WO2022244716A1 - Temperature measuring device, and temperature measuring method - Google Patents

Temperature measuring device, and temperature measuring method Download PDF

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WO2022244716A1
WO2022244716A1 PCT/JP2022/020337 JP2022020337W WO2022244716A1 WO 2022244716 A1 WO2022244716 A1 WO 2022244716A1 JP 2022020337 W JP2022020337 W JP 2022020337W WO 2022244716 A1 WO2022244716 A1 WO 2022244716A1
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temperature
light intensity
quantum dot
light
intensity ratio
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PCT/JP2022/020337
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French (fr)
Japanese (ja)
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博 湯川
司 鳥本
嘉信 馬場
達矢 亀山
諒 都澤
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国立大学法人東海国立大学機構
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Publication of WO2022244716A1 publication Critical patent/WO2022244716A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61B—DIAGNOSIS; SURGERY; IDENTIFICATION
    • A61B5/00—Measuring for diagnostic purposes; Identification of persons
    • A61B5/01—Measuring temperature of body parts ; Diagnostic temperature sensing, e.g. for malignant or inflamed tissue
    • 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

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  • the present disclosure relates to a temperature measurement device and a temperature measurement method using core-shell quantum dots.
  • Conventional methods for measuring cell temperature include the following methods.
  • Non-Patent Document 1 describes a method for measuring cell temperature using the fact that the fluorescence intensity of quantum dots changes with temperature.
  • Non-Patent Document 2 describes measuring the temperature of COS7 cells (monkey kidney-derived cells) using the temperature dependence of fluorescence lifetime changes of polymeric fluorescent probes.
  • Non-Patent Document 3 describes the development of a temperature sensor protein, tsGFP, and the measurement of the temperature distribution in organelles based on changes in the fluorescence of tsGFP.
  • Non-Patent Document 4 describes measuring the intracellular temperature of HepG2 cells (human liver cancer cell line) using a micro thermocouple.
  • Non-Patent Documents 5-7 describe measuring cell temperature using fluorescent nanodiamonds.
  • Non-Patent Document 1 cannot be applied to temperature measurement of cells in vivo because the fluorescence intensity changes depending on the depth in the living body.
  • Non-Patent Documents 2-4 in addition to the problems of fluorescence intensity and light resistance, there is also the problem of biological permeability, and in vivo measurement has not been realized.
  • Non-Patent Documents 5-7 the fluorescence wavelength of the fluorescent nanodiamonds is visible light, and there is a problem of bio-permeability as in Non-Patent Documents 2-4.
  • an object of the present disclosure is to provide a temperature measuring device and a thermometry method capable of measuring cell temperature in vivo.
  • the present disclosure is a core-shell quantum dot that is placed near an object to be measured and has a core with crystal defects and a shell that covers the core, and the quantum dot has a wavelength of energy greater than the bandgap energy of the core.
  • a light irradiation unit that irradiates excitation light
  • a light detection unit that measures the light intensity at the peak wavelength of the band edge emission of the quantum dot, and the light intensity at the peak wavelength of the defect emission of the quantum dot
  • the light detection unit A light intensity ratio calculation unit that calculates the light intensity ratio of the two detected light intensities, a calibration curve storage unit that stores a calibration curve showing the relationship between the light intensity ratio and the temperature around the quantum dot, and a light intensity ratio
  • a temperature measuring device comprising: a temperature calculating section for calculating a temperature of a measurement object from the light intensity ratio calculated by the calculating section and the calibration curve stored in the calibration curve storage section.
  • FIG. 4 is a graph showing the temperature dependence of the light intensity I1 at the peak wavelength of band edge emission and the light intensity I2 at the peak wavelength of defect emission.
  • the temperature measurement principle of the present disclosure will be described.
  • the inventors investigated whether it is possible to measure the temperature inside a living body using quantum dots. Focusing on the energy level structure of core-shell quantum dots, the inventors discovered that the light intensity ratio between the peak of band edge emission and the peak of defect emission changes linearly with the environmental temperature around the quantum dot. did.
  • the temperature measurement device and temperature calculation method of the present disclosure use the correspondence between the light intensity ratio and the temperature as a calibration curve, and calculate the temperature by measuring the light intensity ratio.
  • FIG. 1 is a diagram showing the configuration of the temperature measuring device of the first embodiment.
  • the temperature measurement device of the first embodiment includes a quantum dot 1, a light irradiation unit 2, a light detection unit 3, a light intensity ratio calculation unit 4, a calibration curve storage unit 5, and a temperature calculation 6 and .
  • the quantum dot 1 is used as a temperature measurement probe. By arranging the quantum dot 1 near the object to be measured, the temperature of the object to be measured is measured. Of course, the quantum dot 1 and the object to be measured may be in contact with each other.
  • a quantum dot 1 is a semiconductor particle that exhibits a quantum confinement effect due to its small diameter.
  • a quantum dot 1 having a core-shell structure is used.
  • the core-shell type is composed of a spherical core 10 and a shell 11 covering the core 10 . The shell 11 maintains the energy level of the core 10 and protects it from deactivation.
  • the core 10 of the quantum dot 1 has crystal defects.
  • FIG. 2 shows a band diagram of the core 10 of the quantum dot 1.
  • crystal defects form crystal defect levels in the bandgap. Therefore, some of the carriers generated by irradiation with excitation light (light with energy higher than the bandgap energy) recombine and emit band edge light, and some are trapped in crystal defect levels and then recombine. defect emission. Therefore, the fluorescence spectrum of the core 10 of the quantum dot 1 has a band edge emission peak and a defect emission peak. Further, the defect emission is on the longer wavelength side than the peak of the band edge emission. The peak wavelength of defect emission may change depending on the ambient environment of the quantum dot 1 .
  • the material of the core 10 of the quantum dot 1 may be any semiconductor material in which crystal defects are formed.
  • -III-V group semiconductors, I-II-IV-VI group semiconductors, and the like can be used.
  • AgInS2, AgInSe2 , AgInTe2 , AgInGaS , AgInZnS2 , AgInTe2 , AgInZnTe2 , AgInGaSe2 , CuInS2 , CuInSe2 , InP, GaP, etc. can be used.
  • a material that does not contain highly toxic elements such as Cd and Pb.
  • any material can be used for the shell 11 of the quantum dot 1 as long as it can protect the core 10, and ZnS , GaS, ZnGaS , ZnO, In2S3, GaSx , and the like can be used.
  • the bandgap energy of the core 10 of the quantum dot 1 is determined by the type of material and the diameter of the core 10. Therefore, by changing them, the bandgap energy can be controlled, and the peak wavelength of band edge emission and the peak wavelength of defect emission can be controlled.
  • the bandgap energy can be controlled by the composition ratio.
  • the bandgap energy can be controlled by the composition ratio of Ag, In, and Ga.
  • the difference between the peak wavelength of band edge emission and the peak wavelength of defect emission is preferably 100 nm or more. If it is 100 nm or more, two peaks can be sufficiently separated and detected, and the light intensity ratio I2/I1 can be accurately measured.
  • the diameter of the core 10 of the quantum dot 1 is arbitrary as long as the quantum confinement effect appears, and is, for example, 100 nm or less.
  • the diameter is the diameter of the sphere circumscribing the quantum dot 1 . It is preferably 50 nm or less, more preferably 20 nm or less. Although the lower limit of the diameter is not specified, it is, for example, 1 nm or more.
  • the thickness of the shell 11 of the quantum dot 1 is arbitrary as long as it can protect the core 10 .
  • the shell 11 does not need to be single, and may be double or more.
  • the quantum dot 1 may have various substances bonded to its surface.
  • a hydrophilic group such as a hydroxy group, an amino group, a carboxy group, a sulfo group, or salts thereof may be bound.
  • the quantum dot 1 can be made water-soluble, and the convenience of measuring the temperature of the living body can be enhanced.
  • a membrane-permeable peptide or cationic liposome may be bound to the surface of the quantum dot 1 .
  • the quantum dot 1 can be incorporated into the cell, and the temperature inside the cell can be measured.
  • Membrane permeable peptides are, for example, octaarginine (R8), TAT peptide, and the like.
  • the light irradiation unit 2 is a device that irradiates the quantum dots 1 with excitation light.
  • the wavelength of the excitation light to be irradiated may be any wavelength as long as the wavelength has energy greater than the bandgap energy of the core 10 of the quantum dot 1 .
  • LEDs, LDs, xenon lamps, etc. can be used.
  • infrared light which is highly permeable to the living body, as the excitation light.
  • the excitation light has a wavelength of 700 to 1500 nm, and the diameter and material composition ratio of the quantum dots 1 are adjusted so that the peak wavelength of the band edge emission of the quantum dots 1 is longer than the wavelength of the excitation light.
  • An example of a quantum dot 1 material that can use infrared light as excitation light is AgInGaSe 2 .
  • FIG. 13 is a quote from a paper (Tatsuya Kameyama, et al., ACS Appl. Nano Mater. 2020, 3, 3275-3287), which describes quantum dots made of Ag x In y Ga (1-y) Se 2 It is the graph which showed the fluorescence spectrum.
  • the molar ratio x of Ag is fixed at 0.67, and the molar ratio y of In to the sum of In and Ga is varied from 0.25 to 1.
  • the peak of band edge emission shifts to the longer wavelength side, and when y is 0.6 or more, the peak of band edge emission is 740 nm or more.
  • the defect luminescence peak is seen on the longer wavelength side than the band edge luminescence peak, and the peak is clearly seen when y is 0.25 to 0.5.
  • y is 0.6 or more, the peak of defect emission is not clear but is present. Therefore, when y is 0.6 or more, it is possible to excite band edge emission and defect emission with near-infrared rays.
  • the peak of band edge emission is 740 nm and the peak of defect emission is 840 nm.
  • infrared rays especially wavelengths of 700 to 1500 nm
  • the photodetector 3 is a device that detects band edge luminescence and defect luminescence from the quantum dot 1 and measures the light intensity I1 at the peak wavelength of the band edge luminescence and the light intensity I2 at the peak wavelength of the defect luminescence. For example, the fluorescence spectrum from the quantum dot 1 is measured, and the light intensities I1 and I2 are measured by extracting the band edge emission peak and the defect emission peak.
  • a fluorescence spectrum is obtained by scanning fluorescence wavelengths with a spectroscope and measuring light intensity with a photomultiplier tube or photodiode.
  • a fluorescence intensity meter can be used as the light irradiation unit 2 and the light detection unit 3 .
  • the light intensities I1 and I2 may be measured by obtaining a fluorescence image using a fluorescence imaging device and analyzing the fluorescence image. If there are a plurality of defect luminescence peaks, any one of them may be set as I2, or any two or more peaks may be averaged.
  • the light intensity at the peak position is preferable in terms of accuracy, but the light intensity at a slightly deviated position is acceptable. For example, if the deviation from the peak is within 20 nm, the temperature can be measured with sufficient accuracy.
  • the light intensities I1 and I2 may be averages of light intensities in a predetermined wavelength width including peaks.
  • the predetermined wavelength width is preferably 30 nm or less, more preferably 20 nm or less.
  • the light intensity ratio calculator 4 calculates the light intensity ratio I2/I1 from the light intensity I1 at the peak wavelength of the band edge emission and the light intensity I2 at the peak wavelength of the defect emission measured by the photodetector 3. is.
  • the light intensity ratio calculator 4 is implemented by a computer.
  • the calibration curve storage unit 5 is a device that stores a calibration curve showing the correspondence between the light intensity ratio I2/I1 and the temperature around the quantum dots 1.
  • a plurality of calibration curves are stored for each ambient environment of the quantum dot 1 .
  • a and b are determined by the ambient environment of the quantum dot 1 . More specifically, it is determined by the substances in the route from the light irradiation unit 2 to the quantum dot 1 and the route from the quantum dot 1 to the light detection unit 3 .
  • a and b are experimentally determined according to the ambient environment of the quantum dot 1 and stored in the calibration curve storage unit 5 .
  • the correspondence may be stored as it is, or may be approximated by another function.
  • the temperature calculation unit 6 is a device that calculates the temperature around the quantum dots 1 from the light intensity ratio I2/I1 calculated by the light intensity ratio calculation unit 4 and the calibration curve stored in the calibration curve storage unit 5. be.
  • the temperature calculator 6 is implemented by a computer.
  • the quantum dot 1 is arranged near the object to be measured.
  • the arrangement method is arbitrary.
  • Quantum dots 1 can be easily arranged at a specific site of a living body by injecting an aqueous solution or the like.
  • the wavelength of the excitation light is infrared light, which is highly penetrable to the living body, and has a wavelength of, for example, 700 to 1500 nm. Therefore, a quantum dot 1 with a smaller bandgap energy is used.
  • the quantum dot 1 is irradiated with excitation light by the light irradiation unit 2 . Then, the light from the quantum dot 1 is detected by the photodetector 3, and the light intensity I1 at the peak wavelength of the band edge emission of the quantum dot 1 and the light intensity I2 at the peak wavelength of the defect emission of the quantum dot 1 are measured. If the excitation light is infrared rays, the excitation light passes through the living body and reaches the quantum dots 1 with sufficient intensity even if the quantum dots 1 are inside the living body. Fluorescence from the quantum dots 1 also passes through the living body and reaches the photodetector 3 with sufficient intensity.
  • the light intensity ratio calculator 4 calculates the light intensity ratio I2/I1.
  • the temperature calculation unit 6 calculates the temperature around the quantum dot 1 from the light intensity ratio I2/I1 calculated by the light intensity ratio calculation unit 4 and the calibration curve stored in the calibration curve storage unit 5. . Since the quantum dot 1 is arranged near the object to be measured, the temperature around the quantum dot 1 matches the temperature of the object to be measured.
  • the calibration curve is selected according to the ambient environment of the quantum dots 1 . Selection of the standard curve may be performed manually or may be automated.
  • the temperature of the object to be measured around the quantum dot 1 can be measured.
  • the temperature measurement device of the first embodiment can measure the temperature of a specific site inside the living body, and the temperature of the cell can be measured by arranging the quantum dots 1 inside or around the cell.
  • the organoid internal temperature can be measured.
  • the temperature can be measured with high accuracy, and the temperature can be measured with an error of 0.1K or less.
  • the light intensity ratio I2/I1 is calculated in the first embodiment
  • the light intensity ratio I1/I2 is calculated
  • the temperature is calculated from a calibration curve showing the correspondence between the light intensity ratio I1/I2 and temperature.
  • the quantum dot 1 can also serve as a fluorescent probe for labeling biological samples. Therefore, the temperature measuring device of the first embodiment can measure the temperature of the specific site while visualizing the specific site of the living body by the fluorescence of the quantum dots 1 .
  • a core-shell quantum dot 1 having a core of AgInGaS and a shell of ZnGaS was produced by the following method.
  • the resulting precipitate contained the desired quantum dots, but was subjected to another coating operation to further increase the shell thickness of each particle.
  • Zn(C 17 H 35 COO) 2 100 mg
  • thiourea 12 mg
  • OLA 3.0 mL
  • Methanol 3.0 mL
  • the precipitate was washed by adding ethanol (3.0 mL) to the resulting precipitate, suspending it again, and then centrifuging.
  • a core-shell type quantum dot 1 (average particle size: 5.4 nm) having a core of AgInGaS and a shell of ZnGaS was obtained.
  • quantum dots 1 were dissolved in chloroform (3.0 mL) to obtain a chloroform solution of quantum dots 1 whose surfaces were modified with OLA. Then, 3-mercaptopropionic acid (MPA) (0.10 mL), tetramethylammonium hydroxide (0.73 mL), and ethanol (0.27 mL) were mixed with this solution (1.0 mL), and nitrogen It was heated at 70° C. for 3 hours in an atmosphere. As a result, the OLA on the surface of the quantum dot 1 was replaced with MPA. As described above, quantum dots 1 whose surface was modified with MPA were obtained. Since MPA has a carboxyl group, which is a hydrophilic group, the quantum dots 1 can be dissolved in water by modifying the surface of the quantum dots 1 with MPA.
  • MPA has a carboxyl group, which is a hydrophilic group
  • FIG. 3 is a TEM image of the quantum dot 1.
  • FIG. 3(a) shows quantum dots 1 in chloroform solution (surface modified with OLA), and
  • FIG. 3(b) shows quantum dots 1 in aqueous solution (surface modified with MPA). .
  • FIG. 3 it was found that quantum dots 1 having a particle size of 5.4 nm were obtained.
  • FIG. 4 is a graph showing the results of measuring the fluorescence spectrum of quantum dot 1 (before being covered with a shell).
  • the excitation light was 365 nm.
  • the fluorescence spectrum has two peaks, a band edge emission peak near 550 nm and a defect emission peak near 700 nm.
  • FIG. 5 is a graph showing the spectrum of defect emission, comparing quantum dot 1 in a chloroform solution and quantum dot 1 in an aqueous solution.
  • the excitation light was 365 nm.
  • the peak wavelength of defect luminescence shifted to longer wavelengths in the aqueous solution than in the chloroform solution. From this, it was found that the calibration curve should be set according to the ambient environment of the quantum dots 1 .
  • FIG. 6 is a graph comparing the difference in defect luminescence spectra of quantum dots 1 in an aqueous solution depending on temperature.
  • the excitation light was 365 nm.
  • the temperature was varied from 5°C to 45°C in 10°C increments.
  • the temperature was changed using a heater, and the temperature was measured by bringing a thermocouple into contact with the aqueous solution. As shown in FIG. 6, it was found that the higher the temperature, the lower the light intensity of the defect emission, and the peak wavelength slightly shifted to the shorter wavelength side.
  • FIG. 7 is a graph showing the temperature dependence of the light intensity I1 at the peak wavelength of band edge emission and the light intensity I2 at the peak wavelength of defect emission for quantum dots 1 in an aqueous solution. It was found that both light intensities decreased linearly with temperature change, and that there was a difference in the slope of the decrease.
  • FIG. 8 is a graph showing the temperature dependence of the ratio I2/I1 of the light intensity I2 at the peak wavelength of defect emission to the light intensity I1 at the peak wavelength of band edge emission for quantum dots 1 in an aqueous solution.
  • the wavelength of excitation light and the method of measuring temperature are the same as in FIG.
  • the light intensity ratio I2/I1 and the temperature have a one-to-one correspondence.
  • the corresponding relationship can be approximated by a straight line with high accuracy. Therefore, by using the corresponding relationship between the light intensity ratio I2/I1 and the temperature as a calibration curve, it was found that the temperature can be accurately measured from the light intensity ratio I2/I1 at least in the range of 5 to 45°C.
  • the surface-modified quantum dots 1 prepared in Example 1 with MPA were dissolved in an aqueous solution, octaarginine (R8) (2 mM) was mixed with the aqueous solution, and the mixture was allowed to stand at room temperature for 15 minutes. As a result, a quantum dot 1 whose surface was modified with R8 was produced.
  • R8 is a membrane permeable peptide.
  • the quantum dots 1 whose surface was modified with R8 were dissolved, and the aqueous solution was mixed with the culture medium of mouse adipose tissue-derived stem cells (ASCs) to incorporate the quantum dots 1 into the ASCs. Then, the quantum dot 1 was irradiated with excitation light having a wavelength of 365 nm, and the fluorescence spectrum was measured. The fluorescence spectrum was measured multiple times while changing the temperature of the culture solution with a heater.
  • ASCs mouse adipose tissue-derived stem cells
  • FIG. 9 is a graph showing the temperature dependence of the ratio I2/I1 of the light intensity I2 at the peak wavelength of defect emission to the light intensity I1 at the peak wavelength of band edge emission for quantum dots 1 in ASCs.
  • the light intensity ratio I2/I1 and the temperature have a one-to-one correspondence.
  • the corresponding relationship can be approximated by a straight line with high accuracy. Therefore, it was found that the intracellular temperature can also be measured by using the quantum dot 1.
  • Quantum dots 1 whose surface is modified with MPA prepared in Example 1 are dissolved in an aqueous solution, 50 ⁇ L of the aqueous solution is placed in a container and placed on a heater, and a fluorescent image of the container is captured by a bioimaging device (manufactured by PerkinElmer, Inc.). Acquired using IVIS Lumina K Series III). The aqueous solution was heated by a heater, and fluorescence images were acquired while changing the temperature. A band-pass filter for excitation light was set to 420 to 440 nm. Also, the light intensity I1 at the peak wavelength of the band edge emission was measured from the fluorescence image of the container at wavelengths of 570 to 590 nm. In addition, the light intensity I2 at the peak wavelength of defect emission was obtained from the fluorescence image of the container at wavelengths of 690 to 710 nm.
  • FIG. 10 is a graph showing the temperature dependence of the light intensity ratio I2/I1. As shown in FIG. 10, it has been found that the light intensity ratio I2/I1 and the temperature have a one-to-one correspondence, and the correspondence relationship can be approximated by a straight line with good accuracy. Therefore, it turned out that temperature measurement is possible using a biological imaging device.
  • Quantum dots 1 whose surfaces were modified with MPA prepared in Example 1 were dissolved in an aqueous solution, and 50 ⁇ L of the aqueous solution was subcutaneously injected into BALB/c nude mice. Then, in the same manner as in Example 3, a fluorescence image of the area of the mouse subcutaneously injected with the aqueous solution was acquired using a bioimaging device, and the light intensity ratio I2/I1 was measured.
  • the band-pass filter for excitation light was set to 485 to 515 nm, and other conditions were the same as in Example 3.
  • FIG. 11 is a graph showing the temperature dependence of the light intensity ratio I2/I1. As shown in FIG. 11, it was found that the light intensity ratio I2/I1 corresponds one to one with the temperature of the region of the mouse subcutaneously injected with the aqueous solution, and the correspondence relationship can be approximated by a straight line with good accuracy. Therefore, it was found that temperature measurement of a specific part of a living body is possible using a living body imaging device.
  • Quantum dots 1 whose surfaces were modified with MPA prepared in Example 1 were dissolved in an aqueous solution, and 50 ⁇ L of the aqueous solution was subcutaneously injected into BALB/c nude mice. Then, the mouse was placed in three states of cooling, normal, and heating, and photographed with an infrared thermography camera (FLIR C2, manufactured by FLIR). As a result, the temperature of the region of the mouse subcutaneously injected with the aqueous solution was 21.3°C in the cold state, 32.3°C in the normal state, and 36.7°C in the heated state.
  • FLIR C2 infrared thermography camera
  • Example 4 a fluorescence image of the area of the mouse subcutaneously injected with the aqueous solution was obtained using a bioimaging device. Fluorescence images were obtained for each of the three states of the mouse: the above-described cooling state, normal state, and heating state.
  • the band-pass filter for excitation light was set to 490 to 510 nm, and other conditions were the same as in Example 4. Then, the light intensity ratio I2/I1 was measured from the fluorescence image.
  • FIG. 12 is a graph plotting the light intensity ratio I2/I1 in three states on a calibration curve. From FIG. 12, the temperature of the area of the mouse subcutaneously injected with the aqueous solution was calculated to be 22.0° C. in the cooled state, 32.5° C. in the normal state, and 37.4° C. in the heated state. This result is relatively consistent with the measurement result by the infrared thermography camera, and it was confirmed that the temperature can be measured using the correspondence relationship between the light intensity ratio I2/I1 and the temperature as a calibration curve.
  • the temperature measurement device of the present disclosure is suitable for measuring the temperature of cells inside a living body.
  • Quantum dot 2 Light irradiation unit 3: Light detection unit 4: Light intensity ratio calculation unit 5: Calibration curve storage unit 6: Temperature calculation unit 10: Core 11: Shell

Abstract

[Problem] To provide a temperature measuring device capable of measuring a cell temperature in a living body. [Solution] This temperature measuring device consists of a quantum dot 1, a light emitting unit 2, a light detecting unit 3, a light intensity ratio calculating unit 4, a calibration curve storage unit 5, and a temperature calculating unit 6. The quantum dot 1 has a core-shell type structure. A core 10 of the quantum dot 1 has crystal defects. The light emitting unit 2 is a device for emitting exciting light onto the quantum dot 1. The light detecting unit 3 is a device for measuring a light intensity I1 at a peak wavelength of band edge emission, and a light intensity I2 at a peak wavelength of defect emission. The light intensity ratio calculating unit 4 is a device for calculating a light intensity ratio I2/I1. The calibration curve storage unit 5 is a device for storing a calibration curve representing a correspondence between the light intensity ratio I2/I1 and a temperature at the periphery of the quantum dot 1. The temperature calculating unit 6 is a device for calculating the temperature at the periphery of the quantum dot 1 from the light intensity ratio I2/I1 and the calibration curve.

Description

温度計測装置、および温度計測方法Temperature measuring device and temperature measuring method
　本開示は、コアシェル型の量子ドットを用いた温度計測装置、および温度計測方法に関するものである。 The present disclosure relates to a temperature measurement device and a temperature measurement method using core-shell quantum dots.
　近年、再生医療の技術が著しく進展し、幹細胞や再生細胞の細胞温度が高次元再生機能（再生因子産生能、分化能、生着能、センシング能など）に大きく影響を及ぼすことが分かってきている。そのため、細胞の温度を正確に計る技術が求められている。 In recent years, regenerative medicine technology has made remarkable progress, and it has been found that the cell temperature of stem cells and regenerative cells has a significant effect on high-dimensional regenerative functions (regenerative factor production ability, differentiation ability, engraftment ability, sensing ability, etc.). there is Therefore, there is a demand for a technique for accurately measuring cell temperature.
　従来の細胞温度の計測方法としては、以下の方法がある。 　Conventional methods for measuring cell temperature include the following methods.
　非特許文献１には、量子ドットの蛍光強度が温度により変化することを利用して細胞温度を計測する方法が記載されている。 　Non-Patent Document 1 describes a method for measuring cell temperature using the fact that the fluorescence intensity of quantum dots changes with temperature.
　非特許文献２には、高分子蛍光プローブの蛍光寿命変化の温度依存を利用してＣＯＳ７細胞（サル腎臓由来細胞）の温度を計測することが記載されている。 Non-Patent Document 2 describes measuring the temperature of COS7 cells (monkey kidney-derived cells) using the temperature dependence of fluorescence lifetime changes of polymeric fluorescent probes.
　非特許文献３には、温度センサータンパク質であるｔｓＧＦＰを開発し、ｔｓＧＦＰの蛍光の変化によってオルガネラにおける温度分布を計測することが記載されている。 Non-Patent Document 3 describes the development of a temperature sensor protein, tsGFP, and the measurement of the temperature distribution in organelles based on changes in the fluorescence of tsGFP.
　非特許文献４には、マイクロ熱電対を用いてＨｅｐＧ２細胞（ヒト肝がん細胞株）の細胞内温度を測定することが記載されている。 Non-Patent Document 4 describes measuring the intracellular temperature of HepG2 cells (human liver cancer cell line) using a micro thermocouple.
　非特許文献５－７には、蛍光ナノダイヤモンドを用いて細胞の温度を測定することが記載されている。 Non-Patent Documents 5-7 describe measuring cell temperature using fluorescent nanodiamonds.
　しかし、非特許文献１の方法では、蛍光強度は生体内の深度によって変化するため、生体内の細胞の温度計測には応用することができない。 However, the method of Non-Patent Document 1 cannot be applied to temperature measurement of cells in vivo because the fluorescence intensity changes depending on the depth in the living body.
　また、非特許文献２－４の方法では、蛍光強度や耐光性の問題に加え、生体透過性の問題もあり、生体内での計測は実現されていない。 In addition, in the methods of Non-Patent Documents 2-4, in addition to the problems of fluorescence intensity and light resistance, there is also the problem of biological permeability, and in vivo measurement has not been realized.
　また、非特許文献５－７では、蛍光ナノダイヤモンドの蛍光波長が可視光であり、非特許文献２－４と同様に生体透過性の問題がある。また、電磁波を蛍光ナノダイヤモンドに照射する必要があり、生体深部の特定位置に電磁波を照射することが極めて難しく、現状では生体内の温度計測には利用できない。 Also, in Non-Patent Documents 5-7, the fluorescence wavelength of the fluorescent nanodiamonds is visible light, and there is a problem of bio-permeability as in Non-Patent Documents 2-4. In addition, it is necessary to irradiate fluorescent nanodiamonds with electromagnetic waves, and it is extremely difficult to irradiate electromagnetic waves to a specific position deep inside a living body, and it cannot currently be used to measure the temperature inside a living body.
　そこで本開示の目的は、生体内の細胞温度の計測が可能な温度計測装置および温度計則方法を提供することである。 Therefore, an object of the present disclosure is to provide a temperature measuring device and a thermometry method capable of measuring cell temperature in vivo.
　本開示は、測定対象物の近傍に配置され、結晶欠陥を有したコアとコアを覆うシェルとを有したコアシェル型の量子ドットと、量子ドットに、コアのバンドギャップエネルギーよりも大きなエネルギーの波長である励起光を照射する光照射部と、量子ドットのバンド端発光のピーク波長における光強度と、量子ドットの欠陥発光のピーク波長における光強度とを計測する光検出部と、光検出部により検出した２つの光強度の光強度比を算出する光強度比算出部と、光強度比と量子ドットの周囲の温度との関係を示した検量線を記憶する検量線記憶部と、光強度比算出部により算出した光強度比と、検量線記憶部に記憶された検量線とから、測定対象物の温度を算出する温度算出部と、を有することを特徴とする温度計測装置である。 The present disclosure is a core-shell quantum dot that is placed near an object to be measured and has a core with crystal defects and a shell that covers the core, and the quantum dot has a wavelength of energy greater than the bandgap energy of the core. A light irradiation unit that irradiates excitation light, a light detection unit that measures the light intensity at the peak wavelength of the band edge emission of the quantum dot, and the light intensity at the peak wavelength of the defect emission of the quantum dot, and the light detection unit A light intensity ratio calculation unit that calculates the light intensity ratio of the two detected light intensities, a calibration curve storage unit that stores a calibration curve showing the relationship between the light intensity ratio and the temperature around the quantum dot, and a light intensity ratio A temperature measuring device, comprising: a temperature calculating section for calculating a temperature of a measurement object from the light intensity ratio calculated by the calculating section and the calibration curve stored in the calibration curve storage section.
　本開示によれば、生体内の細胞の温度が計測可能となる。 According to the present disclosure, it is possible to measure the temperature of cells in vivo.
第１実施形態の温度計測装置の構成を示した図。The figure which showed the structure of the temperature measuring device of 1st Embodiment. 量子ドット１のコア１０のバンド図。A band diagram of the core 10 of the quantum dot 1. FIG. 量子ドット１のＴＥＭ像。TEM image of quantum dot 1. 量子ドット１の蛍光スペクトルを測定した結果を示したグラフ。The graph which showed the result of having measured the fluorescence spectrum of the quantum dot 1. FIG. 欠陥発光のスペクトルを示したグラフ。The graph which showed the spectrum of defect emission. 量子ドット１の欠陥発光のスペクトルの温度依存性を示したグラフ。The graph which showed the temperature dependence of the defect luminescence spectrum of the quantum dot 1. FIG. バンド端発光のピーク波長における光強度Ｉ１と、欠陥発光のピーク波長における光強度Ｉ２の温度依存性を示したグラフ。4 is a graph showing the temperature dependence of the light intensity I1 at the peak wavelength of band edge emission and the light intensity I2 at the peak wavelength of defect emission. 水溶液中の量子ドット１について、光強度比Ｉ２／Ｉ１の温度依存性を示したグラフ。The graph which showed the temperature dependence of light intensity ratio I2/I1 about the quantum dot 1 in aqueous solution. ＡＳＣｓ内の量子ドット１について、光強度比Ｉ２／Ｉ１の温度依存性を示したグラフ。The graph which showed the temperature dependence of light intensity ratio I2/I1 about the quantum dot 1 in ASCs. 光強度比Ｉ２／Ｉ１の温度依存性を示したグラフ。The graph which showed the temperature dependence of light intensity ratio I2/I1. 光強度比Ｉ２／Ｉ１の温度依存性を示したグラフ。The graph which showed the temperature dependence of light intensity ratio I2/I1. ３つの状態における光強度比Ｉ２／Ｉ１を、検量線上にプロットしたグラフ。The graph which plotted the light intensity ratio I2/I1 in three states on the calibration curve. 量子ドット１の蛍光スペクトルを測定した結果を示したグラフ。The graph which showed the result of having measured the fluorescence spectrum of the quantum dot 1. FIG.
（温度計測の原理）
　まず、本開示の温度計測原理について説明する。発明者らは、量子ドットを利用して生体内の温度計測ができないか検討した。そして、発明者らはコアシェル型の量子ドットのエネルギー準位構造に注目し、バンド端発光のピークと欠陥発光のピークの光強度比が、量子ドット周囲の環境温度により線形に変化することを発見した。本開示の温度計測装置および温度計則方法は、この光強度比と温度の対応を検量線として利用し、光強度比を測定することで温度を算出するものである。 (Principle of temperature measurement)
First, the temperature measurement principle of the present disclosure will be described. The inventors investigated whether it is possible to measure the temperature inside a living body using quantum dots. Focusing on the energy level structure of core-shell quantum dots, the inventors discovered that the light intensity ratio between the peak of band edge emission and the peak of defect emission changes linearly with the environmental temperature around the quantum dot. did. The temperature measurement device and temperature calculation method of the present disclosure use the correspondence between the light intensity ratio and the temperature as a calibration curve, and calculate the temperature by measuring the light intensity ratio.
（第１実施形態）
　図１は、第１実施形態の温度計測装置の構成を示した図である。図１のように、第１実施形態の温度計測装置は、量子ドット１と、光照射部２と、光検出部３と、光強度比算出部４と、検量線記憶部５と、温度算出部６と、によって構成されている。 (First embodiment)
FIG. 1 is a diagram showing the configuration of the temperature measuring device of the first embodiment. As shown in FIG. 1, the temperature measurement device of the first embodiment includes a quantum dot 1, a light irradiation unit 2, a light detection unit 3, a light intensity ratio calculation unit 4, a calibration curve storage unit 5, and a temperature calculation 6 and .
　量子ドット１は、温度計測のプローブとして使用するものである。量子ドット１を測定対象物の近傍に配置することで、測定対象物の温度を計測する。もちろん、量子ドット１と測定対象物が接していてもよい。量子ドット１は、直径が小さいために量子閉じ込め効果が現れた半導体粒子である。量子ドット１は、コアシェル型の構造のものを用いる。コアシェル型は、球状のコア１０と、コア１０を覆うシェル１１とにより構成されている。シェル１１はコア１０のエネルギー準位を保持し、失活化しないように保護するものである。 The quantum dot 1 is used as a temperature measurement probe. By arranging the quantum dot 1 near the object to be measured, the temperature of the object to be measured is measured. Of course, the quantum dot 1 and the object to be measured may be in contact with each other. A quantum dot 1 is a semiconductor particle that exhibits a quantum confinement effect due to its small diameter. A quantum dot 1 having a core-shell structure is used. The core-shell type is composed of a spherical core 10 and a shell 11 covering the core 10 . The shell 11 maintains the energy level of the core 10 and protects it from deactivation.
　量子ドット１のコア１０は、結晶欠陥を有している。図２は、量子ドット１のコア１０のバンド図を示している。図２のように、結晶欠陥によってバンドギャップ中に結晶欠陥準位が形成される。そのため、励起光（バンドギャップエネルギーよりも大きなエネルギーの光）の照射により生成されたキャリアの一部は再結合してバンド端発光し、一部は結晶欠陥準位にトラップされた後に再結合して欠陥発光する。したがって、量子ドット１のコア１０の蛍光スペクトルは、バンド端発光のピークと欠陥発光のピークを有する。また、欠陥発光は、バンド端発光のピークよりも長波長側となる。欠陥発光のピーク波長は、量子ドット１の周囲環境に応じて変化する場合がある。 The core 10 of the quantum dot 1 has crystal defects. FIG. 2 shows a band diagram of the core 10 of the quantum dot 1. FIG. As shown in FIG. 2, crystal defects form crystal defect levels in the bandgap. Therefore, some of the carriers generated by irradiation with excitation light (light with energy higher than the bandgap energy) recombine and emit band edge light, and some are trapped in crystal defect levels and then recombine. defect emission. Therefore, the fluorescence spectrum of the core 10 of the quantum dot 1 has a band edge emission peak and a defect emission peak. Further, the defect emission is on the longer wavelength side than the peak of the band edge emission. The peak wavelength of defect emission may change depending on the ambient environment of the quantum dot 1 .
　量子ドット１のコア１０の材料は、結晶欠陥が形成される半導体材料であれば任意でよく、ＩＩ－ＶＩ族半導体、ＩＩＩ－Ｖ族半導体、ＩＶ－ＶＩ族半導体、Ｉ－ＶＩ族半導体、Ｉ－ＩＩＩ－Ｖ族半導体、Ｉ－ＩＩ－ＩＶ－ＶＩ族半導体、などを用いることができる。たとえば、ＡｇＩｎＳ２、ＡｇＩｎＳｅ２、ＡｇＩｎＴｅ２、ＡｇＩｎＧａＳ、ＡｇＩｎＺｎＳ２、ＡｇＩｎＴｅ２、ＡｇＩｎＺｎＴｅ２、ＡｇＩｎＧａＳｅ２、ＣｕＩｎＳ２、ＣｕＩｎＳｅ２、ＩｎＰ、ＧａＰ、などを用いることができる。生体の温度計測の場合には、Ｃｄ、Ｐｂなどの強い毒性を有した元素を含まない材料がよい。 The material of the core 10 of the quantum dot 1 may be any semiconductor material in which crystal defects are formed. -III-V group semiconductors, I-II-IV-VI group semiconductors, and the like can be used. For example, AgInS2, AgInSe2 , AgInTe2 , AgInGaS , AgInZnS2 , AgInTe2 , AgInZnTe2 , AgInGaSe2 , CuInS2 , CuInSe2 , InP, GaP, etc. can be used. In the case of measuring the temperature of a living body, it is preferable to use a material that does not contain highly toxic elements such as Cd and Pb.
　量子ドット１のシェル１１の材料は、コア１０を保護できる材料であれば任意であり、ＺｎＳ、ＧａＳ、ＺｎＧａＳ、ＺｎＯ、Ｉｎ２Ｓ３、ＧａＳｘなどを用いることができる。 Any material can be used for the shell 11 of the quantum dot 1 as long as it can protect the core 10, and ZnS , GaS, ZnGaS , ZnO, In2S3, GaSx , and the like can be used.
　量子ドット１のコア１０のバンドギャップエネルギーは、材料の種類やコア１０の直径などによって決まる。よって、それらを変更することによってバンドギャップエネルギーを制御することができ、バンド端発光のピーク波長や欠陥発光のピーク波長を制御することができる。多元系の材料である場合には、組成比によってバンドギャップエネルギーを制御することができる。たとえば、ＡｇＩｎＧａＳを用いる場合、Ａｇ、Ｉｎ、Ｇａの組成比によってバンドギャップエネルギーを制御することができる。 The bandgap energy of the core 10 of the quantum dot 1 is determined by the type of material and the diameter of the core 10. Therefore, by changing them, the bandgap energy can be controlled, and the peak wavelength of band edge emission and the peak wavelength of defect emission can be controlled. In the case of a multicomponent material, the bandgap energy can be controlled by the composition ratio. For example, when using AgInGaS, the bandgap energy can be controlled by the composition ratio of Ag, In, and Ga.
　バンド端発光のピーク波長と欠陥発光のピーク波長の差は１００ｎｍ以上が好ましい。１００ｎｍ以上であれば、２つのピークを十分に分離して検出することができ、光強度比Ｉ２／Ｉ１を精度よく測定することが可能となる。 The difference between the peak wavelength of band edge emission and the peak wavelength of defect emission is preferably 100 nm or more. If it is 100 nm or more, two peaks can be sufficiently separated and detected, and the light intensity ratio I2/I1 can be accurately measured.
　量子ドット１のコア１０の直径は、量子閉じ込め効果が現れる範囲であれば任意であり、たとえば１００ｎｍ以下である。ここで直径は量子ドット１の外接球の直径とする。好ましくは５０ｎｍ以下、さらに好ましくは２０ｎｍ以下である。直径の下限は特に規定しないが、たとえば１ｎｍ以上である。また、量子ドット１のシェル１１の厚さは、コア１０を保護できる範囲であれば任意である。シェル１１は１重である必要はなく、２重以上としてもよい。 The diameter of the core 10 of the quantum dot 1 is arbitrary as long as the quantum confinement effect appears, and is, for example, 100 nm or less. Here, the diameter is the diameter of the sphere circumscribing the quantum dot 1 . It is preferably 50 nm or less, more preferably 20 nm or less. Although the lower limit of the diameter is not specified, it is, for example, 1 nm or more. Moreover, the thickness of the shell 11 of the quantum dot 1 is arbitrary as long as it can protect the core 10 . The shell 11 does not need to be single, and may be double or more.
　量子ドット１は、表面には各種物質を結合させてもよい。たとえば、ヒドロキシ基、アミノ基、カルボキシ基、スルホ基、それらの塩などの親水基を結合させてもよい。これにより、量子ドット１を水溶性とすることができ、生体の温度計測の利便性を高めることができる。また、量子ドット１の表面に膜透過性ペプチドやカチオン性リポソームを結合させてもよい。これにより、細胞に量子ドット１を取り込ませることができ、細胞内の温度を計測可能となる。膜透過性ペプチドは、たとえば、オクタアルギニン（Ｒ８）、ＴＡＴペプチド、などである。 The quantum dot 1 may have various substances bonded to its surface. For example, a hydrophilic group such as a hydroxy group, an amino group, a carboxy group, a sulfo group, or salts thereof may be bound. Thereby, the quantum dot 1 can be made water-soluble, and the convenience of measuring the temperature of the living body can be enhanced. Alternatively, a membrane-permeable peptide or cationic liposome may be bound to the surface of the quantum dot 1 . Thereby, the quantum dot 1 can be incorporated into the cell, and the temperature inside the cell can be measured. Membrane permeable peptides are, for example, octaarginine (R8), TAT peptide, and the like.
　光照射部２は、量子ドット１に対して励起光を照射する装置である。照射する励起光の波長は、量子ドット１のコア１０のバンドギャップエネルギーよりも大きなエネルギーの波長であれば任意の波長でよい。たとえば、ＬＥＤ、ＬＤ、キセノンランプ、などを用いることができる。生体の温度計測を行う場合には、生体透過性の高い赤外線を励起光として用いるとよい。特に、ヘモグロビンと水の双方の透過率が高い波長７００～１５００ｎｍの範囲が好ましい。たとえば、励起光の波長は７００～１５００ｎｍとし、量子ドット１のバンド端発光のピーク波長が励起光の波長よりも長波長となるように、量子ドット１の直径や材料の組成比を調整する。
　励起光として赤外線を使用可能な量子ドット１の材料の一例は、ＡｇＩｎＧａＳｅ２である。図１３は、論文（Tatsuya Kameyama, et al., ACS Appl. Nano Mater. 2020, 3, 3275-3287）からの引用であり、ＡｇｘＩｎｙＧａ（１－ｙ）Ｓｅ２からなる量子ドットの蛍光スペクトルを示したグラフである。ここで、Ａｇのモル比ｘは０．６７に固定し、ＩｎとＧａの合計に対するＩｎのモル比ｙを０．２５から１まで変化させている。図１３のように、ｙが増加するにつれてバンド端発光のピークが長波長側にシフトしており、ｙが０．６以上でバンド端発光のピークは７４０ｎｍ以上となっていることがわかる。欠陥発光のピークは、バンド端発光のピークよりも長波長側に見られ、ｙが０．２５～０．５ではそのピークが明瞭に見られる。ｙが０．６以上では、欠陥発光のピークは明瞭ではないが存在している。したがって、ｙが０．６以上であれば、近赤外線でバンド端発光と欠陥発光を励起可能であることがわかる。たとえば、ｙ＝０．６のとき、バンド端発光のピークは７４０ｎｍ、欠陥発光のピークは８４０ｎｍとなり、波長７００ｎｍ以上７４０ｎｍ未満の励起光で励起可能であることがわかる。このように、量子ドット１の材料や組成比を調整することで赤外線（特に波長７００～１５００ｎｍ）を励起光として使用可能となる。 The light irradiation unit 2 is a device that irradiates the quantum dots 1 with excitation light. The wavelength of the excitation light to be irradiated may be any wavelength as long as the wavelength has energy greater than the bandgap energy of the core 10 of the quantum dot 1 . For example, LEDs, LDs, xenon lamps, etc. can be used. When measuring the temperature of a living body, it is preferable to use infrared light, which is highly permeable to the living body, as the excitation light. In particular, the wavelength range of 700 to 1500 nm, which has high transmittance for both hemoglobin and water, is preferred. For example, the excitation light has a wavelength of 700 to 1500 nm, and the diameter and material composition ratio of the quantum dots 1 are adjusted so that the peak wavelength of the band edge emission of the quantum dots 1 is longer than the wavelength of the excitation light.
An example of a quantum dot 1 material that can use infrared light as excitation light is AgInGaSe 2 . FIG. 13 is a quote from a paper (Tatsuya Kameyama, et al., ACS Appl. Nano Mater. 2020, 3, 3275-3287), which describes quantum dots made of Ag x In y Ga (1-y) Se 2 It is the graph which showed the fluorescence spectrum. Here, the molar ratio x of Ag is fixed at 0.67, and the molar ratio y of In to the sum of In and Ga is varied from 0.25 to 1. As shown in FIG. 13, as y increases, the peak of band edge emission shifts to the longer wavelength side, and when y is 0.6 or more, the peak of band edge emission is 740 nm or more. The defect luminescence peak is seen on the longer wavelength side than the band edge luminescence peak, and the peak is clearly seen when y is 0.25 to 0.5. When y is 0.6 or more, the peak of defect emission is not clear but is present. Therefore, when y is 0.6 or more, it is possible to excite band edge emission and defect emission with near-infrared rays. For example, when y=0.6, the peak of band edge emission is 740 nm and the peak of defect emission is 840 nm. By adjusting the material and composition ratio of the quantum dots 1 in this manner, infrared rays (especially wavelengths of 700 to 1500 nm) can be used as excitation light.
　光検出部３は、量子ドット１からのバンド端発光および欠陥発光を検出し、バンド端発光のピーク波長における光強度Ｉ１と、欠陥発光のピーク波長における光強度Ｉ２を測定する装置である。たとえば、量子ドット１からの蛍光スペクトルを測定し、バンド端発光のピークと欠陥発光のピークを抽出することで光強度Ｉ１、Ｉ２を測定する。蛍光スペクトルは、分光器により蛍光波長を走査し、光電子増倍管やフォトダイオードにより光強度を測定することで得られる。光照射部２および光検出部３として、蛍光強度計を用いることができる。また、蛍光イメージング装置を用いて蛍光画像を取得し、その蛍光画像を解析することにより光強度Ｉ１、Ｉ２を測定してもよい。なお、欠陥発光のピークが複数存在する場合には、それらのうち任意の１つをＩ２としてもよいし、任意の２以上の平均を取ってもよい。 The photodetector 3 is a device that detects band edge luminescence and defect luminescence from the quantum dot 1 and measures the light intensity I1 at the peak wavelength of the band edge luminescence and the light intensity I2 at the peak wavelength of the defect luminescence. For example, the fluorescence spectrum from the quantum dot 1 is measured, and the light intensities I1 and I2 are measured by extracting the band edge emission peak and the defect emission peak. A fluorescence spectrum is obtained by scanning fluorescence wavelengths with a spectroscope and measuring light intensity with a photomultiplier tube or photodiode. A fluorescence intensity meter can be used as the light irradiation unit 2 and the light detection unit 3 . Alternatively, the light intensities I1 and I2 may be measured by obtaining a fluorescence image using a fluorescence imaging device and analyzing the fluorescence image. If there are a plurality of defect luminescence peaks, any one of them may be set as I2, or any two or more peaks may be averaged.
　光強度Ｉ１、Ｉ２は、精度の点でピーク位置での光強度が好ましいが、多少ずれた位置での光強度であっても許容される。たとえば、ピークから２０ｎｍ以内のずれであれば十分な精度で温度計測ができる。また、光強度Ｉ１、Ｉ２は、ピークを含む所定の波長幅の光強度の平均であってもよい。所定の波長幅は、好ましくは３０ｎｍ以下、より好ましくは２０ｎｍ以下である。 For the light intensities I1 and I2, the light intensity at the peak position is preferable in terms of accuracy, but the light intensity at a slightly deviated position is acceptable. For example, if the deviation from the peak is within 20 nm, the temperature can be measured with sufficient accuracy. Further, the light intensities I1 and I2 may be averages of light intensities in a predetermined wavelength width including peaks. The predetermined wavelength width is preferably 30 nm or less, more preferably 20 nm or less.
　光強度比算出部４は、光検出部３によって測定されたバンド端発光のピーク波長における光強度Ｉ１と、欠陥発光のピーク波長における光強度Ｉ２から、その光強度比Ｉ２／Ｉ１を算出する装置である。光強度比算出部４は、コンピュータによって実現される。 The light intensity ratio calculator 4 calculates the light intensity ratio I2/I1 from the light intensity I1 at the peak wavelength of the band edge emission and the light intensity I2 at the peak wavelength of the defect emission measured by the photodetector 3. is. The light intensity ratio calculator 4 is implemented by a computer.
　検量線記憶部５は、光強度比Ｉ２／Ｉ１と、量子ドット１周囲の温度との対応を示す検量線を記憶する装置である。検量線は量子ドット１の周囲環境ごとに複数記憶されている。光強度比Ｉ２／Ｉ１と温度は、１対１に対応し、およそ直線的に対応し、その直線は負の傾きを有する。つまり、温度をＴ（℃）として、Ｔ＝－ａ（Ｉ２／Ｉ１）＋ｂ、ここでａ、ｂは正の実数、で表される。ａ、ｂは、量子ドット１の周囲環境によって決まる。より具体的には、光照射部２から量子ドット１までの経路と、量子ドット１から光検出部３までの経路における物質により決まる。そのため、量子ドット１の周囲環境に応じてａ、ｂを実験的に決めて、検量線記憶部５に記憶しておく。なお、光強度比Ｉ２／Ｉ１と温度の関係を直線で近似せずに、そのまま対応関係を記憶してもよいし、他の関数によって近似してもよい。 The calibration curve storage unit 5 is a device that stores a calibration curve showing the correspondence between the light intensity ratio I2/I1 and the temperature around the quantum dots 1. A plurality of calibration curves are stored for each ambient environment of the quantum dot 1 . The light intensity ratio I2/I1 and temperature have a one-to-one correspondence and approximately linear correspondence, and the straight line has a negative slope. That is, where temperature is T (° C.), T=−a(I2/I1)+b, where a and b are positive real numbers. a and b are determined by the ambient environment of the quantum dot 1 . More specifically, it is determined by the substances in the route from the light irradiation unit 2 to the quantum dot 1 and the route from the quantum dot 1 to the light detection unit 3 . Therefore, a and b are experimentally determined according to the ambient environment of the quantum dot 1 and stored in the calibration curve storage unit 5 . Instead of approximating the relationship between the light intensity ratio I2/I1 and the temperature by a straight line, the correspondence may be stored as it is, or may be approximated by another function.
　温度算出部６は、光強度比算出部４によって算出された光強度比Ｉ２／Ｉ１と、検量線記憶部５に記憶された検量線とから、量子ドット１の周囲の温度を算出する装置である。温度算出部６は、コンピュータによって実現される。 The temperature calculation unit 6 is a device that calculates the temperature around the quantum dots 1 from the light intensity ratio I2/I1 calculated by the light intensity ratio calculation unit 4 and the calibration curve stored in the calibration curve storage unit 5. be. The temperature calculator 6 is implemented by a computer.
　次に、第１実施形態の温度計測装置による温度の計測について説明する。 Next, temperature measurement by the temperature measuring device of the first embodiment will be described.
　まず、量子ドット１を測定対象物の近傍に配置する。配置の方法は任意である。生体の特定部位の温度計測を行う場合、量子ドット１の表面に親水基を結合させ、水に量子ドット１を溶解して水溶液とするとよい。水溶液の注射などによって容易に生体の特定部位に量子ドット１を配置することができる。また、生体内部の温度計測を行う場合、励起光の波長は生体透過性の高い赤外線を用い、たとえば波長７００～１５００ｎｍである。したがって、量子ドット１はバンドギャップエネルギーがそれよりも小さなものを用いる。また、細胞内の温度計測を行う場合、量子ドット１表面に膜透過性ペプチドを結合させ、量子ドット１を細胞内に取り込ませるとよい。 First, the quantum dot 1 is arranged near the object to be measured. The arrangement method is arbitrary. When measuring the temperature of a specific part of a living body, it is preferable to bind a hydrophilic group to the surface of the quantum dot 1 and dissolve the quantum dot 1 in water to obtain an aqueous solution. Quantum dots 1 can be easily arranged at a specific site of a living body by injecting an aqueous solution or the like. Further, when measuring the temperature inside a living body, the wavelength of the excitation light is infrared light, which is highly penetrable to the living body, and has a wavelength of, for example, 700 to 1500 nm. Therefore, a quantum dot 1 with a smaller bandgap energy is used. Moreover, when performing temperature measurement in a cell, it is preferable to bind a membrane-permeable peptide to the surface of the quantum dot 1 and incorporate the quantum dot 1 into the cell.
　次に、光照射部２によって量子ドット１に励起光を照射する。そして、光検出部３によって量子ドット１からの光を検出し、量子ドット１のバンド端発光のピーク波長における光強度Ｉ１と、欠陥発光のピーク波長における光強度Ｉ２を測定する。励起光が赤外線であれば、量子ドット１が生体内部であっても、励起光は生体を透過して量子ドット１に十分な強度で到達する。また、量子ドット１からの蛍光も生体を透過して光検出部３に十分な強度で到達する。 Next, the quantum dot 1 is irradiated with excitation light by the light irradiation unit 2 . Then, the light from the quantum dot 1 is detected by the photodetector 3, and the light intensity I1 at the peak wavelength of the band edge emission of the quantum dot 1 and the light intensity I2 at the peak wavelength of the defect emission of the quantum dot 1 are measured. If the excitation light is infrared rays, the excitation light passes through the living body and reaches the quantum dots 1 with sufficient intensity even if the quantum dots 1 are inside the living body. Fluorescence from the quantum dots 1 also passes through the living body and reaches the photodetector 3 with sufficient intensity.
　次に、光強度比算出部４によって、それらの光強度比Ｉ２／Ｉ１を算出する。 Next, the light intensity ratio calculator 4 calculates the light intensity ratio I2/I1.
　次に、温度算出部６によって、光強度比算出部４で算出した光強度比Ｉ２／Ｉ１と、検量線記憶部５に記憶された検量線とから、量子ドット１の周囲の温度を算出する。量子ドット１は測定対象物の近傍に配置されているので、量子ドット１の周囲の温度は、測定対象物の温度と一致する。ここで、検量線は、量子ドット１の周囲環境に応じたものを選択する。検量線の選択は、手動で行ってもよいし、自動化してもよい。 Next, the temperature calculation unit 6 calculates the temperature around the quantum dot 1 from the light intensity ratio I2/I1 calculated by the light intensity ratio calculation unit 4 and the calibration curve stored in the calibration curve storage unit 5. . Since the quantum dot 1 is arranged near the object to be measured, the temperature around the quantum dot 1 matches the temperature of the object to be measured. Here, the calibration curve is selected according to the ambient environment of the quantum dots 1 . Selection of the standard curve may be performed manually or may be automated.
　以上、第１実施形態の温度計測装置によれば、量子ドット１周囲の測定対象物の温度を計測することができる。特に、第１の実施形態の温度計測装置では、生体内部の特定部位の温度計測が可能であり、細胞の内部あるいはその周辺に量子ドット１を配置することで細胞の温度計測が可能である。たとえば、オルガノイド内部温度を計測可能である。また、第１実施形態の温度計測装置によれば、温度を高精度に計測可能であり、誤差０．１Ｋ以下の精度で計測することができる。 As described above, according to the temperature measuring device of the first embodiment, the temperature of the object to be measured around the quantum dot 1 can be measured. In particular, the temperature measurement device of the first embodiment can measure the temperature of a specific site inside the living body, and the temperature of the cell can be measured by arranging the quantum dots 1 inside or around the cell. For example, the organoid internal temperature can be measured. Moreover, according to the temperature measuring device of the first embodiment, the temperature can be measured with high accuracy, and the temperature can be measured with an error of 0.1K or less.
　なお、第１実施形態では光強度比Ｉ２／Ｉ１を算出しているが、光強度比Ｉ１／Ｉ２を算出し、光強度比Ｉ１／Ｉ２と温度との対応を示す検量線から温度を算出してもよい。 Although the light intensity ratio I2/I1 is calculated in the first embodiment, the light intensity ratio I1/I2 is calculated, and the temperature is calculated from a calibration curve showing the correspondence between the light intensity ratio I1/I2 and temperature. may
　また、量子ドット１は、生体試料標識用の蛍光プローブを兼ねることができる。したがって、第１実施形態の温度計測装置は、生体の特定部位を量子ドット１の蛍光によって可視化しつつ、その特定部位の温度を計測することができる。 In addition, the quantum dot 1 can also serve as a fluorescent probe for labeling biological samples. Therefore, the temperature measuring device of the first embodiment can measure the temperature of the specific site while visualizing the specific site of the living body by the fluorescence of the quantum dots 1 .
　以下、本開示の具体的な実施例について図を参照に説明するが、実施例に限定されるものではない。 Specific examples of the present disclosure will be described below with reference to the drawings, but are not limited to the examples.
　コアをＡｇＩｎＧａＳとし、シェルをＺｎＧａＳとするコアシェル型の量子ドット１を次の方法により作製した。 A core-shell quantum dot 1 having a core of AgInGaS and a shell of ZnGaS was produced by the following method.
　まず、金属源として酢酸銀（Ａｇ（ＯＡｃ））（１３ｍｇ）、インジウムアセチルアセトナ－ト（Ｉｎ（ａｃａｃ）３）（３１　ｍｇ）、ガリウムアセチルアセトナート（Ｇａ（ａｃａｃ）３）（１８　ｍｇ）、硫黄源として粉末硫黄（７．４　ｍｇ）をオレイルアミン（ＯＬＡ、１時間１００℃で減圧乾燥することで脱水したもの）（２．８　ｍＬ）と１－ドデカンチオール（ＤＤＴ）（０．２５　ｍＬ）の混合液に分散させ、窒素雰囲気下で３００℃、１０分間加熱した。８分放冷した後、遠心分離して得られた上澄み溶液を回収し、さらにメンブレンフィルター（孔径０．２０　μｍ）に通して凝集した粒子を除去した。この溶液に、メタノール（３．０　ｍＬ）を加えて遠心分離し、沈殿を得た。得られた沈殿物にエタノール（３．０　ｍＬ）を加えて懸濁させた後、再度、遠心分離することで沈殿を洗浄した。このようにして、コアとなるＡｇＩｎＧａＳ量子ドット（平均粒径：４．３　ｎｍ）を沈殿物として得た。 First, silver acetate (Ag(OAc)) (13 mg), indium acetylacetonate (In(acac) 3 ) (31 mg), and gallium acetylacetonate (Ga(acac) 3 ) (18 mg) as metal sources. , powdered sulfur (7.4 mg) as a sulfur source, oleylamine (OLA, dehydrated by vacuum drying at 100° C. for 1 hour) (2.8 mL) and 1-dodecanethiol (DDT) (0.25 mL) ) and heated at 300° C. for 10 minutes in a nitrogen atmosphere. After standing to cool for 8 minutes, the supernatant solution obtained by centrifugation was collected and passed through a membrane filter (pore size 0.20 μm) to remove aggregated particles. Methanol (3.0 mL) was added to this solution and centrifuged to obtain a precipitate. Ethanol (3.0 mL) was added to the resulting precipitate to suspend it, and the precipitate was washed by centrifugation again. Thus, AgInGaS quantum dots (average particle size: 4.3 nm) serving as cores were obtained as precipitates.
　次に、上記ＡｇＩｎＧａＳ量子ドットをコアとして、以下の方法によりシェル被覆を行った。得られたＡｇＩｎＧａＳ量子ドット（１．０×１０－５　ｍｍｏｌ（粒子））、金属源として、Ｇａ（ａｃａｃ）３（５９　ｍｇ）、ステアリン酸亜鉛（Ｚｎ（Ｃ１７Ｈ３５ＣＯＯ）２）（２００　ｍｇ）、硫黄源としてチオ尿素（４３　ｍｇ）をＯＬＡ（３．０　ｍＬ）に分散させ、窒素雰囲気下１５０℃で１０分間加熱したのち、すぐに２５０℃で３０分間加熱した。４５分間放冷した後、遠心分離によって溶液中に生じた沈殿を回収した。この沈殿にメタノール（３．０　ｍＬ）を加えて懸濁させたのち遠心分離することで沈殿を洗浄した。 Next, using the AgInGaS quantum dots as cores, shell coating was performed by the following method. The resulting AgInGaS quantum dots (1.0×10 −5 mmol (particles)), Ga(acac) 3 (59 mg), zinc stearate (Zn(C 17 H 35 COO) 2 ) (200 mg), and thiourea (43 mg) as a sulfur source was dispersed in OLA (3.0 mL) and heated at 150° C. for 10 minutes under a nitrogen atmosphere, followed immediately at 250° C. for 30 minutes. After standing to cool for 45 minutes, the precipitate generated in the solution was collected by centrifugation. Methanol (3.0 mL) was added to this precipitate to suspend it, and then the precipitate was washed by centrifugation.
　得られた沈殿は、目的とする量子ドットを含むが、さらに各粒子のシェル膜厚を増大させるために、再度被覆操作を行った。１度目のシェル被覆操作で得た沈殿粒子（１．０×１０－５　ｍｍｏｌ（粒子））とともに、金属源として、Ｚｎ（Ｃ１７Ｈ３５ＣＯＯ）２（１００　ｍｇ）、硫黄源としてチオ尿素（１２　ｍｇ）をＯＬＡ（３．０　ｍＬ）に加えて分散させ、窒素雰囲気下、１５０℃で１０分間加熱したのち、すぐに２５０℃で３０分間加熱した。４５分間放冷した後、遠心分離によって溶液中に生じた沈殿を回収した。この沈殿にメタノール（３．０　ｍＬ）を加えて懸濁させたのち遠心分離し、沈殿を回収した。得られた沈殿物にエタノール（３．０　ｍＬ）を加えて、再度懸濁させたのち遠心分離することで、沈殿を洗浄した。このようにして、コアをＡｇＩｎＧａＳとし、シェルをＺｎＧａＳとするコアシェル型の量子ドット１（平均粒径：５．４　ｎｍ）を得た。 The resulting precipitate contained the desired quantum dots, but was subjected to another coating operation to further increase the shell thickness of each particle. Zn(C 17 H 35 COO) 2 (100 mg) as metal source, thiourea ( 12 mg) was added and dispersed in OLA (3.0 mL), heated at 150° C. for 10 minutes under a nitrogen atmosphere, and then immediately heated at 250° C. for 30 minutes. After standing to cool for 45 minutes, the precipitate generated in the solution was collected by centrifugation. Methanol (3.0 mL) was added to this precipitate to suspend it, followed by centrifugation to collect the precipitate. The precipitate was washed by adding ethanol (3.0 mL) to the resulting precipitate, suspending it again, and then centrifuging. Thus, a core-shell type quantum dot 1 (average particle size: 5.4 nm) having a core of AgInGaS and a shell of ZnGaS was obtained.
　次に、得られた量子ドット１をクロロホルム（３．０　ｍＬ）に溶解し、表面がＯＬＡで修飾された量子ドット１のクロロホルム溶液を得た。そして、３－メルカプトプロピオン酸（ＭＰＡ）（０．１０　ｍＬ）、テトラメチルアンモニウムヒドロキシド（０．７３　ｍＬ）、エタノール（０．２７　ｍＬ）をこの溶液（１．０　ｍＬ）に混合し、窒素雰囲気下で７０℃、３時間加熱した。これにより、量子ドット１表面のＯＬＡをＭＰＡで置換した。以上により、表面がＭＰＡで修飾された量子ドット１を得た。ＭＰＡは親水基であるカルボキシ基を有するため、量子ドット１の表面をＭＰＡで修飾したことにより、量子ドット１を水に溶解させることが可能となった。 Next, the obtained quantum dots 1 were dissolved in chloroform (3.0 mL) to obtain a chloroform solution of quantum dots 1 whose surfaces were modified with OLA. Then, 3-mercaptopropionic acid (MPA) (0.10 mL), tetramethylammonium hydroxide (0.73 mL), and ethanol (0.27 mL) were mixed with this solution (1.0 mL), and nitrogen It was heated at 70° C. for 3 hours in an atmosphere. As a result, the OLA on the surface of the quantum dot 1 was replaced with MPA. As described above, quantum dots 1 whose surface was modified with MPA were obtained. Since MPA has a carboxyl group, which is a hydrophilic group, the quantum dots 1 can be dissolved in water by modifying the surface of the quantum dots 1 with MPA.
　図３は、量子ドット１のＴＥＭ像である。図３（ａ）は、クロロホルム溶液中の量子ドット１（表面がＯＬＡで修飾されたもの）、図３（ｂ）は、水溶液中の量子ドット１（表面がＭＰＡで修飾されたもの）である。図３のように、粒径が５．４ｎｍの量子ドット１が得られていることがわかった。 FIG. 3 is a TEM image of the quantum dot 1. FIG. 3(a) shows quantum dots 1 in chloroform solution (surface modified with OLA), and FIG. 3(b) shows quantum dots 1 in aqueous solution (surface modified with MPA). . As shown in FIG. 3, it was found that quantum dots 1 having a particle size of 5.4 nm were obtained.
　図４は、量子ドット１（ただしシェルで覆う前の段階）の蛍光スペクトルを測定した結果を示したグラフである。励起光は３６５ｎｍとした。図４のように、蛍光スペクトルは２つのピークを有し、５５０ｎｍ付近にバンド端発光のピーク、７００ｎｍ付近に欠陥発光のピークを有していることがわかった。 FIG. 4 is a graph showing the results of measuring the fluorescence spectrum of quantum dot 1 (before being covered with a shell). The excitation light was 365 nm. As shown in FIG. 4, it was found that the fluorescence spectrum has two peaks, a band edge emission peak near 550 nm and a defect emission peak near 700 nm.
　図５は、欠陥発光のスペクトルを示したグラフであり、クロロホルム溶液中の量子ドット１と水溶液中の量子ドット１とで比較したものである。励起光は３６５ｎｍとした。図５のように、水溶液中の場合は、クロロホルム溶液中の場合に比べて欠陥発光のピーク波長が長波長側にシフトすることがわかった。このことから、検量線は量子ドット１の周囲環境に応じて設定する必要があることがわかった。 FIG. 5 is a graph showing the spectrum of defect emission, comparing quantum dot 1 in a chloroform solution and quantum dot 1 in an aqueous solution. The excitation light was 365 nm. As shown in FIG. 5, it was found that the peak wavelength of defect luminescence shifted to longer wavelengths in the aqueous solution than in the chloroform solution. From this, it was found that the calibration curve should be set according to the ambient environment of the quantum dots 1 .
　図６は、水溶液中の量子ドット１の欠陥発光のスペクトルについて、温度による違いを比較したグラフである。励起光は３６５ｎｍとした。温度は５℃から４５℃まで１０℃刻みで変化させた。温度はヒータを用いて変化させ、温度測定は熱電対を水溶液に接触させて行った。図６のように、温度が高くなるほど欠陥発光の光強度は低下し、ピーク波長が少し短波長側にシフトすることがわかった。 FIG. 6 is a graph comparing the difference in defect luminescence spectra of quantum dots 1 in an aqueous solution depending on temperature. The excitation light was 365 nm. The temperature was varied from 5°C to 45°C in 10°C increments. The temperature was changed using a heater, and the temperature was measured by bringing a thermocouple into contact with the aqueous solution. As shown in FIG. 6, it was found that the higher the temperature, the lower the light intensity of the defect emission, and the peak wavelength slightly shifted to the shorter wavelength side.
　図７は、水溶液中の量子ドット１について、バンド端発光のピーク波長における光強度Ｉ１と、欠陥発光のピーク波長における光強度Ｉ２の温度依存性を示したグラフである。いずれの光強度も温度変化に対して直線的に減少することがわかり、その減少の傾きに違いがあることがわかった。 FIG. 7 is a graph showing the temperature dependence of the light intensity I1 at the peak wavelength of band edge emission and the light intensity I2 at the peak wavelength of defect emission for quantum dots 1 in an aqueous solution. It was found that both light intensities decreased linearly with temperature change, and that there was a difference in the slope of the decrease.
　図８は、水溶液中の量子ドット１について、バンド端発光のピーク波長における光強度Ｉ１に対する欠陥発光のピーク波長における光強度Ｉ２の比Ｉ２／Ｉ１の温度依存性を示したグラフである。励起光の波長や温度測定の方法は図７と同様である。図８のように、光強度比Ｉ２／Ｉ１と温度は１対１に対応していることがわかった。また、その対応関係は直線で精度よく近似できることがわかった。したがって、光強度比Ｉ２／Ｉ１と温度との対応関係を検量線として用いることで、少なくとも５～４５℃の範囲で光強度比Ｉ２／Ｉ１から温度を精度よく測定できることがわかった。 FIG. 8 is a graph showing the temperature dependence of the ratio I2/I1 of the light intensity I2 at the peak wavelength of defect emission to the light intensity I1 at the peak wavelength of band edge emission for quantum dots 1 in an aqueous solution. The wavelength of excitation light and the method of measuring temperature are the same as in FIG. As shown in FIG. 8, it was found that the light intensity ratio I2/I1 and the temperature have a one-to-one correspondence. In addition, it was found that the corresponding relationship can be approximated by a straight line with high accuracy. Therefore, by using the corresponding relationship between the light intensity ratio I2/I1 and the temperature as a calibration curve, it was found that the temperature can be accurately measured from the light intensity ratio I2/I1 at least in the range of 5 to 45°C.
　実施例１により作製した表面がＭＰＡで修飾された量子ドット１を水溶液に溶解させ、その水溶液にオクタアルギニン（Ｒ８）（２　ｍＭ）を混合し、室温で１５分間静置した。これにより、表面がＲ８で修飾された量子ドット１を作製した。Ｒ８は膜透過性ペプチドである。 The surface-modified quantum dots 1 prepared in Example 1 with MPA were dissolved in an aqueous solution, octaarginine (R8) (2 mM) was mixed with the aqueous solution, and the mixture was allowed to stand at room temperature for 15 minutes. As a result, a quantum dot 1 whose surface was modified with R8 was produced. R8 is a membrane permeable peptide.
　次に、表面がＲ８で修飾された量子ドット１を溶解させ、その水溶液をマウス脂肪組織由来幹細胞（ＡＳＣｓ）の培養液に混合し、ＡＳＣｓ内に量子ドット１を取り込ませた。そして、量子ドット１に波長３６５ｎｍの励起光を照射して蛍光スペクトルを測定した。蛍光スペクトルは、培養液の温度をヒータによって変化させて複数回測定した。 Next, the quantum dots 1 whose surface was modified with R8 were dissolved, and the aqueous solution was mixed with the culture medium of mouse adipose tissue-derived stem cells (ASCs) to incorporate the quantum dots 1 into the ASCs. Then, the quantum dot 1 was irradiated with excitation light having a wavelength of 365 nm, and the fluorescence spectrum was measured. The fluorescence spectrum was measured multiple times while changing the temperature of the culture solution with a heater.
　図９は、ＡＳＣｓ内の量子ドット１について、バンド端発光のピーク波長における光強度Ｉ１に対する欠陥発光のピーク波長における光強度Ｉ２の比Ｉ２／Ｉ１の温度依存性を示したグラフである。図９のように、光強度比Ｉ２／Ｉ１と温度は１対１に対応していることがわかった。また、その対応関係は直線で精度よく近似できることがわかった。したがって、量子ドット１を用いることで細胞内の温度も計測可能であることがわかった。 FIG. 9 is a graph showing the temperature dependence of the ratio I2/I1 of the light intensity I2 at the peak wavelength of defect emission to the light intensity I1 at the peak wavelength of band edge emission for quantum dots 1 in ASCs. As shown in FIG. 9, it was found that the light intensity ratio I2/I1 and the temperature have a one-to-one correspondence. In addition, it was found that the corresponding relationship can be approximated by a straight line with high accuracy. Therefore, it was found that the intracellular temperature can also be measured by using the quantum dot 1.
　実施例１により作製した表面がＭＰＡで修飾された量子ドット１を水溶液に溶解させ、その水溶液５０μＬを容器に入れてヒータ上に配置し、容器の蛍光画像を生体イメージング装置（パーキンエルマー社製、IVIS Lumina K Series III）を用いて取得した。水溶液はヒータにより加熱し、温度を変えて蛍光画像を取得した。励起光のバンドパスフィルタは４２０～４４０ｎｍとした。また、バンド端発光のピーク波長における光強度Ｉ１を、波長５７０～５９０ｎｍにおける容器の蛍光画像から測定した。また、欠陥発光のピーク波長における光強度Ｉ２を、波長６９０～７１０ｎｍにおける容器の蛍光画像から取得した。 Quantum dots 1 whose surface is modified with MPA prepared in Example 1 are dissolved in an aqueous solution, 50 μL of the aqueous solution is placed in a container and placed on a heater, and a fluorescent image of the container is captured by a bioimaging device (manufactured by PerkinElmer, Inc.). Acquired using IVIS Lumina K Series III). The aqueous solution was heated by a heater, and fluorescence images were acquired while changing the temperature. A band-pass filter for excitation light was set to 420 to 440 nm. Also, the light intensity I1 at the peak wavelength of the band edge emission was measured from the fluorescence image of the container at wavelengths of 570 to 590 nm. In addition, the light intensity I2 at the peak wavelength of defect emission was obtained from the fluorescence image of the container at wavelengths of 690 to 710 nm.
　図１０は、光強度比Ｉ２／Ｉ１の温度依存性を示したグラフである。図１０のように、光強度比Ｉ２／Ｉ１と温度は１対１に対応し、その対応関係は直線で精度よく近似できることがわかった。したがって、生体イメージング装置を用いて温度計測が可能であることがわかった。 FIG. 10 is a graph showing the temperature dependence of the light intensity ratio I2/I1. As shown in FIG. 10, it has been found that the light intensity ratio I2/I1 and the temperature have a one-to-one correspondence, and the correspondence relationship can be approximated by a straight line with good accuracy. Therefore, it turned out that temperature measurement is possible using a biological imaging device.
　実施例１により作製した表面がＭＰＡで修飾された量子ドット１を水溶液に溶解させ、その水溶液５０μＬをＢＡＬＢ／ｃヌードマウスに皮下注射した。そして、実施例３と同様に、生体イメージング装置を用いてマウスのうち水溶液が皮下注射された領域の蛍光画像を取得し、光強度比Ｉ２／Ｉ１を測定した。励起光のバンドパスフィルタは４８５～５１５ｎｍとし、それ以外の条件は実施例３と同様とした。 Quantum dots 1 whose surfaces were modified with MPA prepared in Example 1 were dissolved in an aqueous solution, and 50 μL of the aqueous solution was subcutaneously injected into BALB/c nude mice. Then, in the same manner as in Example 3, a fluorescence image of the area of the mouse subcutaneously injected with the aqueous solution was acquired using a bioimaging device, and the light intensity ratio I2/I1 was measured. The band-pass filter for excitation light was set to 485 to 515 nm, and other conditions were the same as in Example 3.
　図１１は、光強度比Ｉ２／Ｉ１の温度依存性を示したグラフである。図１１のように、光強度比Ｉ２／Ｉ１と、マウスのうち水溶液が皮下注射された領域の温度は１対１に対応し、その対応関係は直線で精度よく近似できることがわかった。したがって、生体イメージング装置を用いて生体の特定部位の温度計測が可能であることがわかった。 FIG. 11 is a graph showing the temperature dependence of the light intensity ratio I2/I1. As shown in FIG. 11, it was found that the light intensity ratio I2/I1 corresponds one to one with the temperature of the region of the mouse subcutaneously injected with the aqueous solution, and the correspondence relationship can be approximated by a straight line with good accuracy. Therefore, it was found that temperature measurement of a specific part of a living body is possible using a living body imaging device.
　実施例１により作製した表面がＭＰＡで修飾された量子ドット１を水溶液に溶解させ、その水溶液５０μＬをＢＡＬＢ／ｃヌードマウスに皮下注射した。そして、そのマウスを冷却状態、通常状態、加熱状態の３つの状態にし、赤外線サーモグラフィカメラ（フリアー社製、FLIR C2 ）により撮影した。その結果、マウスのうち水溶液が皮下注射された領域の温度は、冷却状態で２１．３℃、通常状態で３２．３℃、加熱状態で３６．７℃であった。 Quantum dots 1 whose surfaces were modified with MPA prepared in Example 1 were dissolved in an aqueous solution, and 50 μL of the aqueous solution was subcutaneously injected into BALB/c nude mice. Then, the mouse was placed in three states of cooling, normal, and heating, and photographed with an infrared thermography camera (FLIR C2, manufactured by FLIR). As a result, the temperature of the region of the mouse subcutaneously injected with the aqueous solution was 21.3°C in the cold state, 32.3°C in the normal state, and 36.7°C in the heated state.
　次に、実施例４と同様にして、生体イメージング装置を用いてマウスのうち水溶液が皮下注射された領域の蛍光画像を取得した。マウスの状態は上述の冷却状態、通常状態、加熱状態の３つの状態としてそれぞれ蛍光画像を取得した。励起光のバンドパスフィルタは４９０～５１０ｎｍとし、それ以外の条件は実施例４と同様とした。そして、蛍光画像から光強度比Ｉ２／Ｉ１を測定した。 Next, in the same manner as in Example 4, a fluorescence image of the area of the mouse subcutaneously injected with the aqueous solution was obtained using a bioimaging device. Fluorescence images were obtained for each of the three states of the mouse: the above-described cooling state, normal state, and heating state. The band-pass filter for excitation light was set to 490 to 510 nm, and other conditions were the same as in Example 4. Then, the light intensity ratio I2/I1 was measured from the fluorescence image.
　次に、実施例４で求めた図１１の光強度比Ｉ２／Ｉ１との温度の対応の直線を検量線として、冷却状態、通常状態、加熱状態の３つの状態における光強度比Ｉ２／Ｉ１から、温度を算出した。図１２は、３つの状態における光強度比Ｉ２／Ｉ１を、検量線上にプロットしたグラフである。図１２から、マウスのうち水溶液が皮下注射された領域の温度は、冷却状態で２２．０℃、通常状態で３２．５℃、加熱状態で３７．４℃と算出された。この結果は、赤外線サーモグラフィカメラによる測定結果と比較的一致しており、光強度比Ｉ２／Ｉ１と温度の対応関係を検量線として温度計測可能であることが確かめられた。 Next, using the straight line corresponding to the temperature with the light intensity ratio I2/I1 of FIG. , the temperature was calculated. FIG. 12 is a graph plotting the light intensity ratio I2/I1 in three states on a calibration curve. From FIG. 12, the temperature of the area of the mouse subcutaneously injected with the aqueous solution was calculated to be 22.0° C. in the cooled state, 32.5° C. in the normal state, and 37.4° C. in the heated state. This result is relatively consistent with the measurement result by the infrared thermography camera, and it was confirmed that the temperature can be measured using the correspondence relationship between the light intensity ratio I2/I1 and the temperature as a calibration curve.
　本開示の温度計測装置は、生体内の細胞の温度計測に好適である。 The temperature measurement device of the present disclosure is suitable for measuring the temperature of cells inside a living body.
　１：量子ドット
　２：光照射部
　３：光検出部
　４：光強度比算出部
　５：検量線記憶部
　６：温度算出部
　１０：コア
　１１：シェル
  1: Quantum dot 2: Light irradiation unit 3: Light detection unit 4: Light intensity ratio calculation unit 5: Calibration curve storage unit 6: Temperature calculation unit 10: Core 11: Shell

Claims (6)

  1. 　測定対象物の近傍に配置され、結晶欠陥を有したコアと前記コアを覆うシェルとを有したコアシェル型の量子ドットと、
    　前記量子ドットに、前記コアのバンドギャップエネルギーよりも大きなエネルギーの波長である励起光を照射する光照射部と、
    　前記量子ドットのバンド端発光のピーク波長における光強度と、前記量子ドットの欠陥発光のピーク波長における光強度とを計測する光検出部と、
    　前記光検出部により検出した２つの光強度の光強度比を算出する光強度比算出部と、
    　前記光強度比と前記量子ドットの周囲の温度との関係を示した検量線を記憶する検量線記憶部と、
    　前記光強度比算出部により算出した前記光強度比と、前記検量線記憶部に記憶された前記検量線とから、前記測定対象物の温度を算出する温度算出部と、
    　を有することを特徴とする温度計測装置。     A core-shell type quantum dot having a core having a crystal defect and a shell covering the core, which is arranged in the vicinity of an object to be measured;
    A light irradiation unit that irradiates the quantum dots with excitation light having a wavelength of energy greater than the bandgap energy of the core;
    a light detection unit that measures the light intensity at the peak wavelength of the band edge emission of the quantum dot and the light intensity at the peak wavelength of the defect emission of the quantum dot;
    a light intensity ratio calculator that calculates the light intensity ratio of the two light intensities detected by the light detector;
    a calibration curve storage unit that stores a calibration curve showing the relationship between the light intensity ratio and the temperature around the quantum dots;
    a temperature calculation unit that calculates the temperature of the measurement object from the light intensity ratio calculated by the light intensity ratio calculation unit and the calibration curve stored in the calibration curve storage unit;
    A temperature measuring device comprising:
  2. 　前記検量線は直線である、ことを特徴とする請求項１に記載の温度計測装置。 The temperature measuring device according to claim 1, characterized in that the calibration curve is a straight line.
  3. 　前記検量線は、前記量子ドットの周囲環境ごとに設定されている、ことを特徴とする請求項１または請求項２に記載の温度計測装置。 The temperature measuring device according to claim 1 or 2, characterized in that the calibration curve is set for each ambient environment of the quantum dots.
  4. 　前記温度計測装置は、生体内の温度計測用であり、前記励起光の波長は７００～１５００ｎｍであり、前記量子ドットの表面に親水基が結合されている、ことを特徴とする請求項１ないし請求項３のいずれか１項に記載の温度計測装置。 1. The temperature measuring device is for temperature measurement in a living body, the excitation light has a wavelength of 700 to 1500 nm, and hydrophilic groups are bonded to the surfaces of the quantum dots. The temperature measuring device according to claim 3 .
  5. 　前記温度計測装置は、細胞内の温度計測用であり、前記励起光の波長は７００～１５００ｎｍであり、前記量子ドットの表面に膜透過性ペプチドまたはカチオン性リポソームが結合されている、ことを特徴とする請求項１ないし請求項３のいずれか１項に記載の温度計測装置。 The temperature measuring device is for intracellular temperature measurement, the excitation light has a wavelength of 700 to 1500 nm, and a membrane-permeable peptide or cationic liposome is bound to the surface of the quantum dot. The temperature measuring device according to any one of claims 1 to 3.
  6. 　結晶欠陥を有したコアと、前記コアを覆うシェルとを有したコアシェル型の量子ドットを、測定対象物の近傍に配置し、
    　前記量子ドットに励起光を照射し、
    　前記量子ドットのバンド端発光のピーク波長における光強度と、前記量子ドットの欠陥発光のピーク波長における光強度とを計測し、その２つの光強度の光強度比を算出し、
    　前記光強度比と、あらかじめ求めておいた前記光強度比と前記量子ドットの周囲の温度との関係を示した検量線とから、前記量子ドットの周囲の温度を算出する、
    　ことを特徴とする温度計測方法。
          A core-shell type quantum dot having a core with crystal defects and a shell covering the core is arranged in the vicinity of the measurement object,
    irradiating the quantum dots with excitation light,
    Measure the light intensity at the peak wavelength of the band edge emission of the quantum dot and the light intensity at the peak wavelength of the defect emission of the quantum dot, and calculate the light intensity ratio of the two light intensities,
    Calculate the temperature around the quantum dot from the light intensity ratio and a calibration curve showing the relationship between the light intensity ratio obtained in advance and the temperature around the quantum dot,
    A temperature measurement method characterized by:
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