particle layer, and interlayer is the compound substance of DNA layer, i.e. Si/HGN-DNA-HGN structure, and carries out Raman active test:
ReporterDNA is connected on the hollow gold nanometer particle in the hydrosol: by the mixing of the PBS solution of 500 μ LPH=7.1,100 μ L0.1% lauryl sodium sulfate, 10 μ L1mmol/LreporterDNA and the 400 μ L gold hollow ball hydrosol, after this mixed solution being left standstill 6h, carry out salinization.Salinization is: point to add NaCl solution that isopyknic concentration increases progressively successively for 5 times in mixed solution, NaCl solution adds 100 μ L at every turn; The concentration dripping NaCl solution is first 2mol/L, and the concentration gradient of NaCl solution is 0.05mol/L; After adding NaCl solution 5 times, in final solution, NaCl concentration is 0.7mol/L.Mixed liquor is centrifugal after leaving standstill 24h, centrifugal rotational speed 7000rpm/min, and centrifugation time is 7min; After being absorbed by supernatant after centrifugal, add 1mL0.01% lauryl sodium sulfate centrifuge washing again, the PBS buffer solution of the lower sediment 1mL after finally being removed by supernatant is for subsequent use, and it is labeled as reporterDNA-HGN.
Complete the assembling process of reporterDNA-HGN on hollow gold nanometer particle film: the reporterDNA-HGN got after the salinization of 50 μ L is added drop-wise to Si/HGN-probeDNA surface, after incubation at room temperature 24h, with 0.01% lauryl sodium sulfate and water cleaning, after vacuum drying, identified as samples is designated as Si/HGN-DNA-HGN.The SEM of obtained Si/HGN-DNA-HGN sample characterizes as shown in Figure 6.
Obtained Si/HGN-DNA-HGN sample is carried out Raman active test, result after test compares with the Raman test result of Si/HGN-DNA sample, as shown in Figure 7, result display: the intensity of the Raman signal of double-layer hollow golden nanometer particle film sample Si/HGN-DNA-HGN is 6.5 ~ 8.4 times of the intensity of the Raman signal of individual layer hollow gold nanometer particle film.
(L. Fabris compared with job family; M. Dante; G. Braun; S.J. Lee; N.O. Reich; M. Moskovits; T.Q. Nguyen; G.C. Bazan; A Heterogeneous PNA-Based SERS Method for DNA Detection. J. AM. CHEM. SOC. 2007 (129), 6086-6087), the Raman signal of individual layer hollow gold nanometer particle film sample Si/HGN-DNA (complementation) is 2 times of its signal; The peak of the Raman signal of double-layer hollow golden nanometer particle film sample Si/HGN-DNA-HGN is by force 9-10 times of its signal intensity.This research can provide technical scheme support for the work of Related Research Domain.
Above-described embodiment is used for explaining and the present invention is described, instead of limits the invention, and in the protection domain of spirit of the present invention and claim, any amendment make the present invention and change, all fall into protection scope of the present invention.
SEQUENCE LISTING
<110> Institutes Of Technology Of Zhejiang
<120> hollow gold nanometer particle-DNA composite substrate and preparation method thereof
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 20
<212> DNA
<213> Prof. Du Yucang
<400> 1
acatagcaca ttcgaggtag 20
<210> 2
<211> 20
<212> DNA
<213> Prof. Du Yucang
<400> 2
ctacctcgaa tgtgctatgt 20
Claims (6)
1. a hollow gold nanometer particle-DNA composite substrate, it is characterized in that: be made up of the first hollow gold nanometer layer, the second hollow gold nanometer layer and the DNA layer that connects the first hollow gold nanometer layer and the second hollow gold nanometer layer, DNA layer is made up of the probeDNA strand described in SEQ ID NO.1 and the reporterDNA strand described in SEQ ID NO.2.
2. a preparation method for hollow gold nanometer particle-DNA composite substrate according to claim 1, it is characterized in that, the method comprises the following steps:
(1) the probeDNA strand as shown in SEQ ID NO.1 and the reporterDNA strand as shown in SEQ ID NO.2 is designed;
(2) at Si substrate assembles first hollow gold nanometer particle layer;
(3) the probeDNA strand that on the first hollow gold nanometer layer prepared in step (2), modification step (1) designs, Product Labeling is Si/HGN-probeDNA;
(4) step (1) design reporterDNA strand is modified on hollow gold nanometer particle, obtain reporterDNA-HGN solution;
(5) the reporterDNA-HGN solution that step (4) prepares is dripped to Si/HGN-probeDNA surface, after incubation at room temperature 24h, with lauryl sodium sulfate aqueous solution and water cleaning that massfraction is 0.01%, on the first hollow gold nanometer layer, assemble the second hollow gold nanometer layer by two complementary single stranded DNAs, obtain hollow gold nanometer particle-DNA composite substrate.
3. method according to claim 2, is characterized in that, described step 2 is specially:
(2.1) be in 1mmol/L hollow gold nanometer particle aqueous solution to 4mL concentration, dropwise add 300 μ L normal butyl alcohols, form water-oil interface, there is one deck hollow gold nanometer particle film at water-oil interface place;
(2.2) the golden nanometer particle film at water-oil interface place is after aging 10 minutes, hollow gold nanometer particle film step 2.1 obtained with Si substrate picks up, fall the residue on surface after 60 DEG C of oven dry with washes of absolute alcohol, obtain the first hollow gold nanometer particle layer on a si substrate.
4. hollow gold nanometer particle according to claim 2, is characterized in that: the external diameter of described hollow gold nanometer particle is 30 ~ 40nm, and the wall thickness of hollow gold nanometer particle is 6nm ~ 10nm.
5. method according to claim 2, is characterized in that, described step 3 is specially: probeDNA strand 5 terminal modified-(CH
2)
6-SH, obtains probeDNA-(CH
2)
6-SH; Be the probeDNA-(CH of 1 μm of ol/L by enough concentration
2)
6the aqueous solution (PH=7.1) of-SH strand is added drop-wise on the first hollow gold nanometer particle layer prepared by step (2), makes probeDNA-(CH
2)
6-SH infiltrates the first hollow gold nanometer particle layer, incubated at room temperature 24h, then washes unnecessary probe DNA by the lauryl sodium sulfate that massfraction is 0.01%, and 60 DEG C of vacuum drying are for subsequent use.
6. method according to claim 2, is characterized in that, described step 4 is specially: reporterDNA strand 5 terminal modified-(CH
2)
3-SH, obtains reporterDNA-(CH
2)
3-SH; By the PBS solution of 500 μ L PH=7.1,100 μ L massfractions be 0.1% lauryl sodium sulfate, 10 μ L concentration be 1 μm of ol/L reporterDNA-(CH
2)
3the aqueous solution (PH=7.1) of-SH strand and 400 μ L concentration are after the golden hollow ball hydrosol of 1mmol/L mixes, leave standstill 6h, then salinization is carried out, salinization is: point to add NaCl solution that isopyknic concentration increases progressively successively for 5 times in mixed solution, NaCl solution adds 100 μ L at every turn; The concentration dripping NaCl solution is first 2mol/L, and the concentration gradient of NaCl solution is 0.05mol/L; 24h, centrifugal 7min under 7000rpm/min is left standstill after salinization; After removing supernatant, add 1mL massfraction be 0.01% lauryl sodium sulfate again centrifuge washing remove supernatant, the PBS buffer solution of precipitation 1mL, obtains the PBS solution of reporterDNA-HGN.
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