Intracellular quantum measurement device, intracellular quantum measurement method, and cell holding device

Abstract

The present invention achieves a quantometry technique that not only can measure the intracellular state, but also overcomes the disadvantages of quantometry using nanoparticles. The intracellular quantum measurement device (1) is provided with a substrate (10), a nanorod (11) into which a cell (C) is inserted is formed on the surface of the substrate (10), and the tip of the nanorod (11) includes a quantum inductor.

Description

Technical Field

[0001] The present invention relates to an intracellular quantum measurement device and an intracellular quantum measurement method for measuring the intracellular state using a quantum sensor. In addition, the present invention relates to a cell holding device including the intracellular quantum measurement device. Background Art

[0002] When a nitrogen atom exists in a diamond crystal, a vacancy is formed beside the nitrogen atom. At this time, the center composed of the nitrogen atom and the vacancy, that is, the NV (Nitrogen-Vacancy) color center, has the characteristics that its quantum state changes with the surrounding environment such as electric field, magnetic field, and temperature, and that this quantum state can be read by fluorescence measurement. Therefore, the NV color center can be used as a quantum sensor for detecting its surrounding environment. As for the fluorescence microscope used for fluorescence measurement, for example, the fluorescence microscopes described in Patent Documents 1 and 2 are known.

[0003] Such a quantum sensor can be used to measure the cell state. For example, regarding diamond nanoparticles containing an NV color center, if they are introduced into cells, the intracellular state can be measured, so they are expected to be effectively used as "a new generation of biochemical markers". In addition, regarding a diamond nanofilm containing an NV color center, if it is attached to the cell membrane, the state of the cell membrane can be measured, so it is expected to be effectively used as "a new generation of coverslips".

[0004] (Prior Art Documents)

[0005] [Patent Documents]

[0006] Patent Document 1: Japanese Patent No. 5476206

[0007] Patent Document 2: Japanese Patent No. 6117812 Summary of the Invention

[0008] (Problems to be Solved by the Invention)

[0009] However, in the case of quantum measurement using nanoparticles (a new generation of biochemical markers), although it has the advantage of being able to measure the intracellular state, it also has the following disadvantages: (1) It is difficult to strictly set the measurement environment; (2) The luminescence intensity is weak; (3) The spin characteristics are poor; (4) The characteristics of each quantum sensor (NV color center) are not uniform. The main reason for these disadvantages is that the position and orientation of the quantum sensor cannot be fixed. As a result, for example, in terms of the magnetic field applied to the quantum sensor, it is difficult to precisely adjust the magnetic field direction, and in terms of the excitation light irradiated on the quantum sensor, it is difficult to optimally select its pulse shape, etc. Therefore, as a quantum measurement method, the DC measurement method (a measurement method that captures the change in luminescence intensity using the luminescence state) is often used.

[0010] On the other hand, regarding quantum measurement using diamond thin films (new generation cover glasses), it has the following advantages: (1) it is easy to strictly set the measurement environment; (2) it has strong luminescence intensity; (3) it has good spin characteristics; (4) the characteristics of each quantum sensor (NV color center) are uniform. However, it has the disadvantage that it is impossible to measure the intracellular state. The reasons for these advantages mainly lie in the ability to fix the position and orientation of the quantum sensors. As a result, for example, in terms of the magnetic field applied to the quantum sensors, the direction can be easily and precisely adjusted, and in terms of the excitation light irradiated to the quantum sensors, the optimal pulse shape can be easily selected. Therefore, as a quantum measurement method, the AC measurement method (a measurement method that uses the superposition state to capture the phase change of the quantum energy level) is often used.

[0011] One aspect of the present invention is completed in view of the above problems, and its purpose is to realize a quantum measurement technology that can measure the intracellular state and overcome the disadvantages of quantum measurement using nanoparticles.

[0012] (Technical means for solving the problem)

[0013] To solve the above problems, an intracellular quantum measurement device according to one aspect of the present invention is characterized in that: it includes a substrate, and nano-columns for inserting cells are formed on the surface of the substrate, and the front end portion of the nano-column contains a quantum sensor.

[0014] In addition, to solve the above problems, an intracellular quantum measurement method according to one aspect of the present invention is characterized by including: using a substrate on which nano-columns are formed, and performing quantum measurement in a state where the nano-columns are inserted into cells, wherein the front end portion of the nano-column contains a quantum sensor.

[0015] (Effect of the invention)

[0016] According to one aspect of the present invention, it is possible to realize a quantum measurement technology that can measure the intracellular state and overcome the disadvantages of quantum measurement using nanoparticles. Description of the drawings

[0017] Figure 1 Among them, (a) is a top view of an intracellular quantum measurement device according to an embodiment of the present invention, and (b) is a cross-sectional view of the intracellular quantum measurement device.

[0018] Figure 2 Among them, (a) is a top view of a cell holding device including Figure 1 the intracellular quantum measurement device shown, and (b) is a cross-sectional view of the cell holding device.

[0019] Figure 3 is a diagram including Figure 2Structural diagram of the intracellular quantum measurement system of the cell holding device shown.

[0020] Figure 4 is Figure 3 Schematic diagram of an embodiment of the intracellular quantum measurement system shown. (a) is an image obtained by photographing the surface of the intracellular quantum measurement device. (b) is an image obtained by photographing the nanocolumns of the intracellular quantum measurement device. (c) and (d) are images obtained by photographing Hepa1-6 cells cultured in the cell holding device.

[0021] Figure 5 is Figure 3 Graph of an embodiment of the intracellular quantum measurement system shown, showing the τ-dependence of the normalized contrast obtained by Rabi measurement.

[0022] Figure 6 is Figure 3 Graph of an embodiment of the intracellular quantum measurement system shown, showing the τ-dependence of the normalized contrast obtained by NMR measurement.

[0023] Figure 7 is Figure 3 Graph of an embodiment of the intracellular quantum measurement system shown, showing the time-dependence of the normalized contrast.

[0024] Figure 8 In, (a) is a schematic diagram of the nanocolumn, and (b) is an enlarged schematic diagram of the quantum sensor (NV color center) disposed at the front end of the nanocolumn.

[0025] Figure 9 is for Figure 8 Schematic diagram of the pulse sequence for quantum manipulation of the quantum sensor and free radicals near the quantum sensor with respect to the nanocolumn.

[0026] Figure 10 is using Figure 9 Graph of the result of an embodiment obtained using the pulse sequence, showing the Tau-dependence of the contrast obtained by performing ESR measurement using double quantum manipulation. Detailed implementation mode

[0027] (Intracellular quantum measurement device)

[0028] Combined with Figure 1 , the intracellular quantum measurement device 1 of an embodiment of the present invention will be described. In Figure 1 , (a) is a top view of the intracellular quantum measurement device 1, and (b) is a cross-sectional view of the intracellular quantum measurement device 1. Figure 1 The (b) of shows the AA' line of the intracellular quantum measurement device 1 (see Figure 1in cross-section (a))

[0029] The intracellular quantum measurement device 1 includes: a substrate 10 on the surface of which a plurality of nanocolumns 11 are formed. The nanocolumns 11 are columnar or conical structural remnants for inserting into cells C (in this embodiment, a structure with a conical root and a cylindrical front end). The front end 11a of each nanocolumn 11 includes a quantum sensor. The nanocolumns 11 are regularly arranged on the surface of the substrate 10 (in this embodiment, arranged at the lattice points of a square lattice).

[0030] In this embodiment, the substrate 10 and the nanocolumns 11 are composed of diamond crystals. In addition, in this embodiment, the front end 11a of the nanocolumn 11 contains an NV color center composed of one nitrogen atom and one vacancy adjacent to the nitrogen atom, and this NV color center is used as a quantum sensor. Here, in order to accurately adjust the magnetic field applied to the quantum sensor, the

[111] direction of the diamond crystal is controlled to be consistent with the normal direction of the surface of the substrate 10.

[0031] Regarding the height H of the nanocolumn 11 and the diameter D of the front end 11a, based on the physical requirement of sufficiently improving the fluorescence excitation efficiency and the biological requirement of sufficiently reducing the toxicity to cells, the height H and the diameter D are determined in such a way that intracellular measurement can be achieved (for example, it can penetrate the cell membrane and adhere to cells including floating cells, etc.).

[0032] From the above viewpoints, the height H of the nanocolumn 11 may be 1 μm or more and 20 μm or less, preferably 1 μm or more and 5 μm or less, more preferably 2 μm or more and 4 μm or less, and further preferably 2.5 μm or more and 3.5 μm or less. In this embodiment, the height H of the nanocolumn 11 is set to 3.2 μm.

[0033] In addition, from the above viewpoints, the diameter D of the front end 11a of the nanocolumn 11 may be 100 nm or more and 1000 nm or less, preferably 100 nm or more and 500 nm or less, more preferably 200 nm or more and 400 nm or less, and further preferably 250 nm or more and 350 nm or less. In this embodiment, the diameter D of the front end 11a of the nanocolumn 11 is set to 300 nm.

[0034] In addition, from the viewpoint of sufficiently improving the fluorescence excitation efficiency, the thickness T of the substrate 10 is preferably 50 μm or less. In this embodiment, the thickness T of the substrate 10 is set to 40 μm.

[0035] In addition, the interval D (distance between centers) of the adjacent nano-columns 11 is set to be about 1 / 2 of the size of the cell to be measured, so as to increase the probability that a certain nano-column 11 is inserted into the cells cultured in the cell holding device 2 described later.

[0036] From the above viewpoints, the interval D between the adjacent nano-columns 11 is preferably 10 μm or more and 60 μm or less. For example, if the cell to be measured is a somatic cell of a mammal (size about 20 μm to 50 μm), the interval D between the adjacent nano-columns 11 is more preferably 10 μm or more and 25 μm or less. In addition, if the cell to be measured is a germ cell of a mammal (size about 50 μm to 80 μm), the interval D between the adjacent nano-columns 11 is more preferably 25 μm or more and 40 μm or less. In addition, if the cell to be measured is a plant cell (size about 80 μm to 120 μm), the interval D between the adjacent nano-columns 11 is more preferably 40 μm or more and 60 μm or less.

[0037] According to the intracellular quantum measurement device 1 of the present embodiment, quantum measurement can be performed in a state where the front end portion 11a of the nano-column 11 is inserted into the interior of the cell C. Here, quantum measurement means: determining the quantum state of the quantum sensor by measuring the fluorescence emitted from the quantum sensor, and estimating the state around the quantum sensor based on the determined quantum state. In the present embodiment, estimating the state around the quantum sensor means estimating the internal state of the cell C.

[0038] It should be noted here that the nano-columns 11 are not suspended in the cell isolatedly like nano-particles, but are formed on the surface of the substrate like a nano-film. Therefore, similar to the quantum measurement using a diamond film, the AC measurement method can be applied, and high-sensitivity spin analysis techniques such as NMR or ESR can be used to precisely measure the internal state of the cell C. That is, the internal state of the cell C can be measured while overcoming the disadvantages of the quantum measurement using nano-particles.

[0039] Here, although the scheme of forming a plurality of nano-columns 11 on the surface of the substrate 10 is adopted in the present embodiment, the present invention is not limited thereto. That is, a scheme of forming a single nano-column 11 on the surface of the substrate 10 can also be adopted. However, by adopting the scheme of forming a plurality of nano-columns 11 on the surface of the substrate 10 as in the present embodiment, a better effect of simultaneously performing quantum measurement on a plurality of cells (for example, a plurality of cells constituting a living tissue) can be obtained. Regarding such simultaneous measurement, it can be performed in a state where the front end portions of the plurality of nano-columns 11 are inserted into the interiors of the plurality of cells respectively.

[0040] 〔Cell holding device〕

[0041] CombineFigure 2 , to illustrate the cell holding device 2 including the intracellular quantum measurement device 1 described above. Figure 2 Among them, (a) is a top view of the cell holding device 2, and (b) is a cross-sectional view of the cell holding device 2. Figure 2 The (b) of shows the cross-section of the BB' line (see Figure 2 the (a) of ) of the cell holding device 2.

[0042] The cell holding device 2 includes a first substrate 21, a second substrate 22, and a third substrate 23. The first substrate 21 is a substrate that functions as a cover glass. The material of the first substrate 21 is arbitrary, but for example, it can be an insulator (dielectric) through which the excitation light irradiated to the quantum sensor and the fluorescence emitted from the quantum sensor can pass. The second substrate 22 is a substrate formed with an opening 221 that functions as a chamber, and it is laminated on the first substrate 21. The material of the second substrate 22 is arbitrary, but for example, it can be an insulator (dielectric) with low thermal conductivity. The third substrate 23 is a substrate that functions as a chamber cover, and it is laminated on the second substrate 22 after the liquid 26 containing cells (pure water in this embodiment) is injected into the opening 221. The material of the third substrate 23 is arbitrary, but for example, it can be an insulator (dielectric) with low thermal conductivity. In this embodiment, quartz glass is used as the material of the first substrate 21, the second substrate 22, and the third substrate 23.

[0043] The cell holding device 2 further includes the intracellular quantum measurement device 1 (the substrate 10 formed with the nanocolumns 11), a first electrode 24a, and a second electrode 24b. The intracellular quantum measurement device 1 is laminated on the first substrate 21 in such a way that it is inside the opening 221. Here, the intracellular quantum measurement device 1 is laminated on the first substrate 21 with the back surface of the substrate 10 where the nanocolumns 11 are not formed in surface contact with the front surface of the first substrate 21. In addition, the intracellular quantum measurement device 1 can also be fixed on a frame for easy manipulation. In this case, the intracellular quantum measurement device 1 is fixed on the first substrate 21 via this frame.

[0044] The first electrode 24a and the second electrode 24b are respectively strip conductors and are laminated on the first substrate 21. The first electrode 24a is laminated on the first substrate 21 with its outer edge along the first side of the intracellular quantum measurement device 1. The second electrode 24b is laminated on the first substrate 21 with its outer edge along the second side (the opposite side of the first side) of the intracellular quantum measurement device 1. The first electrode 24a and the second electrode 24b are short-circuited to each other through a wire 24c that diagonally crosses the intracellular quantum measurement device 1. When high-frequency current is input to the first electrode 24a and the second electrode 24b, the wire 24c functions as an antenna to emit the electromagnetic wave to be applied to the quantum sensor.

[0045] Openings 222a and 232a are respectively formed on the second substrate 22 and the third substrate 23 so as to enable access to the first electrode 24a from the top surface side of the cell holding device 2. Similarly, openings 222b and 232b are respectively formed on the second substrate 22 and the third substrate 23 so as to enable access to the second electrode 24b from the top surface side of the cell holding device 2. Thus, the high-frequency current source can be easily connected to the first electrode 24a and the second electrode 24b.

[0046] Here, the width W of the first electrode 24a and the second electrode 24b is preferably 0.1 mm or more and 500 mm or less. Thus, an electromagnetic field (electromagnetic wave) with a wide frequency band from several MHz to several GHz can be applied to the cells in the liquid 26. In this embodiment, an electromagnetic field (electromagnetic wave) from 1 MHz to 120 GHz is envisaged, so the width W of the first electrode 24a and the second electrode 24b is set to 2.6 mm.

[0047] In addition, the sum T + U of the thickness U of the first substrate 21 of the cell holding device 2 and the thickness T of the substrate 10 of the intracellular quantum measurement device 1 is preferably 0.3 mm or less. Thus, an objective lens widely used in cell fluorescence observation can be used to irradiate the excitation light and detect the fluorescence. In this embodiment, an objective lens with a working distance Wd of 0.3 mm is assumed. Therefore, the thickness U of the first substrate 21 of the cell holding device 2 and the thickness T of the substrate 10 of the intracellular quantum measurement device 1 are set so that the sum T + U is 0.21 mm.

[0048] If the cell holding device 2 of this embodiment is used, the nanocolumn 11 of the intracellular quantum measurement device 1 can be inserted into the interior of the cells cultured in the liquid 26, so that the internal state of the cells can be easily measured. Moreover, by inputting high-frequency current from the high-frequency current source to the first electrode 24a and the second electrode 24b, the internal state of the cells under the electromagnetic field (electromagnetic wave) can be easily measured.

[0049] Here, the first electrode 24a and the second electrode 24b may not be short-circuited to each other. In this case, when a voltage is applied between the first electrode 24a and 24b, an electric field corresponding to the voltage can be applied to the quantum sensor.

[0050] 〔Intracellular Quantum Measurement System〕

[0051] Combined Figure 3 , the intracellular quantum measurement system 3 including the above cell holding device 2 will be described. Figure 3 is a structural diagram of the intracellular quantum measurement system 3.

[0052] The intracellular quantum measurement system 3 includes a cell holding device 2, a housing 30, a light emitting unit 31, a light receiving unit 32, a single photon counter 33, first to sixth lenses L1 to L6, first to third mirrors M1 to M3, and a pinhole P. The intracellular quantum measurement system 3 has a wide field microscope mode and a confocal microscope mode. To switch between these modes, the first lens L1 and the third mirror M3 are movable.

[0053] The housing 30 is used to house the first to third lenses L1 to L3 and the first to third mirrors M1 to M3. In the present embodiment, as the housing 30, the housing of an existing inverted microscope is used.

[0054] The light emitting unit 31 is used to emit excitation light irradiated to the quantum sensor. In the present embodiment, as the light emitting unit 31, an LED that can emit a laser with a wavelength of 532 nm is used.

[0055] In addition, in the wide field microscope mode, the first lens L1 is placed on the optical path of the excitation light, and the third mirror M3 is deviated from the optical path of the fluorescence. Therefore, the excitation light (collimated light) from the light emitting unit 31 is converted into convergent light by the first lens L1, and then reflected by the first mirror M1 and incident on the second lens L2. The second lens L2 converts the excitation light as convergent light into collimated light and irradiates it onto the intracellular quantum measurement device 1 (more precisely, the surface of the substrate 10) in the cell holding device 2 in a planar manner. The fluorescence (divergent light) from the intracellular quantum measurement device 1 is collimated by the second lens L2, and then reflected by the second mirror M2. The fluorescence reflected by the second mirror M2 is condensed by the third lens L3 and incident on the light receiving unit 32.

[0056] In the confocal microscope mode, the first lens L1 is deviated from the optical path of the excitation light, and the third mirror M3 is placed on the optical path of the fluorescence. Therefore, the excitation light (collimated light) from the light emitting unit 31 is reflected by the first mirror M1 and incident on the second lens L2. The second lens L2 converts the excitation light as collimated light into convergent light and irradiates it onto the intracellular quantum measurement device 1 (more precisely, the surface of the substrate 10) in the cell holding device 2 in a point-like manner. The fluorescence (divergent light) from the intracellular quantum measurement device 1 is collimated by the second lens L2, and then reflected by the third mirror M3. The fluorescence reflected by the third mirror M3 is converted into convergent light by the fourth lens L4, passes through the pinhole P, and is collimated by the fifth lens L5. Then it is condensed by the sixth lens L6 and incident on the single photon counter 33.

[0057] Here, the first mirror M1 needs to both reflect the excitation light and transmit the fluorescence. Therefore, in the present embodiment, a dichroic mirror is used as the first mirror M1. Although the fluorescence from the intracellular quantum measurement device 1 and the excitation light reflected by the cell holding device 2 are both incident on the first mirror M1, the excitation light is reflected by the first mirror M1 and thus is not guided to the light receiving part 32.

[0058] The light receiving part 32 is a structure for receiving the fluorescence emitted from the quantum sensor in the wide-field microscope mode. In the present embodiment, a high-sensitivity imaging camera is used as the light receiving part 32. Specifically, an EMCCD (Electron Multiplying Charge Coupled Device) is used. The single-photon sensor 33 is a structure for receiving the fluorescence emitted from the quantum sensor in the confocal microscope mode. In the present embodiment, a 34APD (Avalanche Photodiode) is used as the single-photon sensor 33.

[0059] Here, when measuring the intracellular state using the intracellular quantum measurement system 3, in each measurement cycle, the light emitting part 31 outputs one pulse of excitation light, and the light receiving part 32 detects one pulse of fluorescence output from the quantum sensor in response to this excitation light. The time required for implementing each measurement cycle is about 5 μs. On the other hand, the frame rate of the image output from the high-sensitivity imaging sensor is about 50 frames / second. That is, the time required to obtain one image from the high-quality imaging sensor (hereinafter, also referred to as the "reading cycle") is about 20 ms, which is about 4000 times the time required for implementing each measurement cycle. In response to this, the intracellular quantum measurement system 3 adopts a scheme in which, in each reading cycle, N measurement cycles (N is a natural number of 2 or more, and N in the present embodiment is about 1000) are executed to make the equivalent sensitivity of the high-sensitivity imaging sensor N times.

[0060] According to the intracellular quantum measurement system 3 of the present embodiment, it is possible to balance the wide-field measurement in the wide-field microscope mode and the high-resolution / high-sensitivity measurement in the confocal microscope mode. Moreover, according to the intracellular quantum measurement system 3 of the present embodiment, it is possible to easily switch between the wide-field microscope mode and the confocal microscope mode without contacting the measurement object.

[0061] Here, the intracellular quantum measurement system 3 may further include a magnetic field defining device 34. In quantum measurement, it is necessary to adjust the direction of the magnetic field acting on the quantum sensor with an accuracy of 10 degrees or less. By using the magnetic field defining device 34, the direction of the magnetic field acting on the quantum sensor can be adjusted with high precision with respect to four directions (3-axis directions + rotation direction). Such adjustment is achieved, for example, through the following process: while changing the direction of the magnetic field using the magnetic field defining device 34, ODMR (Optically Detected Magnetic Resonance) measurement is performed, and the direction of the magnetic field is determined with reference to the contrast and fluorescence intensity as the measurement results. Thus, for example, in structural analysis such as NMR or ESR, a high magnetic field important for achieving high frequency resolution can be applied with high precision.

[0062] 〔Supplementary Explanation of Effects〕

[0063] According to the intracellular quantum measurement system 3, it is possible to perform optimized fluorescence excitation in a state where the quantum measurement material is introduced into the biological measurement sample in various forms. In addition, by using a general-purpose computer (not shown), ODMR (Optically Detected Magnetic Resonance) measurement can be performed on the basis of precisely controlling the environment such as the magnetic field direction, temperature, and humidity. Here, ODMR measurement refers to a measurement method in which while changing the frequency of the microwave, excitation light is irradiated and the fluorescence intensity is measured, thereby measuring the change in the quantum state of the quantum sensor. In this case, the intracellular quantum measurement device 1 is used to introduce the quantum sensor into the cell, the cell holding device 2 is used to apply the microwave to the quantum sensor introduced into the cell, and the intracellular quantum measurement system 3 is used to detect the intensity of the fluorescence emitted from the quantum sensor introduced into the cell. In addition, in ODMR measurement, in order to control the irradiation timing of the excitation light and the application timing of the microwave, and in order to calculate the quantum state of the quantum sensor based on the detected fluorescence intensity, a computer (not shown) is used.

[0064] In addition, by performing microwave application and fluorescence intensity detection with a time resolution of 400 picoseconds or less, advanced quantum manipulations such as rabi or echo can be achieved. In addition, by increasing the number of microwaves to be applied to the spin measurement of the microdomain and controlling the spins other than the quantum sensor, DEER (Double Electron Electron Resonance) can be achieved.

[0065] 〔Examples〕

[0066] The inventor performed intracellular quantum measurement of Hepa1-6 cells using the intracellular quantum measurement system 3. As the intracellular quantum measurement device 1, a device was used in which nanocolumns 11 with a height of 3.2 μm and a diameter of 300 nm were regularly formed at 15-μm intervals on the surface of a substrate with a thickness of 40 μm.

[0067] Figure 4 (a) in [reference] is an image obtained by photographing the surface of the intracellular quantum measurement device 1. Figure 4 (b) in [reference] is an image obtained by photographing the nanocolumns 11 of the intracellular quantum measurement device 1. Figure 4 (c) and (d) in [reference] are images obtained by photographing Hepa1-6 cells cultured in the cell holding device 2. It should be noted that Hepa1-6 cells were previously stained with a cell membrane staining reagent. As the cell membrane staining reagent, Cell Mask (registered trademark) Green was used. From Figure 4 (c) and (d) in [reference], it can be seen that the nanocolumns 11 are inserted into Hepa1-6 cells.

[0068] The inventor performed Rabi measurement using the intracellular quantum measurement system 3 in a state where the nanocolumns 11 were inserted into cells. Figure 5 ([reference]) is a graph showing the τ-dependence of the normalized contrast obtained by this Rabi measurement. Here, the normalized contrast is a dimensionless quantity that expresses the electron spin state transition under microwave control (|0>, |±1> when using the NV color center as a qubit). In addition, τ is a quantity representing the microwave application time, and the unit is n seconds. From Figure 5 the graph shown in [reference], it can be seen that the electron spin state transition corresponding to the microwave application time was achieved. Thus, it was confirmed that quantum control inside cells can be performed with high contrast.

[0069] In addition, the inventor performed NMR measurement using the intracellular quantum measurement system 3 in a state where the nanocolumns 11 were inserted into cells. Figure 6 ([reference]) is a graph showing the τ-dependence of the normalized contrast obtained by this NMR measurement. From Figure 6 the graph shown in [reference], it can be seen that in the region where τ is 164 n seconds or more and 171 n seconds or less, a peak (dip) corresponding to the core spin existing inside the cell appears. Such a peak (dip) does not appear even when performing NMR measurement in a state where the nanocolumns 11 are not inserted into cells. Thus, it was confirmed that quantum measurement of the intracellular state can be achieved using the nanocolumns 11.

[0070] In addition, the inventor measured the time dependence of the normalized contrast using the intracellular quantum measurement system 3 in a state where the nanocolumns 11 were inserted into cells.Figure 7 It is a graph showing the time-dependence of the normalized contrast obtained from this measurement. From Figure 7 the shown graph, it can be seen that the coherence time (T2) is 15.9 μs. Thus, for the quantum sensor included at the front end of the nanorod 11 inserted into the cell, it was confirmed that its quantum property can be maintained for a sufficiently long time.

[0071] As described above, the inventors made it possible to detect the core spins in the intracellular microdomain by using the nanorod 11. In addition, the inventors performed quantum manipulation of the electron spins near the quantum sensor in addition to the quantum manipulation of the quantum sensor. Details will be described below.

[0072] The inventors performed manipulation (dual quantum manipulation) on the quantum state of the quantum sensor (NV color center) inserted into the cell and the electron spins (quantum states) of the molecules, atoms, ions, or free radicals (hereinafter referred to as "free radicals, etc.") existing around the front end portion 11a (quantum sensor) of the nanorod 11 inserted into the cell.

[0073] Figure 8 In (a) of, it is a schematic diagram of the nanorod 11, and (b) is an enlarged schematic diagram of the quantum sensor (NV color center) disposed at the front end portion 11a of the nanorod 11. In this embodiment, the height H of the nanorod 11 is 3.2 μm, and the diameter D of the front end portion 11a of the nanorod 11 is 300 nm. The NV color center is disposed at a position about 10 nm deep from the surface of the front end portion 11a of the nanorod 11, whereby quantum manipulation of the surface free radicals near the front end portion 11a (quantum sensor) can be performed. Here, the periphery of the aforementioned front end portion 11a includes peripheral portions such as the bulging curved surface of the foremost end face constituting the front end portion 11a and the outer peripheral surface of the cylindrical side surface constituting the front end portion 11a.

[0074] Figure 9 is for Figure 8 When performing quantum manipulation on the quantum sensor and free radicals, etc. near the quantum sensor with the nanorod 11, it is a schematic diagram of the sequence. Sequence SQ1 shows the pulse sequence when applying microwaves to the quantum sensor (NV color center), and sequence SQ2 shows the pulse sequence when applying microwaves to free radicals, etc. (their electron spins). Sequence SQ3 shows the pulse sequence during laser reading.

[0075] It can be considered that these sequences SQ1 to SQ3 respectively correspond to the following processes (1) to (3). That is, by processes (1) to (3), an intracellular quantum measurement method can be constructed.

[0076] (1) First quantum manipulation process: A pulse of a first electromagnetic wave (e.g., microwave) is applied to a quantum sensor (NV color center) to manipulate the quantum state of the quantum sensor.

[0077] (2) Second quantum manipulation process: A pulse of a second electromagnetic wave (e.g., microwave) having a frequency different from that of the first electromagnetic wave is applied to intracellular free radicals or the like (at least one of molecules, atoms, ions, free radicals) present around the front end portion 11a to manipulate the quantum state (electronic state) of the free radicals or the like.

[0078] (3) State detection process: An excitation light (e.g., laser pulse) is irradiated onto the quantum sensor and the free radicals or the like, and fluorescence from the quantum sensor and the free radicals or the like is received.

[0079] In sequence SQ1, microwave pulses are applied to the NV color center at a time interval τ, thereby switching the state (π / 2, π) of the NV color center. In sequence SQ2, microwave pulses with a spin manipulation time (pulse width) Tau are applied to external electron spins such as free radicals. As described above, these microwaves (electromagnetic waves) are applied from the wire 24c (antenna).

[0080] In sequence SQ3, a laser (excitation light) with a pulse width of, for example, 300 to 500 ns is irradiated, and fluorescence from the quantum sensor and the free radicals or the like is received. As described above, the excitation light is irradiated from the light emitting unit 31, and the fluorescence is received by the single photon counter 33 or the light receiving unit 32.

[0081] Sequences SQ1 to SQ3 can be controlled independently of each other. By controlling the timing of sequences SQ1 to SQ3, the electron spin manipulation of free radicals or the like near the quantum sensor can be confirmed.

[0082] Figure 10 is a graph showing the results of an embodiment obtained using Figure 9 a pulse sequence, and shows the Tau dependence of the contrast obtained by performing ESR measurement using double quantum manipulation. Comparing the fitting curves Fit1 and Fit2, the intensities of the microwaves applied to the free radicals are different from each other. The intensity of the microwave applied to the free radicals in the fitting curve Fit1 is larger than that in the fitting curve Fit2. For both the fitting curve Fit1 and Fit2, the rotational motion of the free radicals was measured by changing the spin manipulation time Tau. It can be seen that by reducing the intensity of the microwave applied by changing the fitting curve Fit1 to the fitting curve Fit2, the oscillation period of what is called Rabi oscillation is reduced. That is, an ESR response to the microwave was detected, and thus it was confirmed that the quantum manipulation was completed.

[0083] So far, for a microdomain (e.g., about (5 nm) 3Electron spin measurement and quantum manipulation (of volume) are often used for detecting a single protein formed on a thin film and analyzing its structure, and detecting a single DNA and analyzing its structure. However, such measurement or quantum manipulation has not been performed inside cells yet. The inventors made it possible to detect surface free radicals and perform quantum manipulation by forming a single NV color center at the tip of a diamond column.

[0084] The inventors' quantum measurement method performs quantum manipulation on both quantum sensors and free radicals, etc., making it possible to perform various measurements inside cells. As an example thereof, measurement of a single DNA, structural analysis of Cryptochrome protein, which is considered to be related to the orientation perception ability of migratory birds, identification and detection of ions, and structural analysis of various free radicals generated by intracellular oxidation phenomena can be cited.

[0085] 〔Summary〕

[0086] The intracellular quantum measurement device of Aspect 1 includes a substrate, and a nanopillar for inserting a cell is formed on the surface of the substrate, and the tip portion of the nanopillar includes a quantum sensor.

[0087] According to the above solution, a quantum measurement technology that can measure the intracellular state and overcome the disadvantages of quantum measurement using nanoparticles can be realized.

[0088] In the intracellular quantum measurement device of Aspect 2 based on the intracellular quantum measurement device of Aspect 1, a solution is adopted in which the height of the nanopillar is 1 μm or more and 20 μm or less.

[0089] According to the above solution, intracellular quantum measurement can be realized on the basis of taking into account both the physical requirement of sufficiently improving the fluorescence excitation efficiency and the biological requirement of sufficiently reducing the toxicity to cells.

[0090] In the intracellular quantum measurement device of Aspect 3 based on the intracellular quantum measurement device of Aspect 1 or 2, a solution is adopted in which the diameter of the tip portion of the nanopillar is 100 nm or more and 1000 μm or less.

[0091] According to the above solution, intracellular quantum measurement can be realized on the basis of taking into account both the physical requirement of sufficiently improving the fluorescence excitation efficiency and the biological requirement of sufficiently reducing the toxicity to cells.

[0092] In the intracellular quantum measurement device of Aspect 4 based on the intracellular quantum measurement device of any one of Aspects 1 to 3, the following solution is adopted: a plurality of nanopillars for respectively inserting a plurality of cells are formed on the surface of the substrate, and the tip portions of the plurality of nanopillars each include a quantum sensor.

[0093] According to the above scheme, it is possible to measure the states in multiple cells simultaneously.

[0094] In the intracellular quantum measurement device according to aspect 5 of the intracellular quantum measurement device based on aspect 4, a scheme is adopted in which the interval between the adjacent nano-columns is 10 μm or more and 60 μm or less.

[0095] According to the above scheme, it is possible to insert the nano-columns into cells more reliably. As a result, it is possible to perform quantum measurement on the states in cells more reliably.

[0096] In the intracellular quantum measurement device according to aspect 6 of the quantum measurement device based on any one of aspects 1 to 5, a scheme is adopted in which the nano-column and the substrate are made of diamond crystals, and the quantum sensor is an NV color center.

[0097] According to the above scheme, it is possible to perform quantum measurement on the states in cells with high precision.

[0098] In the intracellular quantum measurement device according to aspect 7 of the quantum measurement device based on aspect 6, a scheme is adopted in which the

[111] direction of the diamond crystal is consistent with the normal direction of the surface of the substrate.

[0099] According to the above scheme, it is possible to control the magnetic field applied to the quantum sensor in cells with high precision.

[0100] The cell holding device according to aspect 8 includes: a first substrate; a second substrate laminated on the first substrate and having an opening that functions as a chamber for storing a liquid containing cells; an intracellular quantum measurement device according to any one of aspects 1 to 7, which is laminated on the first substrate and is within the opening; and an electrode, which is laminated with the intracellular quantum measurement device and is within the opening.

[0101] According to the above scheme, it is possible to easily perform quantum measurement on the states in cells cultured in the liquid stored in the chamber.

[0102] The intracellular quantum measurement method according to aspect 9 includes: using a substrate having nano-columns formed on its surface, and performing quantum measurement in a state where the nano-columns are inserted into cells, wherein the front end portion of the nano-columns includes a quantum sensor.

[0103] According to the above scheme, it is possible to realize a quantum measurement technology that can measure the states in cells and overcome the disadvantages of quantum measurement using nanoparticles.

[0104] In the intracellular quantum measurement method of aspect 10, which is based on the intracellular quantum measurement method of aspect 9, the following scheme is adopted: A substrate with a plurality of nanocolumns formed on its surface is used, and quantum measurement is performed in a state where the plurality of nanocolumns are respectively inserted into a plurality of cells, wherein the front end portions of the plurality of nanocolumns each contain a quantum sensor.

[0105] According to the above scheme, it is possible to simultaneously measure the states inside a plurality of cells.

[0106] In the intracellular quantum measurement method of aspect 11, which is based on the intracellular quantum measurement method of aspect 9 or 10, it further includes: a first quantum manipulation process of applying a pulse of a first electromagnetic wave to the quantum sensor to manipulate the quantum state of the quantum sensor; a second quantum manipulation process of applying a pulse of a second electromagnetic wave having a frequency different from that of the first electromagnetic wave to at least one of molecules, atoms, ions, and free radicals existing around the front end portion to manipulate the quantum state of at least one of the molecules, atoms, ions, and free radicals; and a state detection process of irradiating excitation light to at least one of the molecules, atoms, ions, and free radicals and the quantum sensor, and receiving fluorescence from at least one of the quantum sensor, the molecules, atoms, ions, and free radicals and fluorescence from the quantum sensor.

[0107] According to the above scheme, it is possible to obtain various information by performing quantum manipulation on both the quantum sensor and free radicals, etc.

[0108] (Supplementary Note Items)

[0109] The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the inventive concept shown in the entire specification. Embodiments obtained by appropriately combining technical means separately disclosed in different embodiments are also included within the technical scope of the present invention.

[0110] (Explanation of Reference Numerals)

[0111] 1 Intracellular quantum measurement device

[0112] 10 Substrate

[0113] 11 Nanocolumn

[0114] 2 Cell holding device

[0115] 3 Intracellular quantum measurement system

Claims

1. An intracellular quantum measurement device, characterized in that: it includes a substrate, on the surface of which nano-columns for inserting cells are formed, and the front end portion of the nano-column includes a quantum sensor.

2. The intracellular quantum measurement device according to claim 1, characterized in that: the height of the nano-column is 1 μm or more and 20 μm or less.

3. The intracellular quantum measurement device according to claim 1 or 2, characterized in that: the diameter of the front end portion of the nano-column is 100 nm or more and 1000 μm or less.

4. The intracellular quantum measurement device according to any one of claims 1 to 3, characterized in that: on the surface of the substrate, a plurality of nano-columns for respectively inserting a plurality of cells are formed, and the front end portion of each of the plurality of nano-columns includes a quantum sensor.

5. The intracellular quantum measurement device according to claim 4, characterized in that: the interval between adjacent nano-columns is 10 μm or more and 60 μm or less.

6. The intracellular quantum measurement device according to any one of claims 1 to 5, characterized in that: the nano-column and the substrate are composed of diamond crystals, and the quantum sensor is an NV color center.

7. The intracellular quantum measurement device according to claim 6, characterized in that: the [111] direction of the diamond crystal is consistent with the normal direction of the surface of the substrate.

8. A cell holding device, characterized in that, It includes: a first substrate; a second substrate, which is laminated on the first substrate and has an opening that functions as a chamber for storing a liquid containing cells; the intracellular quantum measurement device according to any one of claims 1 to 7, which is laminated on the first substrate and is located within the opening; and an electrode, which is laminated with the intracellular quantum measurement device and is located within the opening.

9. A method for intracellular quantum measurement, characterized in that, It includes: using a substrate with nano-columns formed on its surface, and performing quantum measurement in a state where the nano-columns are inserted into cells, wherein the front end portion of the nano-column includes a quantum sensor.

10. The intracellular quantum measurement method according to claim 9, characterized in that, It includes: using a substrate with a plurality of nano-columns formed on its surface, and performing quantum measurement in a state where the plurality of nano-columns are respectively inserted into a plurality of cells, wherein the front end portion of each of the plurality of nano-columns includes a quantum sensor.

11. The intracellular quantum measurement method according to claim 9 or 10, characterized in that, It further includes: a first quantum control process of applying a pulse of a first electromagnetic wave to the quantum sensor to control the quantum state of the quantum sensor; a second quantum control process of applying a pulse of a second electromagnetic wave with a frequency different from that of the first electromagnetic wave to at least one of molecules, atoms, ions, and free radicals existing around the front end portion to control the quantum state of at least one of the molecules, atoms, ions, and free radicals; and a state detection process of irradiating excitation light to at least one of the molecules, atoms, ions, and free radicals and the quantum sensor, and receiving fluorescence from at least one of the quantum sensor, the molecules, atoms, ions, and free radicals and fluorescence from the quantum sensor.

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