CN104759620A - Method for modifying HS-DNA rapidly on gold nanorods - Google Patents
Method for modifying HS-DNA rapidly on gold nanorods Download PDFInfo
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Abstract
The invention discloses a method for modifying HS-DNA rapidly on gold nanorods. The method comprises the following steps that 1, in the presence of CTAB, the gold nanorods with stable CTAB bilayer are compounded through a growth method of seeds; 2, CTAB molecules on the surface of the god nanorods are replaced through centrifugation and a resuspending method of mPEG-SH and Tween20 mixed liquor, and the mPEG-SH and the Tween20 are modified on the gold nanorods; 3, HS-DNA and acid with high concentration or salt with high concentration are added into gold nanorod solutions which are modified by the mPEG-SH and the Tween20, and aging is conducted for one hour at the temperature of 20-40 DEG C; 4, the gold nanorods which are modified with the HS-DNA can be obtained by removing free HS-DNA through centrifugation and buffer solution resuspending. The method for modifying the HS-DNA rapidly on the gold nanorods is rapid, efficient and economical, high stability of the gold nanorods is achieved, the aging process of slowly increasing salt concentration is avoided, the modification steps of modifying the HS-DNA on the gold nanorods are simplified, and the modification time is shortened.
Description
Technical field
The present invention relates to a kind of method that mPEG-SH and Tween 20 assists Rapid Modification HS-DNA on gold nanorods, belong on gold nanorods and modify HS-DNA Technical Development Area.
Background technology
Bar-shaped gold nano can mediate seed growth simply by CTAB and obtain different size and draw ratio, therefore has the characteristic of much special optics and electricity.These good characteristics of gold nanorods make it have to comprise the extensive use of acology and diagnostics.(1, Khlebtsov, N.; Dykman, L.Biodistribution and toxicity of engineeredgold nanoparticles:a review of in vitro and in vivo studies [J] .Chem.Soc.Rev.2011,40,1647-1671; 2, Khlebtsov, N.; Dykman, L.Biodistribution and toxicity of engineered goldnanoparticles:a review of in vitro and in vivo studies [J] .Chem.Soc.Rev.2011,40,1647-1671) comprise in the middle of these application as assemble nanometer particle, gene therapy, cell-targeting, bio-sensing, medicine carrying and cell adherence, it is a very crucial step that gold nanorods is modified HS-DNA.(3, Grzelczak, M.; Vermant, J.; Furst, E.M.; Liz-Marz á n, L.M.Directed self-assembly of nanoparticles [J] .ACS Nano 2010,4,3591-3605; 4, Chen, C.-C.; Lin, Y.-P.; Wang, C.-W.; Tzeng, H.-C.; Wu, C.-H.; Chen, Y.-C.; Chen, C.-P.; Chen, L.-C.; Wu, Y.-C.DNA-Gold Nanorod Conjugates for Remote Control of LocalizedGene Expression by near Infrared Irradiation [J] .J.Am.Chem.Soc.2006,128,3709-3715; 5, Huang, Y.-F.; Chang, H.-T.; Tan, W.Cancer cell targeting using multiple aptamers conjugated onnanorods [J] .Anal.Chem.2008,80,567-572; 6, Spadavecchia, J.; Barras, A.; Lyskawa, J.; Woisel, P.; Laure, W.; Pradier, C.-M.; Boukherroub, R.; Szunerits, S.Approach for PlasmonicBased DNA Sensing:Amplification of the Wavelength Shift and Simultaneous Detection of thePlasmon Modes of Gold Nanostructures [J] .Anal.Chem.2013,85,3288-3296; 7, Yang, X.; Liu, X.; Liu, Z.; Pu, F.; Ren, J.; Qu, X.Near ?Infrared Light ?Triggered, Targeted Drug Delivery toCancer Cells by Aptamer Gated Nanovehicles [J] .Adv.Mater.2012,24,2890-2895) but, the method of modifying HS-DNA at present on gold nanorods is very consuming time loaded down with trivial details, the gold nanorods that particularly, draw ratio large for particle diameter is little.(8, Vial, S.; Nykypanchuk, D.; Yager, K.G.; Tkachenko, A.V.; Gang, O.LinearMesostructures in DNA-Nanorod Self-Assembly [J] .ACS Nano 2013,7,5437-5445) this is because the synthetic method of current gold nanorods is based on CTAB mediation synthesis mostly, and CTAB can form CTAB bilayer on the surface of gold nanorods.(9, Jana, N.R.; Gearheart, L.; Murphy, C.J.Seed-mediated growth approachfor shape-controlled synthesis of spheroidal and rod-like gold nanoparticles using a surfactanttemplate [J] .Adv.Mater.2001,13,1389; 10, Nikoobakht, B.; El-Sayed, M.A.Evidence forbilayer assembly of cationic surfactants on the surface of gold nanorods [J] .Langmuir 2001,17,6368-6374) the CTAB bilayer of positively charged can hinder and form golden sulfide linkage between gold nanorods and HS-DNA and can not be attached on gold nanorods; Secondly it can nonspecific absorption make gold nanorods reunite with the DNA of negative electricity.In addition, CTAB has very strong cytotoxicity, limits the application of gold nanorods in the middle of biomedicine greatly.(11、Takahashi,H.;Niidome,Y.;Niidome,T.;Kaneko,K.;Kawasaki,H.;Yamada,S.Modification of goldnanorods using phosphatidylcholine to reduce cytotoxicity[J].Langmuir 2006,22,2-5)
Therefore, develop method that is easy, Rapid Modification HS-DNA on gold nanorods and have important meaning.
Summary of the invention
For existing gold nanorods being modified the HS-DNA problem such as loaded down with trivial details consuming time, the first object of the present invention be to propose a kind of fast, method that is simple, protection gold nanorods efficiently.
Second object of invention be by the method for this protection gold nanorods proposed for fast, simple, on gold nanorods, modify HS-DNA efficiently.
The method of described Rapid Modification HS-DNA on gold nanorods, comprises the steps:
(1) under CTAB exists, by the gold nanorods that the method synthesis CTAB bilayer of seed growth is stable;
(2) by the centrifugal CTAB molecule replacing gold nanorods surface again by the method that mPEG-SH is resuspended with the nonionic surface active agent mixed liquor with polyethylene glycol structures, mPEG-SH and surfactant on gold nanorods is modified, wherein, the mixed proportion scope between mPEG-SH and surfactant is 1-25% (w/w);
(3) in the gold nanorods solution being modified with mPEG-SH and surfactant, add acid or the salt of HS-DNA and high concentration, 20-40 DEG C of aging 0.5-5 hour, the acid of described high concentration or salt concentration range are 100-1000mM;
(4) just can obtain with the HS-DNA that the resuspended removal of buffer solution dissociates the gold nanorods being modified with HS-DNA again by centrifugal.
Wherein, step of the present invention (1) is prior art, and its concrete synthetic method can with reference to background technology.Gold nanorods in step (1) is the difform gold nano comprising club shaped structure.
Wherein, the nonionic surface active agent that the present invention has polyethylene glycol structures includes but not limited to TW20, F127, Tween 80 and Triton X100.
Wherein, in step (2), the mixed proportion of mPEG-SH and surfactant ranges preferably from 1-25% (w/w),
The intermixture of mPEG-SH and surfactant and the mixed proportion of gold nanorods range preferably from 2-200:1 (w/w),
Step (2) centrifugal force scope is 8000-14000rpm, or alternate manner obtains gold nanorods precipitation.
Wherein, in step (2), the molecular weight ranges of mPEG-SH can be 0.75-20kDa kDa, and the best is 5kDa, mPEG-SH concentration range is 1-125 μM.
Be in step (2), the preferred Tween 20 of surfactant, its concentration is higher than 0.01wt%, and preferred concentration range is 0.01-1wt%.
Be in step (3), the acid of high concentration can citric acid or phosphoric acid, and salt can be NaCl or MgCl
2in one, preferred MgCl
2.
Described high concentration is higher than 100mM.Preferred concentration range is 400-800mM.
The HS-DNA added can be 0.1-40 μM, and preferable range is 1-10 μM.
Wherein, the preferred centrifugal force scope of step (4) is 8000-14000rpm.
The buffer solution of step (4) can select the one in PBS, Tris-HCl and HEPES.
The resuspended condition of step (2) and (4) is preferably 4000rpm vibration 10s.
The invention has the advantages that: first, undertaken resuspended by centrifugal rear mPEG-SH and Tween 20 mixed liquor, replace the part CTAB on gold nanorods surface with mPEG-SH and Tween 20, enable gold nanorods stable be scattered in high ionic strength and extreme pH condition under; Secondly, can the disposable salt adding HS-DNA and high concentration in the gold nanorods that mPEG-SH and Tween 20 protects, make gold nanorods can complete the modification of HS-DNA in a short period of time; Again, gold nanorods is modified HS-DNA quantity can the ratio by mPEG-SH, Tween 20 and gold nanorods and the kind of aging required salt and concentration regulate and control; In addition, the mPEG-SH on gold nanorods can increase biocompatibility, can carry out Raman image and drug carrier as a protective layer used molecule in stabilized zone positive electricity simultaneously; Finally, Rapid Modification HS-DNA on the difform gold nano grain that such method of modifying can be applicable to comprise bar-shaped gold nano.Compared with conventional method, this method is simple, efficient, highly versatile, for the compound of DNA and gold nanorods improves wide application prospect in bioanalysis and biomedicine.
Accompanying drawing explanation
Fig. 1 is the modification schematic diagram that (A) conventional method and (B) mPEG-SH and Tween 20 auxiliary law modify HS-DNA on gold nanorods.
Fig. 2 is the sign of the gold nanorods synthesizing gold nanorods gold seeds used and different draw ratio.(A) Electronic Speculum characterizes the gold nanorods of gold seeds and different draw ratio; (B) absorption spectrum of UV Vis Spectroscopic Characterization gold seeds; (C) absorption spectrum of the gold nanorods of the different draw ratio of UV Vis Spectroscopic Characterization.
Fig. 3 is the feasibility checking of mPEG-SH and Tween 20 pairs of gold nanorods stabilizations.(A) gold nanorods mPEG-SH, Tween 20 under individualism or mPEG-SH and Tween 20 jointly exist under centrifugal resuspended after dispersiveness in variable concentrations NaCl; (B) mPEG-SH and Tween 20 jointly exist lower centrifugal resuspended after dispersiveness in different pH; (C) dispersiveness after the lower centrifugal resuspended different number of times of the common existence of mPEG-SH and Tween 20.
Fig. 4 be gold nanorods mPEG-SH and 0.01wt%Tween 20 of different size and variable concentrations centrifugal resuspended after dispersiveness in 800mM NaCl.
Fig. 5 be gold nanorods the mPEG-SH of 200nM 5kDa and variable concentrations different surfaces activating agent centrifugal resuspended after dispersiveness in 800mM NaCl.
Fig. 6 is CTAB, mPEG-SH, mPEG-SH and Tween 20 x-ray photoelectron energy spectrogram of gold nanorods of mixed liquor protection.
Fig. 7 is the C of the gold nanorods of (A) CTAB, mPEG-SH, mPEG-SH and Tween 20 mixed liquor protection
1shigh-resolution x-ray photoelectron energy spectrogram, and the peak intensity after corresponding normalization; (B) CTAB, mPEG-SH, mPEG-SH and Tween 20 N of gold nanorods of mixed liquor protection
1shigh-resolution x-ray photoelectron energy spectrogram, and the peak intensity after corresponding normalization.
Fig. 8 is the influence factor of gold nanorods being modified HS-DNA amount.(A) impact of variable concentrations 5kDa mPEG-SH; (B) impact of variable concentrations Tween 20; (C) with 1M NaCl, 100mM MgCl
2with the impact of 100mM citric acid as different ageing time when aging dose.
Detailed description of the invention
The synthesis of embodiment 1 gold nanorods
First synthesize the gold seeds needed for gold nanorods growth, in the CTAB aqueous solution of 0.5mL 0.2M, add 0.5mL0.5mM HAuCl
4mix after the aqueous solution, in above-mentioned mixed liquor, then add the 10mMNaBH of 0.06mL frozen water configuration
4, concuss 2min.At this moment the solution colour can observing gold seeds becomes faint yellow by colourless in 1min, and room temperature places 30min makes unnecessary sodium borohydride decompose.With the pattern and the ultraviolet-visible absorption spectroscopy that project Electronic Speculum and UV Vis Spectroscopic Characterization gold seeds.
The synthetic method of the gold nanorods of bone-shaped is as follows, adds 50mL 0.2M CTAB and 0.6mL 10mM AgNO under the condition stirred continuously in 100mL round-bottomed flask
3, and then add 50mL 1mM HAuCl
4with 0.6mL 0.4M ascorbic acid.Finally add the gold seeds of the above-mentioned middle synthesis of 120uL, trigger the growth of gold nanorods.The synthesis of gold nanorods is completed grow 5h in the water-bath of 27 DEG C after.The synthetic method of the gold nanorods of different-grain diameter and different draw ratio is all described above, the AgNO just added
3, HAuCl
4different with the concentration of ascorbic acid.The synthesis condition of the gold nanorods of different-grain diameter and draw ratio is in table 1.The pattern of the gold nanorods synthesized with projection Electronic Speculum and UV Vis Spectroscopic Characterization and ultraviolet-visible absorption spectroscopy (see Fig. 2).The concentration of this method synthesis gold nanorods is about 0.1nM.
The gold nanorods synthesis condition of table 1, different-grain diameter and draw ratio
Embodiment 2 gold nanorods modifies HS-DNA
To get in 1mL embodiment 1 the gold nanorods solution of synthesis, centrifugal 10min under 10000rpm condition, remove supernatant, add thermal agitation 20s after 50uL 20uM mPEG-SH, then add the resuspended gold nanorods of 1mL 0.01wt%Tween 20.The resuspended process of centrifugal rear mPEG-SH and Tween 20 in above-mentioned repeats 3 times, makes mPEG-SH and Tween20 can replace the CTAB (see Fig. 3,4 and 5) on gold nanorods surface thoroughly.The replacement degree (see Fig. 6 and 7) of mPEG-SH and Tween 20 couples of gold nanorods surface C TAB is analyzed with x-ray photoelectron spectroscopy.The HS-DNA after 8uL 100 μMs activation and 50 μ L, 20 μMs of MgCl is added in the gold nanorods that mPEG-SH and Tween20 protects
2, after aged at room temperature 1h, just can obtain with the centrifugal resuspended HS-DNA dissociated that removes for 3 times of PBS the gold nanorods modifying HS-DNA.
The quantitative gold nanorods of embodiment 3 is modified the amount of HS-DNA
Fluorescein-labeled sulfydryl DNA (sequence: ATT GAC CGC TGT GTG ACG CAA CAC TCAATTTTTTTTTTTTT (FAM)-SH) modifies on gold nanorods by the method in embodiment 2, then adds mercaptoethanol and makes the final concentration of mercaptoethanol reach 20mM.After 37 DEG C of reaction 5h, mercaptoethanol can replace the HS-DNA on gold nano surface, makes gold nanorods occur reunite and solution is become colorless.Remove gold nanorods precipitation by the centrifugal 10min of 10000rpm, and measure the fluorescence intensity of HS-DNA in supernatant.The concentration of modifying the HS-DNA on gold nanorods can obtain by contrasting the fluorescence intensity of HS-DNA and the standard working curve of HS-DNA fluorescence intensity and concentration in supernatant, just can obtain the number (see Fig. 8) of single gold nanorods being modified HS-DNA by the concentration of HS-DNA divided by the concentration of gold nanorods.
Claims (7)
1. a method of Rapid Modification HS-DNA on gold nanorods, comprises the steps:
(1) under CTAB exists, by the gold nanorods of the method synthesis CTAB bilayer of seed growth;
(2) by the centrifugal CTAB molecule replacing gold nanorods surface again by the method that mPEG-SH is resuspended with the nonionic surface active agent mixed liquor with polyethylene glycol structures, mPEG-SH and surfactant on gold nanorods is modified; Wherein, mPEG-SH: the proportion between surfactant is 1-25% (w/w);
(3) in the gold nanorods solution being modified with mPEG-SH and surfactant, acid or the salt of HS-DNA and high concentration is added, 20-40 DEG C of aging 0.5-5 hour; The acid of described high concentration or salt concentration range are 100-1000mM;
(4) obtain with the HS-DNA that the resuspended removal of buffer solution dissociates the gold nanorods being modified with HS-DNA again by centrifugal.
2. the method for Rapid Modification HS-DNA on gold nanorods as claimed in claim 1, is characterized in that the gold nanorods described in step (1) comprises the difform gold nano of club shaped structure.
3. the method for Rapid Modification HS-DNA on gold nanorods as claimed in claim 1, is characterized in that the molecular weight ranges of mPEG-SH in step (2) is 0.75-20kDa.
4. the method for Rapid Modification HS-DNA on gold nanorods as claimed in claim 1, it is characterized in that the nonionic surface active agent with polyethylene glycol structures described in step (2), comprise at least one in Tween 20, F127, Tween 80 and TritonX100.
5. the method for Rapid Modification HS-DNA on gold nanorods as claimed in claim 4, is characterized in that the concentration of Tween 20 in step (2) is higher than 0.01wt%.
6. the method for Rapid Modification HS-DNA on gold nanorods as claimed in claim 1, is characterized in that the acid described in step (3) comprises citric acid or phosphoric acid.
7. the method for Rapid Modification HS-DNA on gold nanorods as claimed in claim 1, is characterized in that the salt in step (3) comprises magnesium chloride.
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