WO2012126281A1 - Graphene fluorescent nano-material, preparation and use thereof - Google Patents
Graphene fluorescent nano-material, preparation and use thereof Download PDFInfo
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- WO2012126281A1 WO2012126281A1 PCT/CN2012/000357 CN2012000357W WO2012126281A1 WO 2012126281 A1 WO2012126281 A1 WO 2012126281A1 CN 2012000357 W CN2012000357 W CN 2012000357W WO 2012126281 A1 WO2012126281 A1 WO 2012126281A1
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- C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
- C09K11/08—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials
- C09K11/65—Luminescent, e.g. electroluminescent, chemiluminescent materials containing inorganic luminescent materials containing carbon
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/001—Preparation for luminescence or biological staining
- A61K49/0063—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres
- A61K49/0069—Preparation for luminescence or biological staining characterised by a special physical or galenical form, e.g. emulsions, microspheres the agent being in a particular physical galenical form
- A61K49/0089—Particulate, powder, adsorbate, bead, sphere
- A61K49/0091—Microparticle, microcapsule, microbubble, microsphere, microbead, i.e. having a size or diameter higher or equal to 1 micrometer
- A61K49/0093—Nanoparticle, nanocapsule, nanobubble, nanosphere, nanobead, i.e. having a size or diameter smaller than 1 micrometer, e.g. polymeric nanoparticle
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- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
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- C01B32/00—Carbon; Compounds thereof
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Definitions
- the present invention relates to a fluorescent nanomaterial and its preparation and use, and in particular to a graphene fluorescent nanomaterial, a preparation method thereof, and application in the field of biology and medicine. Background technique
- Carbon materials are a common and special material on the earth. With the development of nanotechnology, carbon nanomaterials have become the frontier of technological innovation in the past 20 years. Due to the special properties exhibited by the nano-size of the material, it provides a new way for the study of the behavior and interaction mechanism of cells, sub-cells and single-molecule atoms. However, due to its low water solubility and low activity, carbon nanomaterials have limited its application in many research fields such as analytical chemistry, materials science and biotechnology. As one of the carbon materials, graphene is generally described as a flat sheet of a single atom thickness of sp 2 bond carbon atoms densely packed in a honeycomb lattice. 142nm ⁇ The carbon-carbon bond length in the graphene is about 0.
- Graphene is the basic building block of some carbon allotropes, including graphite, carbon nanotubes, and fullerenes.
- the low water solubility and poor biocompatibility of common graphene limits its biological and pharmaceutical applications.
- a graphene fluorescent nanomaterial is prepared by reacting nanographene obtained by electrochemical method with hydrazine hydrate to bond a certain amount of hydrazide groups on the edge carbon atoms of graphene.
- the material has stable fluorescence, high water solubility, good biocompatibility, and the ability to successfully label cells, especially stem cells.
- it can be used not only as a biomarker, imaging and tracer in the biological field, but also as a drug carrier in the medical field and for the detection, diagnosis and treatment of diseases. Summary of the invention
- the invention provides a novel graphene fluorescent nanomaterial.
- the graphene fluorescent nano material of the invention is connected with a hydrazide group on the edge carbon atom of the nano graphene, so that it can emit a special yellow fluorescence, which reduces the agglomeration of the nanoparticles, enhances the luminescence stability, and improves the Biocompatible and water soluble.
- the graphene fluorescent nanomaterial of the present invention preferably has a size of less than 20 nm, more preferably 2 to 18 ⁇ .
- the exemplary general formula of the graphene fluorescent nanomaterial of the present invention is as follows, wherein the hydrazide group is attached to the edge carbon atom of the graphene, the number and position of which are not limited by the following formula.
- the present invention also provides a method of preparing the graphene fluorescent nanomaterial.
- the method for preparing the graphene fluorescent nano material of the present invention comprises the steps of: electrolyzing graphite in an electrolyte solution to obtain nano graphene, and then adding hydrazine hydrate, preferably at room temperature or below, stirring to obtain the graphene fluorescent nanometer. material.
- the preparation process of the graphene fluorescent nanomaterial of the present invention comprises the following steps:
- reaction product (2) adding hydrazine hydrate, preferably at room temperature or below, stirring to obtain a reaction product;
- the graphene fluorescent nano material of the invention since it has good biocompatibility and water solubility and excellent fluorescence stability, it can be used not only as a biomarker, imaging and tracer in the biological field, but also It can be used as a pharmaceutical carrier in the field of medicine as well as for the detection, diagnosis and treatment of diseases.
- FIG. 1 is a transmission electron micrograph of a graphite iridium fluorescent nanomaterial according to an embodiment of the present invention
- FIG. 3 is a schematic view showing the structure of a graphene fluorescent nanomaterial according to an embodiment of the present invention.
- FIG. 4 is a fluorescence spectrum diagram of an aqueous solution of a graphene fluorescent nanomaterial according to an embodiment of the present invention.
- Figure 5 is a graph showing the analysis of cytotoxicity of graphene fluorescent nanomaterials according to an embodiment of the present invention.
- FIG. 6 is a live cell development of graphene fluorescent nanomaterials in accordance with one embodiment of the present invention. detailed description
- High-purity graphite rod (China Steel Group Shanghai New Graphite Material Co., Ltd., 6 X 300 mm) as the working electrode, platinum plate as the counter electrode, 7ml 0.1 mol/L NaOH aqueous solution as the electrolyte, control current intensity is 120mA, room temperature The lower constant current was electrolyzed for 3 hours to obtain a black solution. To the solution, 1 ml of an 80% by weight hydrazine hydrate solution was added, and the magnetization magnetic force was added thereto, and the mixture was stirred at room temperature for 6 hours.
- the product was then centrifuged, and the supernatant was taken out and poured into a dialysis bag (Beijing Huamei Transmission Technology Co., Ltd., MD-25 MW3500).
- the dialysis bag was immersed in 500 ml of distilled water, and distilled water was replaced 5 times in 36 hours.
- An aqueous solution of the reaction product of pure graphene and hydrazine hydrate is obtained. 4 (TC dry to obtain a black solid powder, and 100 ml of the aqueous solution obtained by dialysis can obtain about 1 mg of a black solid powder.
- the transmission electron micrograph of the material shown in FIG. 1 shows that the graphene fluorescence of the present invention
- the nanomaterial is less than 20 nm.
- the graphene fluorescent nanomaterial of the present invention has a certain amount of a hydrazide group attached to the edge of the graphene.
- the fluorescence spectrum of the aqueous solution of the material shown in FIG. 4 shows that: the aqueous solution of the graphene fluorescent nanomaterial of the present invention has a maximum emission wavelength of about 550 nm and a wide range of excitation emission wavelengths when excited by an excitation wavelength of 340-410 nm. Fluorescent luminosity and stable luminescence, different from ordinary carbon nanomaterials.
- the graphene fluorescent nanomaterial of the present invention was prepared in accordance with the procedure of Example 1, in which the reaction of hydrazine hydrate with graphene was carried out at 0 °C.
- An aqueous solution obtained by dialysis of 100 ml can obtain about 1 mg of a black solid powder.
- the characterization results of the obtained product were the same as those of the product prepared in Example 1, and reference may be made to Figs.
- the graphene fluorescent nanomaterial of the present invention was prepared in accordance with the procedure of Example 1, in which the reaction of hydrazine hydrate with graphene was carried out at 60 °C. 100 ml of the aqueous solution obtained by the dialysis gave about 0.4 mg of a black solid powder.
- the characterization results of the obtained product were the same as those of the product prepared in Example 1, and can be referred to Fig. 1 - 4.
- Example 4 Application of Graphene Fluorescent Nanomaterials of the Invention in Biological and Medical Applications
- the graphene fluorescent nanomaterial of the present invention is at a higher concentration (50 ug/mL) against tumor cells such as breast cancer (MCF-7) and glioma cells (SY5Y).
- the growth has a certain inhibitory effect, and is particularly suitable as a carrier material for antitumor drugs.
- Normal cells such as stem cells such as neural stem cells (N), pancreatic stem cells (P), and cardiac stem cells (C) do not exhibit cytotoxicity at concentrations up to 100 ug/mL. This solves the problem of cytotoxic side effects in the current application of commonly used biological imaging agents.
- the cells to be tested were inoculated to a six-well plate in which slides were placed at a density of 1 - 5 X 10 5 /well, and cultured for 24 hours, to be attached.
- the graphene fluorescent nanomaterial of the present invention can enter cells (especially stem cells) and develop in the cytoplasm, thus, the graphene fluorescence of the present invention
- a major feature of nanomaterials is the traceable markers that can be used for stem cells.
- the graphene fluorescent nano material of the present invention has strong fluorescence and stable luminescence, and the excitation emission wavelength range of the material is wide, it can be used together with other cell imaging agents to jointly explore the difference in cytoplasm and nucleus. performance. Thereby, the shortcomings of the current cell imaging agent single, unstable and cytotoxic are overcome.
Abstract
A graphene fluorescent nano-material, obtained by obtaining nano graphene through an electrochemical process, and then reacting nano graphene with a hydrazine hydrate. The graphene fluorescent nano-material has a good biocompatibility, and can be used to label cells, and especially stem cells. The graphene fluorescent nano-material can not only be used as a biomarker, a developing agent, and a tracer in the biological field, but also used as a drug carrier and used for detection, diagnosis, and treatment of diseases in the medical field.
Description
石墨烯荧光纳米材料、 其制备及应用 技术领域 Graphene fluorescent nano material, preparation and application thereof
本发明涉及一种荧光纳米材料及其制备和应用， 具体而言, 是涉 及一种石墨烯荧光纳米材料、 其制备方法、 以及在生物领域和医药领 域的应用。 背景技术 The present invention relates to a fluorescent nanomaterial and its preparation and use, and in particular to a graphene fluorescent nanomaterial, a preparation method thereof, and application in the field of biology and medicine. Background technique
碳材料是一种地球上较普遍而特殊的材料， 随着纳米技术的发展， 近 20年来， 碳纳米材料成为科技创新的前沿领域。 由于该材料纳米尺 寸上表现出的特殊性能， 为细胞、 亚细胞、 单分子原子的行为和相互 作用机理的研究提供了新的途径。 然而， 由于碳納米材料的低水溶性 和低活性等特点， 限制了其在分析化学、 材料科学及生物技术等多个 研究领域的应用。 作为碳材料的一种， 石墨烯通常被描述为密集堆积 在蜂窝状晶格中的 sp2键碳原子的单原子厚度的平面片材。石墨烯中的 碳碳键长度约为 0. 142nm。 石墨烯是一些碳同素异形体（包括石墨、 碳 纳米管和富勒烯） 的基本结构单元。 对石墨烯的功能化及其在化学修 饰电极、 化学电源、 催化剂和以及气体传感器等方面的应用研究较多。 但是， 普通石墨烯的低水溶性和很差的生物兼容性限制了其生物和医 药方面的应用。 Carbon materials are a common and special material on the earth. With the development of nanotechnology, carbon nanomaterials have become the frontier of technological innovation in the past 20 years. Due to the special properties exhibited by the nano-size of the material, it provides a new way for the study of the behavior and interaction mechanism of cells, sub-cells and single-molecule atoms. However, due to its low water solubility and low activity, carbon nanomaterials have limited its application in many research fields such as analytical chemistry, materials science and biotechnology. As one of the carbon materials, graphene is generally described as a flat sheet of a single atom thickness of sp 2 bond carbon atoms densely packed in a honeycomb lattice. 142nm。 The carbon-carbon bond length in the graphene is about 0. 142nm. Graphene is the basic building block of some carbon allotropes, including graphite, carbon nanotubes, and fullerenes. There are many researches on the functionalization of graphene and its applications in chemically modified electrodes, chemical power sources, catalysts, and gas sensors. However, the low water solubility and poor biocompatibility of common graphene limits its biological and pharmaceutical applications.
本发明人经过大量研究， 发现通过将电化学方法得到的纳米石墨 烯与水合肼反应， 在石墨烯的边缘碳原子上连接一定数量的酰肼基团 而制备出一种石墨烯荧光纳米材料。 该材料具有稳定的荧光、 高的水 溶性、 很好的生物相容性， 能够成功标记细胞， 特别是干细胞。 从而 不仅可以在生物领域用作生物标记、 显像和示踪剂， 并且在医药领域 可用作药物载体以及用于疾病的检测、 诊断和治疗。 发明内容 The inventors have found through extensive research that a graphene fluorescent nanomaterial is prepared by reacting nanographene obtained by electrochemical method with hydrazine hydrate to bond a certain amount of hydrazide groups on the edge carbon atoms of graphene. The material has stable fluorescence, high water solubility, good biocompatibility, and the ability to successfully label cells, especially stem cells. Thus, it can be used not only as a biomarker, imaging and tracer in the biological field, but also as a drug carrier in the medical field and for the detection, diagnosis and treatment of diseases. Summary of the invention
一方面， 本发明提供了一种新的石墨烯荧光纳米材料。 本发明的 石墨烯荧光纳米材料是在纳米石墨烯的边缘碳原子上连上酰肼基团， 从而其可以发出特殊的黄色荧光， 降低了纳米颗粒的团聚， 增强了发 光稳定性， 并且提高了生物兼容性和水溶性。
本发明的石墨烯荧光纳米材料尺寸优选为小于 20nm , 更优选 2 - 18謹。 In one aspect, the invention provides a novel graphene fluorescent nanomaterial. The graphene fluorescent nano material of the invention is connected with a hydrazide group on the edge carbon atom of the nano graphene, so that it can emit a special yellow fluorescence, which reduces the agglomeration of the nanoparticles, enhances the luminescence stability, and improves the Biocompatible and water soluble. The graphene fluorescent nanomaterial of the present invention preferably has a size of less than 20 nm, more preferably 2 to 18 Å.
优选地， 本发明的石墨烯荧光纳米材料的示例性通式如下， 其中 酰肼基团连接在石墨烯的边缘碳原子上， 其数量和位置不受下式所限。 Preferably, the exemplary general formula of the graphene fluorescent nanomaterial of the present invention is as follows, wherein the hydrazide group is attached to the edge carbon atom of the graphene, the number and position of which are not limited by the following formula.
另一方面， 本发明还提供了所述石墨烯荧光纳米材料的制备方法。 本发明的石墨烯荧光納米材料的制备方法包括如下步骤： 在电解 质溶液中电解石墨得到纳米石墨烯， 随后加入水合肼， 优选在室温或 低于室温下， 搅拌， 得到所述的石墨烯荧光纳米材料。 In another aspect, the present invention also provides a method of preparing the graphene fluorescent nanomaterial. The method for preparing the graphene fluorescent nano material of the present invention comprises the steps of: electrolyzing graphite in an electrolyte solution to obtain nano graphene, and then adding hydrazine hydrate, preferably at room temperature or below, stirring to obtain the graphene fluorescent nanometer. material.
更具体而言， 本发明的石墨烯荧光纳米材料的制备工艺包括如下 步骤： More specifically, the preparation process of the graphene fluorescent nanomaterial of the present invention comprises the following steps:
(1 ) 在 0.01 -lmol/L的 NaOH水溶液或 pH>7的緩沖溶液 （如 PBS 或硼酸缓沖液） 中， 以 50-200mA的电流强度电解高纯石墨棒 1.5-5小 时， 得到纳米石墨烯； (1) Electrolyzing high-purity graphite rods at a current intensity of 50-200 mA in a 0.01-lmol/L NaOH aqueous solution or a buffer solution of pH>7 (such as PBS or boric acid buffer) for 1.5-5 hours to obtain nano-graphite Alkene
(2) 加入水合肼， 优选在室温或者低于室温下， 搅拌， 得到反应产 物； (2) adding hydrazine hydrate, preferably at room temperature or below, stirring to obtain a reaction product;
(3) 纯化产物， 然后将产物烘干得到石墨烯荧光纳米材料。 (3) Purifying the product, and then drying the product to obtain a graphene fluorescent nanomaterial.
再一方面， 由于本发明的石墨烯荧光纳米材料具有很好的生物兼 容性和水溶性以及优良的荧光稳定性等特性， 不仅可以在生物领域用 作生物标记、 显像和示踪剂， 并且在医药领域可用作药物载体以及用 于疾病的检测、 诊断和治疗。 附图说明 In a further aspect, since the graphene fluorescent nano material of the invention has good biocompatibility and water solubility and excellent fluorescence stability, it can be used not only as a biomarker, imaging and tracer in the biological field, but also It can be used as a pharmaceutical carrier in the field of medicine as well as for the detection, diagnosis and treatment of diseases. DRAWINGS
当与以下附图结合考虑时， 以下对本发明的实施方案的详细描述
将更容易理解， 其中： The following detailed description of embodiments of the invention when considered in conjunction with the following drawings It will be easier to understand, where:
图 1 为根据本发明一个实施方案的石墨浠荧光纳米材料的透射电 镜图； 1 is a transmission electron micrograph of a graphite iridium fluorescent nanomaterial according to an embodiment of the present invention;
图 2 为根据本发明一个实施方案的石墨烯荧光纳米材料的核磁谱 图； 2 is a nuclear magnetic spectrum of a graphene fluorescent nanomaterial according to an embodiment of the present invention;
图 3 为根据本发明一个实施方案的石墨烯荧光纳米材料的结构示 意图； 3 is a schematic view showing the structure of a graphene fluorescent nanomaterial according to an embodiment of the present invention;
图 4 为根据本发明一个实施方案的石墨烯荧光纳米材料的水溶液 的荧光光谱图； 4 is a fluorescence spectrum diagram of an aqueous solution of a graphene fluorescent nanomaterial according to an embodiment of the present invention;
图 5 为根据本发明一个实施方案的石墨烯荧光纳米材料的细胞毒 性测定分析图； Figure 5 is a graph showing the analysis of cytotoxicity of graphene fluorescent nanomaterials according to an embodiment of the present invention;
图 6 为根据本发明一个实施方案的石墨烯荧光纳米材料的活细胞 显像图。 具体实施方式 Figure 6 is a live cell development of graphene fluorescent nanomaterials in accordance with one embodiment of the present invention. detailed description
为了促进对本发明的原理的理解， 现在将参照附图中所示的实施 方案对其描述， 不意于限制本发明的范围。 In order to facilitate an understanding of the principles of the invention, the invention will be described with reference to the accompanying drawings.
实施例 1 : 本发明的石墨烯荧光纳米材料的制备 Example 1 : Preparation of Graphene Fluorescent Nanomaterials of the Invention
以高纯石墨棒（中钢集团上海新型石墨材料有限公司， 6 X 300 mm ) 为工作电极， 铂片为对电极， 7ml 0.1 mol/L的 NaOH水溶液为电解液， 控制电流强度为 120mA, 室温下恒电流电解 3 小时， 得黑色溶液。 向 溶液中加入 1 ml 80% (重量百分比）的水合肼溶液， 加入磁子磁力在室 温下搅拌 6 小时。 然后将产物离心分离， 将上清液取出， 倒入透析袋 (北京华美传导科技有限公司，MD-25 MW3500 )。将透析袋浸入 500ml 蒸馏水中， 36小时内更换 5次蒸馏水。 得到纯净的石墨烯与水合肼的 反应产物的水溶液。 4(TC烘干得到黑色固体粉末， 100ml所述透析得到 的水溶液可得约 lmg黑色固体粉末。 High-purity graphite rod (China Steel Group Shanghai New Graphite Material Co., Ltd., 6 X 300 mm) as the working electrode, platinum plate as the counter electrode, 7ml 0.1 mol/L NaOH aqueous solution as the electrolyte, control current intensity is 120mA, room temperature The lower constant current was electrolyzed for 3 hours to obtain a black solution. To the solution, 1 ml of an 80% by weight hydrazine hydrate solution was added, and the magnetization magnetic force was added thereto, and the mixture was stirred at room temperature for 6 hours. The product was then centrifuged, and the supernatant was taken out and poured into a dialysis bag (Beijing Huamei Transmission Technology Co., Ltd., MD-25 MW3500). The dialysis bag was immersed in 500 ml of distilled water, and distilled water was replaced 5 times in 36 hours. An aqueous solution of the reaction product of pure graphene and hydrazine hydrate is obtained. 4 (TC dry to obtain a black solid powder, and 100 ml of the aqueous solution obtained by dialysis can obtain about 1 mg of a black solid powder.
使用 200 kV 操作的 JEOL JEM 2100 透射电镜 (TEM)上进行成像， 用 100MHz下的 BmkerAvance ill核磁共振波谱仪得到核磁波谱图， 来 表征所得产物， 结果如图 1和图 2所示； 使用 Cary Eclipse荧光分光光 度计测量所得产物水溶液的荧光光谱， 结果如图 4所示。 Imaging was performed on a JEOL JEM 2100 transmission electron microscope (TEM) operating at 200 kV, and a nuclear magnetic spectrum was obtained using a BmkerAvance ill NMR spectrometer at 100 MHz to characterize the resulting product. The results are shown in Figures 1 and 2; using Cary Eclipse The fluorescence spectrum of the obtained product aqueous solution was measured by a fluorescence spectrophotometer, and the results are shown in Fig. 4.
由附图 1 所示的该材料的透射电镜图可知： 本发明的石墨烯荧光
纳米材料小于 20nm。 The transmission electron micrograph of the material shown in FIG. 1 shows that the graphene fluorescence of the present invention The nanomaterial is less than 20 nm.
由附图 2 所示的该材料的核磁谱图可知： 本发明的石, I、烯荧光纳 米材料颗粒在 166.5ppm处有峰， 证明酰肼基团的存在。 From the NMR spectrum of the material shown in Fig. 2, it is known that the stone of the present invention, I, olefin fluorescent material particles have a peak at 166.5 ppm, demonstrating the presence of a hydrazide group.
由附图 3 所示的该材料的结构示意图可知： 本发明的石墨烯荧光 纳米材料是在石墨烯的边缘连有一定量的酰肼基团。 As is apparent from the structural diagram of the material shown in Fig. 3, the graphene fluorescent nanomaterial of the present invention has a certain amount of a hydrazide group attached to the edge of the graphene.
由附图 4 所示的该材料水溶液的荧光光谱图可知： 本发明的石墨 烯荧光納米材料水溶液在 340-410nm 的激发波长激发下， 最高发射波 长均在 550 nm左右， 激发发射波长范围宽， 荧光光度强且发光稳定， 不同于普通的碳纳米材料。 The fluorescence spectrum of the aqueous solution of the material shown in FIG. 4 shows that: the aqueous solution of the graphene fluorescent nanomaterial of the present invention has a maximum emission wavelength of about 550 nm and a wide range of excitation emission wavelengths when excited by an excitation wavelength of 340-410 nm. Fluorescent luminosity and stable luminescence, different from ordinary carbon nanomaterials.
实施例 2: 本发明的石墨烯荧光纳米材料的制备 Example 2: Preparation of Graphene Fluorescent Nanomaterials of the Invention
按照实施例 1 的步骤制备本发明的石墨烯荧光纳米材料， 其中水 合肼与石墨烯的反应是在 0°C进行的。 100ml所述透析得到的水溶液可 得约 lmg黑色固体粉末。 所得产品的表征结果与实施例 1制备的产品 相同， 可参考图 1 - 4。 The graphene fluorescent nanomaterial of the present invention was prepared in accordance with the procedure of Example 1, in which the reaction of hydrazine hydrate with graphene was carried out at 0 °C. An aqueous solution obtained by dialysis of 100 ml can obtain about 1 mg of a black solid powder. The characterization results of the obtained product were the same as those of the product prepared in Example 1, and reference may be made to Figs.
实施例 3 : 本发明的石墨烯荧光纳米材料的制备 Example 3: Preparation of Graphene Fluorescent Nanomaterials of the Invention
按照实施例 1 的步骤制备本发明的石墨烯荧光纳米材料， 其中水 合肼与石墨烯的反应是在 60°C进行的。 100ml 所述透析得到的水溶液 可得约 0.4mg黑色固体粉末。 所得产品的表征结果与实施例 1 制备的 产品相同， 可参考图 1 - 4。 The graphene fluorescent nanomaterial of the present invention was prepared in accordance with the procedure of Example 1, in which the reaction of hydrazine hydrate with graphene was carried out at 60 °C. 100 ml of the aqueous solution obtained by the dialysis gave about 0.4 mg of a black solid powder. The characterization results of the obtained product were the same as those of the product prepared in Example 1, and can be referred to Fig. 1 - 4.
实施例 4:本发明的石墨烯荧光纳米材料在生物及医药方面的应用 Example 4: Application of Graphene Fluorescent Nanomaterials of the Invention in Biological and Medical Applications
1. 本发明的石墨烯荧光纳米材料的细胞毒性测试： 1. Cytotoxicity test of the graphene fluorescent nanomaterial of the present invention:
(1) 以 2000 - 4000个 /孔的密度接种细胞于 96孔培养板中， 培养 24h待贴壁。 (1) Cells were seeded at a density of 2000 - 4000 cells/well in 96-well culture plates and cultured for 24 hours to be attached.
(2) 加入实施例 1制备的石墨烯荧光纳米材料样品至 0.01、 0.1、 1、 10、 50、 100ug/mL。 与细胞共孵育 3天。 (2) The graphene fluorescent nanomaterial samples prepared in Example 1 were added to 0.01, 0.1, 1, 10, 50, 100 ug/mL. Incubate with the cells for 3 days.
(3) 以 15uL/孔将 MTT (四甲基偶氮唑盐）（ 5mg/mL )加入培养板中， 4h后以 lOOuL/孔加入裂解液（10%SDS+0.1%NH4C1)， 避光过夜。 (3) Add MTT (tetramethylazozolium salt) (5mg/mL) to the culture plate at 15uL/well, and add the lysate (10% SDS+0.1% NH 4 C1) at lOOuL/well after 4h. Light overnight.
(4) 次日 , 使用 1420 VICTOR3TMV, 酶标仪 ( PerkinElmer )在 570 nm 处测吸光度值， 以得到石墨烯荧光纳米材料对细胞毒性的测试结 果。 如图 5所示。 (4) The next day, 1420 VICTOR3 TM V, PerkinElmer was used to measure the absorbance at 570 nm to obtain the cytotoxicity test results of graphene fluorescent nanomaterials. As shown in Figure 5.
由附图 5 可知： 本发明的石墨烯荧光纳米材料在较高浓度下 ( 50ug/mL )对肿瘤细胞如乳腺癌（ MCF-7 )和神经胶质瘤细胞（ SY5 Y )
的生长有一定的抑制作用， 特别适用于作为抗肿瘤药物的载体材料。 而对干细胞如神经千细胞 （N ) 、 胰腺干细胞 （P ) 和心肌干细胞 ( C ) 等正常细胞， 在高达 100ug/mL的浓度下仍未表现出细胞毒性。 这解决了目前常用生物显像剂应用时出现细胞毒副作用的问题。 It can be seen from Fig. 5 that the graphene fluorescent nanomaterial of the present invention is at a higher concentration (50 ug/mL) against tumor cells such as breast cancer (MCF-7) and glioma cells (SY5Y). The growth has a certain inhibitory effect, and is particularly suitable as a carrier material for antitumor drugs. Normal cells such as stem cells such as neural stem cells (N), pancreatic stem cells (P), and cardiac stem cells (C) do not exhibit cytotoxicity at concentrations up to 100 ug/mL. This solves the problem of cytotoxic side effects in the current application of commonly used biological imaging agents.
2. 显像具体操作步骤如下： 2. The specific steps of the visualization are as follows:
(1) 将所试细胞按照 1 - 5 X 105个 /孔的密度接种于事先放有玻片 的六孔板中， 培养 24h, 待贴壁。 (1) The cells to be tested were inoculated to a six-well plate in which slides were placed at a density of 1 - 5 X 10 5 /well, and cultured for 24 hours, to be attached.
(2) 于各孔中加入适量实施例 1制备的石墨烯荧光纳米材料样品， 至终浓度为 25ug/mL,与细胞共孵育 12-24h。 (2) An appropriate amount of the graphene fluorescent nanomaterial sample prepared in Example 1 was added to each well to a final concentration of 25 ug/mL, and incubated with the cells for 12-24 hours.
(3) 在 4 °C用 PBS (磷酸緩沖溶液)洗去培养基及细胞表面可能粘附 的样品， 共洗三次。 (3) Wash the medium and samples that may adhere to the cell surface with PBS (phosphate buffer solution) at 4 °C for a total of three washes.
(4) 用 4%多聚甲醛固定细胞 20mm,之后用 PBS洗三次。 (4) The cells were fixed with 4% paraformaldehyde for 20 mm, and then washed three times with PBS.
(5) 取出处理好的样本， 于载玻片上封片， 制得待测样本。 使用 488 nm 激发的 t)FV300荧光共聚焦显微镜进行细胞成像。 结果如图 6 所示。 (5) Take out the processed sample and seal it on the slide to prepare the sample to be tested. Cell imaging was performed using a t) FV300 fluorescence confocal microscope excited at 488 nm. The result is shown in Figure 6.
由附图 6 所示的该材料的激光共聚焦扫描图可知， 本发明的石墨 烯荧光纳米材料能够进入细胞 （尤其是干细胞） ， 并在胞质内显像， 因此， 本发明的石墨烯荧光纳米材料一大特点是可用于干细胞的示踪 标记。 From the laser confocal scanning pattern of the material shown in FIG. 6, it can be seen that the graphene fluorescent nanomaterial of the present invention can enter cells (especially stem cells) and develop in the cytoplasm, thus, the graphene fluorescence of the present invention A major feature of nanomaterials is the traceable markers that can be used for stem cells.
另外, 由于本发明的石墨烯荧光纳米材料荧光光度强且发光稳定， 并且该材料的激发发射波长范围宽， 因此， 可选择与其它细胞显像剂 一同使用， 共同探讨其胞质及核的不同性能。 从而克服了目前细胞显 像剂单一、 不稳定和具有细胞毒性等缺点。 In addition, since the graphene fluorescent nano material of the present invention has strong fluorescence and stable luminescence, and the excitation emission wavelength range of the material is wide, it can be used together with other cell imaging agents to jointly explore the difference in cytoplasm and nucleus. performance. Thereby, the shortcomings of the current cell imaging agent single, unstable and cytotoxic are overcome.
尽管在附图和之前描述中已经详细解释和描述了本发明， 但其本 身应当认为是示例性的而非限制性的。 仅只是显示和描述了某些实施 方案， 而落入此处所述的本发明的精神之内的所有改变、 等价方式和 改进都意于被保护。 此处提供的任何实验、 实施例或实验结果都意于 是本发明的举例， 且不应当被认为对本发明的范围是限定的或限制性 的。 本发明只受所附的权利要求的保护范围的限制。
The invention has been illustrated and described in detail in the drawings and the claims All of the changes, equivalent ways, and improvements within the spirit of the invention as described herein are intended to be protected. The use of any of the experiments, examples, or experimental results provided herein is intended to be illustrative of the invention and should not be construed as limiting or limiting the scope of the invention. The invention is only limited by the scope of the appended claims.
Claims
1. 一种石墨烯荧光纳米材料， 其通过纳米石墨烯与水合肼的反应 而成； A graphene fluorescent nanomaterial obtained by reacting nanographene with hydrazine hydrate;
优选地， 所述纳米石墨烯通过电化学方法得到； · 优选地， 所述反应在室温或者低于室温的温度进行。 Preferably, the nanographene is obtained by an electrochemical method; • Preferably, the reaction is carried out at a temperature of room temperature or below.
2. 如权利要求 1 所迷的石墨烯荧光纳米材料， 其中该石墨烯荧光 纳米材料的尺寸小于 20nm , 优选 2 - 18nm。 2. The graphene fluorescent nanomaterial of claim 1, wherein the graphene fluorescent nanomaterial has a size of less than 20 nm, preferably 2 to 18 nm.
3. 如权利要求 1 或 2所述的石墨烯荧光纳米材料， 其具有以下通 式： 3. The graphene fluorescent nanomaterial of claim 1 or 2 having the following formula:
4. 一种石墨烯荧光纳米材料， 其尺寸小于 20nm , 优选 2 - 18nm, 并且具有以下 4. A graphene fluorescent nanomaterial having a size of less than 20 nm, preferably 2 to 18 nm, and having the following
5. 制备如权利要求 1 -4任一项所述的石墨烯荧光纳米材料的方法， 其通过将纳米石墨烯与水合肼反应而进行。 A method of producing a graphene fluorescent nanomaterial according to any one of claims 1 to 4, which is carried out by reacting nanographene with hydrazine hydrate.
6. 如权利要求 5所述的方法， 其中所述纳米石墨烯通过电化学方 法而得到。 6. The method of claim 5, wherein the nanographene is electrochemically Get it by law.
7. 如权利要求 5或 6所述的方法， 其中所迷反应在室温或低于室 温的温度进行。 7. A method according to claim 5 or 6, wherein the reaction is carried out at or below room temperature.
8. 如权利要求 5所述的方法， 其具体包括以下步骤： 在电解质溶 液中电解石墨得到纳米石墨烯， 随后加入水合肼， 优选在室温或低于 室温下， 搅拌， 得到所述的石墨烯荧光納米材料。 8. The method according to claim 5, comprising the steps of: electrolyzing graphite in an electrolyte solution to obtain nanographene, and subsequently adding hydrazine hydrate, preferably at room temperature or below, stirring to obtain the graphene. Fluorescent nanomaterials.
9. 如权利要求 8所述的方法， 其中所述电解质溶液为浓度为 0.01 - lmol/L的 NaOH溶液或者 pH>7的緩沖溶液。 9. The method according to claim 8, wherein the electrolyte solution is a NaOH solution having a concentration of 0.01 to 1 mol/L or a buffer solution having a pH of 7.
10. 如权利要求 1-4任一项所述的石墨烯荧光纳米材料作为生物显 像剂和示踪剂以及在医药领域的应用。 10. Graphene fluorescent nanomaterials according to any one of claims 1 to 4 as biodisplay agents and tracers and for use in the medical field.
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