Microchamber, microchamber device, and method for manufacturing a microchamber

Abstract

To provide a micro-chamber which enables reaction detection with high spatial resolution and high detection sensitivity, and to provide a micro-chamber device and method of manufacturing the micro-chamber.SOLUTION: A micro-chamber 101 comprises: a diamond substrate 102 having a recess 1021 provided thereon; and a diamond crystal layer 1031 with a Diamond Nitrogen-Vacancy Center (NV center) formed on a side surface 1022 of the recess 1021.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a microchamber, a microchamber device, and a method for manufacturing a microchamber.

Background Art

[0002] An analysis system using a microchamber capable of accommodating a minute sample for various tests is used in chemical analysis. The microchamber can accommodate a sample on the order of femtoliters to nanoliters in volume. Thereby, the microchamber enables a chemical reaction having a high reaction rate. Patent Document 1 shows a microchamber array device for analyzing a bioassay at the cell unit level.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] As described in Patent Document 1, in the measurement of a chemical reaction using a microchamber, the region to be measured is minute, and the absolute amount of the sample becomes small. In this case, it is required to measure the chemical reaction with high sensitivity. For the measurement of a chemical reaction in a microchamber, for example, ELISA (Enzyme Linked ImmunoSolubent Assay) or surface plasmon resonance is utilized. In these methods, since an optical measurement method is used, the spatial resolution and the detection sensitivity are limited.

[0005] Therefore, an object of the present invention is to provide a microchamber, a microchamber device, and a method for manufacturing a microchamber, which enable reaction detection with high spatial resolution and high detection sensitivity. [Means for solving the problem]

[0006] Therefore, the present invention aims to provide a microchamber, a microchamber apparatus, and a method for manufacturing a microchamber that enable reaction detection with high spatial resolution and high detection sensitivity.

[0007] A microchamber according to one aspect of the present invention comprises a diamond substrate having a recess and a diamond crystal layer having NV centers formed on the side surface of the recess.

[0008] NV centers react to the magnetic field of, for example, the nuclear spin of a single molecule in the sample. By forming a diamond crystal layer containing NV centers in a recess, changes in the external magnetic field due to chemical reactions within the recess can be detected as changes in spin. Nuclear magnetic resonance (NMR) is used as a detection method, for example. This enables reaction detection with high spatial resolution and high detection sensitivity.

[0009] In the microchamber according to the above embodiment, a diamond crystal layer having NV centers is formed on the main surface of a diamond substrate, and the surface density of NV centers in the diamond crystal layer formed on the side surface of the recess may be higher than the surface density of NV centers in the diamond crystal layer formed on the main surface of the diamond substrate.

[0010] This allows more NV centers to be provided within the recesses where the chemical reaction takes place, enabling the chemical reaction within the recesses to proceed more effectively.

[0011] In the microchamber of the above embodiment, the surface orientation of the side surface may differ from the surface orientation of the main surface of the diamond substrate. By different surface orientations of the side surface and the main surface, the diamond crystal layer formed on the side surface of the recess contains more NV centers than the diamond crystal layer formed on the main surface of the diamond substrate.

[0012] In the microchamber according to the above embodiment, the diamond crystal layer formed on the side surface of the recess may include diamond grown homoepitaxially from a diamond substrate.

[0013] By homoepitaxially growing the diamond crystal layer formed on the side surface of the recess, the thickness of the diamond crystal layer can be appropriately adjusted while forming the diamond crystal layer. Therefore, the dimensions of the microchamber can be appropriately adjusted.

[0014] The microchamber apparatus according to the above embodiment may further include a channel that communicates with the recess and is formed on the main surface of the diamond substrate. This makes it possible to introduce a sample into the microchamber through the channel, improving the convenience of the microchamber apparatus.

[0015] Furthermore, another embodiment of the present invention comprises a first microchamber and a second microchamber, which are microchambers according to the above embodiment, wherein, in a plan view of the diamond substrate, the diameter of the first microchamber is different from the diameter of the second microchamber.

[0016] By having different diameters for the first and second microchambers, it becomes possible to form microchambers of different dimensions on a single diamond substrate. This allows for the selection of the appropriate microchamber for each reaction in the microchamber apparatus.

[0017] A method for manufacturing a microchamber apparatus according to another aspect of the present invention includes the steps of forming a recess in a diamond substrate and homoepitaxially growing a diamond crystal layer on the diamond substrate while supplying a mixed gas containing nitrogen.

[0018] By this manufacturing method, a diamond crystal layer having NV centers is formed on the side surface of the recess, and a microchamber according to the above embodiment is manufactured. [Effects of the Invention]

[0019] According to the present invention, it is possible to provide a microchamber, a microchamber device, and a method for manufacturing a microchamber that enable reaction detection with high spatial resolution and high detection sensitivity.

Brief Description of the Drawings

[0020] [Figure 1] It is a perspective view of the microchamber according to this embodiment. [Figure 2] It is a cross-sectional view taken along the II-II section of FIG. 1. [Figure 3] It is a diagram for explaining the manufacturing process of the microchamber according to this embodiment. [Figure 4] It is an example of a microchamber manufactured by the manufacturing process according to this embodiment. [Figure 5] It is a diagram for explaining the distribution of NV centers in the microchamber according to this embodiment. [Figure 6] It is a diagram for explaining the quantization axis of the NV centers in the microchamber according to this embodiment. [Figure 7] It is an example of a microchamber of another shape according to this embodiment. [Figure 8] It is a diagram for explaining the distribution of NV centers in the microchamber of another shape according to this embodiment. [Figure 9] It is a plan view of the microchamber device according to this embodiment. [Figure 10] It is a perspective view of a part of the microchamber device according to this embodiment. [Figure 11] It is a diagram for explaining the distribution of NV centers in a part of the microchamber device according to this embodiment. [Figure 12] It is a diagram showing an example of the transport of microbeads in a part of the microchamber device according to this embodiment. [Figure 13] It is a diagram for explaining the NV center. [Figure 14] It is a diagram for explaining the orientation axis of the NV center. [Modes for carrying out the invention]

[0021] A preferred embodiment of the present invention will be described with reference to the attached drawings. In each drawing, components denoted by the same reference numerals have the same or similar configuration.

[0022] Figure 1 shows a schematic diagram of the microchamber 101 according to this embodiment. The microchamber 101 is formed by a diamond substrate 102 and a diamond crystal layer 103 provided on the diamond substrate 102. The microchamber 101 is formed by forming a diamond crystal layer 1031, which is part of the diamond crystal layer 103, on the side surface 1022 of a recess 1021 formed in the diamond substrate 102. The microchamber 101 is open at one end, forming a space inside which a substance can be contained. Although the diamond crystal layer 103 is very thin compared to the thickness of the diamond substrate 102, for illustrative purposes, the thickness of the diamond crystal layer 103 is shown to be larger than it actually is in each figure.

[0023] The diameter φ of the recess 1021 can be varied depending on the intended use, but is typically in the range of 1 to 500 μm. This range is the range that can be formed by process technology using diamond dry etching, and is the range that can be formed on currently available substrate sizes. The depth d of the recess 1021 can also be varied depending on the intended use, but is typically in the range of 0.3 to 10 μm. The planar size of the diamond substrate 102 can be varied depending on the number of recesses 1021 to be formed and the shape of the channels to be formed as needed, but is typically 2 mm square to 12 mm square if the diamond substrate 102 is rectangular, and 12.5 mm in diameter if it is circular. The planar size of the diamond substrate 102 is typically 3 mm square. The thickness of the diamond substrate 102 is set according to the depth d of the recess 1021 to be formed, but is typically in the range of 0.05 to 1 mm. The thickness of the diamond crystal layer 103 is preferably 5 nm or more and 500 nm or less. This is because, at thicknesses of 5 nm or less, the density of NV centers that generate spin is too low, resulting in excessively low sensitivity. At thicknesses of 500 nm or more, abnormal crystal grain growth occurs, abnormal hill-like undulations are created, and step bunching occurs, increasing the crystal imperfections in the diamond crystal layer 103 due to excessive crystal growth.

[0024] The diamond crystal layer 1031 has NV centers, which are described below. Figure 13 shows a structural diagram illustrating the structure of an NV center. An NV center is a composite defect formed by a nitrogen atom N and a vacancy V resulting from the absence of a carbon atom adjacent to the nitrogen atom N, at one of the lattice points of a diamond crystal composed of carbon atoms C. This composite defect can capture an electron e, as conceptually shown in Figure 13. The electron e has a quantum state (spin s). NV centers strongly absorb light of a predetermined wavelength, such as green laser light, and emit red fluorescence from light of other predetermined wavelengths, such as red fluorescence. The intensity of fluorescence from an NV center is characterized by its variation depending on the spin s of the electron e.

[0025] The cross-sectional structure of the microchamber 101 will be described with reference to Figure 2. The microchamber 101 is formed by forming a diamond crystal layer 1031 in a cylindrical recess 1021 with a diameter φ and depth d in the diamond substrate 102. The microchamber 101 has a diamond crystal layer 1031 on the side surface 1022 and a diamond crystal layer 201 located at the bottom. The diamond crystal layer 1031 and the diamond crystal layer 201 are diamond crystal layers having NV centers.

[0026] The manufacturing method for the microchamber 101 will be described with reference to Figure 3. The manufacturing method consists of the following steps (a) to (g). (a) First, an SiO2 film 301 is formed on the diamond substrate 102 using a predetermined film deposition method. When sputtering is applied, sputtering is performed using a sputtering apparatus. There are no particular limitations on the sputtering conditions, but for example, the argon gas flow rate is 30 sccm, the pressure is 0.5 Pa, the RF output is 100 W, and the substrate is not heated.

[0027] Here, for example, a single-crystal type IIa (001) HPHT diamond substrate is used as the diamond substrate 102. In addition, the diamond substrate 102 is pre-treated with wet cleaning (SPM: Sulfuric-acid and hydrogen-peroxide mixture) after skif polishing in order to remove residual minute irregularities and physical deposits on the substrate surface that would be a source of disturbance during microfabrication. Furthermore, the diamond substrate 102 must have resistance to CF4 / O2 etching gas used in the final process, specifically in reactive ion etching (RIE).

[0028] (b) The resist 302 is spin-coated using a spin coater.

[0029] (c) A pattern is drawn on the resist 302 using a photolithography apparatus. A circular pattern is drawn in the microchamber 101. That is, by exposure processing with the pattern applied, the resist 302 in areas other than the circular recess 1021 is cured, and then the resist 302 in areas that have not been chemically cured is removed.

[0030] (d) The patterned resist 302 is used as a mask to perform physical etching on the SiO2 film 301. There are no limitations on the type of physical etching, but for example, an electron cyclotron resonance (ECR) plasma device may be used. There are no particular limitations on the etching conditions, but for example, the etching gas is argon gas, the argon gas flow rate is 6.4 sccm, the pressure is 1 mTorr, the microwave output is 400 W, and the substrate is not heated.

[0031] (e) The diamond substrate 102 is physically etched using the patterned SiO2 film 301 as a mask. There are no limitations on the physical etching of the diamond substrate 102, but for example, the RIE method using a CF4 / O2 mixed gas is used, as it suppresses the generation of residue on the surface of the diamond substrate 102 and allows for the desired level of smoothness. This process forms a diamond substrate 102 in which the crystal orientation of the diamond crystal on the upper surface of the diamond substrate 102 is different from the crystal orientation of the diamond crystal on the side surface 1022.

[0032] (f) Remove the SiO2 film 301 with hydrofluoric acid. Through these steps, recesses 1021 are formed in the diamond substrate 102.

[0033] (g) A diamond crystal layer 103 is formed on a diamond substrate 102 by homoepicatal growth of diamond. The diamond crystal layer 103 is formed by a predetermined crystal growth method, for example, the microwave plasma CVD (Chemical Vapor Deposition) method. In the formation process, there are no limitations on the raw material gas, but for example, a mixed gas of CH4 gas, H2 gas, and N2 gas whose flow rate is controlled by a mass flow controller is used.

[0034] The substrate temperature of the diamond substrate 102 during the formation process is preferably in the range of 300°C to 900°C. More preferably, the substrate temperature is in the range of 750°C to 850°C. For example, the substrate temperature can be 800°C. If the substrate temperature is below 750°C, the yield of NV centers in the diamond crystal layer 103 tends to decrease along with the deterioration of crystallinity. If the substrate temperature is above 850°C, deformation of the substrate and strain on the crystal may occur. Note that the ranges of the above and subsequent parameters may also vary in relation to plasma conditions such as microwave output, gas flow rate, and gas pressure.

[0035] The ratio of nitrogen (N) to carbon (C) in the raw material gas during the formation process, N / C, is preferably in the range of 1% to 18%. More preferably, N / C is 1% to 2%. For example, N / C can be 1.75%. If it is less than 1%, a sufficient amount of nitrogen atoms will not be incorporated, and the surface density of NV centers will decrease. If it is more than 2%, it will exceed the necessary and sufficient amount of nitrogen atoms to be incorporated into the diamond crystal, which may make it difficult to control the formation of NV centers at the desired position in the diamond crystal layer 103. This may impair the crystallinity of the diamond layer.

[0036] The growth rate of the diamond crystal layer 103 in the formation process is preferably in the range of 0.7 nm / h to 500 nm / h. A growth rate in the range of 200 to 300 nm / h is more preferable. This is because if the growth rate is 200 nm / h or less, nitrogen uptake decreases, which may reduce the yield of NV centers in the diamond crystal layer 103. Furthermore, if the growth rate is 300 nm / h or more, defects other than NV centers may be formed in the diamond crystal layer 103, or a change may occur in the growth mode of the diamond crystal layer 103.

[0037] The growth rate Lw of the diamond crystal layer 103 during the formation process can be adjusted by the supply time of the raw material gas. For example, depending on the supply time of the raw material gas, the growth rate Lw of the diamond crystal layer 103 can be adjusted within the range of 25 nm to 250 nm.

[0038] In the process of forming the diamond crystal layer 103, carbon atoms (C) in the diamond crystal formed by homoepicatial growth from the diamond substrate 102 are replaced by nitrogen atoms (N) in the source gas, thereby forming the diamond crystal layer 103 having NV centers.

[0039] Figure 4 shows an example of a recess 1021 in a microchamber 101 formed using the process shown in Figure 3. In Figure 4, the microchamber 101 is cylindrical. The diameters φ and depths d of recesses 1021a to 1021d shown in Figures 4(a) to 4(d) are (a)φ=10μm, d=0.8μm, (b)φ=8μm, d=0.7μm, (c)φ=6μm, d=1.5μm, and (d)φ=2μm, d=1.5μm, respectively. In this way, recesses of desired shapes can be formed by changing the pattern used in the photolithography method.

[0040] Referring to Figure 5, the distribution of NV centers in the microchamber 101 will be explained. Figure 5 shows the visualization of the emission distribution corresponding to the distribution of NV centers in the microchamber. Specifically, the emission distribution was visualized in a microchamber 101 in which a diamond crystal layer 1031 with a growth amount Lw = 250 nm is provided in a recess 1021 with a diameter φ = 6 μm and a depth d = 1.5 μm. In Figure 5, the positions indicated by arrows 1 to 4 are the measurement points of the fluorescence intensity of ODMR (Optically Detected Magnetic Resonance) shown in Figure 6.

[0041] The microchamber 101 is excited by laser light with a wavelength of 532 nm, and the emission from the NV centers of the microchamber 101 is selected in the wavelength range of approximately 594 nm to 815 nm by an optical filter and visualized. The emission intensity (number of photons) shows a strong value at the position corresponding to the diamond crystal layer 1031 of the microchamber 101. This indicates that in the microchamber 101, the diamond crystal layer 1031 formed on the side surface 1022 has a higher distribution of NV centers than other surfaces. The intensity shown in Figure 5 is the sum of the emission from NV centers along the depth direction of the diamond crystal layer 1031. The NV density and quantum retention time (T2) obtained from the measurement results in Figure 5 are 7 to 8 × 10⁻⁶, respectively. 14 cm -3 , 36 μs.

[0042] Referring to Figure 6, the tendency of the orientation axis (quantization axis) of NV centers in the diamond crystal layer 1031 will be explained. Figure 6 shows the fluorescence intensity of ODMR (Photodetector magnetic resonance) at each point indicated by arrows 1 to 4 in Figure 5.

[0043] As shown in Figure 14, generally, in a diamond crystal with NV centers, the possible directions of the vector connecting the vacancy V and nitrogen N are: <111> There are four types in terms of direction. For convenience, in Figure 14, the directions of the vectors connecting the vacancies V and nitrogen N shown in (a) to (d) are denoted as NV1, NV2, NV3, and NV4. In this case, the change in fluorescence intensity of the ODMR in Figure 6 shows that, assuming a static magnetic field in the

[0110] direction penetrating the (110) plane, NV1 and NV2 are dominant at positions 1 and 3, and NV3 and NV4 are dominant at positions 2 and 4. That is, the orientation of NV centers in the diamond crystal layer 1031 at specific positions in the microchamber 101 has order depending on the location.

[0044] In sensing using NV centers, the minimum magnetic detection sensitivity is improved by multiplying it by 1 / √n, where n is the number of NV centers. Typically, the spin at one NV center on one of the four orientation axes is involved in sensing, while signals from other spins become background signals. Therefore, when using NV centers as highly efficient and sensitive sensors, a high degree of order in the orientation axes of the NV centers in the crystal is preferable. In this respect, the microchamber 101 has an ordered orientation of NV centers, and by using the microchamber 101, it becomes possible to detect signals corresponding to chemical reactions within the microchamber 101 with high sensitivity.

[0045] In this embodiment, the reason for the high degree of order of the NV centers is that the diamond crystal layer 1031 was grown by homoepitaxial growth in the microchamber 101. In homoepitaxial growth, the grown layer grows reflecting the crystal structure of the substrate. In the microchamber 101, a diamond crystal layer 1031 having highly ordered NV centers is formed based on the crystal structure of the side surface 1022. Furthermore, by growing the diamond crystal layer 1031 based on the crystal structure of the side surface 1022, it is possible to form a diamond crystal layer 1031 that specifically has many NV centers on the side surface 1022.

[0046] As a result, the surface density of NV centers in the diamond crystal layer 1031 is higher than the surface density of NV centers in the diamond crystal layer 1032 formed on the main surface 1023 of the diamond substrate 102. Furthermore, the surface density of NV centers in the diamond crystal layer 1031 is higher than the surface density of NV centers in the diamond crystal layer 201.

[0047] Figure 7 shows an example of another shape of microchamber 701 according to this embodiment. The microchamber 701 has a honeycomb shape in plan view. Each microchamber 701 is separated from each other by a diamond wall 702. The width w of the microchamber 701 is, for example, 6 μm. The depth of the microchamber 701 is 380 nm. In this way, by adjusting the shape, it is possible to adjust the wettability of the surface on which the microchamber is provided. Such a honeycomb-shaped microchamber 701 is suitable for bioassay systems (microanalysis systems).

[0048] Figure 8 visualizes the light emission from the microchamber 701, similar to Figure 5. As shown in Figure 7, it is also shown that many NV centers exist inside the diamond wall 702, i.e., on the side surface of the microchamber 701.

[0049] Figure 9 shows a plan view of a microchamber apparatus 900 having a microchamber according to this embodiment. The microchamber apparatus 900 has a sample introduction section 901, a flow channel 902, and microchambers 903, 904, 905, 906, and 907. The sample introduction section 901, the flow channel 902, and the microchambers 903, 904, 905, 906, and 907 are recesses formed in the substrate of the microchamber apparatus 900. The microchamber apparatus 900 is formed from a diamond substrate 102 and a diamond crystal layer 103. Note that only the portions of microchambers 903, 904, 905, 906, and 907 are formed from the diamond substrate 102 and the diamond crystal layer 103, and the other portions may be formed from other suitable materials.

[0050] In the microchamber apparatus 900, a sample, such as a solution containing cells, is introduced into the sample introduction section 901. The sample introduced into the sample introduction section 901 travels through a channel 902 extending from the sample introduction section 901 to the microchambers 903, 904, 905, 906, and 907 provided at the end of the channel 902.

[0051] Microchambers 903, 904, and 905 have different diameters. Here, we use the diameter as the diameter of the microchamber. For example, the diameter of each microchamber can be the largest of the side distances. Note that microchambers 906 and 907 have the same diameter as microchambers 903 and 904, respectively.

[0052] Figure 10 shows a perspective view of the microchamber 904 and the channel 902. As shown in Figure 10, in the microchamber 904, the recess 1021 communicates with the channel 902, allowing a sample to be introduced into the microchamber 904 through the channel 902.

[0053] As with the microchamber 904, a diamond crystal layer 1031 may be provided on the side surface 1022 to form a microchamber so that the entire circumference of the recess 1021 is not surrounded, as with the microchamber 101 in Figure 1.

[0054] Figure 11 visualizes the luminescence from microchambers 904 and 905, similar to Figure 5. Microchamber 904 has a diameter of 25 μm, and microchamber 905 has a diameter of 50 μm. As shown in Figure 11, it is shown that many NV centers are present in the diamond crystal layer 1031 on the side surface 1022 in both microchambers 904 and 905. As shown in Figure 11, by patterning numbers indicating the diameter of each microchamber near each microchamber, the shape of the microchamber can be intuitively recognized by the user.

[0055] Figure 12 shows an optical microscope image of a single microbead 1201 being transported from the sample introduction section 901 to the microchamber 905 using a glass tip such as a picopipette capable of transporting picoliters of liquid or single cells / microparticles. The depth of the channel 902 is 3.5 μm. The diffusion and movement of the microbead 1201 are restricted within the microchamber 905. The microbead 1201 reaches the side of the microchamber 905. Since many NV centers are distributed on the side of the microchamber 905, it is possible to measure the effect of the microbead 1201 using the microchamber 905.

[0056] The microchamber having an NV center, as shown in this embodiment, enables highly sensitive and high-resolution reaction measurement in biological tests (bioassays), which are a type of analysis in chemical fields such as fine chemicals, life sciences, or biosciences.

[0057] The embodiments described above are provided to facilitate understanding of the present invention and are not intended to limit its interpretation. The elements of the embodiments, as well as their arrangement, materials, conditions, shapes, and sizes, are not limited to those exemplified and can be modified as appropriate. [Explanation of Symbols]

[0058] 101, 701, 903, 904, 905, 906, 907… Microchamber, 102… Diamond substrate, 1021, 1021a, 1021b, 1021c, 1021d… Recess, 1022… Side surface, 1023… Main surface, 103, 1031, 1032, 201… Diamond crystal layer, 900… Microchamber apparatus

Claims

1. a diamond substrate in which a recess is provided; a diamond crystal layer having an NV center formed on a side surface of the recess; A microchamber comprising:

2. 10. The microchamber of claim 1, a diamond crystal layer formed on the primary surface of the diamond substrate and having NV centers; A microchamber, wherein the surface density of NV centers in the diamond crystal layer formed on the side surface of the recess is higher than the surface density of NV centers in the diamond crystal layer formed on the main surface of the diamond substrate.

3. The microchamber according to claim 1 or 2, A microchamber, wherein the surface orientation of the side surface of the recess is different from the surface orientation of the main surface of the diamond substrate.

4. The microchamber according to any one of claims 1 to 3, The diamond crystal layer formed on the side surface of the recess comprises diamond homoepitaxially grown from the diamond substrate.

5. The microchamber according to any one of claims 1 to 4, a flow channel formed on the main surface of the diamond substrate and communicating with the recess.

6. A microchamber system comprising a first microchamber and a second microchamber, each of which is the microchamber according to any one of claims 1 to 5; a diameter of the recess formed in the first microchamber differs from a diameter of the recess formed in the second microchamber in a plan view of the diamond substrate.

7. forming a recess in a diamond substrate; and homoepitaxially growing a diamond crystal layer on the diamond substrate while supplying a nitrogen-containing mixed gas.

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