Temperature-sensitive probe

Abstract

To provide a temperature-sensitive probe capable of stably and accurately measuring a temperature in a micro space.SOLUTION: An aqueous dispersion comprises a group 14 element-doped nanodiamond having an MV center with an average particle diameter of 1 to 100 nm (M represents a group 14 element selected from a group consisting of Si, Ge, Sn, and Pb, and V represents a vacancy).SELECTED DRAWING: None

Description

[Technical Field]

[0001] This invention relates to a temperature-sensitive probe. [Background technology]

[0002] Many chemical reactions that occur within cells during the process of extracting energy through metabolism are highly temperature-dependent; in other words, intracellular chemical reactions are controlled by the temperature both inside and outside the cell. For example, abnormal heat generation has been reported in certain types of cancer cells. This phenomenon can be used to distinguish between cancer cells with high metabolic activity and normal cells without it by measuring their temperature.

[0003] Furthermore, measuring cell temperature is also important in fermentation using microorganisms. Non-patent document 1 describes temperature measurement using 200 nm-sized fluorescent nanodiamond particles having SiV (silicon-vacancy) centers. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] APPLIED PHYSICS LETTERS, 112, 203102 (2018) [Overview of the project] [Problems that the invention aims to solve]

[0005] While there have been previous studies on temperature measurement in minute regions using the temperature dependence of emission wavelengths of molecules, molecules are generally unstable, leading to problems such as flickering emission. Temperature measurement using NV centers in diamond had been considered, but microwaves were required to manipulate the spin. The object of the present invention is to provide a temperature-sensitive probe that can stably and accurately measure the temperature of a microscopic space. [Means for solving the problem]

[0006] This invention provides the following temperature-sensitive probes. [1] A temperature-sensitive probe containing Group 14 element-doped nanodiamonds having MV centers (where M represents a Group 14 element selected from the group consisting of Si, Ge, Sn, and Pb, and V represents a vacancy) with an average particle size of 1 to 100 nm. [2] The temperature-sensitive probe described in [1], wherein M is Si. [3] A temperature-sensitive probe according to [1] or [2], wherein the standard relative deviation (RSD) of particle size is 25-40%. [4] A temperature-sensitive probe according to any one of items [1] to [3], wherein the shape of the Group 14 element-doped nanodiamond is spherical. [5] The concentration of the MV center is 1 × 10 14 / cm 3 The above is a temperature-sensitive probe as described in any one of items [1] to [4]. [6] Use of Group 14 element-doped nanodiamonds having MV centers with an average particle size of 1 to 100 nm (where M represents a Group 14 element selected from the group consisting of Si, Ge, Sn, and Pb; V represents a vacancy). [7] The use described in [6], wherein the microspace is a cell or intracellular organelle. [8] Introducing the temperature-sensitive probe into cells by mixing Group 14 element-doped nanodiamonds having MV centers (where M represents a Group 14 element selected from the group consisting of Si, Ge, Sn, and Pb, and V represents a vacancy) with an average particle size of 1 to 100 nm with cells in water; irradiating the cells into which the Group 14 element-doped nanodiamonds have been introduced with excitation light and measuring the fluorescence intensity of the ZPL of the MV centers; and measuring the cell temperature from the measured fluorescence intensity. A method for measuring intracellular temperature, including the temperature inside a cell. [Effects of the Invention]

[0007] According to the present invention, it is possible to measure the temperature in a limited microspace typified by intracellular organelles. 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 advantages of being stably present and having high emission intensity. The temperature-sensitive probe of the present invention can be finely set with temperature-responsive regions inside cells by being uniformly dispersed in cells or arranged or bound to specific locations, and can be used as an intracellular fluorescent temperature sensor. [Brief Description of Drawings]

[0008] [Figure 1] Fluorescence spectrum of silicon-doped nanodiamond having SiV center (ZPL=738nm) [Figure 2] Confocal microscope fluorescence image when excitation light (532nm) is irradiated to silicon-doped nanodiamond having SiV center [Figure 3] Fluorescence spectrum of silicon-doped nanodiamond having SiV center, excitation light wavelength: 532nm, excitation light power: 100μW, ND Filter: OD 1.0 [Figure 4] Temperature responsiveness measurement data of SiV fluorescent ND [Figure 5] Confocal microscope apparatus for temperature measurement (equipped with hot plate and lens heater) [Figure 6] Fluorescence spectrum of germanium-doped nanodiamond having GeV center, excitation light wavelength: 532nm, excitation light power: 100μW, ND Filter: OD 1.0 [Figure 7] Temperature responsiveness measurement data of GeV fluorescent ND [Modes for Carrying Out the Invention]

[0009] The temperature-sensitive probe of the present invention includes group 14 element-doped nanodiamond having MV centers (M represents a group 14 element selected from the group consisting of Si, Ge, Sn and Pb, and V represents a vacancy) with an average particle size of 1 to 100 nm. The group 14 element-doped nanodiamond is a nanoparticle, and the upper limit of its average particle size is preferably 100 nm, more preferably 70 nm, still more preferably 50 nm, particularly preferably 30 nm, particularly more preferably 20 nm, and most preferably 10 nm, and the lower limit of the average particle size is preferably 1 nm, more preferably 1.5 nm, still more preferably 2 nm, and particularly preferably 3 nm. A smaller average particle size of the group 14 element-doped nanodiamond is preferred because it does not interfere with the movement or structural change of biomolecules such as intracellular proteins.

[0010] The relative standard deviation (RSD) of the particle size of the group 14 element-doped nanodiamond is preferably 25 to 40%. The average particle size and the relative standard deviation (RSD) can be measured by a small-angle X-ray scattering method (SAXS). The relative standard deviation (%) can be determined by the following formula.

Equation

[0011] The temperature-sensitive probe of the present invention is suitable for temperature measurement in microspaces. For example, it can measure the temperature of an entire cell, as well as distinguish and measure the temperatures of intracellular organelles (e.g., endoplasmic reticulum, mitochondria, Golgi apparatus, peroxisomes, lysosomes, microtubules, and microstructures). Cells that can be measured for temperature include microorganisms such as yeast, animal cells, and plant cells, and the probe can be easily introduced into cells. The temperature-sensitive probe of the present invention utilizes the fact that the position of the fluorescence peak during excitation light irradiation shifts with temperature. The temperature-sensitive probe detects temperature by fluorescence around 738 nm (ZPL) from SiV centers. Since this fluorescence around 738 nm is hardly absorbed by cells or their organelles, it is suitable for measuring intracellular temperature. The fluorescence from GeV centers with ZPL at 602 nm, SnV centers with ZPL at 620 nm, and PbV centers with ZPL at 520 nm and 552 nm has a high intensity, making it possible to measure intracellular temperature.

[0012] In this invention, "cells" refer to prokaryotic and eukaryotic cells, as in general classifications, and are not particularly dependent on the species of organism. For example, prokaryotic cells are divided into bacteria and archaea, and bacteria are further divided into Gram-positive bacteria such as Actinomycetes and Gram-negative bacteria such as Proteobacteria, and the range to which the temperature-sensitive probe can be applied is not limited by the thickness of the peptidoglycan layer, etc. Eukaryotic cells mainly refer to cells belonging to eukaryotes (protists, fungi, plants, and animals). For example, yeast, which is commonly used in molecular biology research and also used industrially, belongs to the fungi. To accurately measure the temperature within a limited microscopic space, such as inside a cell, the peak position of fluorescence (ZPL) around 738 nm can be measured at various temperatures (for example, 35°C to 40°C when the target is a cell), and the temperature of the object can be determined based on these results.

[0013] When measuring the temperature within the microspaces of cells, a temperature-sensitive probe can be applied to or introduced into the object to be measured. After removing any excess temperature-sensitive probe, for example, by washing, the temperature of the object (e.g., cells) can then be measured. For example, by measuring the temperature differences of individual cells within a mammalian cell population, it becomes possible to understand the physiological state of each cell. When measuring temperature within a microspace, a calibration curve can be created by measuring the peak position of fluorescence (ZPL) near the MV center at each temperature, and the temperature can be measured based on this calibration curve. Calibration curves can be created by heating and cooling Group 14 element-doped nanodiamond particles supported or coated on a substrate, but they can also be created in an environment similar to the microspace inside a cell (e.g., ionic strength, pH, etc.). Calibration curves can be created by changing the measurement conditions depending on the object being measured. Specifically, there is no limit to which calibration curve measured under which conditions should be used, but for example, a curve plotting the change in fluorescence intensity of a temperature-sensitive probe with temperature in a potassium chloride solution that mimics the inside of a cell can be used. More specifically, when performing a thermoresponsiveness test using a cell population into which a temperature-sensitive probe has been introduced and plotting the change in the peak position of fluorescence (ZPL), one method is to suspend the cells in a state where they do not actively perform metabolic activity, for example, in water or a buffer containing compounds that cannot be assimilated, and maintain them at a specific temperature for a certain period of time, and measure the fluorescence intensity when the external temperature and the internal cell temperature are considered to have reached equilibrium.

[0014] 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, by adding intracellular temperature, which has been difficult to measure accurately until now, to the analysis parameters in the fermentation production of useful substances using microorganisms, it is expected that the efficiency of examining culture conditions will be improved. The temperature-sensitive probe of the present invention can be applied to a variety of medical uses. For example, by using the temperature-sensitive probe according to the present invention on a portion of a patient's tissue, it is possible to distinguish between cancer cells, which are known to produce a large amount of heat, and normal cells, which do not. Furthermore, this can be applied to develop more effective hyperthermia treatments. Alternatively, by introducing the temperature-sensitive probe according to the present invention into brown adipose tissue, which produces a large amount of heat, and measuring the temperature change when various materials are added to those cells, it is possible to screen for materials that activate brown adipose tissue, which is effective in burning fat through energy consumption. Such materials are effective for weight loss or slimming by promoting fat burning.

[0015] The temperature-sensitive probe of the present invention can also be applied to elucidating various physiological phenomena. For example, by investigating how temperature-sensitive (transient receptor potential, TRP) channels, which are receptors that sense external temperature and trigger biological responses, are related to intracellular temperature, it is possible to explore new approaches to TRP channel activation. Furthermore, by investigating the relationship between intracellular temperature distribution and biological responses occurring inside and outside the cell, it is possible to investigate the influence of local temperature distribution on biological responses, and it may even be possible to control cells by localized heating using infrared lasers or the like. Furthermore, because the temperature-sensitive probe of the present invention is non-toxic, it can be safely used in microorganisms, animal cells, plant cells, or mammalian cells such as human, mouse, and rat cells.

[0016] The present invention further provides a method for measuring intracellular temperature, (a) A step of introducing a temperature-sensitive probe into cells by mixing the temperature-sensitive probe described in any one of items [1] to [4] with cells in water. (b) a step of irradiating cells into which a temperature-sensitive probe has been introduced with excitation light, and measuring the peak position of the ZPL fluorescence (ZPL) of an MV center (M represents a Group 14 element selected from the group consisting of Si, Ge, Sn and Pb; V represents a vacancy), and (c) a step of measuring temperature from the measured fluorescence intensity A method comprising the above steps can be provided. Examples of the wavelength of the excitation light include 532 nm and 633 nm.

[0017] The Group 14 element-doped nanodiamond can preferably be produced by a detonation method. The shape of the Group 14 element-doped nanodiamond is preferably spherical, ellipsoidal, or a polyhedral shape close thereto. The introduction amount of Group 14 element (M) atoms (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 1 to 36%.

[0018] Vacancies can be introduced into the Group 14 element-doped nanodiamond by ion beam irradiation or electron beam irradiation. The upper limit of the concentration of introduced vacancies is preferably 1×10 that can maintain the diamond structure 21 / cm 3 or less, and the lower limit of the vacancy 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 3The above is the explanation. The ion beam is preferably a hydrogen (H) or helium (He) ion beam. For example, 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. When an ion beam or electron beam is irradiated onto a group 14 element-doped nanodiamond, vacancies are formed, and these vacancies (V) and the doped group 14 element (M) form fluorescent MV centers. In the emission spectrum of the resulting MV centers, a sharp peak called the ZPL (Zero Phonon Line) accounts for the majority of the emission, and because the peak width is small, the influence of noise due to autofluorescence is small. The ZPL of the SiV center is 738 nm, which is located in the so-called biological window (the wavelength band in which excitation light and fluorescence penetrate biological tissue), making it an ideal emission center for bioimaging probes that enables external excitation and external measurement. The ZPL at the GeV center is 602nm, the ZPL at the SnV center is 620nm, and the ZPLs at the PbV center are 520nm and 552nm.

[0019] The preferred concentration of MV centers in the Group 14 element-doped nanodiamonds of the present invention is preferably 1 × 10⁻⁶. 14 / cm 3 That is all, more 2 × 10 14 ~1 × 10 19 / cm 3 That is the case. In the preferred Group 14 element-doped nanodiamond particles of the present invention, the center has a diamond structure containing sp3 carbon and doped Group 14 element atoms, and its surface is covered with an amorphous layer composed of sp2 carbon. In a more preferred embodiment, the outside of the amorphous layer may be covered with a graphite oxide layer. Furthermore, a hydration layer may be formed between the amorphous layer and the graphite oxide layer.

[0020] In one preferred embodiment of the present invention, the surface of Group 14 element-doped nanodiamond particles has one or more oxygen-containing functional groups. Examples of oxygen-containing functional groups include OH, C=O, COOH, and -O-. These functional groups can be further modified by etherification, esterification, amidation, etc. In one preferred embodiment of the present invention, the Group 14 element-doped nanodiamond has 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.

[0021] The Group 14 element-doped nanodiamonds of the present invention can be produced by a manufacturing method that includes the steps of mixing an explosive material with a Group 14 element compound such as a silicon compound, germanium compound, tin compound, or lead compound in a sealed container, and exploding the resulting mixture under conditions of negative oxygen balance in the presence of a cooling medium. The explosive material is not particularly limited, and a wide range of known explosive materials can be used. Specific examples include trinitrotoluene (TNT), cyclotrimethylenetrinitramine (Hexogen, RDX), cyclotetramethylenetetranitramine (Octogen), trinitrophenylmethylnitramine (Tetryl), pentaerythritol tetranitrate (PETN), tetranitromethane (TNM), triaminotrinitrobenzene, hexanitrostilbene, and diaminodinitrobenzofloxane, which can be used individually or in combination of two or more. Group 14 element compounds can be broadly used, whether organic or inorganic, as long as they contain Group 14 element atoms such as silicon (Si), germanium (Ge), tin (Sn), and lead (Pb).

[0022] When silicon is the element in Group 14, organic silicon compounds include: Silanes having lower alkyl groups such as acetoxytrimethylsilane, diacetoxydimethylsilane, triacetoxymethylsilane, acetoxytriethylsilane, diacetoxydiethylsilane, triacetoxyethylsilane, acetoxytripropylsilane, methoxytrimethylsilane, dimethoxydimethylsilane, trimethoxymethylsilane, ethoxytrimethylsilane, diethoxydimethylsilane, triethoxymethylsilane, ethoxytriethylsilane, diethoxydiethylsilane, triethoxyethylsilane, and trimethylphenoxysilane. Trichloromethylsilane, dichlorodimethylsilane, chlorotrimethylsilane, trichloroethylsilane, dichlorodiethylsilane, chlorotriethylsilane, trichlorophenylsilane, dichlorodiphenylsilane, chlorotriphenylsilane, dichlorodiphenylsilane, dichloromethylphenylsilane, dichloroethylphenylsilane, chlorodifluoromethylsilane, dichlorofluoromethylsilane, chlorofluorodimethylsilane, chloroethyldifluorosilane, dichloroethylfluorosilane, chlorodifluoropropylsilane, dichlorofluoropropyl Silanes containing halogen atoms, such as pyrusilane, trifluoromethylsilane, difluorodimethylsilane, fluorotrimethylsilane, ethyltrifluorosilane, diethyldifluorosilane, triethylfluorosilane, trifluoropropylsilane, fluorotripropylsilane, trifluorophenylsilane, difluorodiphenylsilane, fluorotriphenylsilane, tribrommethylsilane, dibromdimethylsilane, bromtrimethylsilane, bromtriethylsilane, bromtripropylsilane, dibromdiphenylsilane, and bromtriphenylsilane. Polysilanes such as hexamethyldisilane, hexaethyldisilane, hexapropyldisilane, hexaphenyldisilane, and octaphenylcyclotetrasilane. Silazanes such as triethylsilazane, tripropylsilazane, triphenylsilazane, hexamethyldisilazane, hexaethyldisilazane, hexaphenyldisilazane, hexamethylcyclotrisilazane, octamethylcyclotetrasilazane, hexaethylcyclotrisilazane, octaethylcyclotetrasilazane, and hexaphenylcyclotrisilazane. Aromatic silanes, such as silabenzene and disilabenzene, have silicon atoms incorporated into their aromatic rings. • Hydroxyl group-containing silanes such as trimethylsilanol, dimethylphenylsilanol, triethylsilanol, diethylsilanediol, tripropylsilanol, dipropylsilanediol, triphenylsilanol, and diphenylsilanediol Alkyl or aryl-substituted silanes such as tetramethylsilane, ethyltrimethylsilane, trimethylpropylsilane, trimethylphenylsilane, diethyldimethylsilane, triethylmethylsilane, methyltriphenylsilane, tetraethylsilane, triethylphenylsilane, diethyldiphenylsilane, ethyltriphenylsilane, and tetraphenylsilane. • Carboxyl group-containing silanes such as triphenylsilylcarboxylic acid, trimethylsilylacetic acid, trimethylsilylpropionic acid, and trimethylsilylbutyric acid. Siloxanes such as hexamethyldisiloxane, hexaethyldisiloxane, hexapropyldisiloxane, and hexaphenyldisiloxane. Silanes having an alkyl or aryl group and a hydrogen atom, such as methylsilane, dimethylsilane, trimethylsilane, diethylsilane, triethylsilane, tripropylsilane, diphenylsilane, and triphenylsilane. Tetrakis(chloromethyl)silane, tetrakis(hydroxymethyl)silane, tetrakis(trimethylsilyl)silane, tetrakis(trimethylsilyl)methane, tetrakis(dimethylsilanolyl)silane, tetrakis(tri(hydroxymethyl)silyl)silane, tetrakis(nitratemethyl)silane, These are some examples.

[0023] Inorganic silicon compounds include silicon oxide, silicon oxynitride, silicon nitride, silicon carbide oxide, silicon carbide nitride, silane, or silicon-doped carbon materials. Examples of silicon-doped carbon materials include graphite, activated carbon, carbon black, Ketjenblack, coke, soft carbon, hard carbon, acetylene black, carbon fiber, and mesoporous carbon. Examples of germanium compounds include organic germanium compounds such as methylgermane, ethylgermane, trimethylgermanium methoxide, dimethylgermanium diacetate, tributylgermanium acetate, tetramethoxygermanium, tetraethoxygermanium, tetraphenylgermane, isobutylgermane, alkylgermanium trichloride, and dimethylaminogermanium trichloride; germanium complexes such as nitrotriphenol complex (Ge2(ntp)2O), catechol complex (Ge(cat)2), or aminopyrene complex (Ge2(ap)2Cl2); and germanium alkoxides such as germanium ethoxide and germanium tetrabutoxide.

[0024] Examples of tin compounds include inorganic tin compounds such as tin(II) oxide, tin(IV) oxide, tin(II) sulfide, tin(IV) sulfide, tin(II) chloride, tin(IV) chloride, tin(II) bromide, tin(II) fluoride, tin acetate, and tin sulfate; alkyl tin compounds such as tetramethyltin; monoalkyl tin oxide compounds such as monobutyltin oxide; dialkyl tin oxide compounds such as dibutyltin oxide; aryl tin compounds such as tetraphenyltin; and organotin compounds such as dimethyl tin maleate, hydroxybutyltin oxide, and monobutyltin tris(2-ethylhexanoate). Examples of lead compounds include lead monoxide (PbO), lead dioxide (PbO2), red lead (Pb3O4), lead white (2PbCO3·Pb(OH)2), lead nitrate (Pb(NO3)2), lead chloride (PbCl2), lead sulfide (PbS), lead yellow (PbCrO4, Pb(SCr)O4, PbO·PbCrO4), lead carbonate (PbCO3), lead sulfate (PbSO4), and lead fluoride (P Examples include inorganic lead compounds such as lead bF2, lead tetrafluoride (PbF4), lead bromide (PbBr2), and lead iodide (PbI2), and organic lead compounds such as lead acetate (Pb(CH3COO)2), lead tetracarboxylate (Pb(OCOCH3)4), tetraethyl lead (Pb(CH3CH2)4), tetramethyl lead (Pb(CH3)4), and tetrabutyl lead (Pb(C4H9)4). The above organic or inorganic Group 14 element compounds may be used individually or in combination of two or more.

[0025] The proportion of explosive material in a mixture containing explosive material and a Group 14 element compound is preferably 85 to 99.9% by mass, more preferably 86 to 99% by mass, and the proportion of Group 14 element compound is preferably 0.1 to 15% by mass, more preferably 1 to 14% by mass. Furthermore, the Group 14 element content in the mixture containing explosive material and Group 14 element compound is preferably 0.007 to 4.5% by mass, more preferably 0.06 to 4.3% by mass. The mixture containing explosive material and Group 14 element compound may further contain the above carbon material which does not contain Group 14 elements. The mixture of explosive materials and Group 14 element compounds may be performed by powder mixing if both are solids, or by dissolving or dispersing them in a suitable solvent before mixing. Mixing can be carried out by stirring, bead milling, ultrasound, etc. In one preferred embodiment, the mixture of the explosive material and the group 14 element compound further includes a cooling medium. The cooling medium may be a solid, liquid, or gas. One method of using the cooling medium is to detonate the mixture of the explosive material and the group 14 element compound in the cooling medium. Examples of cooling mediums include inert gases (nitrogen, argon, CO), water, ice, liquid nitrogen, aqueous solutions of group 14 element-containing salts, and crystalline hydrates. Examples of silicon-containing salts included in group 14 element-containing salts include ammonium hexafluorosilicate, ammonium silicate, and tetramethylammonium silicate. When using water or ice, for example, it is preferable to use about five times the amount of cooling medium relative to the weight of the explosive. In one preferred embodiment of the present invention, a mixture containing an explosive material and a Group 14 element compound is converted into diamond by compression due to a shock wave under high-pressure, high-temperature conditions generated by the explosion of the explosive material (detonation method). During the explosion of the explosive material, Group 14 element atoms are incorporated into the diamond lattice. The carbon source of nanodiamonds can be the explosive material and the 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 Group 14 elements, this carbon material can also serve as a carbon source for nanodiamonds. Group 14 element-doped nanodiamonds obtained by detonation can be purified and annealed according to conventional methods. [Examples]

[0026] The present invention will be described more specifically below with reference to examples, but the present invention is not limited to these examples.

[0027] Example 1 (1) Synthesis of silicon-doped nanodiamonds Using TNT and cyclotrimethylentrinitramine (Hexogen, RDX) as explosives, and 0.21 moles of triphenylsilanol (SiPh3OH) per mole of TNT as the silicon compound, silicon-doped nanodiamonds were produced by detonation according to a conventional method under the conditions of temperature (4092K) and pressure (32GPa). As a result, nanodiamonds with SiV centers were obtained, doped with (Si atoms / C atoms) × 100 (%) = 1% silicon. The fluorescence spectra of the obtained silicon-doped nanodiamonds containing SiV centers are shown in Figure 1. For the fluorescence spectrum measurement method, the obtained evaluation sample was prepared as a 1 wt% aqueous dispersion, a few drops were placed on a glass plate, and dried. The dried sample was then irradiated with 532 nm excitation light onto SiV fluorescent nanodiamonds using a micro-Raman spectrometer (product name: Micro-Laser Raman Spectrophotometer LabRAM HR Evolution, manufactured by Horiba, Ltd.) to acquire the fluorescence spectrum. The measurement conditions were excitation light power: 100 μW, exposure time: 1 second, and number of integrations: 1.

[0028] Example 2 The silicon-doped nanodiamonds containing SiV centers obtained in Example 1 were dispersed in water at a concentration of 1% by mass. The resulting dispersion was dropped onto a glass substrate and dried to prepare evaluation samples. The obtained evaluation samples were subjected to high-speed mapping using a confocal microscope (Figure 5) (excitation light wavelength: 532 nm, excitation light power: 100 μW) (Figure 2). Furthermore, the points indicated by the arrows in Figure 2, which are bright spots, were peaked up, and the fluorescence spectra were measured in detail to confirm that the points indicated by the arrows in Figure 2 are SiV fluorescent ZPLs (Figure 3). Subsequently, the temperature was adjusted using the hot plate and lens heater attached to the confocal microscope, and measurements were taken at temperatures of 295K (22℃), 298.5K (25.5℃), 302.5K (29.5℃), 306.5K (33.5℃), 310K (37℃), and 313K (40℃) (excitation light: 532nm, excitation light power: 100μW, ND Filter: OD 1.0) to increase the wavelength resolution in the 732.5736-749nm region. Peak wavelengths were measured at 22℃, 25.5℃, 29.5℃, 33.5℃, 37℃, and 40℃ at the points indicated by the arrows in Figure 2 (Figure 4). Δλ / ΔT = 0.0150 (nm / K).

[0029] Example 3 (1) Synthesis of germanium-doped nanodiamonds Using TNT and cyclotrimethylenetrinitramine (hexogen, RDX) as explosives, and adding 0.6 g of tetraphenylgermane to 60 g of a TNT and RDX mixture, germanium-doped nanodiamonds were produced by detonation according to a conventional method under the conditions of temperature (4092 K) and pressure (32 GPa). This yielded nanodiamonds with GeV centers, doped with (Ge atoms / C atoms) × 100 (%) = 1% germanium.

[0030] Example 4 The germanium-doped nanodiamonds containing GeV centers obtained in Example 3 were dispersed in water at a concentration of 1% by mass. The resulting dispersion was dropped onto a glass substrate and dried to prepare evaluation samples. The obtained evaluation samples were subjected to high-speed mapping using a confocal microscope (Figure 5) (excitation light wavelength: 532 nm, excitation light power: 100 μW). Furthermore, the bright spots were peaked up, and the fluorescence spectra were measured in detail to confirm that these points were GeV fluorescent ZPLs (Figure 6). Subsequently, the temperature was adjusted using the hot plate and lens heater attached to the confocal microscope, and measurements were taken in the 597-607nm region at temperatures of 295K (22℃), 298.5K (25.5℃), 302.5K (29.5℃), 306.5K (33.5℃), 310K (37℃), and 313K (40℃) (excitation light: 532nm, excitation light power: 100μW, ND Filter: OD 1.0) to increase the wavelength resolution. The peak wavelengths at the aforementioned points at 22℃, 25.5℃, 29.5℃, 33.5℃, 37℃, and 40℃ were measured (Figure 7). Δλ / ΔT = 0.0079 (nm / K).

Claims

1. An aqueous dispersion for measuring intracellular temperature, An aqueous dispersion containing Group 14 element-doped nanodiamonds having MV centers (where M represents a Group 14 element selected from the group consisting of Si, Ge, Sn, and Pb, and V represents a vacancy) with an average particle size of 1 to 100 nm, and a temperature of 25.5 to 40°C.

2. The aqueous dispersion according to claim 1, wherein the average particle size is 1 to 10 nm.

3. The aqueous dispersion according to claim 1, wherein the temperature is 29.5 to 40°C.

4. The aqueous dispersion according to claim 1, wherein the temperature is 33.5 to 40°C.

5. The aqueous dispersion according to claim 1, wherein the temperature is 35 to 40°C.

6. The aqueous dispersion according to claim 1, wherein the temperature is 37 to 40°C.

7. The aqueous dispersion according to claim 1, wherein the pH of the aqueous dispersion is within a range of possible pH values ​​within cells.

8. The aqueous dispersion according to claim 1, wherein the ionic strength of the aqueous dispersion is within a range of possible ionic strengths within a cell.

9. An aqueous dispersion comprising Group 14 element-doped nanodiamonds having MV centers (where M represents a Group 14 element selected from the group consisting of Si, Ge, Sn, and Pb) with an average particle size of 1 to 100 nm, and having a temperature of 25.5 to 40°C, and a temperature-measurable object.

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