CN103265950B - Boron nitride nanosheet/gold nanoclusters matrix material and method for making thereof and the application in bioanalysis - Google Patents
Boron nitride nanosheet/gold nanoclusters matrix material and method for making thereof and the application in bioanalysis Download PDFInfo
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- CN103265950B CN103265950B CN201310232523.4A CN201310232523A CN103265950B CN 103265950 B CN103265950 B CN 103265950B CN 201310232523 A CN201310232523 A CN 201310232523A CN 103265950 B CN103265950 B CN 103265950B
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Abstract
A kind of boron nitride nanosheet/gold nanoclusters matrix material.It is that hexagonal boron nitride is peeled off 4 hours at the aqueous solution (PDDA) of PDDA by probe type ultrasonic, after dispersion liquid places 24 hours, with the rotating speeds of 2000 rpms centrifugal 20 minutes, gets supernatant liquid for subsequent use.Get supernatant 10 milliliters and use the rotating speed of 10000 rpms more centrifugal 10 minutes, get subnatant, with intermediate water centrifuge washing 3 times, obtain boron nitride nanosheet.Then with gold nanoclusters by self-assembly effect, after both being mixed, supersound process 30 minutes, with the rotating speeds of 10000 rpms centrifugal 10 minutes, gets subnatant, after intermediate water centrifuge washing three times, obtains boron nitride nanosheet/gold nanoclusters matrix material.Matrix material of the present invention has stronger yellow fluorescence, has low toxicity to HeLa cell simultaneously, and can be applicable to the detection analyses such as biomarker.
Description
Technical field
The present invention relates to boron nitride nanosheet/gold nanoclusters matrix material and method for making thereof and the application in bioanalysis.
Background technology
In recent years, gold nano cluster causes the great interest of researcher.Due to quantum confinement and fringing effect, gold nanoclusters illustrates the chemistry of many novelties and physical properties [see (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), 4217.].In addition, it has low cytotoxicity, fabulous water-soluble, unreactiveness, fluorescent stability, the characteristics such as better surface graft, this makes it to have broad application prospects [see (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, huge progress is achieved [see (e) X.Huang, Z.Y.Zeng, Z.X.Fan in various fields, 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 having many noticeable character, as higher physical strength, high-temperature stability, larger thermal conductivity and lower specific inductivity, obtain huge concern [see (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.].Article has been had to report in addition, the cytotoxicity of boron nitride nano-tube is lower and can with albumen and cell interaction [see (j) X.Chen after functionalization, 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, the Synthesis and applications of nano composite material receives much concern.Compared to independent nano particle, nano composite material illustrates abundanter, adjustable electrical and optical properties [see (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, thus a kind of nano-complex that can be applied to useful for drug delivery, biological detection and living things system of novelty can be prepared as the template of load gold nano bunch.As far as we know, at present about boron nitride nanosheet/gold nanoclusters matrix material and preparation thereof and in bioanalysis application have no bibliographical information.
Summary of the invention
A kind of method that the object of this invention is to provide self-assembly has synthesized novel boron nitride nanosheet/gold nanoclusters matrix material, and relevant bioanalysis application.
Technical scheme of the present invention is as follows:
A method for making for boron nitride nanosheet/gold nanoclusters matrix material, it comprises the steps:
Step 1. takes the hexagonal boron nitride powder of 100 milligrams, be scattered in (PDDA in the aqueous solution of 50 milliliters of PDDA, concentration is 1 milligram every milliliter), by probe type ultrasonic instrument (VC-750,20 kilohertzs, 750W, 30% amplifies, Sonics & Materials company of the U.S.) ultrasonic stripping 4-5 hour, after dispersion liquid places 24 hours, with the rotating speeds of 2000 rpms centrifugal 20 minutes, get supernatant liquid 40 milliliters for subsequent use; Use the rotating speed of 10000 rpms more centrifugal 10 minutes with above-mentioned supernatant liquid 10 milliliters, get subnatant 1 milliliter, wash 3 times with intermediate water, obtain the boron nitride nanosheet that concentration is 1 ± 0.1 mg/ml;
It is [the method for making of gold nanoclusters solution: 0.5 ml concn is the HAuCl of 20 mM/ls in the gold nanoclusters solution of 0.1 mg/litre that above-mentioned obtained boron nitride nanosheet 1 milliliter is scattered in 2-3 ml concn by step 2.
4solution and 0.15 ml concn are that the gsh of 100 mM/ls is mixed in 4.35mL ultrapure water at 25 DEG C, then mixing solutions is placed in 70 DEG C of water-bath mild stirring 24h, namely obtained have fluorescent orange product, concentration is 0.1 ± 0.01 mg/ml. [see: (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 the rotating speeds of 10000 rpms centrifugal 10 minutes, get subnatant 1 milliliter, after intermediate water centrifuge washing three times, obtain the matrix material that concentration is about the boron nitride nanosheet/gold nanoclusters of 1 ± 0.1 mg/ml.
A kind of boron nitride nanosheet/gold nanoclusters matrix material obtained according to above-mentioned method for making.
Above-mentioned boron nitride nanosheet/gold nanoclusters matrix material characterizes through transmission electron microscope (TEM), and result shows that gold nanoclusters is evenly distributed on boron nitride nanosheet, forms obvious composite structure (Figure 1B).
Boron nitride nanosheet of the present invention/gold nanoclusters matrix material characterizes through x-ray photoelectron power spectrum (XPS), and result shows that prepared sample is boron nitride nanosheet/gold nanoclusters mixture (Fig. 2 B).
Boron nitride nanosheet of the present invention/gold nanoclusters matrix material characterizes through fluorescence spectrum, has maximum emission peak at 560nm place, has stronger yellow fluorescence character (Fig. 3 B).
The matrix material of boron nitride nanosheet/gold nanoclusters carries out cytotoxicity test:
Cultivated in 96 orifice plates by cervical carcinoma cell HeLa, density is about 10000, every hole cell.After 24 hours hatch, the substratum fresh culture of the different concns matrix material containing 100 microlitres replaces, remove substratum after a certain time, and add the fresh culture that 100 microlitres contain 20 microlitre tetrazolium bromides (5 milligrams every milliliter), after 4 hours hatch, solution absorbancy characterized by Bio-Rad680 microplate reader, its optical density(OD) (OD) reads at 490nm, it is as follows that versus cell activity presses formulae discovery: and ([OD] test/ [OD] control) × 100% [see: (m) C.Peng, W.Hu, Y.Zhou, C.Fan, Q.Huang, Small.6 (2010), 1686.].Each experiment repeats at least 3 times above.After result shows to hatch 24 and 48 hours in cell, boron nitride nanosheet/gold nanoclusters mixture toxicity is lower, has good biocompatibility (Fig. 4).
The cell in vitro imaging of boron nitride nanosheet/gold nanoclusters complex nano material and HeLa cell:
HeLa cell is cultivated in a circular culture dish, its thickness of sowing is 10000, every hole cell, substratum is the foetal calf serum containing 10%, Pidolidone (2 mmoles often rise), the high glucose substratum of penicillin (10000units/mL) and Streptomycin sulphate (100 milligrams every milliliter), after the cultivation of 24 hours, boron nitride nanosheet/gold nanoclusters the matrix material of 50 micrograms per millilitre joins to be cultivated in base, 2 hours are hatched under 37 degrees Celsius, then cell 10 mmoles often rise, pH is the free matrix material of PBS washing removing of 7.4.Laser Scanning Confocal Microscope photo obtains under the argon laser of 405nm exciting light.
Result shows: HeLa and boron nitride nanosheet occur without any yellow fluorescence after hatching under uv irradiation.And after HeLa and boron nitride nanosheet/gold nanoclusters mixture are hatched, HeLa cell is successfully marked and imaging by yellow fluorescence.Result shows: the boron nitride nanosheet of the present invention/gold nanoclusters mixture with high-specific surface area can be applicable to biomarker etc. and detects analysis (Fig. 5).
The invention provides a kind of boron nitride nanosheet/gold nanoclusters matrix material and preparation method thereof, this preparation method has convenient, fast, high repeatability and other advantages.Nano-complex simultaneously has the good characteristics such as the stronger yellow fluorescence of transmitting, satisfactory stability, water-soluble and low-down cytotoxicity, can be applicable to biomarker etc. and detects and analyze.These show, boron nitride nanosheet/gold nanoclusters mixture is expected to the application being further used for the field such as imaging and treatment in cell.
Accompanying drawing explanation
Fig. 1 is transmission electron microscope (TEM) characterization result (Figure 1B) of boron nitride nanosheet/gold nanoclusters that the present invention obtains and transmission electron microscope (TEM) characterization result (Figure 1A) of boron nitride nanosheet.
Fig. 2 is x-ray photoelectron power spectrum (XPS) characterization result (B) of boron nitride nanosheet/gold nanoclusters that the present invention obtains and x-ray photoelectron power spectrum (XPS) characterization result (A) of boron nitride nanosheet.
Fig. 3 is the fluorescence spectrum figure (B) of boron nitride nanosheet/gold nanoclusters that the present invention obtains and the fluorescence spectrum figure (A) of boron nitride nanosheet.
Fig. 4 be boron nitride nanosheet/gold nanoclusters matrix material of obtaining in the present invention under different concns, hatch the cytotoxicity test of 24 and 48 hours respectively.
The boron nitride nanosheet that Fig. 5 adopts the present invention to obtain/gold nanoclusters matrix material and the HeLa cell incubation Laser Scanning Confocal Microscope of 24 hours characterize, wherein (A) light field picture; (B) fluorescence field picture; (C) light field and fluorescence field compound picture.
Embodiment
The preparation of the matrix material of embodiment 1. boron nitride nanosheets/gold nanoclusters
Take the hexagonal boron nitride powder of 100 milligrams, be scattered in (PDDA in the aqueous solution of 50 milliliters of PDDA, concentration is 1 milligram every milliliter), by probe type ultrasonic instrument (VC-750,20 kilohertzs, 750W, 30% amplifies, Sonics & Materials company of the U.S.) ultrasonic stripping 4 hours.After dispersion liquid places 24 hours, with the rotating speeds of 2000 rpms centrifugal 20 minutes, get supernatant liquid 40 milliliters for subsequent use, the rotating speed of 10000 rpms is used again centrifugal 10 minutes with above-mentioned supernatant liquid 10 milliliters, get subnatant 1 milliliter, wash 3 times with intermediate water, obtain boron nitride nanosheet (concentration is 1 ± 0.1 milligram every milliliter).Above-mentioned obtained boron nitride nanosheet 1 milliliter is scattered in the [method for making of gold nanoclusters solution: 0.5 ml concn is the HAuCl of 20 mM/ls in 2-3 milliliter gold nanoclusters solution
4solution and 0.15 ml concn are that the gsh of 100 mM/ls is mixed in 4.35mL ultrapure water at 25 DEG C, and then mixing solutions is placed in 70 DEG C of water-bath mild stirring 24h, and namely obtained have fluorescent orange product, concentration is 0.1 ± 0.01 mg/ml. [see: (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 the rotating speeds of 10000 rpms centrifugal 10 minutes, get subnatant 1 milliliter, after intermediate water centrifuge washing three times, obtain the matrix material that concentration is 1 ± 0.1 mg/ml boron nitride nanosheet/gold nanoclusters.
The preparation of the matrix material of embodiment 2. boron nitride nanosheets/gold nanoclusters
" hexagonal boron nitride powders of 100 milligrams " of embodiment 1 changed into " hexagonal boron nitride powders of 200 milligrams ", other conditions of preparation are with embodiment 1, and result is with embodiment 1.
The preparation of the matrix material of embodiment 3. boron nitride nanosheets/gold nanoclusters
Embodiment 1 " being scattered in the aqueous solution of 50 milliliters of PDDA " is changed into " being scattered in the aqueous solution of 100 milliliters of PDDA ", other conditions of preparation are with embodiment 1, and result is with embodiment 1.
The preparation of the matrix material of embodiment 4. boron nitride nanosheets/gold nanoclusters
Embodiment 1 " taking the hexagonal boron nitride powder of 100 milligrams; be scattered in the aqueous solution of 50 milliliters of PDDA " is changed into " taking the hexagonal boron nitride powder of 200 milligrams; be scattered in the aqueous solution of 100 milliliters of PDDA ", other conditions of preparation are with embodiment 1, and result is with embodiment 1.
The preparation of the matrix material of embodiment 5. boron nitride nanosheets/gold nanoclusters
" by probe type ultrasonic instrument (VC-750; 20 kilohertzs; 750W, 30% amplifies, Sonics & Materials company of the U.S.) the ultrasonic stripping 4 hours " of embodiment 1 is changed into " by probe type ultrasonic instrument (VC-750; 20 kilohertzs; 750W, 30% amplifies, Sonics & Materials company of the U.S.) ultrasonic stripping 5 hours ", other conditions of preparation are with embodiment 1, and result is with embodiment 1.
The preparation of the matrix material of embodiment 6. boron nitride nanosheets/gold nanoclusters
Embodiment 1 " being scattered in 2 milliliters of gold nanoclusters solution by above-mentioned obtained boron nitride nanosheet " is changed into " being scattered in 3 milliliters of gold nanoclusters solution by above-mentioned obtained boron nitride nanosheet ", other conditions of preparation are with embodiment 1, and result is with embodiment 1.
The preparation of the matrix material of embodiment 7. boron nitride nanosheets/gold nanoclusters
" supersound process 30 minutes " of embodiment 1 is changed into other conditions prepared by " supersound process 60 minutes " with embodiment 1, result is with embodiment 1.
The matrix material of embodiment 8. boron nitride nanosheets/gold nanoclusters is used for cytotoxicity test.
The matrix material of boron nitride nanosheet/gold nanoclusters obtained in Example 1, is made into different concns.Cultivated in 96 orifice plates by cervical carcinoma cell HeLa, density is about 10000, every hole cell.After 24 hours hatch, the substratum fresh culture of the above-mentioned matrix material containing 100 microlitres replaces.Go out substratum after a certain time, and add the fresh culture that 100 microlitres contain 20 microlitre tetrazolium bromides (5 milligrams every milliliter).After 4 hours hatch, solution absorbancy characterized by Bio-Rad680 microplate reader.Its optical density(OD) (OD) reads at 490nm.It is as follows that versus cell activity presses formulae discovery: and ([OD] test/ [OD] control) × 100% [see: (m) C.Peng, W.Hu, Y.Zhou, C.Fan, Q.Huang, Small.6 (2010), 1686.].Each experiment repeats at least 3 times above.Result as shown in Figure 4.
What the matrix material of embodiment 9. boron nitride nanosheets/gold nanoclusters was used for cervical carcinoma cell HeLa is marked as picture
The matrix material of boron nitride nanosheet/gold nanoclusters obtained in Example 1, concentration is 50 micrograms per millilitre.First cultivated in a circular culture dish by HeLa cell, its thickness of sowing is 10000, every hole cell.Substratum is the foetal calf serum containing 10%, Pidolidone (2 mmoles often rise), the high glucose substratum of penicillin (10000units/mL) and Streptomycin sulphate (100 milligrams every milliliter).After the cultivation of 24 hours, above-mentioned materials is joined to cultivate in base under 37 degrees Celsius, hatches 2 hours.Then cell 10 mmoles often rise, and pH is the free matrix material of PBS washing removing of 7.4.Laser Scanning Confocal Microscope photo obtains under the argon laser of 405nm exciting light.Result as shown in Figure 5.
Claims (3)
1. there is a method for making for the boron nitride nanosheet/gold nanoclusters matrix material of fluorescence property, it is characterized in that it comprises the steps:
Step 1. takes the hexagonal boron nitride powder of 100 milligrams, being scattered in 50-100 ml concn is in the aqueous solution of 1 mg/ml PDDA, by probe type ultrasonic instrument ultrasonic stripping 4-5 hour, after dispersion liquid places 24 hours, with the rotating speeds of 2000 rpms centrifugal 20 minutes, get supernatant liquid 40 milliliters for subsequent use; Use the rotating speed of 10000 rpms more centrifugal 10 minutes with above-mentioned 10 milliliters of supernatant liquids, get subnatant 1 milliliter, wash 3 times with intermediate water, obtaining concentration is 1 milligram of every milliliter of boron nitride nanosheet;
It is in the gold nanoclusters solution of 0.1 mg/litre that above-mentioned obtained boron nitride nanosheet 1 milliliter is scattered in 2-3 ml concn by step 2., supersound process 30 minutes, with the rotating speeds of 10000 rpms centrifugal 10 minutes, get subnatant 1 milliliter, after intermediate water centrifuge washing three times, obtain the matrix material of boron nitride nanosheet/gold nanoclusters.
2. boron nitride nanosheet/gold nanoclusters matrix material that method for making is obtained according to claim 1, this matrix material has maximum emission peak near 560nm place, has strong yellow fluorescence character.
3. boron nitride nanosheet according to claim 2/gold nanoclusters mixture is in biomarker, the application detected in analysis.
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