WO2022138854A1 - 温度感受性プローブ - Google Patents
温度感受性プローブ Download PDFInfo
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- WO2022138854A1 WO2022138854A1 PCT/JP2021/047991 JP2021047991W WO2022138854A1 WO 2022138854 A1 WO2022138854 A1 WO 2022138854A1 JP 2021047991 W JP2021047991 W JP 2021047991W WO 2022138854 A1 WO2022138854 A1 WO 2022138854A1
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- AKQNYQDSIDKVJZ-UHFFFAOYSA-N triphenylsilane Chemical compound C1=CC=CC=C1[SiH](C=1C=CC=CC=1)C1=CC=CC=C1 AKQNYQDSIDKVJZ-UHFFFAOYSA-N 0.000 description 1
- FJDWNXHRKLLNHK-UHFFFAOYSA-N triphenylsilylformic acid Chemical compound C=1C=CC=CC=1[Si](C=1C=CC=CC=1)(C(=O)O)C1=CC=CC=C1 FJDWNXHRKLLNHK-UHFFFAOYSA-N 0.000 description 1
- ARBZZGNUESIQEN-UHFFFAOYSA-N tripropyl(tripropylsilyl)silane Chemical compound CCC[Si](CCC)(CCC)[Si](CCC)(CCC)CCC ARBZZGNUESIQEN-UHFFFAOYSA-N 0.000 description 1
- KHQZLUVCZCAMFU-UHFFFAOYSA-N tripropyl(tripropylsilyloxy)silane Chemical compound CCC[Si](CCC)(CCC)O[Si](CCC)(CCC)CCC KHQZLUVCZCAMFU-UHFFFAOYSA-N 0.000 description 1
- ZHOVAWFVVBWEGQ-UHFFFAOYSA-N tripropylsilane Chemical compound CCC[SiH](CCC)CCC ZHOVAWFVVBWEGQ-UHFFFAOYSA-N 0.000 description 1
- FNRQRIRGNYNSHU-UHFFFAOYSA-N tripropylsilyl acetate Chemical compound CCC[Si](CCC)(CCC)OC(C)=O FNRQRIRGNYNSHU-UHFFFAOYSA-N 0.000 description 1
- AYPSYMMFDVSUFP-UHFFFAOYSA-N tris(hydroxymethyl)silylmethanol Chemical compound OC[Si](CO)(CO)CO AYPSYMMFDVSUFP-UHFFFAOYSA-N 0.000 description 1
- 229910052720 vanadium Inorganic materials 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N25/00—Investigating or analyzing materials by the use of thermal means
- G01N25/20—Investigating or analyzing materials by the use of thermal means by investigating the development of heat, i.e. calorimetry, e.g. by measuring specific heat, by measuring thermal conductivity
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01K—MEASURING TEMPERATURE; MEASURING QUANTITY OF HEAT; THERMALLY-SENSITIVE ELEMENTS NOT OTHERWISE PROVIDED FOR
- G01K11/00—Measuring temperature based upon physical or chemical changes not covered by groups G01K3/00, G01K5/00, G01K7/00 or G01K9/00
- G01K11/20—Measuring temperature based upon physical or chemical changes not covered by groups G01K3/00, G01K5/00, G01K7/00 or G01K9/00 using thermoluminescent materials
Definitions
- the present invention relates to a temperature sensitive probe.
- Non-Patent Document 1 temperature measurement is performed using 200 nm size fluorescent nanodiamond particles having a SiV (silicon-vacancy) center.
- An object of the present invention is to provide a temperature sensitive probe capable of stably and accurately measuring the temperature of a minute space.
- the present invention provides the following temperature sensitive probes.
- a MV center having an average particle size of 1 to 100 nm M represents a Group 14 element selected from the group consisting of Si, Ge, Sn and Pb; V represents a vacancy).
- M represents a Group 14 element selected from the group consisting of Si, Ge, Sn and Pb; V represents a vacancy).
- M represents a Group 14 element selected from the group consisting of Si, Ge, Sn and Pb; V represents a vacancy).
- a temperature sensitive probe containing group 14 element-doped nanodiamonds [2] The temperature sensitive probe according to [1], wherein M is Si.
- RSD standard relative deviation
- An MV center having an average particle size of 1 to 100 nm for measuring the temperature of a minute space M is a group 14 element selected from the group consisting of Si, Ge, Sn and Pb, and V is a vacancy. Use of Group 14 element-doped nanodiamonds with (indicating vacancy).
- M is a group 14 element selected from the group consisting of Si, Ge, Sn and Pb
- V is a vacancy.
- a MV center having an average particle size of 1 to 100 nm (M represents a Group 14 element selected from the group consisting of Si, Ge, Sn and Pb; V represents a vacancy).
- the temperature-sensitive probe is introduced into the cell by mixing the group 14 element-doped nanodiamond with the cell in water, and the cell into which the group 14 element-doped nanodiamond is introduced is irradiated with excitation light to irradiate the ZPL of the MV center.
- a method of measuring the intracellular temperature including.
- the present invention it is possible to measure the temperature in a limited microspace such as an organelle in a cell.
- This utilizes the fact that the peak position of the ZPL (Zero Phonon Line) of the MV center shifts with temperature.
- the MV center has the advantage of being stable and having a strong emission intensity.
- the temperature-sensitive probe of the present invention can finely set the temperature response region in the cell by uniformly dispersing it in the cell or arranging or binding it in a specific place, and can be used as an intracellular fluorescent temperature sensor. can.
- the temperature sensitive probe of the present invention represents a Group 14 element selected from the group consisting of MV centers (M is Si, Ge, Sn and Pb) having an average particle size of 1 to 100 nm. V indicates a vacancy.
- Group 14 element-doped nanodiamonds with. Group 14 element-doped nanodiamonds are nanoparticles, the upper limit of their average particle size being preferably 100 nm, more preferably 70 nm, even more preferably 50 nm, particularly preferably 30 nm, particularly more preferably 20 nm.
- the average particle size of the Group 14 element-doped nanodiamond is small because it does not interfere with the movement or structural change of biomolecules such as intracellular proteins.
- the standard relative deviation (RSD) of the particle size of Group 14 element-doped nanodiamonds is preferably 25-40%, and the average particle size and standard relative deviation (RSD) are measured by the Small Angle X-ray Scattering Method (SAXS). can do.
- SAXS Small Angle X-ray Scattering Method
- the relative standard deviation (%) can be calculated by the following formula.
- the temperature-sensitive probe of the present invention is suitable for measuring temperature in a microspace, for example, not only can measure the temperature of the whole cell, but also intracellular small organs (for example, endoplasmic reticulum, mitochondria, Gorgi, peroxysome, lysosome, micro).
- intracellular small organs for example, endoplasmic reticulum, mitochondria, Gorgi, peroxysome, lysosome, micro.
- the temperature of tubes and microscopic bodies can be measured separately.
- cells for which temperature is to be measured include microorganisms such as yeast, animal cells, plant cells, and the like, which can be easily introduced into cells.
- the temperature-sensitive probe of the present invention utilizes the fact that the fluorescence peak position at the time of irradiation with excitation light shifts with temperature.
- the temperature-sensitive probe detects the temperature by fluorescence (ZPL) near 738 nm from the SiV center, but this fluorescence near 738 nm measures the intracellular temperature because it is hardly absorbed by the cell or its organelles. Suitable for. Since the fluorescence of the GeV center with ZPL of 602 nm, the SnV center with ZPL of 620 nm, and the PbV center with ZPL of 520 nm and 552 nm is strong, it is possible to measure the intracellular temperature.
- the "cell” in the present invention consists of prokaryotic cells and eukaryotic cells, which are a general classification, and does not particularly depend on the species of the organism.
- prokaryotic cells are divided into eubacteria and paleobacteria, and eubacteria are broadly divided into gram-positive bacteria such as Radical Bacteria and Gram-negative bacteria such as Proteobacterium, depending on the thickness of the peptidoglycan layer.
- the applicable range of temperature-sensitive probes is not limited.
- eukaryotic cells mainly include cells belonging to eukaryotes (protists, fungi, plants, animals).
- yeast which is generally used in research such as molecular biology and is also used industrially, belongs to fungi.
- the fluorescence (ZPL) near 738 nm at various temperatures (for example, 35 ° C to 40 ° C when the measurement target is a cell)
- the peak position can be measured and the temperature of the object to be measured can be measured based on the result.
- the temperature of the object to be measured (for example, cell) is measured.
- the temperature of the object to be measured can do.
- a calibration curve can be created by measuring the peak position of fluorescence (ZPL) near the ZPL of the MV center at each temperature, and the temperature can be measured based on this calibration curve.
- the calibration curve can be created by heating and cooling the Group 14 element-doped nanodiamond particles carried or coated on the substrate.
- the calibration curve can be created by changing the measurement conditions depending on the measurement target.
- the conditions under which the calibration curve is used are not limited, but for example, a curve plotting the temperature-dependent change in the fluorescence intensity of the temperature-sensitive probe in a potassium chloride solution that mimics the intracellular can be used. can. More specifically, when a temperature-sensitive probe-introduced cell population is used to perform a heat-sensitive response test and plot changes in the peak position of fluorescence (ZPL), the cells actively engage in metabolic activity.
- ZPL peak position of fluorescence
- the cells are kept at a specific temperature for a certain period of time, and the external temperature and the internal temperature of the cells are in an equilibrium state. There is a method of measuring the fluorescence intensity in the situation where it is considered that the temperature has been reached.
- the temperature sensitive probe of the present invention can be applied to various fields of research and development. For example, in the field of biotechnology, in the fermentation production of useful substances using microorganisms, it is expected that the efficiency of examination of culture conditions will be improved by adding the intracellular temperature, which has been difficult to measure accurately, to the analysis parameters. To.
- the temperature sensitive probe of the present invention can be applied to various medical applications. For example, by using the temperature-sensitive probe according to the present invention on a part of a patient's tissue, it is possible to distinguish between cancer cells that are said to produce a large amount of heat and normal cells that do not. be. Furthermore, by applying it, it can be used for the development of more effective hyperthermia treatment methods.
- a temperature-sensitive probe according to the present invention by introducing a temperature-sensitive probe according to the present invention into brown adipocytes having a large amount of heat production and measuring the temperature change due to the addition of various materials to the cells, it is effective for burning fat by consuming energy.
- Materials that activate brown adipocytes can be screened. Such materials are effective for weight loss or slimming by promoting fat burning.
- the temperature-sensitive probe of the present invention can also be applied to elucidate various physiological phenomena. For example, by investigating how temperature-sensitive (Transient Receptor Potential, TRP) channels, which are receptors that sense in vitro temperature and trigger biological reactions, are related to intracellular temperature. Activation of TRP channels with different approaches is conceivable. In addition, by investigating the relationship between the intracellular temperature distribution and the biological reaction that occurs inside and outside the cell, it is possible to investigate the effect of the local temperature distribution on the biological reaction. It is also possible to control. Furthermore, since the temperature-sensitive probe of the present invention is not toxic, it can be safely used for microorganisms, animal cells or plant cells, or mammalian cells such as humans, mice and rats.
- TRP Transient Receptor Potential
- the present invention is further a method for measuring the intracellular temperature.
- a step of introducing the temperature-sensitive probe into the cell by mixing the temperature-sensitive probe according to any one of [1] to [4] with the cell in water.
- B) The cells into which the temperature-sensitive probe has been introduced are irradiated with excitation light, and the MV center (M indicates a Group 14 element selected from the group consisting of Si, Ge, Sn and Pb. V indicates a vacancy.
- Group 14 element-doped nanodiamonds can preferably be produced by the detonation method.
- the shape of the Group 14 element-doped nanodiamond is preferably spherical, ellipsoidal, or a polyhedron close to them.
- the amount of Group 14 element (M) atom introduced (number of doped atoms (M) / number of C atoms) ⁇ 100 [%]) in the Group 14 element-doped nanodiamond of the present invention is preferably 0.5 to 40%. More preferably, it is 1 to 36%.
- Pore can be introduced into the group 14 element-doped nanodiamond by ion beam irradiation or electron beam irradiation.
- the upper limit of the pore concentration to be introduced is preferably 1 ⁇ 10 21 / cm 3 or less capable of retaining the structure of the diamond, and the lower limit of the pore concentration is, for example, 1 ⁇ 10 16 / cm 3 or more, 5 ⁇ 10 16 / Cm 3 or more, 1 ⁇ 10 17 / cm 3 or more, 5 ⁇ 10 17 / cm 3 or more, 1 ⁇ 10 18 / cm 3 or more.
- the ion beam is preferably a hydrogen (H) or helium (He) ion beam.
- the energy of the hydrogen ion beam is preferably 10 to 1500 keV, and the energy of the helium ion beam is preferably 20 to 2000 keV.
- the energy of the electron beam is preferably 500 to 5000 keV.
- the ZPL of the SiV center is 738 nm, which is located in the so-called window of the living body (wavelength band where excitation light and fluorescence pass through the living body), and is an ideal light emitting center that enables external excitation and external measurement as a probe for biological imaging. Is.
- the ZPL of the GeV center is 602 nm
- the ZPL of the SnV center is 620 nm
- the ZPL of the PbV center is 520 nm and 552 nm.
- the concentration of the MV center in the preferred Group 14 element-doped nanodiamond of the present invention is preferably 1 ⁇ 10 14 / cm 3 or more, and more preferably 2 ⁇ 10 14 to 1 ⁇ 10 19 / cm 3 .
- the center of the preferred Group 14 element-doped nanodiamond particles of the present invention has a diamond structure containing sp3 carbon and doped Group 14 element atoms, the surface of which is covered with an amorphous layer composed of sp2 carbon. ing.
- the outside of the amorphous layer may be covered with a graphite oxide layer. Further, a hydration layer may be formed between the amorphous layer and the graphite oxide layer.
- the surface of the Group 14 element-doped nanodiamond particles has one or more oxygen-containing functional groups.
- the Group 14 element-doped nanodiamonds have a positive or negative zeta potential.
- the zeta potential of the Group 14 element-doped nanodiamond is preferably ⁇ 70 to 70 mV, more preferably ⁇ 60 to 30 mV.
- the Group 14 element-doped nanodiamond of the present invention is a step of mixing an explosive material with a Group 14 element compound such as a silicon compound, a germanium compound, a tin compound, and a lead compound in a closed container, and the obtained mixture is used as a cooling medium. It can be manufactured by a manufacturing method comprising the step of exploding under the condition of negative oxygen balance in the presence of.
- the explosive material is not particularly limited, and known explosive materials can be widely used.
- TNT trinitrotoluene
- RDX cyclotrimethylene trinitramine
- octogen cyclotetramethylenetetranitramine
- PTN pentaerythritol tetranitrate
- TNM Tetranitromethane
- TNM triamino-trinitrobenzene, hexanitrostylben, diaminodinitrobenzofuroxane and the like, and these can be used alone or in combination of two or more.
- the Group 14 element compound any organic or inorganic compound is widely used as long as it has a Group 14 element atom such as silicon (Si), germanium (Ge), tin (Sn), and lead (Pb). can do.
- the organic silicon compound is -Acetoxytrimethylsilane, diacetoxydimethylsilane, triacetoxymethylsilane, acetoxytriethylsilane, diacetoxydiethylsilane, triacetoxyethylsilane, acetoxytripropylsilane, methoxytrimethylsilane, dimethoxydimethylsilane, trimethoxymethylsilane, ethoxytrimethylsilane , Silanes with lower alkyl groups such as diethoxydimethylsilane, triethoxymethylsilane, ethoxytriethylsilane, diethoxydiethylsilane, triethoxyethylsilane, trimethylphenoxysilane, -Trichloromethylsilane, dichlorodimethylsilane, chlorotrimethylsilane, trichloroethylsilane, dichlorodiethy
- Aromatic silane in which a silicon atom is incorporated in an aromatic ring such as silabenzene or disilabenzene ⁇ trimethylsilanol, dimethylphenylsilanol, triethylsilanol, diethylsilanediol, tripropylsilanol, dipropylsilanediol, triphenylsilanol, diphenylsilanediol, etc.
- -Silanes having an alkyl group such as methylsilane, dimethylsilane, trimethylsilane, diethylsilane, triethylsilane, tripropylsilane, diphenylsilane, triphenylsilane, or an aryl group and a hydrogen atom
- -Tetrakis (chloromethyl) silane tetrakis (hydroxymethyl) silane, tetrakis (trimethylsilyl) silane, tetrakis (trimethylsilyl) methane
- tetrakis (dimethylsilanolyl) silane tetrakis (tri (hydroxymethyl) silyl) silane, tetrakis (nitrate) Methyl) silane, And so on.
- Examples of the inorganic silicon compound include silicon oxide, silicon oxynitride, silicon nitride, silicon oxide, silicon nitride, silane, and a silicon-doped carbon material.
- Examples of the carbon-doped carbon material include graphite, graphite, activated carbon, carbon black, Ketjen black, coke, soft carbon, hard carbon, acetylene black, carbon fiber, and mesoporous carbon.
- Germanium compounds include methyl germanium, ethyl germanium, trimethyl germanium methoxyd, dimethyl germanium diacetate, tributyl germanium acetate, tetramethoxy germanium, tetraethoxy germanium, tetraphenyl germanium, isobutyl germanium, alkyl germanium trichloride, dimethyl amino germanium trichloride.
- Organic germanium compounds such as, nitrotriphenol complex (Ge 2 (ntp) 2 O), catechol complex (Ge (cat) 2 ) or aminopyrene complex (Ge 2 (ap) 2 Cl 2 ) and the like, germanium ethoxydo , Germanium alkoxides such as germanium tetrabutoxide.
- tin compound examples include tin oxide (II), tin oxide (IV), tin sulfide (II), tin sulfide (IV), tin chloride (II), tin chloride (IV), tin bromide (II), and the like.
- Inorganic tin compounds such as tin fluoride (II), tin acetate, tin sulfate, alkyl tin compounds such as tetramethyltin, monoalkyl tin oxide compounds such as monobutyl tin oxide, dialkyl tin oxide compounds such as dibutyl tin oxide, tetra.
- Examples thereof include aryl tin compounds such as phenyl tin, organotin compounds such as dimethyl tin maleate, hydroxybutyl tin oxide, and monobutyl tin tris (2-ethylhexanoate).
- Examples of lead compounds include lead monoxide (PbO), lead dioxide (PbO 2 ), lead tan (Pb 3 O 4 ), lead white (2PbCO 3 ⁇ Pb (OH) 2 ), and lead nitrate (Pb (NO 3 )).
- Inorganic lead compounds such as lead fluoride (PbF 2 ), lead tetrafluoride (PbF 4 ), lead bromide (PbBr 2 ), lead iodide (PbI 2 ), lead acetate (Pb (CH 3 COO) 2 ), 4 Lead carboxylate (Pb (OCOCH 3 ) 4 ), Tetraethyl lead (Pb (CH 3 CH 2 ) 4 ), Tetramethyl lead (Pb (CH 3 ) 4 ), Tetrabutyl lead (Pb (C 4 H 9 ) 4 ) Examples thereof include organic lead compounds such as.
- the organic or inorganic Group 14 element compound may be used alone or in combination of two
- the proportion of the explosive material in the mixture containing the explosive material and the Group 14 elemental compound is preferably 85 to 99.9% by mass, more preferably 86 to 99% by mass, and the proportion of the Group 14 elemental compound is It is preferably 0.1 to 15% by mass, more preferably 1 to 14% by mass.
- the content of the Group 14 element in the mixture containing the explosive material and the Group 14 element compound is preferably 0.007 to 4.5% by mass, more preferably 0.06 to 4.3% by mass.
- the above carbon material containing no Group 14 element can be further added.
- the mixture of the explosive material and the Group 14 element compound may be powder-mixed when both are solid, or may be dissolved or dispersed in an appropriate solvent and mixed.
- the mixture of explosive material and Group 14 elemental compound further comprises a cooling medium.
- the cooling medium may be a solid, a liquid, or a gas.
- Examples of the method using a cooling medium include a method of detonating a mixture of an explosive material and a Group 14 element compound in the cooling medium.
- Examples of the cooling medium include inert gas (nitrogen, argon, CO), water, ice, liquid nitrogen, an aqueous solution of a Group 14 element-containing salt, and crystalline hydrate.
- the silicon-containing salt contained in the Group 14 element-containing salt examples include ammonium hexafluorosilicate, ammonium silicate, and tetramethylammonium silicate.
- the cooling medium is preferably used about 5 times the weight of the explosive.
- a mixture containing an explosive material and a Group 14 elemental compound is converted to diamond by shock wave compression under high pressure and high temperature conditions produced by the explosion of the explosive material (explosion). Detonation method). During the explosion of explosive materials, Group 14 element atoms are incorporated into the diamond lattice.
- the carbon source of the nanodiamond can be an explosive material and an organic Group 14 element compound, but if the mixture containing the explosive material and the Group 14 element compound further contains a carbon material that does not contain the Group 14 element, this Carbon materials can also be a carbon source for nanodiamonds.
- the Group 14 element-doped nanodiamond obtained by the detonation method can be purified and annealed according to a conventional method.
- Example 1 (1) Synthesis of silicon-doped nanodiamond TNT and cyclotrimethylene trinitramine (hexogen, RDX) were used as explosives, and 0.21 mol of triphenylsilanol (SiPh 3 OH) was used as a silicon compound per 1 mol of TNT, and the temperature was (1).
- SiPh 3 OH triphenylsilanol
- the fluorescence spectrum of the obtained silicon-doped nanodiamond having a SiV center is shown in FIG.
- the obtained evaluation sample was prepared in a 1 wt% aqueous dispersion, a few drops were dropped on a glass plate, and the mixture was dried.
- the dried sample is irradiated with SiV fluorescent nanodiamonds with excitation light of 532 nm using a micro Raman spectroscope (trade name: Micro Laser Raman spectrophotometer LabRAM HR Evolution, manufactured by HORIBA, Ltd.), and the fluorescence spectrum is obtained with. did.
- the measurement conditions were excitation light power: 100 ⁇ W, exposure time 1 second, and integration frequency 1 time.
- Example 2 The silicon-doped nanodiamond having a SiV center obtained in Example 1 was dispersed in water at a concentration of 1% by mass, and the obtained dispersion was dropped onto a glass substrate and dried to prepare an evaluation sample.
- the obtained evaluation sample was subjected to high-speed mapping (excitation light wavelength: 532 nm, excitation light power: 100 ⁇ W) with a confocal microscope (Fig. 5) (Fig. 2). Furthermore, the point indicated by the arrow in FIG. 2, which is the bright point, was peaked up, the fluorescence spectrum was measured in detail, and it was confirmed that the point indicated by the arrow in FIG. 2 was the fluorescence ZPL of SiV (FIG. 3).
- RDX cyclotrimethylene trinitramine
- Example 4 The germanium-doped nanodiamond having a GeV center obtained in Example 3 was dispersed in water at a concentration of 1% by mass, and the obtained dispersion was dropped onto a glass substrate and dried to prepare an evaluation sample.
- the obtained evaluation sample was subjected to high-speed mapping (excitation light wavelength: 532 nm, excitation light power: 100 ⁇ W) with a confocal microscope (Fig. 5). Furthermore, the point that was the bright spot was peaked up, and the fluorescence spectrum was measured in detail, and it was confirmed that the point was the fluorescence ZPL of GeV (FIG. 6).
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Abstract
Description
非特許文献1は、SiV(silicon-vacancy)センターを有する200nmサイズの蛍光ナノダイヤモンド粒子を用いて温度測定を行っている。
本発明の目的は、微小空間の温度を安定して正確に測定できる温度感受性プローブを提供することにある。
〔1〕平均粒子径が1~100 nmのMVセンター(MはSi、Ge、Sn及びPbからなる群から選ばれる14族元素を示す。Vは空孔(vacancy)を示す。)を有する第14族元素ドープナノダイヤモンドを含む温度感受性プローブ。
〔2〕MがSiである、〔1〕に記載の温度感受性プローブ。
〔3〕粒子径の標準相対偏差(RSD)が25~40%である、〔1〕又は〔2〕に記載の温度感受性プローブ。
〔4〕第14族元素ドープナノダイヤモンドの形状が球状である、〔1〕~〔3〕のいずれか1項に記載の温度感受性プローブ。
〔5〕MVセンターの濃度が1×1014/cm3以上である、〔1〕~〔4〕のいずれか1項に記載の温度感受性プローブ。
〔6〕微小空間の温度を測定するための、平均粒子径が1~100 nmのMVセンター(MはSi、Ge、Sn及びPbからなる群から選ばれる14族元素を示す。Vは空孔(vacancy)を示す。)を有する第14族元素ドープナノダイヤモンドの使用。
〔7〕微小空間が細胞又は細胞内小器官である、〔6〕に記載の使用。
〔8〕平均粒子径が1~100 nmのMVセンター(MはSi、Ge、Sn及びPbからなる群から選ばれる14族元素を示す。Vは空孔(vacancy)を示す。)を有する第14族元素ドープナノダイヤモンドを水中で細胞と混合することにより該温度感受性プローブを細胞内に導入すること、前記第14族元素ドープナノダイヤモンドを導入した細胞に励起光を照射してMVセンターのZPLの蛍光強度を測定すること、測定された前記蛍光強度から細胞の温度を測定すること、
を含む細胞内の温度を測定する方法。
本発明の温度感受性プローブは、細胞内に均一に分散または特定の場所に配置又は結合させることによって、細胞内において温度応答領域を細かく設定することが可能であり、細胞内蛍光性温度センサーとして利用できる。
第14族元素ドープナノダイヤモンドはナノ粒子であり、その平均粒子径の上限は、好ましくは100 nm、より好ましくは70 nm、さらに好ましくは50 nm、特に好ましくは30 nm、特により好ましくは20nm、最も好ましくは10nmであり、平均粒子径の下限は、好ましくは1 nm、より好ましくは1.5 nm、さらに好ましくは2 nm、特に好ましくは3 nmである。第14族元素ドープナノダイヤモンドの平均粒子径が小さい方が、細胞内のタンパク質などの生体分子の動きや構造変化に干渉しないので好ましい。
細胞内に代表されるような限られた微小空間内の温度を正確に測定するために、種々の温度(例えば測定対象が細胞の場合35℃~40℃)における738nm付近の蛍光(ZPL)のピーク位置を測定し、その結果に基づき測定対象物の温度を測定することができる。
微小空間内の温度を測定する場合、各温度におけるMVセンターのZPL付近の蛍光(ZPL)のピーク位置を測定して検量線を作成し、この検量線に基づき温度を測定することができる。検量線の作成は、基板に担持または塗布した第14族元素ドープナノダイヤモンド粒子を加熱・冷却することで行うことができるが、例えば細胞内の微小空間に近い環境(例えばイオン強度、pHなど)で行うこともできる。検量線は、測定対象により測定条件を変えて作成することができる。具体的に、どの条件で測定した検量線を使用するかは限定されないが、例えば、細胞内を模倣した塩化カリウム溶液中で温度感受性プローブの蛍光強度の温度による変化をプロットした曲線を用いることができる。さらに具体的には、温度感受性プローブを導入した細胞集団を用いて、感熱応答性試験を行い、蛍光(ZPL)のピーク位置の変化をプロットする際には、細胞は代謝活動を積極的に行わないような状態、例えば、水中や資化することのできない化合物が含まれた緩衝液中に細胞を懸濁した状態で、特定の温度に一定期間保持し、外部温度と細胞内部温度が平衡状態に達したと考えられる状況下で、蛍光強度を測定する方法などが挙げられる。
本発明の温度感受性プローブは、様々な医療用途に応用することができる。例えば、本発明による温度感受性プローブを患者の組織の一部に対して使用することにより、熱産生量が多いとされているがん細胞と、そうでない正常細胞との識別を行うことも可能である。さらにそれを応用する事でより効果的な温熱治療法の開発などにも使用できる。あるいは、熱産生量が多い褐色脂肪細胞に本発明による温度感受性プローブを導入し、その細胞に様々な素材を添加することによる温度変化を測定することにより、エネルギーの消費による脂肪の燃焼に有効な褐色脂肪細胞を活性化する素材をスクリーニングすることができる。このような素材は脂肪の燃焼を促進することで体重減少または痩身に有効である。
さらに本発明の温度感受性プローブは毒性が無いために、微生物、動物細胞または植物細胞、或いはヒト、マウス、ラットなどの哺乳類細胞に安心して使用することができる。
(a)〔1〕~〔4〕のいずれか一項に記載の温度感受性プローブを水中で細胞と混合することにより、該温度感受性プローブを細胞内に導入する工程、
(b)温度感受性プローブを導入した細胞に励起光照射し、MVセンター(MはSi、Ge、Sn及びPbからなる群から選ばれる14族元素を示す。Vは空孔(vacancy)を示す。)のZPLの蛍光(ZPL)のピーク位置を測定する工程、および
(c)測定された前記蛍光強度から温度を測定する工程
を含んでなる、方法を提供することができる。励起光の波長としては、532nm, 633nmが挙げられる。
本発明の第14族元素ドープナノダイヤモンドにおける第14族元素(M)原子導入量(ドープ原子(M)数/C原子数)×100[%])は、好ましくは0.5~40%、より好ましくは1~36%である。
本発明の好ましい第14族元素ドープナノダイヤモンド粒子の中心は、sp3炭素とドープされた第14族元素原子を含むダイヤモンド構造を有し、その表面は、sp2炭素で構成されるアモルファス層で覆われている。さらに好ましい実施形態において、アモルファス層の外側は酸化グラファイト層で覆われていてもよい。また、アモルファス層と酸化グラファイト層の間には水和層が形成されていてもよい。
本発明の好ましい1つの実施形態において、第14族元素ドープナノダイヤモンドは、プラス又はマイナスのゼータ電位を有する。第14族元素ドープナノダイヤモンドのゼータ電位は、好ましくは-70~70mV、より好ましくは-60~30mVである。
爆発性材料としては、特に限定されず、公知の爆発性材料を広く用いることができる。具体例としては、トリニトロトルエン(TNT)、シクロトリメチレントリニトラミン(ヘキソゲン、RDX)、シクロテトラメチレンテトラニトラミン(オクトゲン)、トリニトロフェニルメチルニトラミン(テトリル)、ペンタエリスリトールテトラニトレート(PETN)、テトラニトロメタン(TNM)、トリアミノ-トリニトロベンゼン、ヘキサニトロスチルベン、ジアミノジニトロベンゾフロキサンなどが挙げられ、これらを1種単独で、或いは2種以上を組み合わせて用いることができる。
第14族元素化合物は、ケイ素(Si)、ゲルマニウム(Ge)、スズ(Sn)、鉛(Pb)などの第14族元素原子を有するものであれば、有機もしくは無機のいずれの化合物も広く使用することができる。
・アセトキシトリメチルシラン、ジアセトキシジメチルシラン、トリアセトキシメチルシラン、アセトキシトリエチルシラン、ジアセトキシジエチルシラン、トリアセトキシエチルシラン、アセトキシトリプロピルシラン、メトキシトリメチルシラン、ジメトキシジメチルシラン、トリメトキシメチルシラン、エトキシトリメチルシラン、ジエトキシジメチルシラン、トリエトキシメチルシラン、エトキシトリエチルシラン、ジエトキシジエチルシラン、トリエトキシエチルシラン、トリメチルフェノキシシランなどの低級アルキル基を有するシラン、
・トリクロロメチルシラン、ジクロロジメチルシラン、クロロトリメチルシラン、トリクロロエチルシラン、ジクロロジエチルシラン、クロロトリエチルシラン、トリクロロフェニルシラン、ジクロロジフェニルシラン、クロロトリフェニルシラン、ジクロロジフェニルシラン、ジクロロメチルフェニルシラン、ジクロロエチルフェニルシラン、クロロジフルオロメチルシラン、ジクロロフルオロメチルシラン、クロロフルオロジメチルシラン、クロロエチルジフルオロシラン、ジクロロエチルフルオロシラン、クロロジフルオロプロピルシラン、ジクロロフルオロプロピルシラン、トリフルオロメチルシラン、ジフルオロジメチルシラン、フルオロトリメチルシラン、エチルトリフルオロシラン、ジエチルジフルオロシラン、トリエチルフルオロシラン、トリフルオロプロピルシラン、フルオロトリプロピルシラン、トリフルオロフェニルシラン、ジフルオロジフェニルシラン、フルオロトリフェニルシラン、トリブロムメチルシラン、ジブロムジメチルシラン、ブロムトリメチルシラン、ブロムトリエチルシラン、ブロムトリプロピルシラン、ジブロムジフェニルシラン、ブロムトリフェニルシランなどのハロゲン原子を有するシラン、
・ヘキサメチルジシラン、ヘキサエチルジシラン、ヘキサプロピルジシラン、ヘキサフェニルジシラン、オクタフェニルシクロテトラシランなどのポリシラン
・トリエチルシラザン、トリプロピルシラザン、トリフェニルシラザン、ヘキサメチルジシラザン、ヘキサエチルジシラザン、ヘキサフェニルジシラザン、ヘキサメチルシクロトリシラザン、オクタメチルシクロテトラシラザン、ヘキサエチルシクロトリシラザン、オクタエチルシクロテトラシラザン、ヘキサフェニルシクロトリシラザンなどのシラザン、
・シラベンゼン、ジシラベンゼンなどの芳香環にケイ素原子が組み込まれた芳香族シラン
・トリメチルシラノール、ジメチルフェニルシラノール、トリエチルシラノール、ジエチルシランジオール、トリプロピルシラノール、ジプロピルシランジオール、トリフェニルシラノール、ジフェニルシランジオールなどの水酸基含有シラン
・テトラメチルシラン、エチルトリメチルシラン、トリメチルプロピルシラン、トリメチルフェニルシラン、ジエチルジメチルシラン、トリエチルメチルシラン、メチルトリフェニルシラン、テトラエチルシラン、トリエチルフェニルシラン、ジエチルジフェニルシラン、エチルトリフェニルシラン、テトラフェニルシランなどのアルキルもしくはアリール置換シラン、
・トリフェニルシリルカルボン酸、トリメチルシリル酢酸、トリメチルシリルプロピオン酸、トリメチルシリル酪酸などのカルボキシル基含有シラン、
・ヘキサメチルジシロキサン、ヘキサエチルジシロキサン、ヘキサプロピルジシロキサン、ヘキサフェニルジシロキサンなどのシロキサン、
・メチルシラン、ジメチルシラン、トリメチルシラン、ジエチルシラン、トリエチルシラン、トリプロピルシラン、ジフェニルシラン、トリフェニルシランなどのアルキル基もしくはアリール基と水素原子を有するシラン、
・テトラキス(クロロメチル)シラン、テトラキス(ヒドロキシメチル)シラン、テトラキス(トリメチルシリル)シラン、テトラキス(トリメチルシリル)メタン、テトラキス(ジメチルシラノリル)シラン、テトラキス(トリ(ヒドロキシメチル)シリル)シラン、テトラキス(ニトレートメチル)シラン、
などが挙げられる。
ゲルマニウム化合物としては、メチルゲルマン、エチルゲルマン、トリメチルゲルマニウムメトキシド、ジメチルゲルマニウムジアセテート、トリブチルゲルマニウムアセテート、テトラメトキシゲルマニウム、テトラエトキシゲルマニウム、テトラフェニルゲルマン、イソブチルゲルマン、三塩化アルキルゲルマニウム、三塩化ジメチルアミノゲルマニウム等の有機ゲルマニウム化合物、ニトロトリフェノール錯体(Ge2(ntp)2O)、カテコール錯体(Ge(cat)2) 又はアミノピレン錯体(Ge2(ap)2Cl2)等のゲルマニウム錯体、ゲルマニウムエトキシド、ゲルマニウムテトラブトキシド等のゲルマニウムアルコキシドが挙げられる。
鉛化合物としては、例えば、一酸化鉛(PbO)、二酸化鉛(PbO2)、鉛丹(Pb3O4)、鉛白(2PbCO3・Pb(OH)2)、硝酸鉛(Pb(NO3)2)、塩化鉛(PbCl2)、硫化鉛(PbS)、黄鉛(PbCrO4、Pb(SCr)O4、PbO・PbCrO4)、炭酸鉛(PbCO3)、硫酸鉛(PbSO4)、フッ化鉛(PbF2)、4フッ化鉛(PbF4)、臭化鉛(PbBr2)、ヨウ化鉛(PbI2)等の無機鉛化合物、酢酸鉛(Pb(CH3COO)2)、4カルボン酸鉛(Pb(OCOCH3)4)、テトラエチル鉛(Pb(CH3CH2)4)、テトラメチル鉛(Pb(CH3)4)、テトラブチル鉛(Pb(C4H9)4)等の有機鉛化合物が挙げられる。
上記有機もしくは無機の第14族元素化合物は、1種単独で用いてもよく、2種以上を併用してもよい。
爆発性材料と第14族元素化合物の混合は、両者が固体の場合には粉体混合してもよく、適当な溶媒に溶解ないし分散させて混合してもよい。混合は、撹拌、ビーズミリング、超音波などにより行うことができる。
好ましい1つの実施形態において、爆発性材料と第14族元素化合物の混合物は、さらに冷却媒体を含む。冷却媒体は、固体、液体、気体のいずれであってもよい。冷却媒体を使用する方法として、爆発性材料と第14族元素化合物の混合物を冷却媒体中で起爆する方法が挙げられる。冷却媒体としては、不活性ガス(窒素、アルゴン、CO)、水、氷、液体窒素、第14族元素含有塩の水溶液、結晶水和物などが挙げられる。第14族元素含有塩に含まれるケイ素含有塩としては、ヘキサフルオロケイ酸アンモニウム、ケイ酸アンモニウム、ケイ酸テトラメチルアンモニウムなどが挙げられる。冷却媒体は、例えば水や氷の場合、爆薬重量に対して5倍程度使用することが好ましい。
本発明の1つの好ましい実施形態において、爆発性材料と第14族元素化合物を含む混合物は、爆発性材料の爆発によって生成された高圧高温条件下での衝撃波による圧縮によってダイヤモンドに変換される(爆轟法)。爆発性材料の爆発の際に、ダイヤモンド格子に第14族元素原子が組み込まれる。ナノダイヤモンドの炭素源は、爆発性材料と有機第14族元素化合物であり得るが、爆発性材料と第14族元素化合物を含む混合物が第14族元素を含まない炭素材料をさらに含む場合、この炭素材料もナノダイヤモンドの炭素源となり得る。
爆轟法により得られた第14族元素ドープナノダイヤモンドは、常法に従い精製及びアニーリングを行うことができる。
(1)ケイ素ドープナノダイヤモンドの合成
爆薬としてTNT、シクロトリメチレントリニトラミン(ヘキソゲン、RDX)を用い、ケイ素化合物としてトリフェニルシラノール(SiPh3OH)をTNT1モルに対し0.21モル使用し、温度(4092K)及び圧力(32GPa)の条件で、常法に従い爆轟法によるケイ素ドープナノダイヤモンドの製造を行うと、(Si原子/C原子)×100(%)=1%のケイ素がドープされた、SiVセンターを有するナノダイヤモンドを得た。
得られたSiVセンターを有するケイ素ドープナノダイヤモンドの蛍光スペクトルを図1に示す。
蛍光スペクトルの測定方法として、得られた評価サンプルを1wt%の水分散液に調製し、ガラス板上に数滴滴下し、乾燥した。乾燥後のサンプルを顕微ラマン分光装置(商品名:顕微レーザーラマン分光光度計LabRAM HR Evolution、堀場製作所株式会社製)を用いて、532nmの励起光でSiV蛍光ナノダイヤに照射し、で蛍光スペクトルを取得した。測定条件は、励起光パワー:100μW、露光時間1秒、積算回数1回であった。
実施例1で得られたSiVセンターを有するケイ素ドープナノダイヤモンドを1質量%の濃度で水に分散させ、得られた分散液をガラス基板上に滴下し、乾燥させて評価サンプルを作製した。得られた評価サンプルを共焦点顕微鏡(図5)で高速マッピング(励起光波長:532nm、励起光パワー: 100μW)を行った(図2)。さらに、輝点である図2の矢印で示されるポイントをピークアップし、詳しく蛍光スペクトルを測定し、図2の矢印で示されるポイントがSiVの蛍光ZPLであることを確認した(図3)。その後、共焦点顕微鏡に付いているホットプレート及びレンズヒーターで温度を調整し、295K(22℃)、298.5K(25.5℃)、302.5K(29.5℃)、306.5K(33.5℃)、310K(37℃)及び313K(40℃)の温度で(励起光:532nm、励起光パワー: 100μW、ND Filter: OD 1.0)で732.5736-749nmの領域の波長分解能を上げて測定した。図2の矢印で示されるポイントの22℃、25.5℃、29.5℃、33.5℃、37℃及び40℃におけるピーク波長を測定した(図4)。Δλ/ΔT = 0.0150 (nm/K)。
(1)ゲルマニウムドープナノダイヤモンドの合成
爆薬としてTNT、シクロトリメチレントリニトラミン(ヘキソゲン、RDX)を用い、TNTとRDX混合物60gに対しテトラフェニルゲルマンを0.6g使用し、温度(4092K)及び圧力(32GPa)の条件で、常法に従い爆轟法によるゲルマニウムドープナノダイヤモンドの製造を行うと、(Ge原子/C原子)×100(%)=1%のゲルマニウムがドープされた、GeVセンターを有するナノダイヤモンドを得た。
実施例3で得られたGeVセンターを有するゲルマニウムドープナノダイヤモンドを1質量%の濃度で水に分散させ、得られた分散液をガラス基板上に滴下し、乾燥させて評価サンプルを作製した。得られた評価サンプルを共焦点顕微鏡(図5)で高速マッピング(励起光波長:532nm、励起光パワー: 100μW)を行った。さらに、輝点であるポイントをピークアップし、詳しく蛍光スペクトルを測定し、前記ポイントがGeVの蛍光ZPLであることを確認した(図6)。その後、共焦点顕微鏡に付いているホットプレート及びレンズヒーターで温度を調整し、295K(22℃)、298.5K(25.5℃)、302.5K(29.5℃)、306.5K(33.5℃)、310K(37℃)及び313K(40℃)の温度で(励起光:532nm、励起光パワー: 100μW、ND Filter: OD 1.0)で597-607nmの領域の波長分解能を上げて測定した。前記ポイントの22℃、25.5℃、29.5℃、33.5℃、37℃及び40℃におけるピーク波長を測定した(図7)。Δλ/ΔT = 0.0079 (nm/K)。
Claims (8)
- 平均粒子径が1~100 nmのMVセンター(MはSi、Ge、Sn及びPbからなる群から選ばれる14族元素を示す。Vは空孔(vacancy)を示す。)を有する第14族元素ドープナノダイヤモンドを含む温度感受性プローブ。
- MがSiである、請求項1に記載の温度感受性プローブ。
- 粒子径の標準相対偏差(RSD)が25~40%である、請求項1又は2に記載の温度感受性プローブ。
- 第14族元素ドープナノダイヤモンドの形状が球状である、請求項1~3のいずれか1項に記載の温度感受性プローブ。
- MVセンターの濃度が1×1014/cm3以上である、請求項1~4のいずれか1項に記載の温度感受性プローブ。
- 微小空間の温度を測定するための、平均粒子径が1~100 nmのMVセンター(MはSi、Ge、Sn及びPbからなる群から選ばれる14族元素を示す。Vは空孔(vacancy)を示す。)を有する第14族元素ドープナノダイヤモンドの使用。
- 微小空間が細胞又は細胞内小器官である、請求項6に記載の使用。
- 平均粒子径が1~100 nmのMVセンター(MはSi、Ge、Sn及びPbからなる群から選ばれる14族元素を示す。Vは空孔(vacancy)を示す。)を有する第14族元素ドープナノダイヤモンドを水中で細胞と混合することにより該温度感受性プローブを細胞内に導入すること、前記第14族元素ドープナノダイヤモンドを導入した細胞に励起光を照射してMVセンターのZPLの蛍光強度を測定すること、測定された前記蛍光強度から細胞の温度を測定すること、
を含む細胞内の温度を測定する方法。
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APPLIED PHYSICS LETTERS, vol. 112, 2018, pages 203102 |
NGUYEN CHRISTIAN T.; EVANS RUFFIN E.; SIPAHIGIL ALP; BHASKAR MIHIR K.; SUKACHEV DENIS D.; AGAFONOV VIATCHESLAV N.; DAVYDOV VALERY : "All-optical nanoscale thermometry with silicon-vacancy centers in diamond", APPLIED PHYSICS LETTERS, AMERICAN INSTITUTE OF PHYSICS, 2 HUNTINGTON QUADRANGLE, MELVILLE, NY 11747, vol. 112, no. 20, 15 May 2018 (2018-05-15), 2 Huntington Quadrangle, Melville, NY 11747, XP012228510, ISSN: 0003-6951, DOI: 10.1063/1.5029904 * |
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