CN101224502A - Highly concentrated nano-Au solution, concentrating method and applications thereof - Google Patents
Highly concentrated nano-Au solution, concentrating method and applications thereof Download PDFInfo
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- CN101224502A CN101224502A CNA2007100193907A CN200710019390A CN101224502A CN 101224502 A CN101224502 A CN 101224502A CN A2007100193907 A CNA2007100193907 A CN A2007100193907A CN 200710019390 A CN200710019390 A CN 200710019390A CN 101224502 A CN101224502 A CN 101224502A
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Abstract
The invention discloses a concentration method of a nano gold solution, comprising that a nano gold solution which is prepared by a chlorine gold acid solution is reduced by a reducing agent and further concentrated by a high-speed centrifugation. The centrifugal rotation speed of the high-speed centrifugation ranges from 8000-16000rpm and the centrifugation time ranges from 10-30 minutes. The invention also provides a high-consistency nano gold solution which is prepared by the concentration method; wherein, the nano gold grain granularity is uniformly distributed and the solution has higher salt tolerance. The high-consistency nano gold solution of the invention is applied to a DNA molecule self-assembly and the prepared Au-DNA composite matter has better acid tolerance and stability.
Description
Technical field
The present invention relates to the nm of gold technical field, particularly a kind of method for concentration of nano-Au solution and the highly concentrated nano-Au solution that makes thereof, and the application of this highly concentrated nano-Au solution in the dna molecular self assembly.
Background technology
Since the eighties of last century the nineties, nano material is widely used in fields such as molecular biology, biology sensor owing to have unique physicochemical characteristics, quantum size effect and high-ratio surface effect.Wherein, nano Au particle is owing to having excellent optical property, electrical properties, chemism, bio-compatibility, being most widely used of biological field.Can trace back to the application of the seventies immunity gold in last century in bio-imaging the earliest.1996, Mirkin seminar realized the assembling of sulfydryl modification DNA on the nano Au particle surface first, was prepared into nm of gold-DNA composite nanostructure.Based on this discovery, their seminar has delivered a series of papers on Science, of the regulation and control of biomolecule identifying have been reported for the nm of gold photoelectric property, and the biological detecting method that a series of ultra-high sensitives detect DNA and protein has been proposed on this basis, made up nanometer detection platform based on the nm of gold colorimetric method.Based on this, carried out a series of biological detection relevant again with nm of gold, set up the electrochemical detection method that amplifies based on nm of gold, realized quick, the Sensitive Detection of target substances of multiple types.Compare with detection methods such as traditional ultraviolet, fluorescence, this method need not to depend on large-scale instrument, can directly detect by an unaided eye, be a kind of low cost, simple and efficient detection method, in various fields such as medical diagnosis on disease, food security, biological anti-terrorism, environment, have boundless application prospect.
The physicochemical properties of nano material are big or small directly related with particle.Interactional dynamics of the nanogold particle of different-grain diameter, variable concentrations and DNA and macroscopic property have significant difference.For example, DNA is difficult to effectively assemble on the nm of gold less than 5nm, and particle diameter is when excessive, and particle itself is unstable, and agglomeration takes place easily.Find also in the practice that in addition the assembling of DNA in highly concentrated nano-Au is faster also more stable, and can improve the utilization rate of DNA greatly, these have all proposed higher requirement to the uniformity and the concentration of nm of gold particle diameter.At present commercially available nm of gold is based on Sigma, and its particle diameter is even, but concentration generally about 0.01w/w% (with the gold chloride densimeter) is relatively low, can not satisfy the requirement of detection, and price is higher relatively.Therefore the nm of gold that oneself prepares is still adopted in most of laboratories, and main method is that reduction obtains to gold chloride with reducing agents such as trisodium citrate, tannic acid-trisodium citrate, sodium borohydrides.But the uniformity aspect of particle diameter is not ideal enough when preparing highly concentrated nano-Au at present, and reason is shape and the uniformity coefficient that the size of gold chloride concentration can directly influence nm of gold.And to set up the detection method of high sensitivity and reproducibility, and depending on the quality of nm of gold material to a great extent, the nano-Au solution that therefore can prepare good uniformity, high concentration is most important for subsequent detection.Chinese patent literature discloses a series of nanogold particle, but normally the surface has coated macromolecular material such as PVP etc., though the nanogold particle stability that obtains is higher, but, can't be applied to dna molecular self assembly experiment (referring to the patent documentation of publication number CN1663714A, CN1806973A) owing to changed the character and the electric charge of particle surface.
Summary of the invention
The technical problem to be solved in the present invention is to overcome in the prior art preparation highly concentrated nano-Au solution aspect to exist the uneven shortcoming of size distribution, the nano-Au solution of the highly homogeneous high concentration of a kind of nanogold particle is provided, and makes its method for concentration.
For solving the problems of the technologies described above, the inventor has carried out many experimental studies, discovery will be adopted prior art, the nano-Au solution that promptly utilizes reducing agent reduction chlorauric acid solution to make further adopts supercentrifugal process, carry out centrifugation by certain rotating speed and time, discard after the supernatant of respective volume or the abandoning supernatant again according to required concentration that the solution with respective volume dilutes, can prepare the nanogold particle of high concentration and high homogeneity.
Therefore, the method for concentration of nano-Au solution of the present invention, comprise that will reduce the nano-Au solution that chlorauric acid solution makes by reducing agent further adopts supercentrifugal process to concentrate again, the centrifugal rotational speed of described supercentrifugal process is between 8000-16000rpm, and centrifugation time is 10-30 minute.
The core of method for concentration of the present invention is to control centrifugal rotational speed and centrifugation time, makes the nanogold particle uniform particle diameter that makes.Because the inventor finds after deliberation, the common too high irreversible aggrengation that causes easily of centrifugal speed, too low then part nm of gold can not centrifugal be got off; And oversize reduction operating efficiency of time also can cause irreversible gathering, and too short then part nm of gold can not centrifugal be got off.
Wherein, the present invention's technology of utilizing reducing agent reduction chlorauric acid solution to make nano-Au solution can adopt prior art, technology as described in the background art.The present invention according to document (as Grabar, K.C.Freeman, R.G.Hommer, M.B.Natan, M.J.Anal.Chem.1995,67,735-743) chlorauric acid solution of reduction variable concentrations prepares variable concentrations, the nano-Au solution that the nanogold particle particle diameter is similar, can also be according to document (" practical immunocytochemistry and making nucleic acid molecular hybridization technology ", Cai Wenqin, Wang uncle Yun etc., Chengdu: Sichuan science tech publishing house, 1994,479-582) the reduction mass percent concentration is the nanogold particle that 0.01% chlorauric acid solution prepares average grain diameter 5-150nm.
The reducing agent of above-mentioned 10-150nm nm of gold is selected trisodium citrate (citric acid trisodium) usually for use, and the mass percent concentration of described chlorauric acid solution is generally 0.001%～0.1%, preferably, selects 0.01% for use.The reducing agent of above-mentioned 5-10nm nm of gold preparation is selected the mixed liquor of trisodium citrate or tannic acid and trisodium citrate for use.
Preferably, the average grain diameter of the nanogold particle that the present invention makes is 5-40nm, more preferably is 10-40nm.
The present invention concentrates the nm of gold that prior art makes by centrifugal process, and the low concentration nanogold particle that is about to above-mentioned existing method preparation suspends after carrying out removing the part supernatant behind the high speed centrifugation again, obtains different highly concentrated nano-Aus as required.Specifically, get the low concentration nanogold particle of 1-10mL preparation, under 4 ℃ of conditions, rotating speed when centrifugal is between 8000-16000rpm, behind the centrifugal 10-30min, remove the supernatant of different volumes as required, and use the surplus solution resuspension again, obtain the nanogold particle of concentration from variable concentrations between the 5nM-100nM.Can the nanogold particle of 5-150nm be concentrated according to the method.
Thus, can make the nano-Au solution of high concentration of the present invention.Its not only more original nano-Au solution has improved concentration, and the nanogold particle uniform particle diameter that obtains.The present invention is successfully applied to dna molecular self assembly aspect with it, and the nm of gold that makes-DNA compound has better salt tolerance and stability, lays the foundation in the application of other field for further expanding nm of gold.
The advantage of the method for concentration of nano-Au solution of the present invention is easy and simple to handle, fast, good reproducibility, the nanogold particle narrow particle size distribution that obtains is all showing good stability aspect salt tolerance and the DNA assembling.
Description of drawings
Fig. 1 is the afm image of the 13nm nm of gold of variable concentrations; A is the 3.5nM nm of gold (low concentration group) of prior art for preparing, the 17.5nM nm of gold (concentrate group) of B for adopting method for concentration preparation of the present invention, and C is the 17nM nm of gold of prior art for preparing.
Fig. 2 is the UV spectrum of the 13nm nm of gold of variable concentrations; A is the 3.5nM nm of gold (low concentration group) of prior art for preparing, the 17.5nM nm of gold (concentrate group) of B for adopting method for concentration preparation of the present invention, and C is the 17nM nm of gold of prior art for preparing.
Fig. 3 is the ultraviolet-visible analysis of spectrum (all without volume correction) of nano-Au solution salt tolerance.
Fig. 4 is the UV spectrum (A: low concentration group of two groups of nano-probes centrifugal preceding (dotted line) and centrifugal back (solid line); B: concentrate group).
The specific embodiment
Further specify the present invention with embodiment below, but the present invention is not limited.
3.5nM the preparation of nm of gold (low concentration group) is (according to document Grabar, K.C.Freeman, R.G.Hommer, M.B.Natan, M.J.Anal.Chem.1995,67,735-743): the aqueous solution of chloraurate of 0.01% (mass percent) of 100mL is added in the 250mL round-bottomed flask, after being heated to violent boiling, 1% (mass percent) trisodium citrate aqueous solution that adds fast 3.5mL under the vigorous stirring, continue to add thermal agitation 15min after, stop heating, leave standstill after continuing to stir 30min, room temperature is cooled off naturally.With 0.22 μ m membrane filtration, 4 ℃ of preservations.Afm image is shown in the A figure of Fig. 1, and particle diameter is 12.5 ± 2.3nm, is evenly distributed, and observes a small amount of granule simultaneously and exists.UV spectrum (the A figure of Fig. 2) shows absorption maximum about 516nm, and the absorption value at 520nm place is 0.673.
The 3.5nM nm of gold is concentrated 5 times of highly concentrated nano-Aus (concentrating group) that obtain 17.5nM (not meter loss).The axygen centrifuge tube that the nano-Au solution of getting 1mL 3.5nM is put into 1.5mL carries out centrifugal concentrating.Under 4 ℃ of conditions, and centrifuge (himac CF16RX, Hitachi) rotating speed is controlled at 12000rpm, behind the centrifugal 20min, removes the 0.8mL supernatant, and nm of gold is dispersed in the 0.2mL solution again.AFM figure after centrifugal concentrate shows that particle diameter does not change, and still is 12.5 ± 2.3nm, and original granule disappeared (the B figure of Fig. 1); UV spectrum (the B figure of Fig. 2) shows absorption maximum about 518nm, and the absorption value at 518nm place is 2.955.
List of references (" practical immunocytochemistry and making nucleic acid molecular hybridization technology ", Cai Wenqin, Wang uncle Yun etc., Chengdu: Sichuan science tech publishing house, 1994,479-582), after the chlorauric acid solution of getting 100mL 0.01% (w/w) is heated to violent boiling, add the citric acid three sodium solution of the 10mg/mL of 0.5mL rapidly, after continuing to add thermal agitation 15min, stop heating, continue to stir and naturally cool to room temperature, obtain the nanogold particle solution of 40nm.Behind 0.22 μ m membrane filtration, place 4 ℃ of preservations of refrigerator.It is about 40nm that AFM records average grain diameter.
According to the nanogold particle of the 40nm of method for preparing, concentration is 0.15nM.Concentration step is as follows, and the axygen centrifuge tube that the 40nm nano-Au solution of getting 1mL 0.15nM is put into 1.5mL carries out centrifugal concentrating.Under 4 ℃ of conditions, and centrifuge (himac CF16RX, Hitachi) rotating speed is controlled at 8000rpm, and behind the centrifugal 10min, nanogold particle concentrates on the pipe end, and supernatant is a colorless cleared solution.Remove 0.8mL (or 0.7mL) supernatant, nm of gold is dispersed in 0.2mL (or 0.3mL) solution again.Obtaining concentration is the 40nm nm of gold of 0.75nM (or 0.5nM).
The preparation process of 5nm nanogold particle following (" practical immunocytochemistry and making nucleic acid molecular hybridization technology ", Cai Wenqin, Wang uncle Yun etc., Chengdu: Sichuan science tech publishing house, 1994,479-582): with A liquid (the 1%w/w natrium citricum of 4mL, the 0.1M K of 0.2mL for preparing
2CO
3, 1% tannic acid of 0.7mL, 15.1ml water) and the B liquid (1%HAuCl of 1mL
4, 79mL water) and be heated to 60 ℃ respectively, under stirring state, rapidly A liquid is added B liquid then, continue heating and become vinicolor until collaurum, naturally cool to room temperature, behind 0.22 μ m membrane filtration, place 4 ℃ of preservations of refrigerator.It is about 5nm that AFM records average grain diameter.
According to the nanogold particle of the 5nm for preparing in the said method, concentration is 82nM.Concentration step is as follows, and the axygen centrifuge tube that the 5nm nano-Au solution of getting 1mL 82nM is put into 1.5mL carries out centrifugal concentrating.Under 4 ℃ of conditions, and centrifuge (himac CF16RX, Hitachi) rotating speed is controlled at 16000rpm, and behind the centrifugal 30min, nanogold particle concentrates on the pipe end, and supernatant is a colorless cleared solution.Remove 0.8mL (or 0.92mL) supernatant, nm of gold is dispersed in 0.2mL (or 0.08mL) solution again.Obtaining concentration is the 5nm nm of gold of 410nM (or 1000nM).
Embodiment 4
List of references (" practical immunocytochemistry and making nucleic acid molecular hybridization technology ", Cai Wenqin, Wang uncle Yun etc., Chengdu: Sichuan science tech publishing house, 1994,479-582), after the chlorauric acid solution of getting 100mL 0.01% (w/w) is heated to violent boiling, add the citric acid three sodium solution of the 10mg/mL of 5mL rapidly, after continuing to add thermal agitation 15min, stop heating, continue to stir and naturally cool to room temperature, obtaining average grain diameter is the nanogold particle solution of 10nm.Behind 0.22 μ m membrane filtration, place 4 ℃ of preservations of refrigerator.
According to the nanogold particle of the 10nm for preparing in the said method, concentration is 9.5nM.Concentration step is as follows, and the axygen centrifuge tube that the 5nm nano-Au solution of getting 1mL 9.5nM is put into 1.5mL carries out centrifugal concentrating.Under 4 ℃ of conditions, and centrifuge (himac CF16RX, Hitachi) rotating speed is controlled at 13000rpm, and behind the centrifugal 20min, nanogold particle concentrates on the pipe end, and supernatant is a colorless cleared solution.Remove the 0.8mL supernatant, nm of gold is dispersed in the 0.2mL solution again.Obtaining concentration is the 10nm nm of gold of 44.5nM.
List of references (" practical immunocytochemistry and making nucleic acid molecular hybridization technology ", Cai Wenqin, Wang uncle Yun etc., Chengdu: Sichuan science tech publishing house, 1994,479-582), after the chlorauric acid solution of getting 100mL 0.01% (w/w) is heated to violent boiling, add the citric acid three sodium solution of the 10mg/mL of 2mL rapidly, after continuing to add thermal agitation 15min, stop heating, continue to stir and naturally cool to room temperature, obtaining average grain diameter respectively is the nanogold particle solution of 20nm.Behind 0.22 μ m membrane filtration, place 4 ℃ of preservations of refrigerator.
According to the nanogold particle of the 20nm for preparing in the said method, concentration is 1.16nM.Concentration step is as follows, and the axygen centrifuge tube that the 20nm nano-Au solution of getting 1mL 1.16nM is put into 1.5mL carries out centrifugal concentrating.Under 4 ℃ of conditions, and centrifuge (himac CF16RX, Hitachi) rotating speed is controlled at 11000rpm, and behind the centrifugal 20min, nanogold particle concentrates on the pipe end, and supernatant is a colorless cleared solution.Remove the 0.8mL supernatant, nm of gold is dispersed in the 0.2mL solution again.Obtaining concentration is the 20nm nm of gold of 5.8nM.
The present invention by prior art directly centrifugal the concentrating of low concentration nm of gold of preparation obtain, kept the characteristics that particle diameter is evenly distributed, and controlled the condition when centrifugal, can also effectively remove the granule in the original solution.
The comparative example 1
The direct preparation of 17nM nm of gold is (according to document Grabar, K.C.Freeman, R.G.Hommer, M.B.Natan, M.J.Anal.Chem.1995,67,735-743): the chlorauric acid solution of the 1mM (being equivalent to 0.04% mass percent) of 100mL is added in the 250mL round-bottomed flask, after being heated to violent boiling, the trisodium citrate aqueous solution that adds fast the 38.8mM of 5mL under the vigorous stirring, continue to add thermal agitation 15min after, stop heating, leave standstill after continuing to stir 30min, room temperature is cooled off naturally.With 0.22 μ m membrane filtration, 4 ℃ of preservations.Afm image shows (the C figure of Fig. 1), and particle diameter is near 14 ± 5nm, and it is very inhomogeneous to distribute, and the oarse-grained ratio of statistical result showed surpasses 30% of sum.UV spectrum (the C figure of Fig. 2) shows absorption maximum about 518nm, and the absorption value at 518nm place is 2.346.
Experimental example 1 nm of gold of the present invention is to the comparison of salt tolerance
Colorimetric method: (average grain diameter is similar to get comparative example 1 and the embodiment 1 concentrated nm of gold of organizing, about about 13nm) each 100 μ L, add 0.1M PBS (10mM PB, pH7.0 respectively, 0.1M NaCl) each 5,10,15,20,25 μ L, the change color situation of observation nm of gold.The result shows that embodiment 1 concentrates group after adding PBS, and significant change does not take place color, and comparative example 1 color when adding the 0.1M PBS of 10 μ L begins purpling, has all become blue during 30 μ L.
Spectroscopic methodology: get each 100 μ L of nm of gold that comparative example 1 and embodiment 1 concentrate group respectively, survey uv-vis spectra, and drip 0.1M PBS respectively one by one, each 5 μ L, situation of change with uv-vis spectra monitoring nm of gold absorption spectrum, and do absorption value with the tracing analysis of salt volume, as shown in Figure 3.The high salt concentration that experiment determine to concentrate that group can tolerate is 44mM NaCl, and comparative example 1 can only tolerate the NaCl of 9mM simultaneously.Therefore nano-Au solution of the present invention has improvement significantly at the nano-Au solution that makes than prior art aspect stability, the salt tolerance, and this is extremely important for the application of nm of gold in biology sensor.
Experimental example 2 nm of gold of the present invention are applied to the dna molecular self assembly
List of references (Rosi, N.L.Giljohann, D.A.Thaxton, C.S.Lytton-Jean, A.K.R.Han, M.S.Mirkin, C.A.Science 2006,312,1027-1030.): the sulfydryl DNA of 27bp is assembled on the nanogold particle of comparative example 1 and embodiment 1 concentrated group, carry out centrifugally after aged at room temperature is intact, remove DNA free in the solution.The UV that surveys two groups respectively in centrifugal front and back composes and analyzes.As shown in Figure 4, comparative example 1 is after centrifugal, and the maximum absorption wavelength red shift is to 522nm, and the absorption at long wave place also obviously raises, and illustrates that reunion has to a certain degree taken place the nano-probe particle.Embodiment 1 concentrates group does not then have significant change, and whole centrifugal process does not influence the character of nm of gold, has guaranteed the application of nm of gold dna probe in detection.
Salt tolerance is analyzed: add 5M NaCl solution in above-mentioned two kinds of Au-DNA solution, the high salt concentration of determining that the embodiment of the invention 1 concentrates that group can tolerate is 2.6M; And comparative example 1 is the highest only with tolerance 1.5M.Therefore for the situation of reacting and testing process under high salt concn takes place, nano-Au solution of the present invention has better applicability.Concrete outcome is as shown in table 1 below.
Table 1
Stability analysis: above-mentioned two groups of probes are preserved at 4 ℃ of constant temperature, observed the state variation that prolongs nano-probe in time.Discovery embodiment 1 concentrates the group storage and still stablized in 30 days, and comparative example 1 had the black point-like to be settled out now in the time of 7 days.Therefore the concentrated Au-DNA that makes that organizes of the present invention also is much better than the comparative example aspect stable, helps the nm of gold dna probe is further applied.
Claims (8)
1. the method for concentration of a nano-Au solution, it comprises that the nano-Au solution that will be made by reducing agent reduction chlorauric acid solution further adopts supercentrifugal process to concentrate again, the centrifugal rotational speed of described supercentrifugal process is between 8000-16000rpm, and centrifugation time is 10-30 minute.
2. method for concentration as claimed in claim 1 is characterized in that described reducing agent is the mixture of trisodium citrate or tannic acid and trisodium citrate.
3. method for concentration as claimed in claim 1, the mass percent concentration that it is characterized in that described chlorauric acid solution is 0.001%～0.1%.
4. method for concentration as claimed in claim 3, the mass percent concentration that it is characterized in that described chlorauric acid solution is 0.01%.
5. method for concentration as claimed in claim 1, the average grain diameter that it is characterized in that nanogold particle in this nano-Au solution is 5-150nm.
6. method for concentration as claimed in claim 5, the average grain diameter that it is characterized in that this nanogold particle is 10-40nm.
7. the highly concentrated nano-Au solution that makes as each described method for concentration of claim 1-6.
8. the application of highly concentrated nano-Au solution as claimed in claim 7 in the dna molecular self assembly.
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| CN101987364A (en) * | 2010-09-14 | 2011-03-23 | 江南大学 | Method for preparing functional gold nanoparticles with high stability |
| CN102990082A (en) * | 2012-12-17 | 2013-03-27 | 桂林理工大学 | Method for preparing fluorescence nano gold sol by using PVP (Poly Vinyl Pyrrolidone) through reduction modification under hydrothermal condition |
| CN109295443A (en) * | 2018-10-30 | 2019-02-01 | 华中科技大学 | A kind of nanogold submicron film, its preparation and application |
| CN109940168A (en) * | 2019-05-07 | 2019-06-28 | 郑州轻工业学院 | A method of quickly preparing high salt tolerant nanogold particle |
| CN111906325A (en) * | 2020-07-14 | 2020-11-10 | 广东工业大学 | In-situ nano copper paste and preparation process and application thereof |
| CN111940754A (en) * | 2019-05-17 | 2020-11-17 | 西北民族大学 | Nano gold particles with uniform particle size extracted based on sodium citrate reduction method and preparation method thereof |
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| CN101987364A (en) * | 2010-09-14 | 2011-03-23 | 江南大学 | Method for preparing functional gold nanoparticles with high stability |
| CN101987364B (en) * | 2010-09-14 | 2012-06-20 | 江南大学 | Method for preparing functional gold nanoparticles with high stability |
| CN102990082A (en) * | 2012-12-17 | 2013-03-27 | 桂林理工大学 | Method for preparing fluorescence nano gold sol by using PVP (Poly Vinyl Pyrrolidone) through reduction modification under hydrothermal condition |
| CN109295443A (en) * | 2018-10-30 | 2019-02-01 | 华中科技大学 | A kind of nanogold submicron film, its preparation and application |
| CN109295443B (en) * | 2018-10-30 | 2019-09-24 | 华中科技大学 | A kind of nanogold submicron film, its preparation and application |
| CN109940168A (en) * | 2019-05-07 | 2019-06-28 | 郑州轻工业学院 | A method of quickly preparing high salt tolerant nanogold particle |
| CN111940754A (en) * | 2019-05-17 | 2020-11-17 | 西北民族大学 | Nano gold particles with uniform particle size extracted based on sodium citrate reduction method and preparation method thereof |
| CN111906325A (en) * | 2020-07-14 | 2020-11-10 | 广东工业大学 | In-situ nano copper paste and preparation process and application thereof |
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