Method and system for measuring organelle-specific temperature by using fluorescent nanodiamond

Abstract

A method for measuring organelle temperature, according to the present invention, may comprise the steps of: binding antibodies to a fluorescent nanodiamond so as to prepare an antibody-fluorescent nanodiamond; culturing cells with the antibody-fluorescent nanodiamond so as to prepare a sample; preparing a first source in which a cell fixation solution is provided to the sample, and a second source in which an ATP synthesis inhibitor is provided to the sample; providing a test signal while changing the temperature of the first source, so as to acquire a temperature sensitivity coefficient through a method for measuring a first ESR center-frequency variation of the antibody-fluorescent nanodiamond bound to an organelle of the cell; providing a test signal to the second source so as to measure a second ESR center-frequency variation of the antibody-fluorescent nanodiamond bound to the organelle of the cell; and estimating the temperature of the organelle bound to the antibody-fluorescent nanodiamond in the second source by a method for calculating a temperature variation of the organelle bound to the antibody-fluorescent nanodiamond in the second source by using the temperature sensitivity coefficient and the second ESR center-frequency variation.

Description

Organelle-specific temperature measurement method and temperature measurement system using fluorescent nanodiamonds

[0001] The present invention relates to a method and system for measuring organelle-specific temperature using fluorescent nanodiamonds, and more specifically, to an organelle temperature measuring method that estimates the temperature of a cell organelle through a change in the center frequency of ESR (Electron Spin Resonance) by providing a laser light source and microwaves to an antibody-fluorescent nanodiamond to which a fluorescent nanodiamond and an antibody are bound, and an organelle temperature measuring system comprising a culture chamber, a culture vessel, a laser light source, a microwave application unit, a detection unit that receives a fluorescent signal emitted from the antibody-fluorescent nanodiamond, an analysis unit that derives a temperature change from the center frequency of ESR, and a correction control unit that corrects a change in the position of the antibody-fluorescent nanodiamond.

[0002] Recently, intracellular temperature measurement technology is attracting attention for precisely measuring thermodynamic changes resulting from intracellular metabolic reactions and physiological conditions.

[0003] In particular, various fluorescence-based temperature sensors have been developed to detect minute temperature changes at the level of cellular organelles; however, limitations have existed in long-term precise measurements due to photobleaching, low spatial resolution, and chemical instability.

[0004] To overcome these limitations, a quantum thermometer based on fluorescent nanodiamonds (FNDs) containing nitrogen vacancy defects (NV centers) has been proposed, which enables local intracellular temperature measurement by utilizing the characteristic that the electron spin resonance (ESR) center frequency changes sensitively with temperature variations.

[0005] In particular, FNDs possess excellent photostability and biocompatibility, offering advantages that can compensate for the shortcomings of conventional fluorescent thermometers.

[0006] However, existing FND-based temperature measurement technologies were limited to measuring the average temperature of the entire cell or specific regions, and precise temperature measurement at the level of intracellular organelles was difficult. This was due to a lack of spatial resolution capable of independently distinguishing temperature differences between organelles.

[0007] Accordingly, the present invention relates to a method and system for measuring organelle-specific quantum temperature that can detect organelle-specific temperature changes in real time through changes in the center frequency of ESR by attaching a specific antibody to a fluorescent nanodiamond and precisely positioning it on specific organelles such as mitochondria, the nucleus, and the cell membrane within the cell. The present technology is implemented by comprising a system including a laser light source, a microwave application unit, a single photon detector, a nano-tracking system for position correction, and an ESR signal analysis algorithm, thereby enabling stable temperature measurement even with cell movement or environmental changes.

[0008] The technical problem that the present invention aims to solve is to provide a method for measuring organelle temperature that can non-invasively measure temperature in real time while selectively bound to a specific organelle within a living cell.

[0009] Another technical problem that the present invention aims to solve is to provide a method for measuring organelle temperature that can analyze local temperature changes at the organelle level with high spatial resolution and quantification using antibody-fluorescent nanodiamonds containing NV centers.

[0010] Another technical problem that the present invention aims to solve is to provide a method for measuring organelle temperature that can minimize variations between experimental conditions and cell populations by obtaining a temperature sensitivity coefficient in advance using a first source in a fixed cell state and applying it to a second source in a living cell state.

[0011] Another technical problem that the present invention aims to solve is to provide an organelle temperature measurement system and an organelle temperature measurement method capable of stable organelle temperature kinetic analysis even during long-term measurements by correcting for changes in observation position due to cell migration or morphological changes in real time.

[0012] Another technical problem that the present invention aims to solve is to provide a method for measuring organelle temperature that enables reproducible and reliable temperature measurement for various organelles without complex invasive manipulation, thereby enhancing applicability in the fields of life science and biomedical science.

[0013] The technical problems that the present invention aims to solve are not limited to those described above.

[0014] To solve the above technical problem, the present invention provides a method for measuring organ temperature.

[0015] According to one embodiment, the organelle temperature measurement method comprises the steps of: preparing an antibody-fluorescent nanodiamond by conjugating an antibody to a fluorescent nanodiamond; preparing a sample by culturing the antibody-fluorescent nanodiamond in a cell; preparing a first source in which a cell fixation solution is provided to the sample, and a second source in which an ATP (Adenosine Triphosphate) synthesis inhibitor is provided to the sample; obtaining a temperature sensitivity coefficient by providing a test signal while varying the temperature of the first source to measure the change in the center frequency of the first ESR (Electron Spin Resonance) of the antibody-fluorescent nanodiamond bound to the organelle of the cell; providing a test signal to the second source to measure the change in the center frequency of the second ESR of the antibody-fluorescent nanodiamond bound to the organelle of the cell; and calculating the temperature change amount for the organelle bound to the antibody-fluorescent nanodiamond within the second source using the temperature sensitivity coefficient and the change in the center frequency of the second ESR. It may include a step of estimating the temperature of the organelle combined with the antibody-fluorescent nanodiamond within.

[0016] According to one embodiment, the fluorescent nanodiamond may comprise a nitrogen-vacancy center (NV center) and may comprise an activated ester group (N-hydroxysuccinimide ester) formed on the fluorescent nanodiamond.

[0017] According to one embodiment, in the step of preparing the antibody-fluorescent nanodiamond, the fluorescent nanodiamond and the antibody are reacted so that the activating ester group of the fluorescent nanodiamond and the amine group (NH2) of the protein of the antibody are covalently bonded, and the antibody may be any one of a mitochondrial target antibody, a nuclear target antibody, or a cell membrane target antibody.

[0018] According to one embodiment, the sample is prepared by a method wherein, in the step of preparing the sample, the cell is cultured in a medium, and then the antibody-fluorescent nanodiamond is provided in a dispersed state in a PBS solution to be cultured, and the medium may include DMEM (Dulbecco's Modified Eagle's Medium), 10% FBS (fetal bovine serum), and 1% penicillin / streptomycin.

[0019] According to one embodiment, in the process of preparing the sample, the protein of the organelle of the cell and the antibody of the antibody-fluorescent nanodiamond are antigen-antibody bound, the cell comprises human skin fibroblasts, and the antibody may be any one of a mitochondrial targeting antibody, a nuclear targeting antibody, or a cell membrane targeting antibody.

[0020] According to one embodiment, when the antibody is a mitochondrial target antibody, the Manders' coefficient of the antibody-fluorescent nanodiamond localized to the mitochondria, which are organelles of the cell, is 0.98 ± 0.01; when the antibody is a nuclear target antibody, the Manders' coefficient of the antibody-fluorescent nanodiamond localized to the nucleus, which is an organelle of the cell, is 0.91 ± 0.05; and when the antibody is a cell membrane target antibody, the Manders' coefficient of the antibody-fluorescent nanodiamond localized to the cell membrane is 0.67 ± 0.10.

[0021] According to one embodiment, the concentration of the antibody-fluorescent nanodiamond in the PBS solution may be controlled to be greater than 3 μg / mL and less than 7 μg / mL.

[0022] According to one embodiment, the sample comprises a medium, the medium comprises DMEM (Dulbecco's Modified Eagle's Medium), 10% FBS (fetal bovine serum), and 1% penicillin / streptomycin, and in the step of preparing the first source, the medium is removed from the sample before providing the cell fixation solution to the sample, and the organelles of the cell are physically fixed by the cell fixation solution.

[0023] According to one embodiment, the ATP synthesis inhibitor comprises FCCP (Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone), and the concentration of the ATP synthesis inhibitor may be controlled to 50 μM.

[0024] According to one embodiment, the test signal includes a continuous laser light source and a microwave, the wavelength of the laser light source is 532 nm, and the intensity is 5.0 μW / cm² 2 It includes that, and the output of the microwave may be -23dBm.

[0025] According to one embodiment, the cell comprises human skin fibroblasts, the organelles of the cell comprise mitochondria, a nucleus, and a cell membrane, the antibody comprises any one of a mitochondrial target antibody, a nuclear target antibody, or a cell membrane target antibody, and among the organelles of the cell, the one with the greatest temperature change in mitochondria may be included.

[0026] To solve the above technical problem, the present invention provides a small organ temperature measuring system for measuring the temperature of a small organ by the method described above.

[0027] According to one embodiment, the organelle temperature measuring system may include a culture chamber, a culture vessel containing a source in which an antibody-fluorescent nanodiamond is cultured in cells inside the culture chamber, a laser light source that photoexcites the antibody-fluorescent nanodiamond inside the source, a microwave application unit that provides microwaves to induce electron spin resonance of the antibody-fluorescent nanodiamond inside the source, a detector unit that receives a fluorescence signal emitted from the antibody-fluorescent nanodiamond excited by the laser light source, an analysis unit that calculates a change in the center frequency of the ESR (Electron Spin Resonance) of the antibody-fluorescent nanodiamond based on the fluorescence signal received from the detector unit and derives a temperature change from the change in the center frequency of the ESR, and a correction control unit for correcting a change in the position of the antibody-fluorescent nanodiamond.

[0028] According to one embodiment, the correction control unit detects a positional change of the antibody-fluorescent nanodiamond using a temporal change of the fluorescent signal received from the detection unit, and includes a piezo stage positioned at the bottom of the culture vessel to control displacement so that the antibody-fluorescent nanodiamond can be analyzed at a preset observation position according to the detected positional change, and the correction control unit may include calculating a change in the ESR center frequency of the antibody-fluorescent nanodiamond while the positional correction is performed in a signal-linked manner with the analysis unit.

[0029] According to one embodiment, the piezo stage is driven in real time, and the piezo stage may be a 3-axis piezo stage capable of being driven independently in the x-axis, y-axis, and z-axis directions.

[0030] According to one embodiment, the sample is prepared by a method of providing and culturing the antibody-fluorescent nanodiamond dispersed in a solution in PBS after culturing the cells in the medium of the culture vessel, wherein the medium comprises DMEM (Dulbecco's Modified Eagle's Medium), 10% FBS (fetal bovine serum), and 1% penicillin / streptomycin, and the source within the culture vessel may comprise a first source in which a cell fixation solution is provided after removing the medium from the sample, and a second source in which an ATP (Adenosine Triphosphate) synthesis inhibitor is provided to the sample.

[0031] According to one embodiment, the concentration of the antibody-fluorescent nanodiamond in the PBS solution may be greater than 3 μg / mL and less than 7 μg / mL.

[0032] The organelle temperature measurement method according to the present invention can non-invasively measure temperature in real time while selectively bound to a specific organelle within a living cell using an antibody-fluorescent nanodiamond. In particular, since local temperature changes at the organelle level can be quantitatively analyzed through NV center-based ESR measurement of the antibody-fluorescent nanodiamond, it can provide high spatial resolution and quantification compared to conventional fluorescent temperature sensors.

[0033] Furthermore, the present invention reliably calculates organelle temperature changes occurring in a biological environment by obtaining a temperature sensitivity coefficient in advance using a first source in a fixed cell state and applying it to a second source in a living cell state. Accordingly, highly reproducible organelle temperature measurements become possible while minimizing the influence of variations caused by experimental conditions or individual cell populations.

[0034] Furthermore, through a correction control unit including a piezo stage of the organelle temperature measurement system, changes in observation position caused by cell migration or morphological changes occurring during long-term measurement can be corrected in real time. Accordingly, stable fluorescence and ESR signals can be continuously acquired at the same organelle location, enabling long-term analysis of organelle temperature dynamics.

[0035] Consequently, the present invention provides a technology capable of quantitatively analyzing temperature changes in organelles such as mitochondria, the nucleus, or the cell membrane with high reliability in a living cell environment, and can be usefully applied in various life science and biomedical fields, such as cell metabolism research, disease diagnosis, and drug response evaluation.

[0036] FIG. 1 is a schematic diagram illustrating a small organ temperature measuring system according to one embodiment of the present invention.

[0037] FIG. 2 is a flowchart illustrating a method for measuring organ temperature according to one embodiment of the present invention.

[0038] Figure 3 is a diagram for specifically explaining the features of a small organ temperature measuring system according to Experimental Example 1 of the present invention.

[0039] FIG. 4 is a diagram illustrating the thermodynamic reaction occurring in a sample when an ATP (Adenosine Triphosphate) synthesis inhibitor is added to a sample provided in a culture vessel of an organelle temperature measuring system according to Experimental Example 1 of the present invention.

[0040] FIGS. 5 and 6 are drawings illustrating the method of manufacturing antibody-fluorescent nanodiamonds used in the organelle temperature measurement system according to Experimental Example 1 of the present invention and the organelle-specific binding characteristics.

[0041] Figures 7 and 8 are diagrams illustrating the organelle temperature kinetics of human skin fibroblasts when an ATP synthesis inhibitor is administered using the organelle temperature measurement system according to Experimental Example 1 of the present invention.

[0042] Figure 9 is an experimental result for explaining the method of calculating the temperature sensitivity coefficient of a fluorescent nanodiamond used in an organelle temperature measurement system according to Experimental Example 1 of the present invention.

[0043] FIG. 10 is a diagram illustrating the intracellular introduction characteristics and organelle temperature measurement stability according to the concentration of antibody-fluorescent nanodiamond introduced into human skin fibroblasts (WS1) in the organelle temperature measurement system according to Experimental Example 1 of the present invention.

[0044] Hereinafter, preferred embodiments of the present invention will be described in detail with reference to the attached drawings. However, the technical concept of the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the embodiments introduced herein are provided to ensure that the disclosed content is thorough and complete and to ensure that the concept of the present invention is sufficiently conveyed to those skilled in the art.

[0045] In this specification, when a component is described as being on another component, it means that it may be formed directly on the other component or that a third component may be interposed between them. Additionally, in the drawings, the thicknesses of the films and regions are exaggerated for the effective description of the technical content.

[0046] Additionally, although terms such as first, second, third, etc., have been used to describe various components in the various embodiments of this specification, these components should not be limited by such terms. These terms are used merely to distinguish one component from another. Accordingly, what is referred to as the first component in one embodiment may be referred to as the second component in another embodiment. Each embodiment described and illustrated herein also includes its complementary embodiment. Furthermore, in this specification, "and / or" is used to mean including at least one of the components listed before and after it.

[0047] In the specification, singular expressions include plural expressions unless the context clearly indicates otherwise. Furthermore, terms such as "include" or "have" are intended to specify the existence of the features, numbers, steps, components, or combinations thereof described in the specification, and should not be understood as excluding the existence or addition of one or more other features, numbers, steps, components, or combinations thereof. Additionally, in this specification, "connection" is used to include both indirectly connecting multiple components and directly connecting them.

[0048] Furthermore, in describing the present invention below, if it is determined that a detailed description of related known functions or configurations could unnecessarily obscure the essence of the invention, such detailed description will be omitted.

[0049]

[0050] FIG. 1 is a schematic diagram illustrating a small organ temperature measuring system according to one embodiment of the present invention.

[0051] Referring to FIG. 1, the organelle temperature measuring system of the present invention may include a culture chamber (10), a source (20) containing an antibody-fluorescent nanodiamond, a culture vessel (30), a laser light source (40), a microwave application unit (50), a detection unit (60), an analysis unit (70), and a correction control unit (80).

[0052] The above culture chamber (10) can be configured to allow living cells to be stably maintained for a long time under physiological conditions by placing the culture vessel (30) containing the source (20) containing the sample in which cells are cultured inside, and by providing an environment in which temperature, humidity, and carbon dioxide concentration are controlled.

[0053] And, the source (20) comprises a sample in which the antibody-fluorescent nanodiamond is introduced into the cell, and the antibody-fluorescent nanodiamond may have a structure in which an antibody is covalently bonded to a fluorescent nanodiamond containing a nitrogen-vacancy center (NV center). In this case, the antibody may be any one of a mitochondrial targeting antibody, a nuclear targeting antibody, or a cell membrane targeting antibody, and accordingly, the antibody-fluorescent nanodiamond may selectively bind to a specific organelle of the cell (e.g., mitochondria, nucleus, cell membrane, etc.).

[0054] And, the laser light source (40) is configured to photoexcite the antibody-fluorescent nanodiamond in the source (20), and, for example, can provide a laser of a wavelength of 532 nm. Accordingly, the NV centers inside the fluorescent nanodiamond are transitioned to an excited state and a fluorescent signal can be emitted.

[0055] And, the microwave application unit (50) provides microwaves to induce electron spin resonance (ESR) at the NV center of the antibody-fluorescent nanodiamond, and, for example, the microwaves can be applied through a PCB on which a coplanar waveguide (CPW) is formed. Accordingly, the zero-field splitting value of the NV center changes with temperature change, and this change may appear as a shift in the ESR center frequency.

[0056] Furthermore, the detection unit (60) receives the fluorescent signal emitted from the antibody-fluorescent nanodiamond excited by the laser light source (40), and the analysis unit (70) calculates the change in the ESR center frequency of the antibody-fluorescent nanodiamond based on the fluorescent signal received from the detection unit (60), and can quantitatively derive the amount of temperature change of the corresponding organelle (e.g., mitochondria, nucleus, cell membrane, etc.) by applying a previously acquired temperature sensitivity coefficient. Accordingly, temperature changes in organelles such as mitochondria, nucleus, or cell membrane within the living cell can be analyzed in real time.

[0057] Meanwhile, the correction control unit (80) may be configured to correct for changes in the position of the antibody-fluorescent nanodiamond due to movement or shape change of the cell that may occur during a long-term measurement process. Additionally, the correction control unit (80) may detect changes in the position of the antibody-fluorescent nanodiamond using temporal changes in the fluorescent signal received from the detection unit (60), and correct the observation position by driving the piezo stage (81) located at the bottom of the culture vessel (30) according to the detected changes in position. Specifically, the piezo stage (81) may be a 3-axis piezo stage that can be driven independently in the x-axis, y-axis, and z-axis directions, and continuous analysis may be possible at the same organelle location by driving in real time.

[0058] In addition, the correction control unit (80) may be configured to be signal-linked with the analysis unit (70) so that the change in the ESR center frequency of the antibody-fluorescent nanodiamond is calculated when position correction is performed.

[0059] With such a configuration, the organelle temperature measuring system according to an embodiment of the present invention can non-invasively measure the temperature in real time while selectively bound to a specific organelle of the cell using the antibody-fluorescent nanodiamond, and can provide a technical effect that enables stable organelle temperature kinetic analysis over a long period of time while minimizing measurement errors caused by cell movement.

[0060] In conclusion, the organelle temperature measurement system according to an embodiment of the present invention can non-invasively measure temperature in real time while selectively bound to a specific organelle within the cell using the antibody-fluorescent nanodiamond. Furthermore, local temperature changes at the organelle level can be quantitatively analyzed through ESR-based measurement using the laser light source and the microwave application unit. Moreover, by correcting changes in measurement location due to cell movement or morphological changes in real time by the correction control unit including the piezo stage, stable signal acquisition can be achieved even during long-term measurements. Accordingly, the effect of analyzing temperature dynamics by organelle with high reliability in a living cell environment can be provided.

[0061]

[0062] Hereinafter, with reference to FIG. 2, a method for measuring the temperature of a small organ using the small organ temperature measuring system described above with reference to FIG. 1 is explained.

[0063] FIG. 2 is a flowchart illustrating a method for measuring organ temperature according to one embodiment of the present invention.

[0064] Referring to FIG. 2, the organelle temperature measurement method comprises the steps of: preparing an antibody-fluorescent nanodiamond by conjugating an antibody to a fluorescent nanodiamond; preparing a sample by culturing the antibody-fluorescent nanodiamond in the cell; preparing a first source in which a cell fixation solution is provided to the sample, and a second source in which an ATP (Adenosine Triphosphate) synthesis inhibitor is provided to the sample; obtaining a temperature sensitivity coefficient by providing a test signal while varying the temperature of the first source to measure the change in the center frequency of the first Electron Spin Resonance (ESR) of the antibody-fluorescent nanodiamond bound to the organelle of the cell; providing a test signal to the second source to measure the change in the center frequency of the second ESR of the antibody-fluorescent nanodiamond bound to the organelle of the cell; and using the temperature sensitivity coefficient and the change in the center frequency of the second ESR, for the organelle bound to the antibody-fluorescent nanodiamond within the second source A method for calculating the amount of temperature change may include the step of estimating the temperature of the organelle combined with the antibody-fluorescent nanodiamond within the second source.

[0065] In the antibody-fluorescent nanodiamond preparation step described above, as described in detail below with reference to FIG. 5, the fluorescent nanodiamond is prepared to include a nitrogen-vacancy center (NV center), and a carboxyl group and an activated ester group (N-hydroxysuccinimide ester) may be formed on the surface. Subsequently, the fluorescent nanodiamond can be prepared as the antibody-fluorescent nanodiamond by forming an activated ester group through an EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) / NHS (N-Hydroxysuccinimide) reaction and covalently bonding with an amine group (-NH2) contained in the lysine residue of the antibody. In this case, the antibody may be any one of a mitochondrial target antibody (e.g., anti-TOMM20), a nuclear target antibody (e.g., anti-Lamin B1), or a cell membrane target antibody (e.g., anti-CD98), and the antibody-fluorescent nanodiamond prepared accordingly may have bio-specificity capable of selectively binding to a specific organelle.

[0066] Next, in the step of preparing the sample, the antibody-fluorescent nanodiamond may be prepared by providing the cell with the antibody-fluorescent nanodiamond dispersed in a PBS solution and culturing it. For example, the cell may be a human skin fibroblast (WS1). For example, the cell may be cultured for 24 hours at 37°C and 5% CO₂ conditions in a medium containing DMEM (Dulbecco's Modified Eagle's Medium), 10% FBS, and 1% penicillin / streptomycin. Accordingly, during the process of preparing the sample, the antibody-fluorescent nanodiamond is localized to a specific organelle within the cell through antigen-antibody binding, and organelle-specific fluorescent signals and ESR signals may be provided in subsequent organelle temperature measurements.

[0067] According to one embodiment, the concentration of the antibody-fluorescent nanodiamond in the PBS solution can be controlled to be greater than 3 μg / mL and less than 7 μg / mL.

[0068] Below the above concentration range, the number of antibody-fluorescent nanodiamonds introduced into the cell is limited, so the fluorescent signal at the organelle location is not sufficiently secured; consequently, a problem may arise in which the continuous tracking of temporal changes in the ESR center frequency becomes unstable. In such cases, the quantification and reproducibility of the organelle temperature change amount may be reduced.

[0069] On the other hand, if the concentration range exceeds the above, the antibody-fluorescent nanodiamonds may be excessively introduced into the cell, leading to localized accumulation or aggregation. Consequently, fluorescence signal saturation, distortion of the ESR spectrum, or a decrease in the signal-to-noise ratio may be observed. Furthermore, in some cells, morphological changes or physiological stress may be induced, resulting in a condition unsuitable for stable organelle temperature measurements over extended periods.

[0070] Accordingly, according to an embodiment of the present application, in the step of preparing the sample, the concentration of the antibody-fluorescent nanodiamond can be controlled to a range of greater than 3 μg / mL and less than 7 μg / mL. Accordingly, uniform introduction into the cell and organelle-specific binding efficiency can be secured, and at the same time, stable time tracking of the fluorescence signal and ESR center frequency can be achieved. As a result, a technical effect can be achieved in which the quantity, reproducibility, and long-term stability of the measurement of organelle temperature changes are secured in balance.

[0071] Next, in the preparation steps of the first source and the second source, the first source for obtaining the temperature sensitivity coefficient and the second source for measuring the actual organ temperature can each be prepared from the sample.

[0072] Specifically, the first source can be prepared by providing a cell fixation solution after removing the medium from the sample. For example, the cell fixation solution may be an aqueous solution containing 4% formaldehyde. Accordingly, the structure of the cell and the position of the antibody-fluorescent nanodiamond can be physically fixed so as not to change over time.

[0073] On the other hand, the second source can be prepared by providing the ATP (Adenosine Triphosphate) synthesis inhibitor to the sample. For example, the ATP synthesis inhibitor may be FCCP (Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone), and its concentration may be controlled to 50 μM. Accordingly, thermogenesis in mitochondria may be induced within the living cell.

[0074] Next, in the step of obtaining the temperature sensitivity coefficient using the first source, the test signal including a laser light source and microwaves may be provided while gradually changing the external temperature with respect to the first source. At this time, the laser light source may be a continuous wave laser with a wavelength of 532 nm, and the microwaves may be applied to induce electron spin resonance at the NV center. For example, the intensity of the laser light source may be 5.0 μW / cm². For example, the microwave output may be -23 dBm.

[0075] Accordingly, as described in detail below with reference to FIG. 9, the first ESR (Electron Spin Resonance) spectrum of the antibody-fluorescent nanodiamond is measured under each temperature condition, and the first ESR center frequency (D) can be extracted therefrom.

[0076] In addition, by analyzing the change in the first ESR center frequency (ΔD) according to the externally applied change in temperature (ΔT), a linear relationship between ΔD and ΔT is derived, and the temperature sensitivity coefficient (ΔD / ΔT) can be obtained as the slope. The temperature sensitivity coefficient can subsequently be used as a reference value for calculating the actual change in the organ temperature.

[0077] Next, in the organelle temperature estimation step using the second source, the change in the second ESR center frequency of the antibody-fluorescent nanodiamond can be measured over time while the same test signal as the first source is provided to the second source. At this time, due to heat generation induced by the ATP synthesis inhibitor, the local temperature change of the organelle to which the antibody-fluorescent nanodiamond is bound may appear as a shift in the second ESR center frequency.

[0078] That is, the first ESR center frequency is the ESR center frequency measured at a reference temperature in a fixed cell state and is a reference value for calculating the temperature sensitivity coefficient, and the second ESR center frequency is the ESR center frequency measured in a living cell state and may be a value reflecting the actual temperature change of the organelle.

[0079] Subsequently, the change in temperature of the organelle to which the antibody-fluorescent nanodiamond is bound can be calculated using the change in the second ESR center frequency measured at the second source and the temperature sensitivity coefficient obtained from the first source. Accordingly, the temperature of organelles such as mitochondria, the nucleus, or the cell membrane within the living cell can be quantitatively estimated.

[0080] In conclusion, the organelle temperature measurement method according to an embodiment of the present invention may include the step of preparing the sample by manufacturing the antibody-fluorescent nanodiamond and introducing it into the cell, obtaining the temperature sensitivity coefficient using the first source, and estimating the temperature of the organelle using the second source. The first source is used to derive a correlation between the first ESR center frequency and the temperature in the cell fixation state, and the second source may be used to measure the change in the second ESR center frequency due to heat generation induced by the ATP synthesis inhibitor. Through this, the local temperature change of the organelle to which the antibody-fluorescent nanodiamond is bound can be quantitatively calculated. Accordingly, the present invention can provide a method for non-invasively estimating the temperature of a specific organelle within a living cell in real time.

[0081]

[0082] Hereinafter, experimental examples and characteristic evaluation results regarding an organ temperature measurement system according to an embodiment of the present invention and a method for measuring organ temperature using the same are described.

[0083]

[0084] Organelle temperature measurement system according to Experimental Example 1

[0085] The organelle temperature measurement system according to Experimental Example 1 includes, as shown in FIG. 1, a culture chamber, a laser light source, a microwave application unit, a fluorescence signal detection unit, an analysis unit, and a correction control unit for position correction.

[0086] First, the culture chamber includes a culture vessel for receiving a sample in which cells are cultured, and is configured to control temperature, humidity, and CO2 concentration. Through this, human skin fibroblasts (WS1) can be stably maintained under physiological conditions, and long-term experiments are possible with the introduction of antibody-fluorescent nanodiamonds.

[0087] Next, the laser light source and the microwave application unit are configured to excite the NV (Nitrogen-Vacancy) centers of the antibody-fluorescent nanodiamond and induce electron spin resonance. Specifically, the laser light source excites the NV centers of the antibody-fluorescent nanodiamond by irradiating light with a wavelength of 532 nm, and the microwave application unit induces a spin state transition of the NV centers by applying microwaves through a PCB integrated with a CPW (Coplanar Waveguide). Accordingly, a shift in the ESR center frequency occurs due to changes in temperature.

[0088] In addition, the detection and analysis units receive the fluorescence signal emitted from the antibody-fluorescent nanodiamond excited by a laser light source and calculate the change in the ESR center frequency based on the signal. The analysis unit applies the temperature sensitivity coefficient (ΔD / ΔT) derived from fixed cell experiments to convert the change in the ESR center frequency into the actual temperature change of the organelle, thereby quantitatively determining the temperature change for each organelle, such as mitochondria, the nucleus, or the cell membrane.

[0089] Meanwhile, the correction control unit is configured to correct for positional changes in the antibody-fluorescent nanodiamond that may occur due to cell movement or morphological changes. The correction control unit detects positional changes in the antibody-fluorescent nanodiamond using temporal changes in the fluorescence signal and corrects the observation position by driving a 3-axis piezo stage placed at the bottom of the culture vessel in real time. Through this, it is possible to acquire a stable ESR signal at the same organelle location even during long-term measurement processes.

[0090] With this configuration, the organelle temperature measurement system according to Experimental Example 1 can measure temperature in real time in a living cell environment using antibody-fluorescent nanodiamonds that are selectively bound to specific organelles. In particular, by controlling the concentration of antibody-fluorescent nanodiamonds to be greater than 3 μg / mL and less than 7 μg / mL, organelle-specific introduction efficiency and stability of fluorescence and ESR signals can be simultaneously secured, thereby providing the effect of analyzing organelle-level temperature kinetics with high reliability.

[0091]

[0092] Figure 3 is a diagram for specifically explaining the features of a small organ temperature measuring system according to Experimental Example 1 of the present invention.

[0093] Referring to Fig. 3(a), this is a schematic diagram illustrating the Zero-Field Splitting (ZFS) value of the fluorescent nanodiamond according to the spin state and temperature change of the NV (Nitrogen-Vacancy) center of the fluorescent nanodiamond using the organelle temperature measurement system according to Experimental Example 1.

[0094] As can be seen in Figure 3(a), the NV center of the fluorescent nanodiamond has a ZFS corresponding to an energy interval between spin states ms = 0 and ms = ±1.

[0095] And, when excited by a 532nm laser light source, the NV center of the fluorescent nanodiamond can be initialized to the ms = 0 state through a non-radiative path.

[0096] Furthermore, the ZFS value of the fluorescent nanodiamond changes with temperature variations, and this change in ZFS can be detected as a shift in the center frequency of the Electron Spin Resonance (ESR) of the fluorescent nanodiamond. Accordingly, the amount of change in ZFS corresponds linearly to temperature, thereby enabling the measurement of the temperature of intracellular organelles.

[0097] Referring to Fig. 3(b), it can be seen that the organelle temperature measuring system according to Experimental Example 1 includes a temperature, humidity, and CO2 control system at the top of a culture vessel provided in an incubating chamber.

[0098] In addition, it can be seen that the microwave application unit of the above-mentioned organ temperature measurement system according to Experimental Example 1 is configured on a PCB integrated with a Coplanar Waveguide (CPW), and that the microwave is applied to the CPW by a wire bonding method.

[0099] Referring to Fig. 3(c), a sample was prepared by culturing human skin fibroblasts (WS1) (37°C, 5% CO2, 24 hours) with an antibody (mitochondrial outer membrane protein TOMM20) bound to an antibody in the culture vessel of the organelle temperature measuring system according to Experimental Example 1, and the fluorescence of the sample was measured using a confocal microscope.

[0100] Specifically, the human skin fibroblasts may have been cultured for 24 hours in advance at 37°C and 5% CO2 conditions in a medium containing 10% FBS and 1% penicillin / streptomycin in DMEM (Dulbecco's Modified Eagle's Medium).

[0101] As can be seen in Figure 3(c), the cell outline (white dotted line), nucleus (blue dotted line), mitochondria, and the location of the antibody-fluorescent nanodiamond within the sample can be clearly observed. This means that the antibody-fluorescent nanodiamond is stably introduced into the outer membrane of the mitochondria, which are organelles of the cell, and that its location can be tracked through a fluorescent signal.

[0102] Referring to FIG. 3 (d) and (e), a piezo stage capable of independent driving in three axes (x-axis, y-axis, z-axis) included in the calibration control unit of the organelle temperature measurement system according to Experimental Example 1 was operated to analyze the fluorescence signal of the NV center center position of the antibody-fluorescent nanodiamond and the change in ESR center frequency according to cell movement in the sample described in FIG. 3 (c) for 12 hours.

[0103] As can be seen in Figures 3 (d) and (e), the position of the antibody-fluorescent nanodiamond, which moves according to the movement of the cell by the operation of the piezo stage of the correction control unit, was tracked in real time, and by continuously correcting the measurement position accordingly, it can be seen that the fluorescence signal of the antibody-fluorescent nanodiamond and the change in the ESR center frequency were stably measured without distortion or loss.

[0104]

[0105] FIG. 4 is a diagram illustrating the thermodynamic reaction occurring in a sample when an ATP (Adenosine Triphosphate) synthesis inhibitor is added to a sample provided in a culture vessel of an organelle temperature measuring system according to Experimental Example 1 of the present invention.

[0106] Referring to Fig. 4(a), this is a schematic diagram explaining the mechanism of heat generation when FCCP (Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone, 50 μM), an ATP synthesis inhibitor, is added to the sample (human skin fibroblasts (WS1) and antibody-fluorescent nanodiamond) described in Fig. 3(c).

[0107] As can be seen in Figure 4 (a), the ATP synthesis inhibitor can be seen to induce heat generation by inhibiting ATP synthesis through the breakdown of the proton concentration gradient of the mitochondrial inner membrane.

[0108] Referring to FIGS. 4(b) to (d), the results of comparing fluorescence intensity and ESR spectrum for about 1 hour immediately after the ATP synthesis inhibitor was added to the sample described in FIG. 4(a) are shown.

[0109] As can be seen in Figures 4 (b) to (d), after the ATP synthesis inhibitor is added to the sample, the change in the center frequency (△D) of the ESR spectrum and the corresponding change in temperature (△T) remain relatively constant for about 40 minutes, then increase after about 40 minutes, and then return to the initial state after about 50 minutes.

[0110]

[0111] FIGS. 5 and 6 are drawings illustrating the method of manufacturing antibody-fluorescent nanodiamonds used in the organelle temperature measurement system according to Experimental Example 1 of the present invention and the organelle-specific binding characteristics.

[0112] Referring to FIG. 5 (a) and (b), a fluorescent nanodiamond (1, FND) was surface-treated (75°C, 48 hours) by providing it to a mixed acidic solution (H2SO4:HNO3:HClO4 = 1:1:1 v / v / v), then washing and neutralizing it with a base (0.1 M NaOH) and an acid (0.1 M HCl), centrifuging it with distilled water, and washing it three times to form carboxyl groups (-COOH) on the surface of the fluorescent nanodiamond (2, FND-COOH).

[0113] Subsequently, EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide) and NHS (N-hydroxysuccinimide) were provided to the fluorescent nanodiamond having a carboxyl group, and then reacted with a Poly-L-lysine (PLL, MW = 30,000) solution to form a positively charged PLL layer having an amine group (-NH2) covalently bonded to the carboxyl group (3, FND-PLL).

[0114] Then, the PLL layer of the fluorescent nanodiamond with the PLL layer formed was treated with succinic anhydride to form a carboxyl group (-COOH) at the end (4, FND-PLL-COOH).

[0115] Subsequently, a fluorescent nanodiamond having a carboxyl group at the end was treated with EDC and NHS to activate the carboxyl group (-COOH), thereby forming an activated ester group (-CO-O-NHS) (5, FND-PLL-NHS).

[0116] Then, an antibody (mitochondrial outer membrane protein, anti-TOMM20) was applied to a fluorescent nanodiamond having an activated ester group (fluorescent nanodiamond:antibody = 1:100 v / v, mixed in PBS for 2 hours) to prepare an antibody-fluorescent nanodiamond in which the activated ester group (-CO-O-NHS) of the fluorescent nanodiamond and the lysine residue (-NH2) of the antibody were amide-conjugated (6, FND-Bioconjugates, Mito-FND).

[0117] In addition, FT-IR analysis was performed on the above 1(FND), 2(FND-COOH), 3(FND-PLL), 4(FND-PLL-COOH), 5(FND-PLL-NHS), and 6(FND-Bioconjugates, Mito-FND).

[0118] As can be seen in Figures 5(a) and 5(b), major peaks corresponding to the respective terminal functional groups are observed for 1 to 5, and an additional peak of the NH amide series is observed for 6. Through this, it can be confirmed that the antibody is bound to the fluorescent nanodiamond.

[0119] Referring to Figures 5(c) and (d), instead of the culture vessel of the organelle temperature measuring system according to Experimental Example 1, specimens were prepared by culturing 1 (FND) and 6 (Mito-FND) described in Figure 5(a) at a concentration of 5 μg / mL each on a glass slide (1 cm x 1 cm, hydrophilic surface, 2,000 WS1 cells, overnight culture) in which human skin fibroblasts (WS1) were cultured.

[0120] Then, the mitochondria and nuclei within the human skin fibroblasts (WS1) of the above specimen were stained with MitoTracker (green) and DAPI (4',6-diamidino-2-phenylindole, blue), respectively, and the self-fluorescence (red) of the fluorescent nanodiamonds was imaged using a confocal microscope by irradiating them with a laser light source (532 nm) of the organelle temperature measurement system according to Experimental Example 1. Subsequently, the Manders' coefficient was calculated using ImageJ-JAcoP.

[0121] As can be seen in Figures 5 (c) and (d), 1 (FND) has a limited area of ​​overlap with mitochondria (green) within human skin fibroblasts, whereas 6 (Mito-FND) overlaps along the mitochondrial network. The Mandus coexistence coefficient, which quantifies this, also shows that 6 (Mito-FND) is 0.98 ± 0.01, which is significantly higher than that of 1 (FND, 0.13 ± 0.11).

[0122] Referring to Figures 5(e) and 6(a), the Z-stack function of a confocal microscope was used to analyze whether mitochondria and 6 (Mito-FND) overlap three-dimensionally (XY, XZ, YZ) within human skin fibroblasts.

[0123] As can be seen in Fig. 5(e) and Fig. 6(a), 6(Mito-FND) is shown to be three-dimensionally superimposed along the mitochondrial network within human skin fibroblasts.

[0124] Referring to Figures 6(b) and (c), 7(Nuc-FND) and 8(Mem-FND) were prepared by changing only the antibodies in the method for preparing 6(Mito-FND) described in Figures 5(a) and (b) to a nuclear target antibody (anti-Lamin B1) and a cell membrane target antibody (anti-CD98), respectively. Then, the Mandus coexistence coefficients for 7(Nuc-FND) and 8(Mem-FND) were calculated in the same manner as described in Figures 5(c) and (d).

[0125] For reference, the cell membrane was stained with blue-green fluorescence by conjugating Alexa Fluor 488-conjugated anti-rabbit IgG to anti-CD98.

[0126] As can be seen in Figures 6 (b) and (c), 7 (Nuc-FND) overlaps with the nucleus (blue) within human skin fibroblasts, and 8 (Mem-FND) overlaps with the cell membrane (cyan-green fluorescence). The Mandus coexistence coefficients quantified for this are 0.91±0.05 for 7 (Nuc-FND) and 0.67±0.10 for 8 (Mem-FND).

[0127] As a comparison to this, it can be seen that the Mandus coexistence coefficient of 1(FND) for the nucleus is 0.08±0.12 and the Mandus coexistence coefficient of 1(FND) for the cell membrane is 0.22±0.10.

[0128]

[0129] Figures 7 and 8 are diagrams illustrating the organelle temperature kinetics of human skin fibroblasts when an ATP synthesis inhibitor is administered using the organelle temperature measurement system according to Experimental Example 1 of the present invention.

[0130] Referring to Fig. 7(a), antibody-fluorescent nanodiamonds (Mito-FND, Nuc-FND, Mem-FND, concentration 5 μg / mL) bound to a mitochondrial targeting antibody (TOMM20), a nuclear targeting antibody (anti-Lamin B1), and a cell membrane targeting antibody (anti-CD98), respectively, were cultured in human skin fibroblasts (WS1) (37℃, 5% CO2, 24 hours) to prepare the sample.

[0131] Subsequently, an ATP synthesis inhibitor (FCCP, 50 μM) was added to the sample, and a laser light source (532 nm, 5.0 μW / cm²) and microwaves (-23 dBm) were applied to measure the change in the center frequency of the fluorescent nanodiamonds' ESR. Then, the temperature change (△T) of the organelle was estimated using the temperature sensitivity coefficient (△D / △T = 100.3 kHz / ℃) measured in the fixed cells (human skin fibroblasts) described later in Fig. 9.

[0132] As shown in Figure 7(a), the temperature change of Mito-FND was 8.91±0.78℃, the temperature change of Nuc-FND was 2.14±2.72℃, the temperature change of Mem-FND was 1.56±2.14℃, and the temperature change of FND was 1.43±1.62℃, indicating that the temperature change of Mito-FND was the largest. Accordingly, it can be seen that the main location for heat generation in human skin fibroblasts upon inhibition of ATP synthesis is the mitochondria.

[0133] Referring to Fig. 7(b), the results of comparing the latency from the time of injection of the ATP synthesis inhibitor until the temperature begins to rise while performing the experiment described in Fig. 7(a) are shown.

[0134] As can be seen in Figure 7(b), the average delay time of Mito-FND, Nuc-FND, and Mem-FND is in the range of about 15 to 25 minutes, and it can be seen that there is no significant difference between the organelles.

[0135] Referring to Fig. 7(c), the results of comparing the duration of the temperature change after the administration of the ATP synthesis inhibitor are shown while performing the experiment described in Fig. 7(a).

[0136] As can be seen in Figure 7(c), the average duration of temperature change for Mito-FND is the longest at 34.6±8.0 minutes, whereas the average duration of temperature change for Nuc-FND and Mem-FND is relatively short and unstable. Accordingly, it can be seen that mitochondria within human skin fibroblasts function as a source and reservoir of heat generation.

[0137] Referring to Figures 8 (a) to (c), while performing the experiment described in Figure 7 (a), the X, Y, and Z axis stack imaging functions of a confocal microscope were used after administering an ATP synthesis inhibitor to calculate the Mandus coexistence coefficients of Mito-FND, Nuc-FND, and Mem-FND relative to mitochondria within human skin fibroblasts, and the distance of each organelle from mitochondria was analyzed.

[0138] As can be seen in Figures 8 (a) to (c), the Mandus coexistence coefficients based on mitochondria are 0.98±0.01 for Mito-FND, 0.19±0.17 for Nuc-FND, and 0.26±0.04 for Mem-FND, respectively. That is, while mitochondria and Mito-FND practically overlap completely, Nuc-FND and Mem-FND show a significantly lower level of spatial overlap with mitochondria.

[0139] In addition, it can be seen that the nucleus is separated from the mitochondria by an average of several μm or more, and the cell membrane is located at a greater distance than the nucleus. Accordingly, since the nucleus and the cell membrane are separated from the mitochondria, which are the heat source, it can be seen that the temperature change of the nucleus and the cell membrane observed in Fig. 7 (a) is due to the indirect transfer effect of heat generated in the mitochondria.

[0140]

[0141] Figure 9 is an experimental result for explaining the method of calculating the temperature sensitivity coefficient of a fluorescent nanodiamond used in an organelle temperature measurement system according to Experimental Example 1 of the present invention.

[0142] Referring to Fig. 9, fixed cells containing fluorescent nanodiamonds (FND) were prepared, and the Electron Spin Resonance (ESR) spectrum of the fluorescent nanodiamonds was measured while controlling the external temperature of the fixed cells. Specifically, the fluorescent nanodiamonds were introduced into human skin fibroblasts in advance, and then the cells were fixed using paraformaldehyde, etc., so that the structure and position of the fluorescent nanodiamonds within the human skin fibroblasts did not change over time.

[0143] Subsequently, a heating device was attached to a culture vessel containing the fixed cells, and the ESR spectrum of fluorescent nanodiamonds was measured under each temperature condition while varying the temperature stepwise. At this time, fluorescent nanodiamonds were excited using a laser light source with a wavelength of 532 nm, and microwaves were applied to obtain ESR signals according to the spin state of the NV (Nitrogen-Vacancy) centers.

[0144] As shown in Fig. 9, the ESR center frequency (D) of the fluorescent nanodiamond was extracted from the ESR spectrum obtained under each temperature condition, and it was confirmed that the ESR center frequency gradually shifts as the external temperature increases. This is a result of the Zero-Field Splitting (ZFS) value occurring at the NV center of the fluorescent nanodiamond changing with temperature change.

[0145] In addition, when plotting the change in the ESR center frequency (△D) against the externally applied temperature change (△T), it can be seen that a linear correlation is formed between △D and △T. Accordingly, the temperature sensitivity coefficient (△D / △T) of the fluorescent nanodiamond contained in the fixed cell was derived as a linear relationship with a constant slope, and it can be seen that the average value is approximately -100.3 kHz / ℃.

[0146] Accordingly, this experimentally verified that the change in the center frequency of fluorescent nanodiamonds in a fixed cell environment responds linearly to changes in temperature, and the temperature sensitivity coefficient derived from this can be used as a reference value to convert the change in the center frequency of ESR into the actual change in temperature in subsequent organelle temperature measurement experiments performed in living cells.

[0147]

[0148] FIG. 10 is a diagram illustrating the intracellular introduction characteristics and organelle temperature measurement stability according to the concentration of antibody-fluorescent nanodiamond introduced into human skin fibroblasts (WS1) in the organelle temperature measurement system according to Experimental Example 1 of the present invention.

[0149] Referring to FIG. 10, antibody-fluorescent nanodiamonds conjugated with organelle-targeting antibodies at different concentrations were introduced into human skin fibroblasts (WS1), and then fluorescence imaging using a confocal microscope and center frequency measurement of ESR (Electron Spin Resonance) were performed as described in FIG. 3 to 9. At this time, the antibody-fluorescent nanodiamonds may be in a form conjugated with at least one of a mitochondrial-targeting antibody (TOMM20), a nuclear-targeting antibody (anti-Lamin B1), or a cell membrane-targeting antibody (anti-CD98), and the cell culture conditions were maintained at 37°C, 5% CO2, and for 24 hours. Specifically, in the experiment, the detection stability of the fluorescence signal within WS1 cells was analyzed while varying the concentration of the antibody-fluorescent nanodiamonds.

[0150] As shown in Figure 10, when the concentration of antibody-fluorescent nanodiamonds is 3 μg / mL or less, the number of antibody-fluorescent nanodiamonds introduced into WS1 cells is limited, resulting in weak detection of fluorescence signals at organelle locations or a tendency for continuous tracking of the ESR center frequency to become unstable. Consequently, it can be seen that a problem arises in which the reliability and reproducibility of measuring organelle temperature changes are reduced.

[0151] On the other hand, when the concentration of antibody-fluorescent nanodiamonds was 7 μg / mL or higher, excessive antibody-fluorescent nanodiamonds were introduced into the cells, showing a tendency for localized accumulation or aggregation, which resulted in the observation of fluorescence signal saturation or ESR spectrum distortion. In addition, changes in cell morphology or increased physiological stress were observed in some WS1 cells, indicating that these conditions are unsuitable for stable organelle temperature measurements over a long period.

[0152] In contrast, in the range where the concentration of antibody-fluorescent nanodiamonds exceeds 3 μg / mL and is less than 7 μg / mL (5 μg / mL), antibody-fluorescent nanodiamonds are uniformly introduced into WS1 cells, and clear fluorescent signals are observed at organelle-specific locations, while the temporal change of the ESR center frequency is maintained stably. Furthermore, within the above concentration range, as described in Figures 7 and 8, the analysis of the amount of temperature change, latency, and duration of temperature change per organelle could be performed reproducibly.

[0153] Accordingly, from the experimental results shown in FIG. 10, when applying the organelle temperature measurement system according to Experimental Example 1 of the present invention to WS1 cells, the concentration of antibody-fluorescent nanodiamond can be controlled to a range of more than 3 μg / mL and less than 7 μg / mL. In this concentration range, the organelle-specific introduction efficiency of antibody-fluorescent nanodiamond, the stability of fluorescence and ESR signals, and the reliability of organelle temperature measurement can be ensured in balance.

[0154]

[0155] Although the present invention has been described in detail using preferred embodiments, the scope of the invention is not limited to specific embodiments and should be interpreted by the appended claims. Furthermore, those skilled in the art will understand that many modifications and variations are possible without departing from the scope of the invention.

[0156] The organelle-specific temperature measurement method and temperature measurement system using fluorescent nanodiamonds according to the present invention provide a technology capable of quantitatively analyzing temperature changes in organelles such as mitochondria, the nucleus, or the cell membrane in a cellular environment with high reliability, and can be usefully applied in various life science and biomedical fields such as cell metabolism research, disease diagnosis, and drug response evaluation.

Claims

1. A step of manufacturing an antibody-fluorescent nanodiamond by conjugating an antibody to a fluorescent nanodiamond; A step of preparing a sample by culturing the antibody-fluorescent nanodiamond in cells; A step of preparing a first source in which a cell fixation solution is provided to the sample, and a second source in which an ATP (Adenosine Triphosphate) synthesis inhibitor is provided to the sample; A method for measuring the change in the center frequency of the first ESR (Electron Spin Resonance) of the antibody-fluorescent nanodiamond bound to the organelle of the cell by providing a test signal while varying the temperature of the first source, comprising the step of obtaining a temperature sensitivity coefficient; A step of providing a test signal to the second source to measure the change in the second ESR center frequency of the antibody-fluorescent nanodiamond bound to the organelle of the cell; and A method for measuring organelle temperature, comprising the step of estimating the temperature of an organelle bound to the antibody-fluorescent nanodiamond in the second source by calculating the amount of temperature change for the organelle bound to the antibody-fluorescent nanodiamond in the second source using the temperature sensitivity coefficient and the amount of change in the second ESR center frequency.

2. In Paragraph 1, The above fluorescent nanodiamond contains NV centers (Nitrogen-Vacancy Centers), and A method for measuring organelle temperature comprising having an activated ester group (N-hydroxysuccinimide ester) formed on the fluorescent nanodiamond.

3. In Paragraph 2, In the step of manufacturing the above antibody-fluorescent nanodiamond, The method comprises reacting the fluorescent nanodiamond with the antibody to form a covalent bond between the activated ester group of the fluorescent nanodiamond and the amine group (NH2) of the protein of the antibody. A method for measuring organelle temperature, wherein the above antibody comprises any one of a mitochondrial target antibody, a nuclear target antibody, or a cell membrane target antibody.

4. In Paragraph 1, In the step of preparing the above sample, The method comprises preparing the sample by providing the antibody-fluorescent nanodiamond dispersed in a PBS solution and culturing the cells after culturing them in a culture medium. A method for measuring organelle temperature, wherein the above medium comprises DMEM (Dulbecco's Modified Eagle's Medium), 10% FBS (fetal bovine serum), and 1% penicillin / streptomycin.

5. In Paragraph 4, In the process of preparing the above sample, The protein of the organelle of the cell and the antibody of the antibody-fluorescent nanodiamond are included in antigen-antibody binding, The above cells include human skin fibroblasts, A method for measuring organelle temperature, wherein the above antibody comprises any one of a mitochondrial target antibody, a nuclear target antibody, or a cell membrane target antibody.

6. In Paragraph 5, In the case where the above antibody is a mitochondrial-targeting antibody, the Manders' coefficient of the antibody-fluorescent nanodiamond localized to the mitochondria, which are organelles of the cell, is 0.98 ± 0.01, and In the case where the above antibody is a nuclear target antibody, the Manders' coefficient of the antibody-fluorescent nanodiamond localized to the nucleus, an organelle of the cell, is 0.91 ± 0.05, and A method for measuring organelle temperature, comprising, when the antibody is a cell membrane target antibody, that the Manders' coefficient of the antibody-fluorescent nanodiamond localized to the cell membrane of the cell is 0.67 ± 0.

10.

7. In Paragraph 4, A method for measuring organelle temperature, comprising controlling the concentration of the antibody-fluorescent nanodiamond in the PBS solution to be greater than 3 μg / mL and less than 7 μg / mL.

8. In Paragraph 1, The above sample includes a culture medium, and The above medium comprises DMEM (Dulbecco's Modified Eagle's Medium), 10% FBS (fetal bovine serum), and 1% penicillin / streptomycin, and The step of preparing the first source comprises removing the medium from the sample before providing the cell fixation solution to the sample, and A method for measuring organelle temperature, comprising physically fixing the organelles of the cell by the cell fixation solution.

9. In Paragraph 1, The above ATP synthesis inhibitor comprises FCCP (Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone), and A method for measuring organelle temperature, comprising controlling the concentration of the above ATP synthesis inhibitor to 50 μM.

10. In Paragraph 1, The above test signal includes a continuous laser light source and microwaves, and The wavelength of the above laser light source is 532 nm, and the intensity is 5.0 μW / cm² 2 Includes, A method for measuring organelle temperature, comprising that the output of the microwave is -23dBm.

11. In Paragraph 1, The above cells include human skin fibroblasts, The organelles of the cell mentioned above include mitochondria, a nucleus, and a cell membrane, and The above antibody includes any one of a mitochondrial targeting antibody, a nuclear targeting antibody, or a cell membrane targeting antibody, and A method for measuring organelle temperature, including the fact that among the organelles of the cell above, the temperature change of mitochondria is the largest.

12. Culture chamber; A culture vessel containing a source containing a sample in which antibody-fluorescent nanodiamonds are cultured in cells inside the culture chamber; A laser light source that photoexcites the antibody-fluorescent nanodiamond within the above source; A microwave application unit for providing microwaves to induce electron spin resonance of the antibody-fluorescent nanodiamond within the above source; A detector that receives a fluorescence signal emitted from the antibody-fluorescent nanodiamond excited by the laser light source; An analysis unit that calculates a change in the center frequency of the Electron Spin Resonance (ESR) of the antibody-fluorescent nanodiamond based on the fluorescent signal received from the detection unit, and derives a temperature change from the change in the center frequency of the ESR; and An organelle temperature measurement system comprising a correction control unit for correcting the position change of the antibody-fluorescent nanodiamond.

13. In Paragraph 12, The correction control unit detects a change in the position of the antibody-fluorescent nanodiamond using a temporal change in the fluorescence signal received from the detection unit, and A piezo stage positioned at the bottom of the culture vessel to control displacement so that the antibody-fluorescent nanodiamond can be analyzed at a preset observation position according to a detected position change is included. An organelle temperature measurement system comprising a correction control unit that is signal-linked with an analysis unit, wherein a change in the ESR center frequency of the antibody-fluorescent nanodiamond is calculated when position correction is performed.

14. In Paragraph 13, The above piezo stage includes being operated in real time, and A system for measuring organ temperature, comprising the above-mentioned piezo stage being a 3-axis piezo stage capable of driving independently in the x-axis, y-axis, and z-axis directions.

15. In Paragraph 12, The above sample comprises a method of preparing the above sample by providing and culturing the above antibody-fluorescent nanodiamond dispersed in a solution in PBS after culturing the above cells in the medium of the above culture vessel. The above medium comprises DMEM (Dulbecco's Modified Eagle's Medium), 10% FBS (fetal bovine serum), and 1% penicillin / streptomycin, and An organelle temperature measuring system comprising, wherein the source within the culture vessel comprises a first source in which a cell fixation solution is provided after removing the medium from the sample, and a second source in which an ATP (Adenosine Triphosphate) synthesis inhibitor is provided to the sample.

16. In Paragraph 15, An organelle temperature measuring system comprising the concentration of the antibody-fluorescent nanodiamond in the PBS solution being greater than 3 μg / mL and less than 7 μg / mL.

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