CN103265950A - Boron nitride nano sheet/gold nano cluster composite material, as well as preparation method and application thereof in bioanalysis - Google Patents
Boron nitride nano sheet/gold nano cluster composite material, as well as preparation method and application thereof in bioanalysis Download PDFInfo
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Abstract
The invention discloses a boron nitride nano sheet/gold nano cluster composite material. The boron nitride nano sheet/gold nano cluster composite material is prepared through the following steps of: executing probe type ultrasonic stripping on hexagonal boron nitride in a water solution (PDDA) of phthalic acid diethylene glycol diacrylate for 4 hours, after standing the dispersion liquid for 24 hours, centrifuging the dispersion liquid for 20 minutes at the speed of 2000rpm (revolutions per minute), and obtaining the supernatant liquid for use; obtaining 10ml of the supernatant liquid, further centrifuging for 10 minutes at the speed of 10000rpm, obtaining the subnatant, centrifugally washing the subnatant for 3 times with the secondary water, and then, getting the boron nitride nano sheet; and then, mixing the boron nitride nano sheet with the gold nano cluster under self-assembly action, performing ultrasonic treatment for 30 minutes, centrifuging for 10 minutes at the speed of 10000rpm, obtaining the subnatant, and centrifugally washing the subnatant for 3 times with the secondary water, thus, getting the boron nitride nano sheet/gold nano cluster composite material. The composite material provided by the invention has stronger yellow fluorescence, has lower toxicity to HeLa cell, and can be used in detection analysis, such as bio-labeling.
Description
Technical field
The present invention relates to boron nitride nanosheet/gold nano bunch matrix material and method for making thereof and the application in bioanalysis.
Background technology
In recent years, gold nano cluster has caused the very big interest of researcher.Because quantum confinement and fringing effect, gold nano bunch has shown the chemistry of many novelties and physical properties [referring to (a) A.Lechtken, D.Schooss, J.R.Stairs, M.N.Blom, F.Furche, N.Morgner, O.Kostko, B.von Issendorff and M.M.Kappes, Angew.Chem.Int.Ed.46 (2007), 2944. (b) B.S.Gonzalez, M.J.Rodriguez, C.Blanco, J.Rivas, M.A.Lopez-Quintela and J.M.G.Martinho, Nano Lett 10 (2010), and 4217.].In addition, it has low cytotoxicity, fabulous water-soluble, unreactiveness, fluorescent stability, characteristics such as better surface graft, this makes it wide application prospect be arranged [referring to (c) T.H.Lee in fields such as photoelectric device, sensor, bio-imagings, J.I.Gonzalez, J.Zheng and R.M.Dickson, Acc.Chem.Res.38 (2005), 534. (d) C.C.Huang, C.K.Chiang, Z.H.Lin, K.H.Lee and H.T.Chang, Anal.Chem.80 (2008), 1497].On the other hand, because two dimension (2D) crystal has unique character and very high specific surface area, this makes it have numerous potential application.Graphene is a kind of well-known two-dimentional SP
2Key carbon thin slice, its extremely superior character has caused studies interest widely, in various fields, obtained huge progress [referring to (e) X.Huang, Z.Y.Zeng, Z.X.Fan, J.Q.Liu, H.Zhang, Adv Mater24 (2012), 5979. (f) X.Li, H.Wang, J.T.Robinson, H.Sanchez, G.Diankov and H.Dai, J.Am.Chem.Soc.131 (2009), 15939. (g) D.Chen, L.H.Tang, J.H.Li, Chem.Soc.Rev.39 (2010], 3157.].As a kind of novel class grapheme material, boron nitride nanosheet is owing to have many noticeable character, as higher physical strength, high-temperature stability, bigger thermal conductivity and lower specific inductivity, obtained huge concern [referring to (h) Y.Kubota, K.Watanabe, O.Tsuda, and T.Taniguchi, Science317 (2007), 932. (i) K.Watanabe, T.Taniguchi, T.Niiyama, K.Miya, and M.Taniguchi, Nat.Photonics3 (2009), 591.].The article report has been arranged in addition, after the lower and functionalization of the cytotoxicity of boron nitride nano-tube can with albumen and cell interaction [referring to (j) X.Chen, P.Wu, M.Rousseas, D.Okawa, Z.Gartner, A.Zettl, and C.R.Bertozzi, J.Am.Chem.Soc.131 (2009), 890.].Along with the development of nanosecond science and technology, preparation and the application of nano composite material receive much concern.Than independent nano particle, nano composite material has shown abundanter, adjustable optics and electrical properties [referring to (k) T.Mokari, C.G.Sztrum, A.Salant, E.Rabani, and U.Banin, Nat.Mater.4 (2005), 855.].Because boron nitride nanosheet has higher specific surface area and chemical stability, thereby thereby the template that can be used as load gold nano bunch prepare a kind of nano-complex that can be applied to useful for drug delivery, biological detection and living things system of novelty.Understand according to us, at present about boron nitride nanosheet/gold nano bunch matrix material and preparation thereof with in bioanalysis, use and do not see bibliographical information.
Summary of the invention
The purpose of this invention is to provide a kind of method with self-assembly and synthesized novel boron nitride nanosheet/gold nano bunch matrix material, and relevant bioanalysis is used.
Technical scheme of the present invention is as follows:
The method for making of a kind of boron nitride nanosheet/gold nano bunch matrix material, it comprises the steps:
Step 1. takes by weighing 100 milligrams hexagonal boron nitride powder, is scattered in the aqueous solution of 50 milliliters of PDDA (PDDA, concentration is 1 milligram every milliliter), by probe type ultrasonic instrument (VC-750,20 kilohertzs, 750W, 30% amplifies U.S. Sonics﹠amp; Materials company) ultrasonic peeling off 4-5 hour, dispersion liquid were placed after 24 hours, and with 2000 rpms rotating speed centrifugal 20 minutes, it was standby to get 40 milliliters of supernatant liquids; Use 10000 rpms centrifugal 10 minutes of rotating speed again with 10 milliliters of above-mentioned supernatant liquids, get 1 milliliter of subnatant, wash 3 times with secondary water washing, obtaining concentration is the boron nitride nanosheet of 1 ± 0.1 mg/ml;
Step 2. 1 milliliter of the above-mentioned boron nitride nanosheet that makes is scattered in the gold nano bunch solution that the 2-3 ml concn is 0.1 mg/litre [method for making of gold nano bunch solution: 0.5 ml concn be 20 mmoles/liter HAuCl
4Solution and 0.15 ml concn be 100 mmoles/liter gsh be mixed in the 4.35mL ultrapure water at 25 ℃, mixing solutions places 70 ℃ of water-bath mild stirring 24h then, namely make and have the fluorescent orange product, concentration is 0.1 ± 0.01 mg/ml .[referring to (1) Z.Luo, X.Yuan, Y.Yu, Q.Zhang, D.Leong, J.Lee, J.Xie C.Peng, J.Am.Chem.Soc.134 (2012), 16662.]], supersound process 30 minutes with 10000 rpms rotating speed centrifugal 10 minutes, is got 1 milliliter of subnatant, behind secondary water centrifuge washing three times, namely get the matrix material that concentration is about the boron nitride nanosheet/gold nano bunch of 1 ± 0.1 mg/ml.
A kind of boron nitride nanosheet/gold nano bunch matrix material that makes according to above-mentioned method for making.
Above-mentioned boron nitride nanosheet/gold nano bunch matrix material characterizes through transmission electron microscope (TEM), and the result shows that gold nano bunch is evenly distributed on the boron nitride nanosheet, forms tangible composite structure (B among Fig. 1).
Boron nitride nanosheet/gold nano of the present invention bunch matrix material characterizes through x-ray photoelectron power spectrum (XPS), and the result shows that prepared sample is boron nitride nanosheet/gold nano bunch mixture (B among Fig. 2).
Boron nitride nanosheet/gold nano of the present invention bunch matrix material characterizes through fluorescence spectrum, at the 560nm place maximum emission peak is arranged, and has stronger yellow fluorescence character (B among Fig. 3).
The matrix material of boron nitride nanosheet/gold nano bunch carries out the cytotoxicity test:
Cervical cancer HeLa cell is cultivated in 96 orifice plates, and density is about 10000 cells in every hole.Through after 24 hours hatch, substratum replaces with the fresh culture of the different concns matrix material that contains 100 microlitres, behind certain hour, remove substratum, and add the fresh culture that 100 microlitres contain 20 microlitre tetrazolium bromides (5 milligrams every milliliter), through after 4 hours hatch, solution absorbancy characterize by the Bio-Rad680 microplate reader, its optical density(OD) (OD) reads at 490nm, relatively cytoactive by formula is calculated as follows: ([OD] test/[OD] control) * 100%[is referring to (m) C.Peng, W.Hu, Y.Zhou, C.Fan, Q.Huang, Small.6 (2010), 1686.].More than each experiment repeat at least 3 times.The result show in cell, hatch 24 and 48 hours after, boron nitride nanosheet/gold nano bunch mixture toxicity is lower, has excellent biological compatibility (Fig. 4).
Boron nitride nanosheet/gold nano bunch mixture nano material and HeLa cells in vitro cell imaging:
The HeLa cell is cultivated in a circular culture dish, its thickness of sowing is 10000 cells in every hole, substratum is to contain 10% foetal calf serum, L-L-glutamic acid (every liter of 2 mmole), the high dextrose culture-medium of penicillin (10000units/mL) and Streptomycin sulphate (100 milligrams every milliliter), after 24 hours cultivation, boron nitride nanosheet/gold nano that 50 micrograms are every milliliter bunch matrix material joins to be cultivated in the base, under 37 degrees centigrade, hatched 2 hours, then cell is with every liter of 10 mmole, and pH is that 7.4 PBS washs and removes free matrix material.The Laser Scanning Confocal Microscope photo obtains under the argon laser of 405nm exciting light.
The result shows: HeLa and boron nitride nanosheet are hatched the back and are occurred without any yellow fluorescence under ultraviolet radiation.And after HeLa and boron nitride nanosheet/gold nano bunch mixture are hatched, the success of HeLa cell by yellow fluorescence mark and imaging.The result shows: the boron nitride nanosheet/gold nano of the present invention bunch mixture with high-specific surface area can be applicable to detection analyses (Fig. 5) such as biomarker.
The invention provides a kind of boron nitride nanosheet/gold nano bunch matrix material and preparation method thereof, this preparation method has convenient, fast, high repeatability and other advantages.Simultaneously prepared nano-complex has good characteristics such as the stronger yellow fluorescence of emission, satisfactory stability, water-soluble and low-down cytotoxicity, can be applicable to biomarker etc. and detects and analyze.These show that boron nitride nanosheet/gold nano bunch mixture is expected to be further used for the application in field such as imaging and treatment in the cell.
Description of drawings
Transmission electron microscope (TEM) characterization result (B among Fig. 1) of boron nitride nanosheet/gold nano that Fig. 1 obtains for the present invention bunch and transmission electron microscope (TEM) characterization result (A among Fig. 1) of boron nitride nanosheet.
X-ray photoelectron power spectrum (XPS) characterization result (B) of boron nitride nanosheet/gold nano that Fig. 2 obtains for the present invention bunch and x-ray photoelectron power spectrum (XPS) characterization result (A) of boron nitride nanosheet.
The fluorescence spectrum figure (B) of boron nitride nanosheet/gold nano that Fig. 3 obtains for the present invention bunch and the fluorescence spectrum figure (A) of boron nitride nanosheet.
Fig. 4 for boron nitride nanosheet/gold nano bunch matrix material of obtaining among the present invention under different concns, hatch 24 and 48 hours cytotoxicity test respectively.
Fig. 5 adopts the Laser Scanning Confocal Microscope that boron nitride nanosheet/gold nano bunch matrix material that the present invention obtains and HeLa cell are hatched 24 hours to characterize, wherein (A) light field picture; (B) fluorescence field picture; (C) the compound picture of light field and fluorescence field.
Embodiment
The preparation of the matrix material of embodiment 1. boron nitride nanosheets/gold nano bunch
Take by weighing 100 milligrams hexagonal boron nitride powder, be scattered in the aqueous solution of 50 milliliters of PDDA (PDDA, concentration is 1 milligram every milliliter), by probe type ultrasonic instrument (VC-750,20 kilohertzs, 750W, 30% amplifies U.S. Sonics﹠amp; Materials company) ultrasonic peeling off 4 hours.Dispersion liquid was placed after 24 hours, with 2000 rpms rotating speed centrifugal 20 minutes, it is standby to get 40 milliliters of supernatant liquids, use 10000 rpms centrifugal 10 minutes of rotating speed again with 10 milliliters of above-mentioned supernatant liquids, get 1 milliliter of subnatant, wash 3 times with secondary water washing, obtain boron nitride nanosheet (concentration is 1 ± 0.1 milligram every milliliter).1 milliliter of the above-mentioned boron nitride nanosheet that makes is scattered in the 2-3 milliliter gold nano bunch solution [method for making of gold nano bunch solution: 0.5 ml concn be 20 mmoles/liter HAuCl
4Solution and 0.15 ml concn be 100 mmoles/liter gsh be mixed in the 4.35mL ultrapure water at 25 ℃, mixing solutions places 70 ℃ of water-bath mild stirring 24h then, namely makes to have the fluorescent orange product, concentration is 0.1 ± 0.01 mg/ml .[referring to (1) Z.Luo, X.Yuan, Y.Yu, Q.Zhang, D.Leong, J.Lee, J.Xie C.Peng, J.Am.Chem.Soc.134 (2012), 16662.]], supersound process 30 minutes.With 10000 rpms rotating speed centrifugal 10 minutes, get 1 milliliter of subnatant, behind secondary water centrifuge washing three times, namely get concentration and be the matrix material of 1 ± 0.1 mg/ml boron nitride nanosheet/gold nano bunch.
The preparation of the matrix material of embodiment 2. boron nitride nanosheets/gold nano bunch
Embodiment 1 " 100 milligrams hexagonal boron nitride powders " are changed into " 200 milligrams hexagonal boron nitride powders ", and other conditions of preparation are with embodiment 1, and the result is with embodiment 1.
The preparation of the matrix material of embodiment 3. boron nitride nanosheets/gold nano bunch
Embodiment 1 " being scattered in the aqueous solution of 50 milliliters of PDDA " changed into " being scattered in the aqueous solution of 100 milliliters of PDDA ", other conditions of preparation are with embodiment 1, and the result is with embodiment 1.
The preparation of the matrix material of embodiment 4. boron nitride nanosheets/gold nano bunch
Embodiment 1 " taking by weighing 100 milligrams hexagonal boron nitride powder; be scattered in the aqueous solution of 50 milliliters of PDDA " changed into " taking by weighing 200 milligrams hexagonal boron nitride powder; be scattered in the aqueous solution of 100 milliliters of PDDA ", other conditions of preparation are with embodiment 1, and the result is with embodiment 1.
The preparation of the matrix material of embodiment 5. boron nitride nanosheets/gold nano bunch
With embodiment 1 " by the probe type ultrasonic instrument (VC-750,20 kilohertzs, 750W, 30% amplifies, U.S. Sonics﹠amp; Materials company) ultrasonic peeling off 4 hours " change into " by probe type ultrasonic instrument (VC-750,20 kilohertzs, 750W, 30% amplification, U.S. Sonics﹠amp; Materials company) ultrasonic peeling off 5 hours ", other conditions of preparation are with embodiment 1, and the result is with embodiment 1.
The preparation of the matrix material of embodiment 6. boron nitride nanosheets/gold nano bunch
Embodiment 1 " the above-mentioned boron nitride nanosheet that makes is scattered in 2 milliliters of gold nano bunch solution " changed into " the above-mentioned boron nitride nanosheet that makes is scattered in 3 milliliters of gold nano bunch solution ", other conditions of preparation are with embodiment 1, and the result is with embodiment 1.
The preparation of the matrix material of embodiment 7. boron nitride nanosheets/gold nano bunch
Other conditions that change " supersound process 30 minutes " of embodiment 1 into " supersound process 60 minutes " preparation are with embodiment 1, and the result is with embodiment 1.
The matrix material of embodiment 8. boron nitride nanosheets/gold nano bunch is used for the cytotoxicity test.
Get the matrix material of prepared boron nitride nanosheet/gold nano among the embodiment 1 bunch, be made into different concns.Cervical cancer HeLa cell is cultivated in 96 orifice plates, and density is about 10000 cells in every hole.Through after 24 hours hatch, substratum replaces with the fresh culture of the above-mentioned matrix material that contains 100 microlitres.Behind certain hour, go out substratum, and add the fresh culture that 100 microlitres contain 20 microlitre tetrazolium bromides (5 milligrams every milliliter).Through after 4 hours hatch, solution absorbancy characterize by the Bio-Rad680 microplate reader.Its optical density(OD) (OD) reads at 490nm.Relatively cytoactive by formula is calculated as follows: ([OD] test/[OD] control) * 100%[is referring to (m) C.Peng, W.Hu, Y.Zhou, C.Fan, Q.Huang, Small.6 (2010), 1686.].More than each experiment repeat at least 3 times.The result as shown in Figure 4.
The matrix material of embodiment 9. boron nitride nanosheets/gold nano bunch is used for the picture that is marked as of cervical cancer HeLa cell
Get the matrix material of prepared boron nitride nanosheet/gold nano among the embodiment 1 bunch, concentration is every milliliter of 50 microgram.Earlier the HeLa cell is cultivated in a circular culture dish, its thickness of sowing is 10000 cells in every hole.Substratum is to contain 10% foetal calf serum, L-L-glutamic acid (every liter of 2 mmole), the high dextrose culture-medium of penicillin (10000units/mL) and Streptomycin sulphate (100 milligrams every milliliter).After 24 hours cultivation, above-mentioned materials joined to cultivate in the base under 37 degrees centigrade, hatched 2 hours.Then cell is with every liter of 10 mmole, and pH is that 7.4 PBS washs and removes free matrix material.The Laser Scanning Confocal Microscope photo obtains under the argon laser of 405nm exciting light.The result as shown in Figure 5.
Claims (3)
1. the method for making of boron nitride nanosheet/gold nano bunch matrix material is characterized in that it comprises the steps:
Step 1. takes by weighing 100 milligrams hexagonal boron nitride powder, be scattered in the aqueous solution that the 50-100 ml concn is 1 mg/ml PDDA, peeled off 4-5 hour by the probe type ultrasonic instrument is ultrasonic, dispersion liquid was placed after 24 hours, with 2000 rpms rotating speed centrifugal 20 minutes, it was standby to get 40 milliliters of supernatant liquids; Use 10000 rpms centrifugal 10 minutes of rotating speed again with 10 milliliters of above-mentioned supernatant liquids, get 1 milliliter of subnatant, wash 3 times with secondary water washing, obtaining concentration is the boron nitride nanosheet of 1 ± 0.1 mg/ml;
It is in 0.1 ± 0.01 mg/ml gold nano bunch solution that step 2. is scattered in the 2-3 ml concn with 1 milliliter of the above-mentioned boron nitride nanosheet that makes, supersound process 30 minutes, with 10000 rpms rotating speed centrifugal 10 minutes, get 1 milliliter of subnatant, behind secondary water centrifuge washing three times, namely get concentration and be the matrix material of 1 ± 0.1 mg/ml boron nitride nanosheet/gold nano bunch.
2. boron nitride nanosheet/gold nano bunch matrix material that makes according to the described method for making of claim 1.
3. the application of the described boron nitride nanosheet/gold nano of claim 2 bunch mixture in biomarker, detection are analyzed.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104889382A (en) * | 2015-04-27 | 2015-09-09 | 中国科学院深圳先进技术研究院 | Preparation method of deposition nano-silver particulate composite materials of hexagonal boron nitride nanosheet |
| CN104889382B (en) * | 2015-04-27 | 2017-03-29 | 中国科学院深圳先进技术研究院 | Hexagonal boron nitride nanosheet deposits the preparation method of nano-Ag particles composite |
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|---|---|
| CN103265950B (en) | 2015-09-02 |
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