WO2020042295A1 - 一种单分散胺化纳米金刚石胶体溶液的制备方法及其二次分散工艺和在细胞标记中的应用 - Google Patents

一种单分散胺化纳米金刚石胶体溶液的制备方法及其二次分散工艺和在细胞标记中的应用 Download PDF

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WO2020042295A1
WO2020042295A1 PCT/CN2018/110699 CN2018110699W WO2020042295A1 WO 2020042295 A1 WO2020042295 A1 WO 2020042295A1 CN 2018110699 W CN2018110699 W CN 2018110699W WO 2020042295 A1 WO2020042295 A1 WO 2020042295A1
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diamond
nano
aminated
monodispersed
nanodiamond
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French (fr)
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曹郁
方明新
蔡小娟
史淑瑞
刘翠
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华中师范大学
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
    • G01N33/587—Nanoparticles
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/0004—Preparation of sols
    • B01J13/0008—Sols of inorganic materials in water
    • C—CHEMISTRY; METALLURGY
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    • C01B—NON-METALLIC ELEMENTS; COMPOUNDS THEREOF; METALLOIDS OR COMPOUNDS THEREOF NOT COVERED BY SUBCLASS C01C
    • C01B32/00—Carbon; Compounds thereof
    • C01B32/25—Diamond
    • C01B32/28—After-treatment, e.g. purification, irradiation, separation or recovery
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
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    • C01—INORGANIC CHEMISTRY
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    • C01P2002/00—Crystal-structural characteristics
    • C01P2002/70—Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data
    • C01P2002/72—Crystal-structural characteristics defined by measured X-ray, neutron or electron diffraction data by d-values or two theta-values, e.g. as X-ray diagram
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
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    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
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    • C01P2004/60—Particles characterised by their size
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    • C01—INORGANIC CHEMISTRY
    • C01P—INDEXING SCHEME RELATING TO STRUCTURAL AND PHYSICAL ASPECTS OF SOLID INORGANIC COMPOUNDS
    • C01P2004/00—Particle morphology
    • C01P2004/60—Particles characterised by their size
    • C01P2004/61—Micrometer sized, i.e. from 1-100 micrometer
    • C—CHEMISTRY; METALLURGY
    • C01—INORGANIC CHEMISTRY
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    • C01P2004/00—Particle morphology
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    • C01P2004/62—Submicrometer sized, i.e. from 0.1-1 micrometer
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    • C01—INORGANIC CHEMISTRY
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    • C01P2004/60—Particles characterised by their size
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    • C01P2006/00—Physical properties of inorganic compounds
    • C01P2006/90—Other properties not specified above
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks

Definitions

  • the invention belongs to the technical field of nano-diamond processing, and particularly relates to a method for preparing a monodispersed aminated nano-diamond colloid solution, its secondary dispersion process, and its application as a fluorescent probe in cell labeling.
  • nanodiamonds are widely used as substrate materials for biosensors. Its unique chemical composition and electrical properties make nanodiamonds more conducive to the development of bioelectronics and biosensors than other traditional substances, such as gold, silicon, and glass. Most biomedical applications require efficient and reliable diamond surface functions. Since biomolecules are generally composed of amino acid sequences, many researchers have noted that amino-functionalized nanodiamonds have a very promising application in biosensors.
  • the patent application publication number CN 103173267 A discloses "a nanodiamond derivative with a terminal group as an amino group, and a preparation method and use thereof"
  • the invention uses purified, mixed acid oxidation-treated nanodiamonds, first reacts with sulfoxide, and then carries out nucleophilic substitution reaction with diamine compounds and trichloromethanetriazine respectively; and then reacts with diamine compounds, trichloride Chlorotriazine is prepared by iterative reaction, but the method has a complicated preparation process and harsh reaction conditions, and the nucleophilic substitution reaction involved in the method needs to be performed under the protection of an inert gas, the production cost is high, and the product obtained It also contains impurities.
  • M.Khan, N.Shahzad et al. also disclosed the use of different grinding media (such as NH 4 HCO 3 , NaCl or sucrose) for ball milling of detonated nanodiamonds (DNDs), and studied the different grinding media in different solvents (ethanol , M.Khan, N. Shahzad, et al. Dispersion behavior and the influences of ball milling technique on functionalization of detonated nano-diamonds [J] .Diamond & Related Materials 61 (2016) 32–40.), But the aminated nanodiamonds prepared in this study still have the problem of introducing impurities. In addition, although the grinding medium assisted aminated nanodiamonds in this study have better performance in epoxy resins. Dispersibility, but still can not get monodisperse nano-diamond colloid.
  • grinding media such as NH 4 HCO 3 , NaCl or sucrose
  • the object of the present invention is to propose a method for preparing a monodispersed aminated nanodiamond colloid solution and the secondary method based on the technical problem that impurities existing during the direct amination process of the nanodiamond surface are introduced and monodispersed nanodiamond colloid cannot be obtained.
  • the method of the present invention enables simple amination modification of the surface of the nano-diamond, and the obtained monodispersed colloid solution can be dispersed again.
  • the present invention adopts the following technical solutions:
  • a method for preparing a monodispersed aminated nano-diamond colloid solution includes the following steps:
  • the purified nano-diamond powder in the above technical solution of the present invention may be a commercially available product, or may be purified nano-diamond synthesized by detonation.
  • the purity of the purified nano-diamond powder is> 90%, and preferably the purity is> 95%; the particle size of the purified nano-diamond powder is 30 nm to 100 ⁇ m.
  • the mass ratio of the purified nanodiamond and deionized water is 1: (20-2000), and the mass ratio of the ammonium chloride and sodium chloride is 1: (2-100).
  • the mass ratio of the purified nano-diamond powder and the ammonium chloride is 1: (0.1-100).
  • the above technical solution adds ball milling beads during ball milling.
  • the ball milling beads are non-metal ball milling beads.
  • the ball milling beads include, but are not limited to, agate, corundum, zirconia, and silica. Made of agate beads.
  • the diameter of the ball milling beads is greater than 1 mm, preferably greater than 4 mm.
  • the ball milling beads may be ball milling beads of a single diameter or may be composed of mixed ball milling beads having different diameters.
  • the diameter ratio of the mixed ball milling beads is preferably 1.5 to 200, more preferably 2 to 10.
  • the ball milling is performed at room temperature, and an efficient planetary ball mill is used.
  • the ball mill has a line speed of 20 to 600 r / min and a line speed of 20 to 600 m / min.
  • the ball milling time is from 0.1 to 20 hours.
  • the speed of the ball mill is 512r / min.
  • the amount of ammonium chloride also has a significant impact on the amino content of the final product. The smaller or greater the quality of the ammonium chloride, The smaller the amount of amino groups on the surface of the obtained nanodiamond.
  • the ratio of the aminating reagent (mass ratio of nanodiamond to ammonium chloride) is 1: 1, 1: 5, 1:10, 1:15, 1:20, and the ratio of the aminating reagent
  • the monodispersed aminated nanodiamond colloid solution prepared by the method of the present invention can be dispersed again.
  • the specific steps of the secondary dispersion process adopted by the present invention are as follows:
  • step (ii) Disperse the aminated nano-diamond powder obtained in step (i) in a solvent again under the action of ultrasound or shear, and shake to obtain a clear and transparent black colloidal solution.
  • the drying treatment in step (i) may be performed by any one of rotary evaporation drying, spray drying, and freeze drying.
  • the drying temperature of the rotary evaporator in step (i) is not higher than 80 ° C, and may be 60 ° C, 65 ° C, 70 ° C, 80 ° C, and generally not more than 80 ° C.
  • the solvent described in step (ii) is any one or more of water, dimethyl sulfoxide (DMSO), ethanol, ethylene glycol or dimethylformamide (DMF). Or other composite solvents containing more than 20% by volume of the solvent.
  • the ultrasound described in step (ii) may utilize a 100-1000W ultrasonic cleaner, and the shear dispersion (> 500 rpm) processing time is 15 minutes.
  • a third object of the present invention is to provide the application of the aminated nanodiamond described above.
  • the aminated nanodiamond of the present invention can be used as a fluorescent probe to label cells.
  • the concentration of the aminated nanodiamond as a fluorescent probe to label cells is 0.5 to 2 mg / ml.
  • the present invention relates to a method for preparing a monodispersed aminated nanodiamond colloid solution, its secondary dispersion process, and its application in cell labeling, which have the following beneficial effects:
  • the preparation method of the invention is simple, easy to operate, does not require special reaction equipment, the entire reaction process does not need to be performed under an inert gas atmosphere, and the reagents used are cheap and readily available, the production cost is low, and industrial production is easy to achieve;
  • the aminated nanodiamond produced by the ball milling method of the present invention has a high yield, higher than 90%, up to 97%, and the prepared aminated nanodiamond colloid has monodispersity;
  • aminated nano-diamond powder obtained by the present invention can realize secondary dispersion, make the dispersed nano-diamond more convenient in transportation, and further expand the application field of nano-diamond;
  • the invention has no adverse impact on the environment during the experiment.
  • the acid waste liquid produced by the invention is relatively simple to treat and can be further concentrated or recycled for reuse.
  • FIG. 1 are XRD comparative spectra of the nano-diamond raw material, the purified nano-diamond, and the amination-modified nano-diamond in Example 3 of the present invention;
  • Example 2 is a FTIR spectrum of a nano-diamond sample before and after amination in Example 3 of the present invention, wherein: (a) a FTIR spectrum of a purified nano-diamond; (b) a FTIR spectrum of a nano-diamond after modification;
  • FIG. 4 is a comparison diagram of the particle size distribution of the aminated nanodiamond colloid solution prepared under different amination reagent ratio conditions in Examples 1 to 5 of the present invention
  • FIG. 5 is a comparison chart of the Zeta potential-pH relationship curve of the nano-diamond colloidal solution prepared under the conditions of different amination reagent ratios in Examples 1 to 5 of the present invention
  • Example 6 is a real digital photograph of a colloidal solution obtained by performing secondary dispersion of the aminated nano-diamond prepared in Example 3 of the present invention in different solvents;
  • FIG. 7 is a comparison diagram of particle size distributions of colloidal solutions obtained by performing secondary dispersion of the aminated nanodiamonds prepared in Example 3 of the present invention in different solvents.
  • Example 8 is a transmission electron microscope photograph of the aminated nanodiamond prepared in Example 3 of the present invention.
  • FIG. 9 is a real-time photograph of the aminated nano-diamond mixed liquid cultured in 5 different cells for 30 min in Example 6, wherein: a) HepG2 cells, b) HeLa cells, c) L02 cells, d) H3B cells, e) NIH 3T3 cells. Bright field picture on the left and fluorescent field picture on the right.
  • the nano-diamonds in each of the following examples are purified by the following process, the steps are as follows:
  • the 10g nano-diamond gray powder was reacted with 30g-50g concentrated sulfuric acid and 10g potassium permanganate in a reaction kettle, and the reaction temperature in the reaction kettle was controlled to be 220 ° C and the reaction time was 8h, so that the nano-diamond gray powder was concentrated.
  • the sulfuric acid medium is subjected to oxidation and purification treatment with potassium permanganate at high temperature, and then the product is ultrasonically washed to neutrality with deionized water, centrifuged, and dried to obtain purified nano-diamond powder; the nano-diamond gray powder is a commercially available product,
  • the diamond content in the nano-diamond gray powder is greater than 90%; the mass fraction of the concentrated sulfuric acid is 98%.
  • a method for preparing a monodispersed aminated nano-diamond colloidal solution in this embodiment includes the following steps:
  • the clarified and transparent nano-diamond black colloid solution obtained in this example was removed by a rotary evaporator (model: N-1001, Shanghai Ailang Instrument Co., Ltd.), and a water bath (model: SB-2000, Shanghai Ailang Instrument Co., Ltd.) The temperature was 80 ° C to obtain aminated nano-diamond powder.
  • the obtained aminated nano-diamond powder was dissolved in DMF under the action of ultrasound and sonicated for 15 min to obtain a clear and transparent black colloidal solution of nano-diamond with a higher concentration, which was characterized by dynamic light scattering, as shown in Figure 7 As shown.
  • the median average particle size of the aminated nanodiamonds in the colloidal solution prepared in this embodiment is about 50nm, and the average particle size of the aminated nanodiamonds in the colloidal solution obtained by secondary dispersion is about 60nm.
  • a method for preparing a monodispersed aminated nano-diamond colloidal solution in this embodiment includes the following steps:
  • the nano-diamond dispersed after the amination modification was 0.473 g, and the yield was 94.6%.
  • the obtained aminated nano-diamond powder was dissolved in water under the action of ultrasound and sonicated for 15 minutes to obtain a clear and transparent black colloidal solution of nano-diamond with a higher concentration, which was characterized by dynamic light scattering, as shown in FIG. 7
  • the average particle size of the aminated nanodiamonds in the colloidal solution prepared in this embodiment is about 50nm
  • the average particle size of the aminated nanodiamonds in the colloidal solution obtained by secondary dispersion is about 50nm. .
  • a method for preparing a monodispersed aminated nano-diamond colloidal solution in this embodiment includes the following steps:
  • the clarified and transparent nano-diamond black colloid solution obtained in this example was removed by a rotary evaporator (model: N-1001, Shanghai Ailang Instrument Co., Ltd.), and a water bath (model: SB-2000, Shanghai Ailang Instrument Co., Ltd.) The temperature was 80 ° C to obtain aminated nano-diamond powder.
  • the XRD test was performed on the aminated nano-diamond powder obtained in this embodiment, and the test result is shown in FIG. 1.
  • FIG. 1 the aminated nano-diamond powder obtained in this embodiment, the nano-diamond raw material, and the purified nano-diamond
  • the XRD diffraction peaks are basically the same, which proves that the structure of the nanodiamonds modified by amination is basically unchanged.
  • the colloidal solution after secondary dispersion obtained in this embodiment is characterized by dynamic light scattering, and the test result is shown in FIG. 7.
  • the median average particle size of the aminated nanodiamonds in the colloidal solution prepared in this embodiment is about 50nm, and the average particle size of the aminated nanodiamonds in the colloidal solution obtained by secondary dispersion is about 60nm.
  • a method for preparing a monodispersed aminated nano-diamond colloidal solution in this embodiment includes the following steps:
  • the clarified and transparent nano-diamond black colloid solution obtained in this example was removed by a rotary evaporator (model: N-1001, Shanghai Ailang Instrument Co., Ltd.), and a water bath (model: SB-2000, Shanghai Ailang Instrument Co., Ltd.) The temperature was 80 ° C to obtain aminated nano-diamond powder.
  • a method for preparing a monodispersed aminated nano-diamond colloidal solution in this embodiment includes the following steps:
  • the clarified and transparent nano-diamond black colloid solution obtained in this example was removed by a rotary evaporator (model: N-1001, Shanghai Ailang Instrument Co., Ltd.), and a water bath (model: SB-2000, Shanghai Ailang Instrument Co., Ltd.) The temperature was 80 ° C to obtain aminated nano-diamond powder.
  • the nano-diamond gray powder and the purified nano-diamond were subjected to XRD tests, and the test results are shown in FIG. 1. It can be seen from the curve (a) in FIG. 1 that the diffraction peaks appearing at about 20-30 ° in the unpurified nano-diamond raw materials are the peaks of impurities such as graphite (100) and amorphous carbon, and the noise is high, indicating that they have not been purified.
  • the nanocrystalline diamond has defects, imperfections and a certain amount of non-diamond carbon; after oxidation and purification of potassium permanganate, the amorphous carbon and graphite diffraction peaks in the middle of 20-30 ° in the graph (b) disappear, It shows that potassium permanganate can effectively remove the amorphous carbon, graphite and other impurities in the nano-diamond crystals, and the nano-diamond powder with high purity and perfect crystal form is obtained by oxidation treatment. In addition, due to the strong oxidation of potassium permanganate, the nano-diamonds were etched, which reduced the carbon content of the (111) and (220) crystal forms.
  • Figure (c) is the nanometer modified by ball milling amination in Example 1.
  • the XRD spectrum of diamond shows from Figure (c) that the (111) crystal plane and (220) crystal plane of the nanodiamond after modification are intact and have little change in strength with respect to the nanodiamond after purification, indicating the ball milling amination modification process Medium does not have much effect on the nano-diamond crystal form, and the amination process maintains the integrity of the nano-diamond crystal form.
  • Example 2 is a FTIR spectrum of a nanodiamond sample before and after amination in Example 1 of the present invention, wherein: (a) a FTIR spectrum of a purified nanodiamond; (b) a FTIR spectrum of a nanodiamond after modification;
  • the surface of the purified nano-diamond has carboxyl groups.
  • the broad absorption peak near 3332 cm -1 is a stretching vibration absorption peak of -OH
  • the absorption peak near 1629 cm -1 is -OH.
  • the bending vibration absorption peak of -COOH appears near 1791cm -1 as the stretching vibration absorption peak of -COOH, which indicates that the surface of the oxidized nanodiamond has carboxyl and hydroxyl groups
  • the anti-symmetric stretching vibration peak of COC appears near 1259cm -1 .
  • the broad absorption peak appearing near 3413cm -1 is a stretching vibration peak of -NH
  • the new absorption peak appearing at 1401cm -1 is a stretching vibration peak of -CN.
  • This characteristic peak indicates that the amino group was bonded to the surface of the nano-diamond after modified by ball milling.
  • the carbonyl stretching vibration peak of nanodiamond modified red shifted from 1791cm -1 to around 1779cm -1 , and it also showed that ball milling amination modified nanodiamond can be effectively functionalized.
  • Nano-diamond which shows the success of amination modified nano-diamond.
  • Fig. 3 is a digital photo of the colloidal solution of nanodiamonds prepared under different conditions of amination reagent ratios in Examples 1 to 5 above.
  • the curves a to e in FIG. 4 are the particle diameters of the aminated nanodiamond colloidal solution prepared under different amination reagent ratios (mass ratio of nanodiamond to amination reagent ammonium chloride) in Examples 1 to 5 of the present invention.
  • the surface of the nano-diamond has more hydrophilic groups, and the nano-diamond has better dispersibility in water.
  • the stability of the system can be enhanced. Therefore, the nanodiamonds modified by amination can be stably stored for a long time.
  • the amination reagent ratio When the amination reagent ratio is increased from 1: 0 to 1:10, the corresponding potential value of each pH value decreases, and when the amination reagent ratio is increased to 1:20, the corresponding potential value of each pH value increases. It shows that the surface of the nano-diamonds modified by amination has more hydrophilic groups, and the electrostatic repulsion between particles is enhanced to achieve the dispersion effect.
  • FIG. 6 is a real digital photo of the colloidal solution obtained by secondary dispersion of the aminated nano-diamond prepared in Example 1 of the present invention in different solvents. It can be seen from FIG. 6 that the nano-diamond colloid solution dispersed in the water phase after being modified by amination is dried to obtain nano-diamond particles, which can be dispersed again in water, DMSO, ethanol, DMF, etc. under the action of ultrasound. Colloids are formed in the solvent. Through the DLS test, the average particle size does not change much, and it can be stored stably for a long time. It has a wide range of applications in drug carriers, thermal interface materials, optoelectronic materials, and lubricating materials. The experiment is: the grinding time is 2h, the grinding medium ratio is 1: 5, the grinding material ratio is 1:10, and the grinding aid ratio (NaCl) is 1: 5.
  • FIG. 7 is a comparison diagram of the particle size distribution of the colloidal solution obtained by secondary dispersion of the aminated nanodiamonds prepared in Example 3 of the present invention in different solvents;
  • FIG. 8 is the aminated nanodiamonds prepared in Example 3 of the present invention TEM picture of diamond. It can be seen from FIG. 7 that the nano-diamonds modified by amination are re-dispersed in water, ethanol, DMSO, and DMF and are in a colloidal state. According to DLS analysis, the average particle size of the red-dispersed nanoparticles in water is 48.9 nm, 53.6 nm in ethanol, 58.4 nm in DMSO, and 68.7 nm in DMF. The particle diameters are all below 100nm, indicating that the nano-diamond modified or re-dispersed is better.
  • Example 6 Application of the aminated nanodiamond prepared in Example 1 as a fluorescent probe in cell labeling.
  • the aminated nanodiamond itself has a certain fluorescence intensity, it will be found that the fluorescence intensity is not obvious.
  • the surface of the aminated nanodiamond particle contains many amino functional groups, as the cell absorbs the particle, The distance between intracellular particles gradually decreases, and many hydrogen bonds are formed.
  • the structure of the aminated nanodiamond is relatively stable, and it is not easy to perform intramolecular motion and rotation to release the energy to return the molecule to a steady state like ordinary organic molecules. Instead, it may release energy in a weak vibration mode, but the formation of hydrogen bonds is inhibited. Higher energy is released by vibration to return to steady state. At this time, the energy released in the form of light energy is increased, and the fluorescence intensity is increased.

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Abstract

提供一种单分散胺化纳米金刚石胶体溶液的制备方法，包括：在室温条件下，将纯化纳米金刚石粉体、氯化铵、氯化钠混合后置于球磨机中进行干法球磨，将球磨后的混合物经去离子水洗涤，超声分散，离心，制得单分散胺化纳米金刚石胶体溶液。还提供一种单分散胺化纳米金刚石胶体溶液的二次分散方法，步骤如下：将胺化纳米金刚石胶体溶液干燥后得到胺化纳米金刚石粉体，在超声或剪切作用下再次分散在溶剂中。还提供一种该制备方法制得的单分散胺化纳米金刚石溶液用于细胞标记的应用。

Description

一种单分散胺化纳米金刚石胶体溶液的制备方法及其二次分散工艺和在细胞标记中的应用 技术领域
本发明属于纳米金刚石加工技术领域，具体涉及一种单分散胺化纳米金刚石胶体溶液的制备方法及其二次分散工艺和在作为荧光探针在细胞标记中的应用。
背景技术
纳米金刚石由于具有独特的半导体特性和良好的生物相容性，被广泛应用于生物传感器的基体材料。其独特的化学组成和电学性能使得纳米金刚石相比于其他传统的物质，如金、硅、玻璃等更有利于生物电子学和生物传感器的发展，大多数生物医学应用需要高效可靠的金刚石表面功能化，由于生物分子一般由氨基酸序列组成，许多研究工作者注意到氨基功能化的纳米金刚石应用于生物传感器具有十分理想的前景。
现有技术中已有纳米金刚石功能化的相关研究成果报道，例如，专利申请公布号为CN 103173267 A的专利公开了“一种末端基团为氨基的纳米金刚石衍生物及其制备方法和用途”，该发明采用经纯化、混酸氧化处理后的纳米金刚石，先与氯化亚砜反应，然后分别与二元胺化合物、三氯均三嗪进行亲核取代反应；再与二元胺化合物、三氯均三嗪进行迭代反应制得，但是该方法具体制备工艺繁琐，反应条件苛刻，且该方法中涉及的亲核取代反应需要在惰性气体保护条件下进行，生产成本高，且制得的产物中还含有杂质。另外，M.Khan，N.Shahzad等也公开了采用不同的研磨介质(如NH 4HCO 3，NaCl或蔗糖)对爆轰纳米金刚石(DNDs)进行球磨，研究了不同研磨介质在不同溶剂(乙醇，水或二甲基亚砜)中对纳米金刚石分散行为的影响(M.Khan，N.Shahzad，et al.Dispersion behavior and the influences of ball milling technique on functionalization of detonated nano-diamonds[J].Diamond&Related Materials 61(2016)32–40.)，但是该研究制得的胺化纳米金刚石仍然存在杂质引入的问题，另外，虽然该研究中研磨介质辅助的胺化纳米金刚石在环氧树脂中具有较好的分散性，但仍不能得到单分散的纳米金刚石胶体。
基于现有技术存在的上述技术问题，特提出本申请。
发明内容
本发明的目的是基于目前纳米金刚石表面直接胺化过程中还存在的杂质引入且不能得到单分散纳米金刚石胶体的技术问题，提出一种单分散胺化纳米金刚石胶体溶液的制备方法及其二次分散工艺和在细胞标记中的应用，本发明方法能够使纳米金刚石表面 简单胺化改性，且得到的单分散胶体溶液能再次分散。
为了实现本发明的上述第一个目的，本发明采用如下技术方案：
一种单分散胺化纳米金刚石胶体溶液的制备方法，所述方法包括如下步骤：
选择合适粒径的纯化纳米金刚石粉体，在室温条件下将所述纯化纳米金刚石粉体、氯化铵(胺化试剂)和氯化钠(研磨助剂)分别置于球磨机中进行干法球磨，球磨后的混合物经去离子水洗涤、超声分散、离心、即可制得本发明所述的单分散胺化纳米金刚石黑色胶体溶液。
本发明上述所述技术方案中的纯化纳米金刚石粉体可以是市售产品，也可以是爆轰合成的纯化后的纳米金刚石。在一个优选的实施方案中，所述纯化纳米金刚石粉体的纯度>90％，优选纯度>95％；所述纯化纳米金刚石粉体的粒度为30nm～100μm。
在一个优选的实施方案中，所述纯化纳米金刚石和去离子水的质量比为1:(20～2000)，所述氯化铵和氯化钠的质量比为1:(2～100)。
在一个优选的实施方案中，所述纯化纳米金刚石粉体和所述氯化铵的质量比为1:(0.1～100)。
在一个优选的实施方案中，上述技术方案在球磨过程中加入了球磨珠，所述球磨珠为非金属球磨珠，所述球磨珠包括但不限于玛瑙，刚玉，氧化锆，二氧化硅，优选采用玛瑙珠。
进一步优选地，所述球磨珠的直径大于1mm，优选大于4mm，球磨珠可以是单一直径的球磨珠，也可以是由直径大小不同的混合球磨珠组成，混合球磨珠珠子直径比优选为1.5～200，更优选为2～10。
在一个优选的实施方案中，所述球磨在室温下进行的，采用的是一种高效的行星式球磨机，所述球磨机线转速为20～600r/min，线速度为20～600m/min，所述球磨时间为0.1～20h。
在一个优选的实施方案中，所述球磨机转速为512r/min，在优化过程中发现，氯化铵的用量对最终产物的氨基含量也有着显著的影响，氯化铵质量越小或越大，所获得的纳米金刚石表面氨基含量越少。
在一个优选的实施方案中，所述胺化试剂比(纳米金刚石与氯化铵质量比)比为1：0，1：5，1：10，1：15，1：20，胺化试剂比越小，使体系中胺化试剂氯化铵分解氨基浓度小，胺化改性不充分，胺化试剂比越大，分解出的氨基浓度大，使得胺化反应平衡提前，氨基浓度降低。
本发明方法上述制得的单分散胺化纳米金刚石胶体溶液能再次分散，为了实现本发明的上述第二个目的，本发明采用的二次分散工艺具体步骤如下：
(i)将上述方法制得的胺化纳米金刚石黑色胶体溶液干燥，得到胺化纳米金刚石粉体；
(ii)将步骤(i)制得的胺化纳米金刚石粉体在超声或剪切作用下再次分散在溶剂中，振荡，得到澄清透明的黑色胶体溶液。
在一个优选的实施方案中，步骤(i)中所述干燥处理可以采用旋转蒸发仪干燥、喷雾干燥或冷冻干燥中的任一种方式。
在一个优选的实施方案中，步骤(i)中旋转蒸发仪干燥温度不高于80℃，可以是60℃，65℃，70℃，80℃，一般不超过80℃。
在一个优选的实施方案中，步骤(ii)中所述的溶剂为水、二甲基亚砜(DMSO)、乙醇、乙二醇或二甲基甲酰胺(DMF)中的任一种或多种或含有上述溶剂体积比超过20％的其它复合溶剂。
在一个优选的实施方案中，步骤(ii)中所述的超声可利用100～1000W超声清洗机，所述剪切分散(>500rpm)处理时间为15min。
本发明的第三个目的在于提供上述所述的胺化纳米金刚石的应用，本发明的胺化纳米金刚石可作为荧光探针对细胞进行标记。
在一个优选的实施方案中，胺化纳米金刚石作为荧光探针对细胞进行标记的浓度为0.5～2mg/ml。
与现有技术相比，本发明涉及的一种单分散胺化纳米金刚石胶体溶液的制备方法及其二次分散工艺和在细胞标记中的应用具有如下有益效果：
(1)本发明制备方法简单、易操作，无需特殊的反应设备，整个反应过程无需在惰性气体氛围下进行，且采用的试剂廉价易得，生产成本低，容易实现工业化生产；
(2)本发明使用球磨法制得的胺化纳米金刚石产率高，高于90％，最高可达97％，且制得的胺化纳米金刚石胶体具有单分散性；
(3)本发明获得的胺化纳米金刚石粉体能实现二次分散，使分散好的纳米金刚石在运输上更加方便，进一步拓宽了纳米金刚石的应用领域；
(4)本发明在实验过程中未对环境产生不良影响，本发明产生的酸废液处理比较简单，可进一步浓缩或回收再利用。
附图说明
图1中(a)、(b)、(c)分别为本发明实施例3中纳米金刚石原料、纯化后的纳米金刚石、胺化改性后的纳米金刚石的XRD对比谱图；
图2为本发明实施例3中胺化前后纳米金刚石样品的FTIR对比谱图，其中：(a)纯化后纳米金刚石的FTIR谱图；(b)胺化改性后纳米金刚石的FTIR谱图；
图3为本发明实施例1～5中不同胺化试剂比条件下制得的纳米金刚石后胶体溶液的实物数码照片；
图4为本发明实施例1～5中不同胺化试剂比条件下制得的胺化纳米金刚石胶体溶液的粒径分布对比图；
图5为本发明实施例1～5中不同胺化试剂比条件下制得的纳米金刚石胶体溶液Zeta电位-pH关系曲线对比图；
图6为本发明实施例3中制得的胺化纳米金刚石在不同溶剂中进行二次分散获得的胶体溶液的实物数码照片；
图7为本发明实施例3中制得的胺化纳米金刚石在不同溶剂中进行二次分散获得的胶体溶液的粒径分布对比图。
图8为本发明实施例3中制得的胺化纳米金刚石的透射电镜照片；
图9为实施例6中胺化纳米金刚石混合液培养5种不同cell在30min的实时拍摄图，其中：a)HepG2细胞，b)HeLa细胞，c)L02细胞，d)H3B细胞，e)NIH 3T3细胞。左侧明场图片，右侧荧光场图片。
具体实施方式
下面结合附图对本发明的实施案例作详细说明。本实施案例在本发明技术方案的前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施案例。
根据本申请包含的信息，对于本领域技术人员来说可以轻而易举地对本发明的精确描述进行各种改变，而不会偏离所附权利要求的精神和范围。应该理解，本发明的范围不局限于所限定的过程、性质或组分，因为这些实施方案以及其他的描述仅仅是为了示意性说明本发明的特定方面。实际上，本领域或相关领域的技术人员明显能够对本发明实施方式作出的各种改变都涵盖在所附权利要求的范围内。
为了更好地理解本发明而不是限制本发明的范围，在本申请中所用的表示用量、百分比的所有数字、以及其他数值，在所有情况下都应理解为以词语“大约”所修饰。因此，除非特别说明，否则在说明书和所附权利要求书中所列出的数字参数都是近似值， 其可能会根据试图获得的理想性质的不同而加以改变。各个数字参数至少应被看作是根据所报告的有效数字和通过常规的四舍五入方法而获得的。
为了提高胺化纳米金刚石产率，下述各实施例中的纳米金刚石均采用下述工艺进行纯化处理，步骤如下：
将10g纳米金刚石灰色粉体与30g～50g浓硫酸、10g高锰酸钾在反应釜中反应，控制所述反应釜中的反应温度为220℃，反应时间为8h，使纳米金刚石灰色粉体浓硫酸介质中经高锰酸钾高温下氧化纯化处理，然后用去离子水将产物超声清洗至中性，离心、干燥，获得纯化纳米金刚石粉体；所述纳米金刚石灰色粉体为市售产品，纳米金刚石灰色粉体中金刚石含量大于90％；所述浓硫酸的质量分数为98％。
实施例1
本实施例的一种单分散胺化纳米金刚石胶体溶液的制备方法，所述方法包括如下步骤：
(1)取球磨珠(玛瑙珠大珠直径6.8mm与小珠直径4.2mm质量比1：5)放入体积为100ml大小的球磨罐中，加入0.5g上述纯化处理的纳米金刚石和2.5g氯化钠，不加氯化铵，设置球磨机(型号：QM-1SP2，南京大学仪器厂)线速度为512m/min，球磨时间为2h，关闭球磨机，隔30min后取下球磨罐。
(2)取出球磨罐中的球磨珠，用去离子水洗涤(250～300ml，可适当加大水的体积)，在超声的作用下(8000W超声一般不超过1min，360W超声一般15min)分散，在10000r/min的离心速度下离心，时间设置5min，重复超作4～5次，获得澄清透明的单分散胺化纳米金刚石黑色胶体溶液，并对其进行动态光散射(DLS)表征，如图4所示。
本实施例上述制得的单分散胺化纳米金刚石胶体溶液的二次分散工艺具体步骤如下：
(1)将本实施例上述获得的澄清透明的纳米金刚石黑色胶体溶液用旋转蒸发仪(型号：N-1001，上海爱朗仪器有限公司)除去水溶剂，设置水浴锅(型号：SB-2000，上海爱朗仪器有限公司)温度为80℃，得到胺化纳米金刚石粉体。
(2)将得到的胺化纳米金刚石粉体溶在超声作用下溶解在DMF中，超声15min，得到浓度较大的纳米金刚石澄清透明的黑色胶体溶液，对其进行动态光散射表征，如图7所示。本实施例制得的胶体溶液中胺化纳米金刚石的平均粒径大小中值在50nm左右，二次分散获得的胶体溶液中胺化纳米金刚石平均粒径在60nm左右。
实施例2
本实施例的一种单分散胺化纳米金刚石胶体溶液的制备方法，所述方法包括如下步骤：
(1)取球磨珠(玛瑙珠大珠直径6.8mm与小珠直径4.2mm质量比1：5)放入体积为100ml大小的球磨罐中，加入0.5g上述纯化处理的纳米金刚石和2.5g氯化铵，2.5g氯化钠，设置球磨机(型号：QM-1SP2，南京大学仪器厂)线速度为512m/min，球磨时间为2h，关闭球磨机，隔30min后取下球磨罐。
(2)取出球磨罐中的球磨珠，用去离子水洗涤(250～300ml，可适当加大水的体积)，在超声的作用下(8000W超声一般不超过1min，360W超声一般15min)分散，在10000r/min的离心速度下离心，时间设置5min，重复超作4～5次，获得澄清透明的单分散胺化纳米金刚石黑色胶体溶液，并对其进行动态光散射(DLS)表征，如图4所示。将其进行透射电镜分析，其形貌如图8所示。
本实施例上述制得的单分散胺化纳米金刚石胶体溶液的二次分散工艺具体步骤如下：
(1)将本实施例上述获得的澄清透明的纳米金刚石黑色胶体溶液用旋转蒸发仪(型号：N-1001，上海爱朗仪器有限公司)除去水溶剂，设置水浴锅(型号：SB-2000，上海爱朗仪器有限公司)温度为80℃，得到胺化纳米金刚石粉体。称其重量，按公式计算得出胺化改性后的纳米金刚石收率：
Yield＝m 1/m 0×100％
式中：m o-纳米金刚石原料质量，单位g；
m 1-胺化纳米金刚石干燥后的质量，单位g；
胺化改性后分散的纳米金刚石为0.473g，收率为94.6％。
(2)将得到的胺化纳米金刚石粉体在超声作用下溶解在水中，超声15min，得到浓度较大的纳米金刚石澄清透明的黑色胶体溶液，对其进行动态光散射表征，如图7所示，由图7可以看出，本实施例制得的胶体溶液中胺化纳米金刚石的平均粒径大小中值在50nm左右，二次分散所得胶体溶液中胺化纳米金刚石的平均粒径在50nm左右。
实施例3
本实施例的一种单分散胺化纳米金刚石胶体溶液的制备方法，所述方法包括如下步骤：
(1)取球磨珠(玛瑙珠大珠直径6.8mm与小珠直径4.2mm质量比1：5)放入体积为100ml大小的球磨罐中，加入0.5g上述纯化处理的纳米金刚石和5g氯化铵，2.5g氯 化钠，设置球磨机(型号：QM-1SP2，南京大学仪器厂)转速为512r/min，球磨时间为2h，关闭球磨机，隔30min后取下球磨罐。
(2)取出球磨罐中的球磨珠，用去离子水洗涤(250～300ml，可适当加大水的体积)，在超声的作用下(8000W超声一般不超过1min，360W超声一般15min)分散，在10000r/min的离心速度下离心，时间设置5min，重复超作4～5次，获得了澄清透明的单分散胺化纳米金刚石黑色胶体溶液，并对其进行动态光散射(DLS)表征，如图4所示。
本实施例上述制得的单分散胺化纳米金刚石胶体溶液的二次分散工艺具体步骤如下：
(1)将本实施例上述获得的澄清透明的纳米金刚石黑色胶体溶液用旋转蒸发仪(型号：N-1001，上海爱朗仪器有限公司)除去水溶剂，设置水浴锅(型号：SB-2000，上海爱朗仪器有限公司)温度为80℃，得到胺化纳米金刚石粉体。
(2)将得到的胺化纳米金刚石粉体溶在超声作用下溶解在DMSO中，超声15min，得到浓度较大的纳米金刚石澄清透明的黑色胶体溶液。
将本实施例得到的胺化纳米金刚石粉体进行XRD测试，测试结果如图1所示，由图1可知，本实施例得到的胺化纳米金刚石粉体与纳米金刚石原料、纯化后的纳米金刚石的XRD衍射峰基本一致，证明胺化改性后的纳米金刚石结构基本未发生变化。
将本实施例得到的二次分散后的胶体溶液进行动态光散射表征，测试结果如图7所示。本实施例制得的胶体溶液中胺化纳米金刚石的平均粒径大小中值在50nm左右，二次分散获得的胶体溶液中胺化纳米金刚石平均粒径在60nm左右。
实施例4
本实施例的一种单分散胺化纳米金刚石胶体溶液的制备方法，所述方法包括如下步骤：
(1)取球磨珠(玛瑙珠大珠直径6.8mm与小珠直径4.2mm质量比1：5)放入体积为100ml大小的球磨罐中，加入0.5g上述纯化处理的纳米金刚石和7.5g氯化铵，2.5g氯化钠，设置球磨机(型号：QM-1SP2，南京大学仪器厂)线速度为512m/min，球磨时间为2h，关闭球磨机，隔30min后取下球磨罐。
(2)取出球磨罐中的球磨珠，用去离子水洗涤(250～300ml，可适当加大水的体积)，在超声的作用下(8000W超声一般不超过1min，360W超声一般15min)分散，在10000r/min的离心速度下离心，时间设置5min，重复超作4～5次，获得澄清透明的单 分散胺化纳米金刚石黑色胶体溶液，并对其进行动态光散射(DLS)表征，表征结果如图4所示。
本实施例上述制得的单分散胺化纳米金刚石胶体溶液的二次分散工艺具体步骤如下：
(1)将本实施例上述获得的澄清透明的纳米金刚石黑色胶体溶液用旋转蒸发仪(型号：N-1001，上海爱朗仪器有限公司)除去水溶剂，设置水浴锅(型号：SB-2000，上海爱朗仪器有限公司)温度为80℃，得到胺化纳米金刚石粉体。
(2)将得到的胺化纳米金刚石粉体溶在超声作用下溶解在乙醇中，超声15min，得到浓度较大的纳米金刚石澄清透明的黑色胶体溶液，对其进行动态光散射表征，表征结果如图7所示。本实施例制得的胶体溶液中胺化纳米金刚石的平均粒径大小中值在50nm左右，二次分散所得胶体溶液中胺化纳米金刚石的平均粒径在60nm左右。
实施例5
本实施例的一种单分散胺化纳米金刚石胶体溶液的制备方法，所述方法包括如下步骤：
(1)取球磨珠(玛瑙珠大珠直径6.8mm与小珠直径4.2mm质量比1：5)放入体积为100ml大小的球磨罐中，加入0.5g上述纯化处理的纳米金刚石和10.0g氯化铵，2.5g氯化钠，设置球磨机(型号：QM-1SP2，南京大学仪器厂)线速度为512m/min，球磨时间为2h，关闭球磨机，隔30min后取下球磨罐。
(2)取出球磨罐中的球磨珠，用去离子水洗涤(250～300ml，可适当加大水的体积)，在超声的作用下(8000W超声一般不超过1min，360W超声一般15min)分散，在10000r/min的离心速度下离心，时间设置5min，重复超作4～5次，获得澄清透明的单分散胺化纳米金刚石黑色胶体溶液，并对其进行动态光散射(DLS)表征，表征结果如图4所示。
本实施例上述制得的单分散胺化纳米金刚石胶体溶液的二次分散工艺具体步骤如下：
(1)将本实施例上述获得的澄清透明的纳米金刚石黑色胶体溶液用旋转蒸发仪(型号：N-1001，上海爱朗仪器有限公司)除去水溶剂，设置水浴锅(型号：SB-2000，上海爱朗仪器有限公司)温度为80℃，得到胺化纳米金刚石粉体。
(2)将得到的胺化纳米金刚石粉体溶在超声作用下溶解在乙醇中，超声15min，得到浓度较大的纳米金刚石澄清透明的黑色胶体溶液。
将上述纳米金刚石灰色粉体、纯化后的纳米金刚石分别进行XRD测试，测试结果如图1所示。由图1中(a)曲线可知，未经纯化的纳米金刚石原料中在20～30°左右出现的衍射峰是石墨(100)和无定形碳等杂质的峰且噪声较高，说明未经纯化的纳米金刚石晶型有缺陷，不完美且存在一定含量的非金刚石碳；经高锰酸钾氧化纯化处理后，图(b)曲线中20～30°中间的无定型碳和石墨衍射峰消失，说明高锰酸钾能有效去除纳米金刚石晶体中的无定型碳和石墨等杂质，氧化处理得到了纯度较高、晶型完美的纳米金刚石粉体。另外，由于高锰酸钾氧化性较强，纳米金刚石被刻蚀，使得其(111)和(220)晶型碳含量降低，图(c)是实施例1中经球磨胺化改性后纳米金刚石的XRD谱图，由图(c)可知，胺化改性后纳米金刚石(111)晶面和(220)晶面完整且与纯化后纳米金刚石强度变化不大，说明球磨胺化改性过程中对纳米金刚石晶型无太大影响，胺化过程保持了纳米金刚石晶型的完整性。
图2为本发明实施例1中胺化前后纳米金刚石样品的FTIR对比谱图，其中：(a)纯化后纳米金刚石的FTIR谱图；(b)胺化改性后纳米金刚石的FTIR谱图；
通过FTIR光谱图可知，纯化后的纳米金刚石表面带有羧基基团，图中3432cm -1附近出现的宽的吸收峰为-OH的伸缩振动吸收峰，1629cm -1附近出现的吸收峰为-OH的弯曲振动吸收峰，1791cm -1附近出现的为-COOH的伸缩振动吸收峰，说明氧化后的纳米金刚石表面带有羧基和羟基，在1259cm -1附近出现的为C-O-C的反对称伸缩振动峰。通过胺化改性后的红外光谱图可知，在3413cm -1附近出现的宽的吸收峰为-NH的伸缩振动峰，在1401cm -1处出现的新的吸收峰为-C-N的伸缩振动峰，此特征峰说明了经球磨改性处理后的纳米金刚石表面键接了氨基基团。对比改性前后红外光谱图可以知道，胺化改性后纳米金刚石的羰基伸缩振动峰由1791cm -1红移至1779cm -1附近，同时也表明了球磨胺化改性纳米金刚石可以有效地功能化纳米金刚石，说明胺化改性纳米金刚石成功。
表1 红外谱图分析表
Figure PCTCN2018110699-appb-000001
图3为上述实施例1～5中不同胺化试剂比条件下制得的纳米金刚石后胶体溶液的 实物数码照片。图4中曲线a～e分别为本发明实施例1～5中不同胺化试剂比(纳米金刚石与胺化试剂氯化铵的质量比)条件下制得的胺化纳米金刚石胶体溶液的粒径分布对比图；由图3和4可以看出，当不加入胺化试剂比即胺化试剂与纳米金刚石质量比为1:0时，纳米金刚石分散效果不好，水溶液中呈乳浊液，且平均粒径为516nm，当加入胺化改性粒子后，纳米金刚石分散较好，当胺化试剂的量从1:5增加到1:10时，纳米金刚石最小粒径达43.4nm。同时，纳米金刚石表面胺化后使得纳米金刚石表面带有更多的亲水性基团，纳米金刚石在水中分散性能更好，氨基在纳米金刚石表面固定后，能增强体系稳定性。所以，胺化改性后的纳米金刚石能长期稳定的保存。
图5为本发明实施例1～5中不同胺化试剂比条件下制得的纳米金刚石胶体溶液Zeta电位-pH关系曲线对比图。由图5可以看出，当胺化试剂比比设置为1:10时，电位降低最明显。在pH＝3左右，出现等电点。不同pH对应下的电位值最小，当pH＝10时，此时Zeta电位有最小值为-49.9mV。当胺化试剂比从1:0增加至1:10时，各pH值对应下电位值降低，当胺化试剂比增加到1:20时，各pH值对应电位值增加。说明胺化改性后纳米金刚石表面带有更多的亲水性基团，颗粒间静电斥力增强，达到分散效果。
图6为本发明实施例1中制得的胺化纳米金刚石在不同溶剂中进行二次分散获得的胶体溶液的实物数码照片。由图6可以看出，经过胺化改性后分散在水相中的纳米金刚石胶体溶液经干燥后得到纳米金刚石颗粒，此颗粒在超声的作用下能再次分散在水、DMSO、乙醇、DMF等溶剂中形成胶体。通过DLS测试，其平均粒径变化不大，且能长久稳定的保存。在药物载体、热界面材料、光电材料、润滑材料中具有很广的应用价值。实验即：研磨时间2h，研磨介质比1:5，研磨物料比1:10，研磨助剂比(NaCl)1:5的样品进行实验。
图7为本发明实施例3中制得的胺化纳米金刚石在不同溶剂中进行二次分散获得的胶体溶液的粒径分布对比图；图8为本发明实施例3中制得的胺化纳米金刚石的透射电镜照片。由图7可以看出，经胺化改性后的纳米金刚石二次分散在水、乙醇、DMSO、DMF中再次分散且呈胶体状态，通过DLS分析可知，其再次分散在水中的平均粒径为48.9nm，乙醇中为53.6nm，DMSO中为58.4nm，DMF中为68.7nm。其粒径均在100nm以下，说明胺化改性或的纳米金刚石、再次分散较好。
实施例6 实施例1制得的胺化纳米金刚石作为荧光探针在细胞标记中的应用。
1、细胞的培养与标记
(1)先接种好需要的五种细胞，每种细胞都接种一个六孔细胞培养板，每孔中细 胞密度：5×10 5±0.05×10 5个，在37℃、5％CO 2培养箱中培养24h后待用，实验所需不同的三个染色时间，每个时间平行两组实验；
(2)配置好待用的胺化纳米金刚石和培养基混合染色液，其中：胺化纳米金刚石浓度为1mg/ml；
(3)取一种事先培养好的六孔细胞培养板，先用移液枪吸出培养基，再往六孔细胞培养板的每个孔中都添加3ml混合液到细胞中染色，继续于培养箱中培养。
2、细胞标记图片的采集
(1)在加入胺化纳米金刚石混合液后的5min、15min、30min，分别吸出混合液备用；
(2)吸出混合液以后，用PBS冲洗细胞三次，往六孔细胞培养板的每个孔中都添加1ml新鲜培养基；
(3)在显微镜下观察，取细胞形态较好的视野分别采集同一视野下明场和荧光场的图片。
3、胺化纳米金刚石进入活细胞内的实时拍摄图如图9中(a)～(e)所示。从胺化纳米金刚石染色30min的实时拍摄图片和之前细胞外的荧光强度对比可以看出，胺化纳米金刚石颗粒在进入细胞以前荧光强度比较弱，但在实时拍摄图片中发现荧光强度有所增强。可能是当胺化纳米金刚石进入细胞后密度增大，在细胞范围内达到了聚集的效果，在细胞内震动受到了限制，辐射衰减比例增加，荧光强度增强，能够作为荧光探针对细胞进行标记。
虽然胺化纳米金刚石本身就具有一定的荧光强度，但会发现荧光强度并不明显，当进入到细胞内时，由于胺化纳米金刚石颗粒表面含有许多的氨基官能团，随着细胞对颗粒的吸收，细胞内颗粒之间的距离会逐渐减小，会形成许多氢键。胺化纳米金刚石的结构较为稳定，不容易像一般的有机分子一样进行分子内运动和旋转释放能量使分子回归稳态，而是可能以微弱的震动方式释放能量，但由于氢键的形成抑制了以震动方式释放出较高的能量使之回归稳态，这时会增加了以光能的形式释放能量，荧光强度会有所增加。

Claims (10)

  1. 一种单分散胺化纳米金刚石胶体溶液的制备方法，其特征在于：所述方法包括如下步骤：
    选择合适粒径的纯化纳米金刚石粉体，在室温条件下将所述纳米金刚石粉体、氯化铵和氯化钠混合后置于球磨机中进行干法球磨，球磨后的混合物经去离子水洗涤、超声分散、离心、即可制得本发明所述的单分散胺化纳米金刚石黑色胶体溶液。
  2. 根据权利要求1所述的单分散胺化纳米金刚石胶体溶液的制备方法，其特征在于：所述纯化纳米金刚石粉体的粒度为30nm～100μm。
  3. 根据权利要求1所述的单分散胺化纳米金刚石胶体溶液的制备方法，其特征在于：所述纯化纳米金刚石和去离子水的质量比为1:20～2000，所述氯化铵和氯化钠的质量比为1:2～100。
  4. 根据权利要求1所述的单分散胺化纳米金刚石胶体溶液的制备方法，其特征在于：所述纯化纳米金刚石粉体和所述氯化铵的质量比为1:0.1～100。
  5. 根据权利要求1所述的单分散胺化纳米金刚石胶体溶液的制备方法，其特征在于：在球磨过程中加入了球磨珠，所述球磨珠为非金属球磨珠，所述球磨珠包括但不限于玛瑙，刚玉，氧化锆，二氧化硅。
  6. 权利要求1～5所述方法制得的单分散胺化纳米金刚石胶体溶液的二次分散方法，其特征在于：所述二次分散工艺具体步骤如下：
    (i)将权利要求1～5所述方法制得的胺化纳米金刚石黑色胶体溶液干燥，得到胺化纳米金刚石粉体；
    (ii)将步骤(i)制得的胺化纳米金刚石粉体在超声或剪切作用下再次分散在溶剂中，振荡，得到澄清透明的黑色胶体溶液。
  7. 根据权利要求6所述的单分散胺化纳米金刚石胶体溶液的二次分散方法，其特征在于：步骤(i)中所述干燥处理可以采用旋转蒸发仪干燥、喷雾干燥或冷冻干燥中的任一种方式。
  8. 根据权利要求6所述的单分散胺化纳米金刚石胶体溶液的二次分散方法，其特征在于：步骤(ii)中所述的溶剂为水、二甲基亚砜、乙醇、乙二醇或二甲基甲酰胺中的任一种或多种或含有上述溶剂体积比超过20％的其它复合溶剂。
  9. 权利要求1～5所述方法制得的单分散胺化纳米金刚石胶体溶液的应用，其特征在于：所述胺化纳米金刚石可应用于细胞标记。
  10. 权利要求9所述的单分散胺化纳米金刚石胶体溶液的应用，其特征在于：所述 胺化纳米金刚石的浓度为0.1～2mg/ml。
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