Fine particle filling method and fine particle filling system

Abstract

Provided is a technique for accurately aligning the tip end of a nozzle and a micro reaction tank when filling the micro reaction tank with fine particles. The present disclosure is a fine particle filling method for filling a container with fine particles, the method comprising: aligning a nozzle and the container by using an image captured by an imaging device; and filling the container with fine particles from the nozzle. The alignment includes aligning the tip end of the nozzle with the horizontal center position of an opening of the container by using (a) a horizontal distance difference from a reference position in the image captured by the imaging device to the horizontal center position of the tip end of the nozzle, and (b) coordinates at which the horizontal center position of the opening of the container is disposed at the reference position.

Description

Fine particle filling method and fine particle filling system

[0001] The present disclosure relates to a particulate loading method and system.

[0002] Generally, single-cell analysis is one of the analytical methods in life science, and is a technique for analyzing and quantifying biomolecules in individual cells. In particular, single-cell analysis is a method for determining the base sequences of DNA and RNA (especially mRNA) in individual cells and measuring the number of molecules using a DNA sequencer.

[0003] Specific methods for single cell analysis include a method using a tube (a plastic reaction vessel of approximately 0.2 mL to 2 mL) or a titer plate (a plate-like arrangement of a specified number of resin reaction vessels, such as 96 or 384 vessels) (Non-Patent Document 1), a method using a microchannel (Non-Patent Document 2), a method using an emulsion (Non-Patent Document 3), a method using a microwell (Non-Patent Document 4), and a method using a through-hole microwell (Non-Patent Document 5).

[0004] In both methods, a large number of cells to be analyzed are separated into single cells, and a cell-specific DNA base sequence (hereinafter referred to as a "barcode") is introduced into the nucleic acid extracted from each cell. The nucleic acid samples obtained from these multiple cells are then combined and subjected to DNA sequencing. DNA sequence information for each barcode sequence information is separated from the sequence data obtained by DNA sequencing, thereby obtaining single-cell analysis data.

[0005] In particular, in the methods described in Non-Patent Documents 3 to 5, barcodes are introduced after cell isolation and before nucleic acid amplification, making it possible to prepare DNA sequencer samples from a large number of cells using a small amount of reagents and obtain sequence data.

[0006] The single-cell analysis method described in Non-Patent Document 5 uses a larger number of microparticles for analyzing a single cell than the methods described in Non-Patent Documents 3 and 4 (Non-Patent Documents 3 and 4 use only one microparticle). Therefore, the number of DNA probes for capturing nucleic acids used for analyzing a single cell can be increased by more than 100 times. Furthermore, because the cell lysate solution passes through a porous structure composed of a large number of microparticles, nucleic acids from single cells can be captured on the microparticle surface with high efficiency in a short period of time.

[0007] In this way, in order to use a large number of microparticles for the analysis of one cell, it is necessary to fill a large number of microparticles into a micro-reaction chamber.

[0008] In Non-Patent Document 5, a micro-reaction chamber is filled with microparticles by dispensing a micro-particle suspension into the micro-reaction chamber and discharging excess water through the through-holes, utilizing the through-holes in the bottom of the micro-reaction chamber. In addition, an inkjet device is used because the diameter of the micro-reaction chamber is small, at 50 to 100 μm.

[0009] Patent Document 1 describes a technology in which microparticles are concentrated at the tip of a nozzle in advance to a concentration close to the closest packing density, and then the nozzle is lowered and, by utilizing surface tension (wettability), only the concentrated microparticle solution is separated and filled into a micro-reaction tank (container).

[0010] International Publication No. 2021 / 181467

[0011] F. Tang, C. Barbacioru, Y. Wang, E. Nordman, C. Lee, N. Xu, X. Wang, J. Bodeau, B. B. Tuch, A. Siddiqui, K. Lao , M. A. Surani, “mRNA-Seq whole-transcriptome analysis of a single cell,” Nature Methods, 6, 377-382, 2009.A. K. Shalek, R. Satija, X. Adiconis, R. S. Gertner, J. T. Gaublomme, R. Raychowdhury, S. Schwartz, N. Yosef, C. Malboeuf, D. Lu, J. J. Trombetta, D. Gennert, A. Gnirke, A. Goren, N. Hacohen, J. Z. Levin, H. Park , A. Regev, “Single-cell transcriptomics reveals bimodality in expression and splicing in immune cells,” Nature, 498, 7453, p.236-240, 2013.A. Klein, L. Mazutis, I. Akartuna, N. Tallapragada, A. Veres, V. Li, L. Peshkin, D. Weitz, M. Kirschner, “Droplet barcoding for single-cell transcriptomics applied to embryonic stem cells,” Cell, 161, 5, p. 1187-1201, 2015.H. Fan, G. Fu , S. Fodor, “Expression profiling. Combinatorial labeling of single cells for gene expression cytometry,” Science, 347, 6222, p.1258367, 2015.Masataka Shirai, Koji Arikawa, Kiyomi Taniguchi, Maiko Tanabe, Tomoyuki Sakai, “Vertical Flow Array Chip reliably identify cell types from single-cell mRNA sequencing experiments” Scientific Reports, 6, 36014 DOI: 10.1038 / srep36014 (2016).

[0012] The above-mentioned method of filling micro-reaction chambers (containers) with microparticles using an inkjet device as in Non-Patent Document 5 has the following problems.

[0013] First, to fill microparticles into a micro-reaction chamber, it is necessary to dispense a solution containing suspended microparticles into the micro-reaction chamber and then drain the excess solution from the micro-reaction chamber. In order to drain the solution from the micro-reaction chamber while leaving the microparticles behind, a membrane with a mesh finer than the microparticles must be used to prevent the microparticles from leaking out. However, when the diameter of the microparticles is small, on the order of a few micrometers, the size of the mesh openings must be smaller than the diameter of the microparticles. It is extremely difficult to fabricate a membrane with a mesh that is both small and strong. If a membrane with such a mesh size is used, the membrane thickness must be on the order of millimeters in order to maintain strength. This results in a large pressure loss, making it difficult to quickly aspirate the solution. Furthermore, to rapidly remove the solution by applying high pressure, a membrane with a stronger mesh would be required, making it impossible to maintain membrane strength while also improving the solution aspirating speed.

[0014] Second, in order to control the number of particles after filling, it is necessary to stabilize the particle density of the particle suspension and control the particle number by the amount of solution. However, when the specific gravity of the particles is greater than that of the solvent, not only does the particle density change due to the settling of the particles, but the settling rate also changes depending on the degree of micro-aggregation of the particles, making it difficult to maintain a stable particle density.

[0015] Furthermore, to stably eject droplets, the density of microparticles inside the ejection nozzle must be stable and constant. If the physical properties of the solution become inconsistent, not only will the ejection volume and ejection direction become unstable, but ejection itself will frequently stop. When ejection stops, the microparticle suspension must be prepared again and refilled into the inkjet head. Therefore, the time required to fill the many micro-reaction chambers with microparticles is lengthened by the amount of replacement work.

[0016] The technology of Patent Document 1 has been made to solve the problems in the method of filling fine particles using an inkjet device as described above.

[0017] Here, in the method described in Patent Document 1, in order to successfully fill the micro-reaction chamber (container) with a highly concentrated microparticle solution accumulated at the tip of the nozzle, it is necessary to reliably lower the tip of the nozzle into the interior of the micro-reaction chamber 101.

[0018] FIG. 1A shows a cross-sectional schematic diagram (left side) of the state in which the tip of the nozzle 109 and the micro-reaction chamber 101 have been successfully aligned, and an image (right side) of the micro-reaction chamber 101 taken from above in a state in which the micro-reaction chamber 101 has been successfully filled with microparticles. As shown in FIG. 1A, when the tip of the nozzle 109 and the micro-reaction chamber 101 have been successfully aligned, the tip of the nozzle 109 descends into the interior of the micro-reaction chamber 101, allowing the microparticles (high-concentration microparticle solution) to be filled. In the image on the right side of FIG. 1A, the microparticles inside the micro-reaction chamber 101 appear black. However, because the size of the micro-reaction chamber 101 is small, ranging from 50 to 100 μm, the first challenge in successfully filling the microparticles is the need to accurately align the tip of the nozzle with the target micro-reaction chamber to be filled to within 1 μm.

[0019] 1B is a cross-sectional schematic diagram (left side) showing a state in which the tip of the nozzle 109 and the micro-reaction chamber 101 have failed to be aligned, and an image (right side) taken from above showing a state in which the filling of microparticles has failed. As shown in FIG. 1B, when the tip of the nozzle 109 and the micro-reaction chamber 101 have failed to be aligned, the nozzle 109 cannot be properly lowered into the micro-reaction chamber 101. As a result, as shown in the image on the right side of FIG. 1B, the microparticles are discharged outside the micro-reaction chamber 101, causing a filling failure.

[0020] Therefore, the present disclosure provides a technique for accurately aligning the tip of a nozzle with a micro-reaction chamber when filling the micro-reaction chamber with fine particles.

[0021] In order to solve the above problem, the present disclosure provides a particle filling method for filling a container with particle, which includes aligning a nozzle and a container using an image captured by an imaging device, and filling the container with particle from the nozzle, wherein the aligning includes aligning the tip of the nozzle with the horizontal center position of the opening of the container using (a) a horizontal differential distance from a reference position of the image captured by the imaging device to the horizontal center position of the tip of the nozzle, and (b) coordinates where the horizontal center position of the opening of the container is located at the reference position.

[0022] Further features related to the present disclosure will become apparent from the description of this specification and the accompanying drawings. Also, aspects of the present disclosure are achieved and realized by the elements and combinations of various elements and the aspects of the following detailed description and the appended claims. The description of this specification is merely exemplary and does not limit the scope or application of the claims of the present disclosure in any way.

[0023] According to the technology of the present disclosure, the tip of the nozzle and the micro-reaction chamber can be accurately aligned when filling the micro-reaction chamber with fine particles. Problems, configurations, and effects other than those described above will become clear from the description of the following embodiments.

[0024] 1 is a diagram showing a state in which alignment between the tip of the nozzle and the micro-reaction chamber and micro-particle filling are successful; FIG. 2 is a diagram showing a state in which alignment between the tip of the nozzle and the micro-reaction chamber and micro-particle filling are unsuccessful; FIG. 3 is a diagram for explaining the filling process when filling the interior of the micro-reaction chamber with a high-concentration micro-particle solution is successful; FIG. 4 is a diagram for explaining the filling process when filling the interior of the micro-reaction chamber with a high-concentration micro-particle solution is unsuccessful; FIG. 5 is a schematic diagram showing a micro-particle filling system according to the first embodiment; FIG. 6 is a side view showing an implementation example of the micro-particle filling system according to the first embodiment; FIG. 7 is a cross-sectional schematic diagram of a flat substrate having a plurality of micro-reaction chambers; FIG. 8 is a perspective view showing an implementation example of a sensor system; FIG. 9 is a diagram for explaining an alignment method in the micro-particle filling method according to the first embodiment; FIG. 10 is a diagram for explaining step (ii) in the alignment method according to the first embodiment; FIG. 11 is a photographed image for explaining step (ii) in the alignment method according to the first embodiment; FIG. 12 is a diagram for explaining step (iii) in the alignment method according to the first embodiment; FIG. 13 is a diagram for explaining step (iv) in the alignment method according to the first embodiment. FIG. 1 is a photographed image for explaining step (iv) in the alignment method according to the first embodiment. FIG. 2 is a bird's-eye view for explaining step (iv) in the alignment method according to the first embodiment. FIG. 3 is a photographed image for explaining step (iv) in the alignment method according to the first embodiment. FIG. 4 is a bird's-eye view for explaining step (iv) in the alignment method according to the first embodiment. FIG. 5 is a photographed image for explaining step (iv) in the alignment method according to the first embodiment. FIG. 6 is a bird's-eye view for explaining step (v) in the alignment method according to the first embodiment. FIG. 7 is a photographed image for explaining step (v) in the alignment method according to the first embodiment. FIG. 8 is a schematic view for explaining a particle filling method including the alignment method according to the first embodiment. FIG. 9 is a schematic cross-sectional view for explaining the principle of the particle filling method according to the first embodiment. FIG. 10 is a flowchart showing the operation of the particle filling system according to the first embodiment.1 is a schematic top view of a single cell analysis chip according to a second embodiment to be filled with microparticles. FIG. 2 is a schematic cross-sectional view of a portion of a single cell analysis chip according to the second embodiment to be filled with microparticles. FIG. 3 is a bird's-eye view of a single cell analysis chip according to the second embodiment to be filled with microparticles. FIG. 4 is a diagram illustrating a workflow for filling microparticles into a single cell analysis chip according to the second embodiment. FIG. 5 is a diagram for explaining image processing in filling microparticles into a single cell analysis chip according to the second embodiment. FIG. 6 is a photographed image of a chip showing the results of microparticle filling according to the second embodiment. FIG. 7 is a graph showing the waiting time until a high-concentration microparticle solution is formed when natural sedimentation due to gravity or accelerated sedimentation due to magnetic force is used in the microparticle filling system according to the second embodiment. FIG. 8 is a conceptual diagram for explaining a first image processing technique in alignment according to the third embodiment. FIG. 9 is a conceptual diagram for explaining a first image processing technique in alignment according to the third embodiment. 10 is a diagram showing predicted values ​​and detected values ​​obtained by predicting the XY coordinate positions of micro-reaction chambers in advance in the first image processing technique according to the third embodiment. FIG. 11 is a diagram showing detected values ​​and interpolated values ​​of the XY coordinate positions of micro-reaction chambers in the first image processing technique according to the third embodiment. FIG. 12 is a flowchart showing the first image processing technique according to the third embodiment. FIG. 13 is a diagram showing the results of acquiring XY coordinate position information of each micro-reaction chamber for 20 chips using the first image processing technique according to the third embodiment. FIG. 14 is a conceptual diagram for explaining the second image processing technique according to the fourth embodiment. FIG. 15 is a diagram showing the results of precise correction of the XY coordinates of 400 micro-reaction chambers in 20 chips using the second image processing technique according to the fourth embodiment. FIG. 16 is a schematic cross-sectional view for explaining a method of forming a nozzle shape trace according to the fifth embodiment. FIG. 17 is a schematic top view of a nozzle shape trace. FIG. 18 is a conceptual diagram for explaining image processing using the nozzle shape trace according to the fifth embodiment. FIG. 19 is a photographed image showing an example of a plurality of nozzle shape traces (formed at 100 μm pitch intervals).10 is a graph showing the result of the correction amount for "the horizontal direction difference distance from the reference position of the captured image to the horizontal direction center position of the tip of the nozzle" in the fifth embodiment. FIG. 11 is a cross-sectional schematic diagram of a flat substrate according to the sixth embodiment. FIG. 12 is a cross-sectional schematic diagram showing a state immediately before the tip of the nozzle is lowered into the inside of a micro-reaction chamber. FIG. 13 is a cross-sectional schematic diagram showing an example in which the shape of the tip of the nozzle is changed according to the sixth embodiment. FIG. 14 is a cross-sectional schematic diagram showing an example in which the shape of the tip of the nozzle is changed according to the sixth embodiment. FIG. 15 is a cross-sectional schematic diagram showing a chip having a micro-reaction chamber in the shape of an inverted truncated cone according to the sixth embodiment. FIG. 16 is a side observation image of a nozzle having a tip in the shape of an inverted truncated cone according to the sixth embodiment. FIG. 17 is a table showing the result of the filling success rate when the difference between the circular diameter of the bottom of the micro-reaction chamber and the circular outer diameter of the tip of the nozzle is changed according to the seventh embodiment. FIG. 18 is a cross-sectional schematic diagram showing a state in which a high-concentration micro-particle solution inside the nozzle according to the eighth embodiment is pressurized. FIG. 19 is a table showing the result of the filling success rate when the pressure inside the tip of the nozzle is increased according to the eighth embodiment. FIG. 19 is a cross-sectional schematic diagram showing the state in which the high-concentration micro-particle solution inside the nozzle according to the eighth embodiment is pressurized. 18 is a schematic diagram for explaining a particle filling method according to a tenth embodiment. FIG. 19 is a schematic diagram for explaining a particle filling method according to a tenth embodiment. FIG. 20 is a schematic diagram for explaining a particle filling method according to a tenth embodiment. FIG. 21 is a schematic diagram for explaining a particle filling method according to a tenth embodiment. FIG. 22 is a diagram for explaining a particle filling method according to a tenth embodiment. FIG. 23 is a diagram for explaining a work flow for detecting unfilled micro-reaction chambers according to the tenth embodiment. FIG. 24 is a table showing the results of the particle filling success rate when refilling of particles is performed according to the tenth embodiment. FIG. 25 is a bird's-eye observation image of a resin sheet provided with well-filled micro-reaction chambers according to the tenth embodiment. FIG. 26 is a schematic diagram for explaining a particle filling method of a continuous filling method according to an eleventh embodiment. FIG. 27 is a diagram showing a work flow of the continuous filling method according to the eleventh embodiment. FIG. 28 is a graph showing the filling time per one micro-reaction chamber when the work flow of FIG. 18 in the second embodiment is used. FIG. 29 is a graph showing the filling time per one micro-reaction chamber when the continuous filling method according to the eleventh embodiment is used.

[0025] [Definition of Terms] First, definitions of terms used in each embodiment of the present disclosure will be explained.

[0026] "Micro-reaction chamber": A concave-shaped container formed on a flat substrate. Also called a microwell. Its diameter is typically several μm to several hundred μm, and particularly several tens of μm. This includes those with through-holes formed in the bottom surface for capturing cells, as described in Non-Patent Document 5.

[0027] "Nozzle": A device with a hollow structure through which solutions and particles can move, and whose tip can be inserted into a micro-reaction chamber.

[0028] "Alignment": Aligning the horizontal center position of the nozzle tip with the horizontal center position of the micro-reaction chamber.

[0029] "Microparticle": A particle having a specific gravity greater than that of a solution and a diameter of several hundred nanometers to several hundred micrometers, or a particle having magnetic, electric, or dielectric properties and a diameter of several nanometers to several hundred micrometers.

[0030] "Buffer": A solution in which microparticles are suspended. The pH or salt concentration may be controlled to maintain the suspension of the microparticles. Furthermore, a surfactant may be mixed in the solution as needed to control the wettability (contact angle) of the solution with the outer wall of the nozzle and the inner wall of the micro-reaction chamber.

[0031] "Contact": The solution component (buffer) of the microparticle solution sucked into the tip of the nozzle comes into contact with the inner wall (bottom and side wall) of the micro-reaction chamber. In particular, this refers to a state in which a contact angle can be defined between the solution and the inner wall.

[0032] "Filling": Supplying the required number of microparticles (required microparticle concentration) into the micro-reaction chamber.

[0033] "Concentration": To increase the particle concentration (number of particles per unit volume (number density)) of a particle suspension until it reaches a particle concentration that is 10% or more of the maximum concentration while maintaining the dispersed state of the particles.

[0034] "Settling": A deviation from the particle concentration of the particle solution stored in the storage container, resulting in an increase in particle concentration in some areas, particularly at the tip of the nozzle.

[0035] "Determination": A region where the concentration of microparticles is increased (high-concentration suspension) is formed at the tip of the nozzle, and it is determined whether the concentration of microparticles and the volume of the region have reached a level where the number of microparticles occupies 10% or more of the volume of the micro-reaction chamber.

[0036] "High concentration": The state in which the concentration of particles required for filling has been reached. This refers to a particle concentration that is 10% or more of the maximum concentration while maintaining the dispersed state of the particles.

[0037] [Problems of the Present Disclosure] The first problem to be solved by the technology of the present disclosure has been described as accurately aligning the nozzle tip with the micro-reaction chamber. Here, the second problem to be solved by the technology of the present disclosure will be described. Even if the nozzle tip is accurately aligned with the micro-reaction chamber and the nozzle tip can be lowered into the micro-reaction chamber, filling the micro-reaction chamber with a high-concentration microparticle solution does not necessarily result in successful filling. As will be described later, the principle of microparticle filling in this method is the spreading of liquid driven by Laplace pressure due to the surface tension (wettability) of the microparticle solution on the nozzle and the micro-reaction chamber. If this spreading is insufficient, there is a risk that the microparticle solution will not be filled at all or will only be partially filled.

[0038] 2A is a cross-sectional schematic diagram (upper side) for explaining the filling process when the high-concentration microparticle solution is successfully filled into the micro-reaction chamber 101, and a photographed image (lower side) of the micro-reaction chamber 101 when the microparticles are successfully filled. As shown in FIG. 2A, when the high-concentration microparticle solution can be spread up to the side wall of the micro-reaction chamber 101, the filling of the microparticles is successful.

[0039] 2B is a cross-sectional schematic diagram (left side) for explaining the filling process when filling of a high-concentration microparticle solution into the inside of the micro-reaction chamber 101 fails, and a photographed image (right side) of the micro-reaction chamber when filling of the microparticles fails. As shown in FIG. 2B, if the microparticle solution cannot spread to the side wall of the micro-reaction chamber 101, it will be partially unfilled (voids will be generated), resulting in filling failure. In the upper part of FIG. 2B, the microparticle solution does not reach the side wall of the micro-reaction chamber 101, and is retained only in the center of the micro-reaction chamber 101. In the lower part of FIG. 2B, the microparticle solution reaches only one side wall of the micro-reaction chamber.

[0040] As described above, the second problem of the present disclosure is the need to increase the probability (yield) of successfully filling a micro-reaction chamber with a high-concentration microparticle solution accumulated at the tip of the nozzle. Only after solving the first and second problems can microparticles be successfully filled into the interior of the micro-reaction chamber 101 as shown in Figures 1A and 2A. Thus, in order to successfully fill a micro-reaction chamber with a microparticle solution, it is necessary to accurately align the tip of the nozzle with the micro-reaction chamber and then increase the probability of successfully filling a micro-reaction chamber with a high-concentration microparticle solution accumulated at the tip of the nozzle.

[0041] [First Embodiment] <Particle Filling System> Fig. 3A is a schematic diagram showing a particle filling system 100 according to a first embodiment. Fig. 3B is an overhead view showing an implementation example of the particle filling system 100. Fig. 3C is a side view showing an implementation example of the particle filling system 100. The particle filling system 100 is an apparatus that automatically performs a method for aligning a micro-reaction chamber and a nozzle, and a particle filling method, which will be described later. As shown in Fig. 3A, the particle filling system 100 includes a first drive stage 106 (first drive mechanism), a first image capture device 107, a second drive stage 108 (second drive mechanism), a nozzle 109, a second image capture device 114, a drive mechanism 115, a sensor system 116, a dispensing mechanism 117, a plurality of storage containers 118, a stirring mechanism 119, and a control device 120.

[0042] The first drive stage 106 is configured to be fixed in position in the vertical direction and movable only in the horizontal direction. The planar substrate 102 is fixedly disposed on the first drive stage 106. The planar substrate 102 is provided with a plurality of micro-reaction chambers (containers). The first drive stage 106 has a table on which the planar substrate 102 is placed. The table has a space below (directly below) the planar substrate 102, and this space is configured to be able to hold a plurality of magnets 121.

[0043] The first imaging device 107 is disposed directly above the first drive stage 106 in the vertical direction. The first imaging device 107 is fixed in position in the horizontal direction. The first imaging device 107 is configured to be able to capture an image of the planar substrate 102 from above in a bird's-eye view. The first imaging device 107 is used to acquire XY coordinates on the first drive stage 106 in the alignment method and particle filling method described below.

[0044] The second driving stage 108 is configured to be fixed in position in the horizontal direction and movable only in the vertical direction. The nozzle 109 is provided on the second driving stage 108. The nozzle 109 is configured to be able to suck in the microparticle solution 110.

[0045] The drive mechanism 115 automatically controls the first drive stage 106 and the second drive stage 108 via a control board circuit. The first drive stage 106 and the second drive stage 108 are used for aligning the micro-reaction chamber 101 with the nozzle 109, accessing the nozzle 109 to the various storage containers 118, etc.

[0046] The second imaging device 114 is used to observe the concentration state of the microparticle suspension in the nozzle 109 that has been moved to immediately above the micro-reaction chambers. The second imaging device 114 may be a camera system that takes an image of the tip of the nozzle 109, or may be an optical system that irradiates the tip of the nozzle 109 with light and analyzes scattered light, absorbed light, or transmitted light.

[0047] The sensor system 116 detects contact between the nozzle 109 and the micro-reaction chambers. The sensor system 116 typically has a pressure sensor, a load sensor, or a strain sensor. The sensor system 116 determines contact between the nozzle 109 and the micro-reaction chambers by detecting, with the sensor, an upward force applied to the tip of the nozzle 109 that is generated by contact between the nozzle 109 and the micro-reaction chambers. Alternatively, the sensor system 116 may be configured to detect contact by optical means such as a camera.

[0048] The dispensing mechanism 117 has, for example, a syringe pump, and controls the suction and discharge of liquid into the nozzle 109. The dispensing mechanism 117 particularly controls the suction of the microparticle solution 110 into the nozzle 109. The nozzle 109 is connected to the dispensing mechanism 117 by piping (for example, a PEEK piping tube). An electromagnetic valve, typically a solenoid valve, can be provided as one of the components of the dispensing mechanism 117, and is used to release atmospheric pressure inside the dispensing mechanism and control pressurization and depressurization by realizing a closed system. The syringe pump in the dispensing mechanism 117 may be controlled as part of the driving mechanism 115, for example.

[0049] The plurality of storage containers 118 include a storage container for storing the microparticle solution, a storage container for storing the cleaning solution, and a waste liquid container for collecting waste liquid. In particular, the storage container for storing the microparticle solution is mounted on a stirring mechanism 119, which stirs the solution at appropriate times and controls the stirring so as to always maintain a good dispersion state.

[0050] The magnet 121 is typically a neodymium magnet. When the nozzle 109 is placed directly above the micro-reaction chamber 101, the magnetic field can move the magnetic microparticles in a short time, efficiently forming a highly concentrated suspension. The magnetization direction of the magnet can be set in the direction in which the microparticles are desired to settle, i.e., perpendicular to the flat substrate, thereby allowing the microparticles to settle efficiently.

[0051] The control device 120 controls the operations of the first imaging device 107, the second imaging device 114, the drive mechanism 115, the sensor system 116, the dispensing mechanism 117, and the stirring mechanism 119. The control device 120 also receives detection signals from the first imaging device 107, the second imaging device 114, and the sensor system 116, and executes necessary arithmetic processing.

[0052] FIG. 4 is a schematic cross-sectional view of a planar substrate 102 having multiple micro-reaction chambers 101. The planar substrate 102 is, for example, a chip for single-cell analysis, and has a structure that is a miniaturized version of a so-called microtiter plate. The planar substrate 102 has multiple micro-reaction chambers 101 (containers). For simplicity of illustration, only three micro-reaction chambers 101 are shown in FIG. 4, but the number of micro-reaction chambers 101 can be four or more. The micro-reaction chambers 101 have an upper opening 103a and a bottom 103b, and a through-hole 105 extending from the bottom 103b to the bottom surface 104 of the planar substrate 102 is formed. Here, an example of a linear cylindrical shape of the micro-reaction chamber 101 is illustrated, but other shapes may be used as in the examples described below. The through-hole 105 may be a linear cylindrical shape or a truncated cone shape.

[0053] FIG. 5 is a perspective view showing an example of an implementation of the sensor system 116. As shown in FIG. 5, the sensor system 116 includes two ferrules 92, a block 93, two leaf springs 94 (elastic bodies), a fixing jig 95, and a strain sensor 96. The two ferrules 92 connect the nozzle 109 to the piping. The ferrules 92 are fixed to a block 93 (made of, for example, PMMA). One longitudinal end of each of the two leaf springs is fixed to the block 93 by screws. The other longitudinal end of the leaf spring 94 is fixed to a metal fixing jig. With this configuration, when stress is applied to the nozzle 109, the stress is transmitted to the leaf spring 94 via the block 93, causing the leaf spring 94 to deform.

[0054] A strain sensor 96 is fixed to each leaf spring 94 so as to fit along the surface of the leaf spring 94. When the tip of the nozzle 109 comes into contact with the bottom surface of the micro-reaction chamber 101 and an upward force (stress) is applied to the nozzle 109, the leaf spring 94 is distorted and the amount of this distortion is detected by the strain sensor 96. When the amount of distortion reaches a predetermined value or more, it can be determined that the nozzle 109 has come into contact with the micro-reaction chamber 101.

[0055] <Method for Aligning Micro-Reaction Chambers and Nozzles> A method for aligning micro-reaction chambers and nozzles associated with the micro-particle filling method according to the first embodiment will be described with reference to Figures 6 to 12B. As will be described below, the alignment method according to this embodiment includes the following steps (i) to (v).

[0056] (i) Preparing Micro-Reaction Chambers First, the user prepares the planar substrate 102 having the above-described plurality of micro-reaction chambers 101 thereon.

[0057] (ii) Obtaining Position Information of Micro-Reaction Chambers FIG. 6 is an overhead view showing the state in which the planar substrate 102 is fixed on the first drive stage 106. As shown in FIG. 6, the user fixes the planar substrate 102 at a predetermined position on the first drive stage 106. Here, the horizontal direction of the drive direction of the first drive stage 106 is defined as the X coordinate, and the vertical direction is defined as the Y coordinate. The initial position of the first drive stage 106 is set as the origin (0,0) of the XY coordinate. Furthermore, the drive direction of the second drive stage 108 is defined as the Z coordinate. The initial position of the second drive stage 108 is set as the origin (0) of the Z coordinate.

[0058] 7A is an overhead view showing the state in which the planar substrate 102 is placed directly below the first imaging device 107. As shown in FIG. 7A, the first drive stage 106 is driven to place the planar substrate 102 directly below the first imaging device 107. Then, the first imaging device 107 is used to capture an image of the multiple micro-reaction chambers 101. The control device 120 processes the captured image to obtain positional information within the image for each of the multiple micro-reaction chambers 101.

[0059] 7B is a captured image for explaining a method for acquiring in-image position information of a plurality of micro-reaction chambers 101. First, as shown in FIG. 7B, in-image position information (relative position with respect to the reference position) of the micro-reaction chambers 101 is acquired from the reference position of the captured image. The reference position of the captured image can be, for example, the center position of the captured image (left side of FIG. 7B) or the edge of the captured image (right side of FIG. 7B). Hereinafter, in this disclosure, the reference position of the captured image will be described as the center position.

[0060] Next, the position information of the micro-reaction chamber 101 in the image is converted into the XY coordinates of the first drive stage 106. At this time, the reference position of the captured image and the XY coordinates of the first drive stage 106 are set so as to be linearly linked. In other words, the settings are made so that linear conversion can be performed using a coefficient that converts position information of a specific pixel in the image into XY coordinate information of the first drive stage 106. With such settings, the image information of the first image capture device 107 and the position information of the first drive stage 106 can be linked. The above coefficient is generally determined by the resolution of the first image capture device 107.

[0061] In this way, the horizontal center position of the micro-reaction chamber 101 when the planar substrate 102 is placed directly below the first imaging device 107 can be obtained as "the coordinates at which the horizontal center position of the container opening is located at the reference position of the captured image" on the XY coordinates of the first drive stage 106. Specifically, the horizontal center position of the micro-reaction chamber 101 can be calculated by the following formula (1).

[0062]

[0063] Here, X ｗｅｌｌＮｔｈ＿ｉｎｉｔｉａｌ , Y ｗｅｌｌＮｔｈ＿ｉｎｉｔｉａｌ are the X and Y coordinates on the first driving stage at which the initially detected N-th micro-reaction chamber 101 is placed at the reference position of the image captured by the first imaging device 107. ｓｔａｇｅ＿ｒｅｆｅｒ , Y ｓｔａｇｅ＿ｒｅｆｅｒare the X and Y coordinates on the first drive stage that are used as a reference when the micro-reaction chamber 101 is photographed. ΔxwellNth_image and ΔywellNth_image are the X and Y coordinates (relative coordinates from the reference position) of the Nth micro-reaction chamber 101 in the image photographed by the first image pickup device 107. α is a coefficient that converts pixel position information in the image photographed by the first image pickup device 107 into X and Y coordinate information of the first drive stage 106. For example, the reference position of the photographed image is set to the center position, and X ｓｔａｇｅ＿ｒｅｆｅｒ , Y ｓｔａｇｅ＿ｒｅｆｅｒ are the X and Y coordinates on the current first drive stage at the time of imaging, and ΔxwellNth_image and ΔywellNth_image are the X and Y coordinates (relative coordinates from the reference position) in the image of the Nth micro-reaction chamber on the captured image. ｗｅｌｌＮｔｈ＿ｉｎｉｔｉａｌ , Y ｗｅｌｌＮｔｈ＿ｉｎｉｔｉａｌ can be obtained.

[0064] Furthermore, a specific micro-reaction chamber is determined in advance as a reference position, and the X and Y coordinates on the first driving stage when the micro-reaction chamber is positioned at the reference position (center position) of the image captured by the first imaging device 107 are defined as X ｓｔａｇｅ＿ｒｅｆｅｒ , Y ｓｔａｇｅ＿ｒｅｆｅｒ In this case, ΔxwellNth_image and ΔywellNth_image are set as relative positions from a specific micro-reaction chamber on the captured image. Even with such a setting, X ｗｅｌｌＮｔｈ＿ｉｎｉｔｉａｌ , Y ｗｅｌｌＮｔｈ＿ｉｎｉｔｉａｌ can be obtained.

[0065] (iii) Defining the differential distance between the first imaging device 107 and the nozzle 109 As described above, it is necessary to align the desired micro-reaction chamber 101 in the horizontal direction directly below the nozzle 109. Because the first imaging device 107 and the nozzle 109 are fixed in position in the horizontal direction, the "horizontal differential distance from the reference position of the captured image to the horizontal center position of the nozzle tip" between the first imaging device 107 and the nozzle 109 is basically a fixed value. This horizontal differential distance is determined in advance by the layout information in the mechanical design.

[0066] 8 is a bird's-eye view for explaining the difference in distance between the first imaging device 107 and the nozzle 109. As shown in FIG.ｎｏｚｚｕｌｅ＿ｉｎｉｔｉａｌ , ΔY ｎｏｚｚｕｌｅ＿ｉｎｉｔｉａｌ is defined as the difference distance in the X and Y coordinate directions on the first drive stage, which is the distance from the reference position of the image captured by the first image capture device 107 to the horizontal center position of the tip of the nozzle 109.

[0067]

[0068] (iv) Correcting the differential distance between the first imaging device 107 and the nozzle 109. The "horizontal differential distance from the reference position of the captured image to the horizontal center position of the nozzle tip" described above may deviate slightly by several to several tens of micrometers in actual operation due to misalignment of mechanical components caused by temperature fluctuations and the degree of freedom of horizontal movement of the nozzle tip 109. The dimensional difference between the micro-reaction chamber 101 and the tip of the nozzle 109 must be adjusted to within several micrometers. Therefore, if this minute deviation is not corrected, it can cause a descent failure (see FIG. 1B ) during alignment. Therefore, by correcting the differential distance value at an appropriate time rather than leaving it as a fixed value, descent failure can be prevented.

[0069] 9A is an overhead view for explaining a method for correcting the differential distance between the first imaging device 107 and the nozzle 109. First, a planar substrate 112 for forming a nozzle shape mark 111 that is the same as the tip shape of the nozzle 109 is prepared and fixed on the first driving stage 106. Next, the first driving stage 106 is used to move the XY coordinates so that the planar substrate 112 for form a shape mark is positioned directly below the first imaging device 107, and the "location on the planar substrate 112 for form a nozzle shape mark 111" is set as the reference position of the captured image.

[0070] 9B is a photographed image of the planar substrate 112 for pattern tracing. As shown in FIG. ｓｔａｍｐ＿ｂｅｆｏｒｅ , Y ｓｔａｍｐ＿ｂｅｆｏｒｅ are set as the X and Y coordinates on the first driving stage of the "place where the nozzle shape trace 111 should be provided on the shape trace flat substrate 112."

[0071]

[0072] 10A is an overhead view illustrating the movement of the first drive stage 106 to form the nozzle shape mark 111. As shown in FIG. 10A, a preset "horizontal differential distance from the reference position in the captured image to the horizontal center position of the nozzle tip" is added to the XY coordinates of the first drive stage 106, and the first drive stage 106 is moved so that the nozzle 109 is positioned directly above the "location where the nozzle shape mark 111 should be formed on the shape mark flat substrate 112." Specifically, the first drive stage 106 is moved to the X and Y coordinates calculated by the following equation (2).

[0073]

[0074] Here, X ｓｔａｍｐ＿ｎｏｚｚｕｌｅ , Y ｓｔａｍｐ＿ｎｏｚｚｕｌｅ are the X and Y coordinates on the first driving stage 106 where the nozzle 109 is placed directly above the "place where the nozzle shape trace 111 should be provided on the shape trace flat substrate 112."

[0075] 10B is an image captured by the second imaging device 114 when forming the nozzle shape mark 111. As shown in FIG. 10B , the second drive stage 108 is used to lower the nozzle 109 to the Z coordinate where it contacts the planar substrate 112 for the shape mark, and the nozzle shape mark 111 is formed on the planar substrate 112 for the shape mark.

[0076] 11A is an overhead view for explaining the movement of the first drive stage 106 for photographing the nozzle shape mark 111. As shown in Fig. 11A, the first drive stage 106 is used to return the XY coordinates to "the location where the nozzle shape mark 111 should be provided on the shape mark flat substrate 112," and the nozzle shape mark 111 is photographed.

[0077] FIG. 11B shows a captured image of the nozzle shape mark 111. If the reference position of the captured image and the center position of the nozzle shape mark 111 in the image match, no correction is necessary. However, if the positions do not match, correction of the corresponding misalignment amount is required. Correct correction can be performed by correcting the pixel misalignment amount in the image using a conversion coefficient that converts pixel values ​​of the image into coordinate values ​​on the drive stage and adding it to the pre-registered "horizontal differential distance from the reference position of the captured image to the horizontal center position of the nozzle tip" between the first image capture device 107 and the nozzle 109. Specifically, the coordinate after correcting the differential distance between the first image capture device 107 and the nozzle 109 can be calculated using the following equation (3).

[0078]

[0079] Here, Δx ｓｔａｍｐ , Δy ｓｔａｍｐ are the X and Y coordinates (relative coordinates from the reference position) of the newly formed nozzle shape mark 111 on the image captured by the first imaging device 107. ｎｏｚｚｕｌｅ＿ｍｏｄｉｆｉｅｄ , ΔY ｎｏｚｚｕｌｅ＿ｍｏｄｉｆｉｅｄ is the difference distance after correction in the X and Y coordinate directions on the first drive stage from the reference position of the image captured by the first image capturing device 107 to the horizontal center position of the tip of the nozzle 109.

[0080] The nozzle trace 111 can be formed, for example, by using ink (such as an aqueous amido black solution). The nozzle 109 can suck the aqueous amido black solution and apply a small amount of the aqueous amido black solution to a predetermined position on the flat substrate 112 for forming the nozzle trace 111. Alternatively, the flat substrate 112 for forming the nozzle trace may be made of a resin having mechanical plasticity. In this case, the nozzle trace 111 can be formed by pressing the tip of the nozzle 109 against the flat substrate 112 for forming the nozzle trace.

[0081] (v) Aligning the micro-reaction chamber and the nozzle The horizontal center positions of the micro-reaction chamber 101 and the nozzle 109 can be aligned by using the "coordinates at which the horizontal center position of the container opening is located at the reference position of the photographed image" on the XY coordinates of the first driving stage 106 obtained in steps (ii) to (iv) and the "horizontal differential distance from the reference position of the photographed image to the horizontal center position of the tip of the nozzle."

[0082] 12A is a bird's-eye view illustrating the movement of the first drive stage 106 when aligning the horizontal center positions of the micro-reaction chamber 101 and the nozzle 109. As shown in FIG. 12A, by moving the first drive stage 106 to the XY coordinates obtained by adding the above two variables, the horizontal center position of the micro-reaction chamber 101 can be moved to a position that coincides with the horizontal center position of the nozzle 109. For example, the target XY coordinates are calculated using the following equation (4):

[0083]

[0084] Here, X ｗｅｌｌＮｔｈ＿ｉｎｉｔｉａｌ , Y ｗｅｌｌＮｔｈ＿ｉｎｉｔｉａｌ are the X and Y coordinates on the first drive stage where the Nth micro-reaction chamber is placed directly below the nozzle.

[0085] 12B is an image captured by the second imaging device 114 in a state where the nozzle 109 and the micro-reaction chamber 101 are aligned. With the above-described precise alignment achieved, the second drive stage 108 is moved to the Z coordinate where the nozzle 109 descends into the micro-reaction chamber 101, thereby positioning the tip of the nozzle 109 directly above the micro-reaction chamber 101. Thereafter, the nozzle 109 is accurately lowered into the micro-reaction chamber 101, and the micro-particle solution can be filled into the micro-reaction chamber 101 from inside the nozzle 109.

[0086] The nozzle 109 is typically a glass capillary, and has a shape that tapers toward the tip. It is sufficient that the nozzle 109 can be inserted into the micro-reaction chamber 101 until the tip of the nozzle 109 contacts the bottom 103b of the micro-reaction chamber 101, and the nozzle 109 may have a constant outer diameter.

[0087] <Method for filling particles> Fig. 13 is a schematic diagram for explaining the method for filling particles including the alignment method of this embodiment. As shown in Fig. 13, the method for filling particles of this embodiment includes basic steps (a) to (f) for filling the inside of the micro-reaction chamber 101 with the micro-particle solution 110.

[0088] (a) Suction of the microparticle solution 110 by the nozzle 109 The first drive stage 106 is used to move the nozzle 109 to an XY coordinate position where a solution tube holding the microparticle solution 110 is located directly below the nozzle 109. Next, the second drive stage 108 is used to lower the nozzle 109 to a Z coordinate position where the nozzle 109 contacts the microparticle solution 110, and the microparticle solution 110 is sucked into the nozzle 109. Thereafter, the second drive stage 108 is moved to the origin coordinate, and this step is completed.

[0089] The solution tube may be a single PCR tube, an 8-tube PCR tube, or a 96-well or 384-well microplate. By using an appropriate automatic stirrer to stir these solution tubes immediately before aspirating the solution in this step, the microparticles can be kept well dispersed.

[0090] The nozzle 109 sucks the microparticle solution 110 by immersing the empty nozzle 109 in the solution and utilizing capillary action to suck the microparticle suspension. Alternatively, a syringe pump or the like may be connected to a pipe or the like at the tip of the empty nozzle 109 opposite to the tip at which the microparticle suspension is dispensed, and the microparticle solution 110 may be discharged and sucked through discharge and suction operations performed by pressurizing and depressurizing.

[0091] (b) Alignment of the micro-reaction chamber 101 and the nozzle 109 Using the alignment method described above, the first drive stage 106 is moved so that the horizontal center positions of the micro-reaction chamber 101 and the nozzle 109 are aligned. Then, the second drive stage 108 is moved so that the tip of the nozzle 109 is positioned directly above the micro-reaction chamber 101, approximately 100 to 300 μm away in the Z direction.

[0092] (c) Concentration (sedimentation) of microparticles After leaving the solution for a predetermined time, the microparticles in the microparticle solution 110 are concentrated by gravity to the tip of the nozzle 109, thereby obtaining a high-concentration microparticle solution 113 of a predetermined volume and concentration. Alternatively, if the microparticles are magnetic microparticles, an external magnetic force is applied to move the microparticles to the tip of the nozzle 109, thereby concentrating the microparticle solution 113, thereby obtaining a high-concentration microparticle solution 113. The predetermined volume is set to a value such that, when the high-concentration microparticle solution 113 is filled into the micro-reaction chamber 101, the solution contains a number of microparticles that occupy 20% or more of the volume of the micro-reaction chamber 101. As described above, the predetermined concentration (high concentration) is a microparticle concentration that is 10% or more of the maximum concentration while maintaining the microparticles in a dispersed state.

[0093] Once the state of step (c) is established, for example, the tip of the nozzle 109 is photographed with the second imaging device 114, and temporal and spatial changes of the microparticles in the nozzle 109 are observed. In this manner, the volume of the high-concentration microparticle solution 113 in the nozzle 109 is estimated. At this time, when the height of the liquid surface of the high-concentration microparticle solution 113 in the nozzle 109 reaches a predetermined value, it can be determined that a predetermined volume of the high-concentration microparticle solution 113 has been obtained. At this time, by installing the second imaging device 114 in a lateral direction relative to the nozzle 109, it becomes easy to observe the microparticles in the nozzle 109.

[0094] Alternatively, it is also possible to determine whether a predetermined volume of the high-concentration microparticle solution 113 has been obtained by irradiating the tip of the nozzle 109 with light and measuring the amount or distribution of scattered light, absorbed light, or transmitted light. In this case, it is necessary to appropriately control the spot size and position of the irradiated light near the nozzle 109. Alternatively, it is also possible to irradiate light at a position at a predetermined height of the nozzle 109 and determine whether a predetermined volume of the high-concentration microparticle solution 113 has been obtained based on the scattered light, absorbed light, or transmitted light at that position.

[0095] (d) Lowering the nozzle 109 into the micro-reaction chamber 101: When a predetermined volume of the high-concentration microparticle solution 113 is obtained in step (c), the nozzle 109 is lowered, and the surface tension of the high-concentration microparticle solution 113 at the tip of the nozzle 109 causes the high-concentration microparticle solution 113 to come into contact with the bottom 103b or the side wall surface 103c of the micro-reaction chamber 101. This causes the high-concentration microparticle solution 113 to wet and spread within the micro-reaction chamber 101. Then, the high-concentration microparticle solution 113 seeps out to at least a part of the side wall surface 103c of the micro-reaction chamber 101, and the high-concentration microparticle solution 113 comes into contact with the bottom 103b and the side wall surface 103c of the micro-reaction chamber 101.

[0096] (e) Raising the nozzle 109 When the high-concentration microparticle solution 113 comes into contact with the bottom 103b and the side wall surface 103c of the micro-reaction chamber 101 in step (d), the nozzle 109 is raised at a predetermined speed to fill the high-concentration microparticle solution 113 into the micro-reaction chamber 101. The principle of this filling will be described later.

[0097] (f) Completion of Filling The state in which the micro-reaction chamber 101 is filled with the high concentration microparticle solution 113 is regarded as completion of filling.

[0098] 14A and 14B are cross-sectional schematic views for explaining the principle of the microparticle filling method according to this embodiment, showing an enlarged view of the tip of the nozzle 109 and the internal space of the micro-reaction chamber 101. Fig. 14A shows the state immediately after the high-concentration microparticle solution 113 in the nozzle 109 comes into contact with the bottom 103b of the micro-reaction chamber 101. As shown in Fig. 14A, in step (d), the nozzle 109 is lowered and, when a part of the lower end of the nozzle 109 comes into contact with the bottom 103b of the micro-reaction chamber 101, the high-concentration microparticle solution 113 wets and spreads over (comes into contact with) the bottom 103b of the micro-reaction chamber 101.

[0099] 14B shows a state in which the high-concentration microparticle solution 113 also contacts the side wall surface 103c of the micro-reaction chamber 101. As shown in FIG. 14B, the high-concentration microparticle solution 113 seeps out until it contacts at least a part of the side wall surface 103c.

[0100] Here, the radius of the bottom 103b of the micro-reaction chamber 101 is Rw, the radius of the outer wall 109a at the tip of the nozzle 109 is Rc, the contact angle of the high-concentration microparticle solution 113 with the inner wall of the micro-reaction chamber 101 is θw, and the contact angle of the high-concentration microparticle solution 113 with the nozzle outer wall 109a is θc.

[0101] When the suction pressure (ΔP) expressed by the following formula (5) is positive, the high-concentration microparticle solution 113 can be drawn out from the nozzle 109. Therefore, in step (e), by raising the nozzle 109 at a predetermined speed while maintaining the state of ΔP>0, the high-concentration microparticle solution 113 can be filled into the micro-reaction chamber 101. The raising speed of the nozzle 109 can be constant, or may be raised while accelerating or decelerating, as long as it is within a range in which ΔP>0 can be maintained.

[0102]

[0103] If the particle concentration and volume of the high-concentration particle solution 113 are set to appropriate conditions, they can be set to 50% or more of the maximum concentration while maintaining the dispersed state of the high-concentration particle solution 113, and the volume of the high-concentration particle solution 113 can be made equal to or greater than the volume of the micro-reaction chamber 101, thereby filling at least 50% or more of the volume of the micro-reaction chamber 101. If the particle concentration and volume of the high-concentration particle solution 113 are set to even more appropriate conditions, the microparticles can occupy 80% or more of the volume of the micro-reaction chamber 101 in a dry state. By appropriately setting the concentration and volume in this way, there is no need to additionally discharge the high-concentration particle solution 113 filled in the micro-reaction chamber 101.

[0104] The volume of the high-concentration microparticle solution 113 formed in the nozzle 109 can be made larger than the volume of the micro-reaction chamber 101. Assuming that the micro-reaction chamber 101 is formed without error, the opening angle is formed at 90°, and there is no adhesion on the inner wall (shape shown in FIG. 4 ), when the nozzle 109 is pulled up in step (e), when the seeping high-concentration microparticle solution 113 reaches the upper end of the micro-reaction chamber 101, the contact angle θw is less than 90°. Substituting this contact angle θw into equation (1) results in ΔP<0, and the discharge of the high-concentration microparticle solution 113 from the nozzle 109 stops. Therefore, by setting the conditions appropriately, even if the volume of the high-concentration microparticle solution 113 is larger than the volume of the micro-reaction chamber 101, the discharge stops at the upper end of the micro-reaction chamber 101, so the high-concentration microparticle solution 113 does not overflow from the micro-reaction chamber 101.

[0105] Although the micro-reaction chamber 101 shown in Figure 4 has a through-hole 105 for capturing cells, which is necessary for the single-cell analysis chip, the through-hole 105 is not essential for the principle of micro-particle filling described above. In fact, micro-particles can be filled into a container without a through-hole 105 according to the above principle. That is, the container into which micro-particles are filled by the technology of this embodiment is not limited to the micro-reaction chamber 101 of the single-cell analysis chip, but may be any container into which a nozzle 109 can be inserted. The cross-sectional shape of the container into which micro-particles are filled is also not limited to a rectangle, and other shapes, such as a trapezoid whose bottom is narrower than the opening, can be used.

[0106] The above has described a method for filling a single micro-reaction chamber 101 with microparticles. When filling multiple micro-reaction chambers 101 with microparticles, the above operation can be repeated. Specifically, after completing the filling of a first micro-reaction chamber according to the above steps (a) to (f), the above steps (a) to (f) are also carried out for a second micro-reaction chamber, and the steps for filling microparticles are similarly repeated thereafter.

[0107] The same operation is performed when filling multiple types of microparticles into different microreaction chambers 101. For example, to produce the single cell analysis chips described in Non-Patent Document 5 and Patent Document 1, an example will be described in which multiple types of microparticles, to which DNA probes of different sequences are immobilized, are individually filled into preset microreaction chambers.

[0108] First, solution tubes containing the microparticle solutions 110 in which various types of microparticles are suspended are prepared, and step (a) is performed on the solution tube containing the desired solution. Then, steps (b) to (f) are performed to complete filling of the desired micro-reaction chambers 101 with the desired microparticle solutions 110.

[0109] Next, the first drive stage 106 is moved to the XY coordinate position where the nozzle 109 and the waste liquid tube are located, and by driving the second drive stage 108 and operating a syringe pump or the like, the excess microparticle solution 110 in the nozzle 109 is discarded into the waste liquid tube.

[0110] Next, to clean the nozzle 109, the nozzle 109 is moved to an XY coordinate position where multiple solution tubes containing multiple types of cleaning liquid are arranged. Then, by driving the second drive stage 108 and operating a syringe pump or the like, the nozzle 109 is immersed, and the cleaning liquid is repeatedly sucked and discarded. This completely cleans and removes the remaining microparticle solution and adhering microparticles from the nozzle 109. For example, pure water such as filtered ion-exchanged water or alcohol such as ethanol can be used as the cleaning liquid.

[0111] The above procedure is repeated to fill the next type of microparticles into the micro-reaction chamber at the next position.

[0112] <Operation of the Particle Filling System> FIG. 15 is a flowchart showing the operation of the particle filling system 100.

[0113] In step S1, the control device 120 establishes an initial state for the entire particulate filling system 100. Specifically, first, the user places the planar substrate 102 on the first drive stage 106 and places the storage container 118 for the particulate solution 110 and the storage container 118 for the cleaning solution in predetermined positions. After that, when the user inputs an instruction to start operation, for example, from an input device of the control device 120, the control device 120 checks whether the planar substrate 102, the nozzle 109, and the storage container 118 are all located in their predetermined initial positions. If any of these are not located in their initial positions, the drive mechanism 115 moves them to their initial positions.

[0114] In step S2, the control device 120 establishes an initial state for each micro-reaction chamber 101. Specifically, the control device 120 drives the drive mechanism 115 and the dispensing mechanism 117 to aspirate a cleaning liquid into the nozzle 109 and discharge it into a waste liquid container (not shown), thereby cleaning the nozzle 109. Thereafter, the control device 120 causes the nozzle 109 to aspirate the microparticle suspension in step (a), and moves the nozzle 109 to a position directly above a predetermined micro-reaction chamber 101 in step (b).

[0115] Step (c) is performed in step S3. The control device 120 determines whether the particles in the particle solution 110 in the nozzle 109 have settled, forming a predetermined volume of a high-concentration particle solution 113 with a predetermined particle concentration. Here, the high-concentration particle solution 113 is optically dense, so it can be observed by the second imaging device 114 as a dark area compared to low-concentration areas. The control device 120 calculates the size of the dark area based on the contrast of the image received from the second imaging device 114, and then calculates the concentration and volume of the high-concentration particle solution 113 based on that. Alternatively, the control device 120 calculates the size of the dark area of ​​the high-concentration particle solution 113 by detecting the scattering surface at its boundary, and then similarly calculates the concentration and volume. If the particles are magnetic, the magnetic field of the magnet 121 can be used to establish the state of step S2, and then the state can be maintained for, for example, 3 to 10 seconds, thereby determining that the concentration and volume of the high-concentration particle solution 113 have reached the predetermined values. If gravity is used, this maintenance time is approximately 50 to 120 seconds.

[0116] Step (d) is performed in step S4. The control device 120 drives the drive mechanism 115 to lower the nozzle 109, bringing the tip of the nozzle 109 into contact with the bottom 103b of the micro-reaction chamber 101. This contact causes the high-concentration microparticle solution 113 at the tip of the nozzle 109 to wet the bottom 103b and side wall surface 103c of the micro-reaction chamber 101. In the present disclosure, contact of the nozzle 109 is defined as the high-concentration microparticle solution 113 wetting the inner wall of the micro-reaction chamber 101. This is because it is necessary and essential for the high-concentration microparticle solution 113 to wet both the inner wall of the micro-reaction chamber 101 and the outer wall 109a of the nozzle 109, forming an appropriate contact angle, and for the high-concentration microparticle solution 113 in the nozzle 109 to be drawn into the micro-reaction chamber 101. However, in practice, the descent of the nozzle 109 must be stopped at some point, and therefore a sensor system 116 is provided to detect contact.

[0117] After contact between the nozzle 109 and the micro-reaction chamber 101, for example, within 1 second, the interface of the micro-particle suspension penetrates between the nozzle 109 and the sidewall surface 103c of the micro-reaction chamber 101 and rises. Step (e) is performed in step S5. The control device 120 drives the drive mechanism 115 to lift the nozzle 109 at a speed of, for example, approximately 1 to 130 μm / s. This moves the high-concentration micro-particle solution 113 in the nozzle 109 to the micro-reaction chamber 101, filling the micro-reaction chamber 101 with the micro-particles. Typically, the volume of the high-concentration micro-particle solution 113 filled in the micro-reaction chamber 101 is 1 nL or less, so it evaporates within a few seconds, leaving only the micro-particles in the micro-reaction chamber 101. During the drying process, the high-density micro-particles attract each other and aggregate due to the surface tension of the solution and the interfacial interaction between the micro-particles, leaving the micro-reaction chamber 101 with densely packed micro-particles.

[0118] In step S6, the control device 120 determines whether or not all the micro-reaction chambers 101 have been filled with microparticles.

[0119] If the answer is NO in step S6, the control device 120 drives the drive mechanism 115 and the dispensing mechanism 117 to fill the next micro-reaction chamber 101 with microparticles, and discards any remaining micro-particle solution 110 in the nozzle 109 into a waste container.

[0120] Next, returning to step S2, the control device 120 drives the drive mechanism 115 and the dispensing mechanism 117 to aspirate and dispense two types of cleaning liquid, thereby cleaning the nozzle 109. Next, the control device 120 drives the drive mechanism 115 and the dispensing mechanism 117 to aspirate a suspension of microparticles different from those previously filled (for example, microparticles having a different cell recognition tag sequence than the previous one) into the nozzle 109, and moves it to directly above a micro-reaction chamber 101 different from the previous one. This establishes an initial state for the next micro-reaction chamber 101. Thereafter, steps S3 to S6 are executed in the same manner as above.

[0121] Steps S2 to S6 are repeated for the second and subsequent micro-reaction chambers 101 in the same manner, and if the answer is YES in step S6, the control device 120 ends the operation of the micro-particle filling system 100.

[0122] In order for the microparticles to be filled into the micro-reaction chamber 101, the microparticles must be concentrated in step S4 (step (c)), and the microparticle solution 110 must wet the microparticles in the following steps S4 and S5 (steps (d) and (e)) and reach the side wall surface 103c of the micro-reaction chamber 101, so that the contact angle between the inner wall of the micro-reaction chamber 101 and the microparticle solution 110 and the contact angle between the outer wall of the nozzle 109 and the microparticle solution 110 must satisfy the condition of formula (1). To satisfy this condition, it is necessary to appropriately select the materials that determine the hydrophilicity of the inner wall of the micro-reaction chamber 101 and the hydrophilicity of the nozzle outer wall 109a, as well as the inner diameter of the micro-reaction chamber 101 and the outer diameter of the tip of the nozzle 109.

[0123] When the target microparticles are magnetic microparticles, a time reduction using a magnetic field is necessary to shorten the filling time. In this case, a magnet 121 can be placed directly below the planar substrate 102 having the micro-reaction chamber 101. As a result, when the nozzle 109 approaches the micro-reaction chamber 101 during a series of processes, the microparticles can be automatically moved to the tip of the nozzle 109 by the remote action of the magnetic field. On the other hand, when this configuration is adopted, a realistic method for capturing an overhead image of the micro-reaction chamber 101 is to capture a reflected image. Therefore, the first imaging device 107 needs to be installed on the same vertical side as the nozzle 109. Under the restrictive conditions of such a configuration, the technology of this embodiment is particularly useful for achieving a quick and accurate alignment method.

[0124] <Technical Effects> As described above, the microparticle filling method according to the first embodiment, which uses a method for aligning the micro-reaction chamber and the nozzle, ensures that the tip of the nozzle 109 is lowered into the interior of the micro-reaction chamber, which leads to an increased probability of success in the subsequent microparticle filling method using the microparticle filling principle.

[0125] [Second Embodiment] In the first embodiment, a technique for successfully filling microparticles by accurately aligning a micro-reaction chamber formed on a planar substrate with a nozzle was described. In the second embodiment, an example will be described in which the technique of the present disclosure is applied to filling microparticles into a micro-reaction chamber provided on a chip (substrate) of a single-cell analysis device.

[0126] <Chip Configuration Example> FIG. 16A is a top view showing a single-cell analysis chip 122 (substrate). FIG. 16B is a cross-sectional view showing a portion of the cross section of the chip 122. FIG. 16C is an image of the chip 122 captured by the first imaging device 107. 10 x 10 (100 in total) micro-reaction chambers 123 are formed at equal intervals on the chip 122. The micro-reaction chambers 123 are provided with through-holes 124 for cell capture, resulting in a chip structure capable of cell isolation for single-cell analysis. A chip 122 with such a structure can be molded using imprinting technology using, for example, dimethylpolysiloxane. Alternatively, it can be molded by molding using an elastomer material. The through-holes 124 can be drilled using laser processing, but other particle beam processing or molding using a mold may also be used.

[0127] Various materials can be used as the material for the chip 122, including, for example, various resin materials (polycarbonate, polycycloolefin, polyolefin, polypropylene, polyethylene, acrylic, acrylonitrile, polystyrene resin, polydimethylsiloxane resin, etc.), metal materials, semiconductor materials such as silicon, oxide materials such as quartz and alumina, and amorphous materials such as glass. A combination of these materials can also be used for processing or to control surface wettability.

[0128] 16A and 16B show an example of the dimensions of the chip 122. In the example shown, the micro-reaction chambers 123 are cylindrical with a diameter of 75 μm and a depth of 70 μm. The center-to-center spacing between adjacent micro-reaction chambers 123 is 105 μm. The through-holes 124 have a smallest diameter of 3 μm and a length of 30 μm. Note that the above size is an example of a size suitable for a single-cell analysis device, and the size of the micro-reaction chambers 123 to be filled with microparticles may range from several μm to several hundred μm in both diameter and depth. Furthermore, the shape of the micro-reaction chambers 123 does not have to be cylindrical. As long as they have an opening that can be filled with microparticles, the micro-reaction chambers 123 may be in the form of a polygonal prism such as a square prism, or a tapered shape such as a truncated cone or a square truncated cone.

[0129] In the single-cell analysis device, the microparticles filled in the micro-reaction chambers 123 of the chip 122 are used for gene expression analysis, which measures the amount of mRNA in a single cell for each gene sequence. By changing the molecules immobilized on the microparticles depending on the purpose of analysis, the device can be applied to various single-cell analyses.

[0130] In this embodiment, magnetic microparticles (1 μm in diameter) with numerous streptavidin molecules immobilized on their surfaces are used. For single-cell analysis, a DNA probe is immobilized on the microparticle surface to capture the mRNA to be measured and introduce a barcode sequence for cell identification into the nucleic acid sample. Biotin is immobilized at the 5' end of the DNA probe to firmly bind to streptavidin.

[0131] The DNA probe sequence has, from the 5' end, a consensus sequence for PCR, a tag sequence for molecular recognition, a tag sequence for cell identification (7 bases), and a continuous sequence of T's for mRNA capture at the 3' end. The tag sequence for cell identification is a 7-base sequence of 100 known types, and the tag sequence for molecular recognition is a 7-base random sequence.

[0132] The length and position of the cell identification tag sequence may be different from those described above. For example, three types of known 7-base sequences may be arranged so as to sandwich two known 3-base sequences, or the tag sequence may be arranged as a random sequence rather than a known sequence. Similarly, the length and position of the molecular recognition tag sequence may be changed.

[0133] The method for immobilizing DNA probes on microparticles is as follows. First, the microparticles and DNA probes are mixed in 1x B&W buffer (5 mM Tris-HCl (pH 7.5), 0.5 mM EDTA, 1 M NaCl), taking care to avoid mixing of DNA probes with different cell identification tag sequences, and a binding reaction is allowed to occur at room temperature for approximately 30 minutes. After that, the microparticles are washed twice with 1x B&W buffer, and then resuspended in 10 mM Tris pH 8.0 buffer to obtain a microparticle solution. 100 types of microparticle solutions with different cell identification tag sequences were prepared in 100 different storage tubes.

[0134] <Loading of DNA probe-immobilized microparticles into chip> A method for loading microparticles with DNA probes immobilized into chip 122 will be described.

[0135] The storage container 118 containing the above-mentioned microparticle solution to which the 100 different types of cell identification tags are fixed is placed on the first drive stage 106. As storage containers for containing cleaning solutions, two containers containing filtered ion-exchanged water (pure water) and two containers containing a buffer solution in which the microparticles have been resuspended are installed. The storage containers are, for example, PCR microtubes or 96-well microplates. Instead of the buffer solution in which the microparticles have been resuspended, the cleaning solution may be 100% EtOH.

[0136] Using the particle filling system 100, steps S1 to S6 are performed in accordance with a control program. At this time, 20 chips each having 100 micro-reaction chambers are installed. The particle filling system 100 determines which of the 100 types of particle solutions should be filled into each of the total 2,000 micro-reaction chambers. The particle filling system 100 then executes the operations of predetermined steps (a) to (f) in a manner basically similar to the first embodiment. At this time, each chip having 100 micro-reaction chambers is filled with one of the 100 corresponding particle solutions.

[0137] The nozzle 109 used was a straight cylindrical glass capillary with an outer diameter of 71 μm and an inner diameter of 54 μm. The predetermined amount of the high-concentration suspension to be determined in step (c) was 61 μm. The nozzle 109 was raised at a rate of 8 μm / s in step (e).

[0138] Fig. 17 is a diagram showing a workflow for filling particles according to the second embodiment. Fig. 17 shows in more detail steps S2 to S5 described in the first embodiment. In the above-described configuration, particle filling into the tip 122 was attempted according to the workflow of Fig. 17.

[0139] In step S2-0, the tip of the nozzle 109 is cleaned with cleaning liquid. After that, a nozzle shape trace is formed, and the "horizontal direction difference distance from the reference position of the captured image to the horizontal center position of the nozzle tip" is detected and corrected. The nozzle shape trace at this time is formed using ink.

[0140] In the next step S2-1, the microparticle solution was aspirated from the No. i solution tube into the nozzle. In step S2-2, the No. i micro-reaction chamber in the No. j chip was moved directly below the first imaging device. In step S2-3, the "coordinates at which the horizontal center position of the container opening is located at the reference position of the captured image" of the No. i micro-reaction chamber was detected or corrected. In step S2-4, the tip of the nozzle 109 and the No. i micro-reaction chamber were aligned using the first drive stage, and the tip of the nozzle 109 was lowered to directly above the micro-reaction chamber. Steps S3 to S5 were performed as in the first embodiment described above. By attempting to continuously fill the micro-reaction chambers to be filled with the same microparticle solution in this way, the number of cleanings can be reduced so that cleaning only needs to be performed when replacing the solution tube.

[0141] FIG. 18 is a diagram for explaining image processing during microparticle filling into a single-cell analysis chip according to the second embodiment. The upper left of FIG. 18 shows an image captured by the first imaging device 107 in step S2-3. The lower left and upper right of FIG. 18 show images captured by the second imaging device in steps S3 and S4, respectively. The lower right of FIG. 18 shows a schematic diagram illustrating the region where a nozzle-shaped trace was formed in step S2-0. As shown in the lower right of FIG. 18, the chip 122 was attached and fixed to a cover glass, and the region where a nozzle-shaped trace was formed was specified in an arbitrary region of the cover glass.

[0142] Fig. 19 is a photographed image of the chip 122 showing the results of filling with microparticles according to the second embodiment. As shown in Fig. 19, it can be seen that microparticles can be accurately filled into each of the 100 micro-reaction chambers of the chip 122 without misalignment.

[0143] <Concentration Time of Microparticle Solution> Next, the results of comparing the concentration time of the microparticle solution in the nozzle 109 when the magnet 121 is used and when it is not used will be described. First, when the concentration is 1.0×10 ９ A microparticle solution of 5.0×10 particles / mL was used and attracted into the nozzle 109. A rectangular neodymium magnet with a surface magnetic flux density of 540 mT, 2.8 mm square, and 10 mm length was used as the magnet 121 and placed directly below the chip 122. The tip of the nozzle 109 was stopped 220 μm directly above the micro-reaction chamber 123, and the distance from the surface of the rectangular neodymium magnet to the tip of the nozzle 109 was kept stationary at 540 μm (magnetic flux density 400 mT). The waiting time until the high-concentration microparticle solution 113 reached the target height of 61 μm was measured. At the same time, a concentration of 5.0×10 １０ A microparticle solution of 50 times the concentration of microparticles / mL (concentrated 50 times using a magnet) was used and allowed to settle naturally by gravity. The waiting time for the high-concentration microparticle solution 113 to reach the target height of 61 μm was also measured. The results are shown in FIG. 20.

[0144] Fig. 20 is a graph showing the measurement results of the concentration time of the microparticle solution by magnetic force or gravity. As shown in Fig. 20, it was confirmed that the concentration time for natural sedimentation by gravity was 52 seconds on average, but that the concentration time for accelerated sedimentation by the magnetic field was 5 seconds on average.

[0145] [Third Embodiment] In the third embodiment, a first image processing technique for detecting the horizontal positions (positions on the XY coordinate system) of the micro-reaction chambers on the first drive stage, which is necessary for alignment, will be described. Specifically, an image processing technique for simultaneously detecting the horizontal positions of multiple micro-reaction chambers in a short time, in step (ii) of the first embodiment, will be described.

[0146] 21A to 21E are conceptual diagrams illustrating a first image processing technique for alignment according to the third embodiment. A planar substrate 102 with multiple micro-reaction chambers 101 mounted thereon is moved directly below a first imaging device 107 mounted on top of a first drive stage 106. A group of multiple micro-reaction chambers is captured as a bird's-eye view by the first imaging device 107 ( FIG. 21A ). For each micro-reaction chamber included in this captured image, pattern matching is performed using an image of the micro-reaction chamber previously registered as a reference image ( FIG. 21B ). The positions of micro-reaction chambers in the captured image that are determined to be similar to the reference image by the pattern matching method are simultaneously detected ( FIG. 21C ). While any pattern matching algorithm may be used, one example is a method in which the original image is converted to binary and then pattern matched, as shown in FIG. 21C .

[0147] Next, the position of the detected micro-reaction vessel on the captured image is calculated as the amount of pixel upward displacement from the reference position of the captured image (center position: also a predetermined XY coordinate position on the first drive stage 106). Next, using a conversion coefficient that converts pixel values ​​of the image into coordinate values ​​on the first drive stage, position information is converted into XY coordinates of each micro-reaction vessel on the first drive stage 106. In this way, the horizontal positions of the micro-reaction vessels can be detected all at once from the captured image (FIG. 21D). Specifically, this calculation can be performed using equation (1) described in the first embodiment. For example, for convenience, the micro-reaction vessel in FIG. 21A is set as number 1 in advance as a specific micro-reaction vessel, and the micro-reaction vessel number 1 is determined as the reference position. The X and Y coordinates on the first drive stage when the micro-reaction vessel number 1 is positioned at the reference position (center position) of the captured image of the first imaging device 107 are calculated as X and Y coordinates. ｓｔａｇｅ＿ｒｅｆｅｒ , Y ｓｔａｇｅ＿ｒｅｆｅｒ and ΔxwellNth_image and ΔywellNth_image are set as relative positions from the micro-reaction chamber number 1 on the photographed image, thereby determining the X-axis of each micro-reaction chamber in the photographed image. ｗｅｌｌＮｔｈ＿ｉｎｉｔｉａｌ , Y ｗｅｌｌＮｔｈ＿ｉｎｉｔｉａｌ can be obtained.

[0148] In this case, to improve the accuracy of determining the horizontal position of the micro-reaction chambers 101, if the dimensional information of the arrangement grid on which the multiple micro-reaction chambers are arranged is known in advance, that information can be used. For example, when the micro-reaction chambers are arranged in an array at equal intervals (such as a square grid or a trigonal grid), the XY coordinate position information of each micro-reaction chamber on the first actuation stage 106 can be theoretically predicted by providing the XY coordinate position information of certain reference points (e.g., four points at the four corners) on the first actuation stage 106 as prior information. In reality, the planar substrate 102 having the micro-reaction chambers 101 often exhibits elastic deformation characteristics, and when the planar substrate 102 is installed and fixed on the first actuation stage 106, the planar substrate 102 often expands and contracts, resulting in an arrangement that is no longer equally spaced. In such cases, a theoretical prediction can be made, taking into account the effects of expansion and contraction, by further applying a homography transformation matrix to the XY coordinate position information using a homography transformation matrix calculated based on the position coordinates of the four corner points.

[0149] 22A is a diagram showing predicted and detected values ​​of the XY coordinate position of each micro-reaction chamber on the first drive stage 106 in a certain chip. The theoretically predicted XY coordinate position information of the micro-reaction chamber obtained as described above is used as reference information. If the position information of the actual micro-reaction chamber detected by the pattern matching method is included within a certain threshold value from this position information, it can be determined that the detected position information of the micro-reaction chamber is a correct value. By using such reference information, the risk of erroneous detection of the horizontal position of the micro-reaction chamber can be reduced.

[0150] Furthermore, even if it is not possible to obtain horizontal position information for some micro-reaction chambers locally due to reasons such as unclear contrast in the captured image, this can be compensated for by using the horizontal position information for multiple actual micro-reaction chambers obtained as described above.

[0151] 22B is a diagram showing the detected values ​​and interpolated values ​​of the XY coordinate position of each micro-reaction chamber. As shown in FIG. 22B, when the position information of a certain micro-reaction chamber is not detected by the pattern matching method, it is also possible to interpolate the position information by linear interpolation using the position information of multiple correctly detected surrounding micro-reaction chambers. This method can be used when the original micro-reaction chambers are arranged at equal intervals.

[0152] When the contrast of a captured image is unclear, in order to increase the probability of detecting micro-reaction vessels in the captured image using the pattern matching method, it is also effective to capture multiple images at the same position while continuously changing the parameters of the imaging device to increase the probability of obtaining good contrast. Specifically, multiple images are acquired while continuously changing the analog gain value, digital gain value, or exposure time of the imaging device, and then the above-mentioned pattern matching method is used to attempt to detect micro-reaction vessels in the captured image from these images (Figure 21E). The position information of multiple micro-reaction vessels obtained from multiple images is synthesized and merged. If overlapping position information of micro-reaction vessels is obtained, the position information with the highest judgment score in the pattern matching method is used to degenerate to a single point, thereby obtaining position information of the micro-reaction vessels with high accuracy. In this case, it is useful for subsequent processing to simultaneously acquire the imaging condition information of the analog gain value, digital gain value, or exposure time of the imaging device with the highest judgment score in the pattern matching method.

[0153] In the above series of processes, if the number of micro-reaction chambers is large and they cannot all fit within one field of view of the captured image, it is possible to perform the above series of processes in multiple fields of view and synthesize and obtain the horizontal positions of the multiple micro-reaction chambers obtained. Specifically, multiple images are captured at equal intervals so that approximately 10 to 20 micro-reaction chambers overlap in one field of view, and processing can be performed so that no micro-reaction chambers are missed within the field of view (Figure 21A).

[0154] 23 is a flowchart showing a first image processing technique according to the third embodiment. The first image processing technique is executed by the control device 120. In step S101, the XY coordinates of the four corners on the first drive stage are acquired as advance information. In step S102, the area in which the chip is imaged is divided into N×M parts, and the chip is moved to an imaging area where its position is detected using a pattern matching method (repeated N×M times).

[0155] In step S103, the setting values ​​(analog gain value, etc.) of the first imaging device are switched. In step S104, a pattern matching method is performed on the captured image. In step S105, the detected positions of the micro-reaction chambers on the image are converted into XY coordinates on the first drive stage. In step S106, it is determined whether processing for all setting values ​​has been completed. If YES in step S106, the process returns to step S102. If NO in step S106, the process returns to step S103.

[0156] In step S107, it is determined whether or not the detection of the positions of the micro-reaction chambers for all divided regions has been completed. If the answer is YES in step S107, the process proceeds to step S108. If the answer is NO in step S107, the process returns to step S102.

[0157] In step S108, all the obtained XY coordinate information is merged and overlapping information is reduced to one point. In step S109, each detected value is assigned to the micro-reaction vessel numbered i to be detected. In step S110, it is determined whether the detected value is within a threshold from the predicted value of the micro-reaction vessel numbered i to be detected. If the answer is YES in step S110, the process proceeds to step S111. If the answer is NO in step S110, the process proceeds to step S112.

[0158] In step S111, it is determined that the detection is successful. The detected values ​​are then adopted as the X and Y coordinates on the first drive stage of the micro-reaction chamber numbered i. In step S112, it is determined that the detection is unsuccessful. The linearly interpolated values ​​from the detected values ​​of the surrounding micro-reaction chambers are then adopted as the X and Y coordinates on the first drive stage of the micro-reaction chamber numbered i.

[0159] <Technical Effects> As described above, in the third embodiment, the horizontal positions (positions on the XY coordinate system) of a plurality of micro-reaction chambers on the drive stage, which are necessary for alignment, can be detected all at once in a short time.

[0160] <Example of the Third Embodiment> A specific example of the third embodiment will be described below. First, a planar substrate-like resin sheet, on which 20 chips (chip numbers 1 to 20) each had 400 micro-reaction chambers, was placed on the first drive stage. The micro-reaction chambers were arranged at equal intervals in a triangular lattice pattern. A monochrome CCD camera installed above the resin sheet was used as the first imaging device. The first drive stage 106 was moved so that chip number 1, which had micro-reaction chambers in the resin sheet, was positioned directly below the monochrome CCD camera. Since 400 micro-reaction chambers could not be accommodated in one field of view of the monochrome CCD camera, the chip was divided into four sections to accommodate 400 micro-reaction chambers per chip. First, 10 consecutive images were taken of division number 1 of chip number 1, with the gain value of the monochrome CCD camera ranging from low to high. A pattern matching method was performed on each captured image to simultaneously detect multiple micro-reaction chambers. During this process, the amount of deviation of each detected micro-reaction chamber from the reference position (center position) of the captured image was detected by image processing. At this time, the XY coordinates on the first drive stage 106 of the micro-reaction chambers at the four corners of each chip number were obtained in advance, and the XY coordinates of the micro-reaction chamber (number 1) at the upper left corner of each chip were set as reference coordinates.

[0161] The XY coordinates of each micro-reaction chamber on the first driving stage 106 were calculated using equation (1) using the relative position on the image of each micro-reaction chamber detected by the pattern matching method for micro-reaction chamber number 1, a conversion coefficient (1 pixel = 1.1625 μm) for converting the pixel value of the image into a coordinate value on the driving stage, and the above-mentioned reference coordinates.

[0162] Next, the first drive stage 106 was moved to the positions of division numbers 2 to 4 of chip number 1, and the same processing was performed on each. The obtained XY coordinates of the multiple micro-reaction chambers were merged, and if overlapping position information for the micro-reaction chambers was obtained, the position information with the highest judgment score value in the pattern matching method was adopted, thereby degenerating to single point information. In this series of processing, if there were any micro-reaction chambers that could not be detected by the pattern matching method, linear interpolation was performed using the position information of the surrounding detected micro-reaction chambers. In this way, the XY coordinate position information for 400 micro-reaction chambers on chip number 1 was finally obtained. The same processing was performed on chips number 2 to 20.

[0163] 24 shows the results of acquiring XY coordinate position information of each micro-reaction chamber for 20 chips. As shown in FIG. 24, it was possible to acquire XY coordinate position information of a total of 8,000 micro-reaction chambers provided on the resin sheet.

[0164] [Fourth Embodiment] In the fourth embodiment, a second image processing technique for detecting the horizontal position (position on the XY coordinate system) of the micro-reaction chamber on the drive stage, which is necessary for alignment, will be described. Specifically, the image processing technique for precisely correcting the horizontal position of the micro-reaction chamber immediately before filling with microparticles, which is performed in step S2-3 in the second embodiment, will be described.

[0165] 25 is a conceptual diagram for explaining the second image processing technique according to the fourth embodiment. The method of this embodiment assumes that the horizontal positions of a plurality of micro-reaction chambers 101 have been acquired in advance using the technique described in the third embodiment. The first drive stage 106 is used to move to the XY coordinates of the horizontal center position of a specific numbered target micro-reaction chamber among the planar substrate 102 on which a group of a plurality of micro-reaction chambers is provided, and is then moved so that the horizontal center position of the specific numbered target micro-reaction chamber is positioned directly below the first imaging device 107, specifically at the reference position (center position) of the captured image.

[0166] Next, a bird's-eye view image is captured by the first imaging device 107, and a threshold value centered on the reference position is set to restrict the image processing range, resulting in an image limited to only the micro-reaction vessels with specific numbers (left side of Figure 25). As an example, this narrowing process is performed using a circular area. From this image with the narrowed range, an edge detection method is used to detect the center position of the micro-reaction vessel with a specific number on the captured image (center of Figure 25). Next, the position of the detected micro-reaction vessel on the captured image is calculated as the pixel shift amount from the reference position (center position) of the captured image. Next, using a conversion coefficient that converts the pixel shift amount into XY coordinate values ​​on the first drive stage 106, the XY coordinate information of each micro-reaction vessel on the first drive stage 106 is converted (right side of Figure 25).

[0167] In this manner, the horizontal position of a micro-reaction chamber with a specific number can be detected and corrected from the captured image. Specifically, the horizontal position of a micro-reaction chamber with a specific number can be calculated using the following equation (6).

[0168]

[0169] Here, X ｗｅｌｌＮｔｈ＿ｆｉｎａｌ , Y ｗｅｌｌＮｔｈ＿ｆｉｎａｌ are the X and Y coordinates on the first drive stage at which the precisely corrected Nth micro-reaction chamber is placed at the reference position in the image captured by the first imaging device 107. ΔxwellNth_diff_image and ΔywellNth_diff_image are the X and Y coordinates (relative coordinates from the reference position) in the image of the Nth micro-reaction chamber on the image captured by the first imaging device 107 detected by the processing of this embodiment.

[0170] The processing of this embodiment can improve alignment accuracy by being performed immediately before filling the micro-particle solution into the micro-reaction chambers 101. The reason is as follows: If filling multiple micro-reaction chambers with the micro-particle solution takes a long time, the relative positioning of each component within the device may change due to environmental factors such as temperature fluctuations. For example, the horizontal positions of multiple micro-reaction chambers acquired in advance using the technology described in the third embodiment may fluctuate by several to several tens of micrometers due to temperature fluctuations. Therefore, by performing the processing of this embodiment immediately before filling the micro-particle solution into the micro-reaction chambers 101, the horizontal positions of the micro-reaction chambers can be precisely corrected immediately beforehand, improving alignment accuracy.

[0171] An example of an edge detection method used in the processing of this embodiment is a circular edge detection method. Specifically, a binary conversion process is performed on the image, the circular edge of the upper opening of the micro-reaction chamber is emphasized, the interior of the circle is filled, and finally, the circular edge detection method is performed. This method is effective for micro-reaction chambers having a structure with a single through-hole that is smaller than the diameter of the upper opening of the micro-reaction chamber. If the above processing is not performed, the presence of the through-hole may result in erroneous edge detection. However, if the above processing is performed, it is possible to eliminate the influence of the through-hole, thereby increasing the success rate of edge detection.

[0172] Furthermore, when an image is acquired by the imaging device in the processing of this embodiment, the imaging conditions can be adjusted at the same time using imaging condition information such as the analog gain value, digital gain value, or exposure time of the imaging device, which corresponds to the horizontal position information of the micro-reaction chambers acquired in advance by the technology of the third embodiment. This increases the probability of obtaining good contrast even when the image contrast of the imaging subject is non-uniform, thereby increasing the success rate of edge detection.

[0173] <Technical Effects> As described above, in the fourth embodiment, the horizontal position (position on the XY coordinate system) of a micro-reaction chamber with a specific number on the drive stage, which is necessary for alignment, can be precisely corrected.

[0174] <Verification of the Effects of the Fourth Embodiment> The effects of the fourth embodiment will be verified below. The same device configuration and measurement object as those of the second and third embodiments were used. Using horizontal position information of the micro-reaction vessels previously acquired using the technology of the third embodiment, the drive stage was moved to the XY coordinates of the horizontal position of a specific numbered micro-reaction vessel, and then moved directly below the monochrome CCD camera. At this time, the drive stage was moved so that the horizontal center position of the specific numbered micro-reaction vessel was positioned at the image reference position (center position) of the camera. With this positioning, an image was captured using the previously acquired imaging condition information, and the center position of the specific numbered micro-reaction vessel in the captured image was attempted to be detected using the above-mentioned circular edge detection method. At this time, the pixel deviation of each detected micro-reaction vessel from the reference position (center position) of the captured image was detected by image processing. Image processing was performed by calculating the number of pixels by which the image position of each micro-reaction chamber detected by the technology of the third embodiment was shifted from the center position of the image, and correcting the position by converting it into XY coordinates on the first drive stage 106 using a conversion coefficient of 1 pixel = 1.1625 μm, which converts the pixel shift amount into XY coordinate values ​​on the first drive stage 106. In this way, correction processing was performed on a total of 8,000 micro-reaction chambers using a planar substrate-like resin sheet on which 20 chips, each with 400 micro-reaction chambers per chip, were provided. This processing was performed immediately before filling with the microparticle solution.

[0175] Fig. 26 shows the results of precise correction of the XY coordinates of 400 micro-reaction chambers in 20 chips using the second image processing technique. As shown in Fig. 26, it was possible to obtain the amount of deviation in the X and Y coordinate directions of all micro-reaction chambers, and it was confirmed that the horizontal positions of all of them were correctly corrected.

[0176] Fifth Embodiment In the fifth embodiment, an example of a technique for forming the nozzle-shaped mark described above will be described. More specifically, a technique will be described in which the tip of the nozzle 109 is brought into contact with a resin sheet 125 having mechanical plasticity, and a mechanical load is applied until the resin sheet is deformed, thereby forming a nozzle-shaped mark 111 on the resin sheet that is identical to the tip of the nozzle 109. The formation of the nozzle-shaped mark is important in the correction step of the "horizontal differential distance from the reference position in the captured image to the horizontal center position of the nozzle tip," which is necessary for alignment.

[0177] FIG. 27A is a cross-sectional schematic diagram illustrating a method for forming a nozzle-shaped mark according to the fifth embodiment. First, a resin sheet 125 (a planar substrate for forming a nozzle-shaped mark) having mechanical plasticity is prepared. A mechanical load greater than a predetermined threshold is applied to the resin sheet 125. As a result, the location where the load is applied exceeds the elastic deformation region and reaches the plastic deformation region, resulting in deformation. Taking advantage of this phenomenon, as shown in FIG. 27A, the tip of the nozzle 109 is pressed against the resin sheet 125 with a predetermined mechanical load. This allows a nozzle-shaped mark 111 having the same shape as the tip of the nozzle 109 to be formed on the surface of the resin sheet 125.

[0178] Fig. 27B is a schematic top view of the nozzle-shaped mark 111. Fig. 27C is a measured view of the nozzle-shaped mark 111. As shown in Figs. 27B and 27C, the tip of the nozzle 109 is generally hollow and circular, and by using this technique, it is possible to form with good reproducibility a nozzle-shaped mark 111 having a double circle with an outer diameter and an inner diameter at the tip of the nozzle 109.

[0179] FIG. 27D is a conceptual diagram for explaining image processing using the nozzle shape mark 111. As shown in FIG. 27D, both circles of the double circle of the nozzle shape mark 111 are detected, and the position coordinates of the tip of the nozzle 109 on the captured image are accurately detected using the position information of the centers of both circles. This makes it possible to accurately correct the "horizontal differential distance from the reference position of the captured image to the horizontal center position of the tip of the nozzle 109." Specific correction processing can be performed using the same processing as described in step (iv) of the first embodiment. When the nozzle shape mark 111 is formed, the inside of the nozzle may be empty or may be filled with pure water or a buffer.

[0180] In this case, the imaging device for capturing a bird's-eye view of the nozzle mark 111 from above can be configured with a coaxial epi-illumination system and an optical system that captures only the linear light reflected from the target. This configuration allows the target resin sheet to be clearly captured with high contrast, even if it is a transparent body. Furthermore, by using this optical system, the shape mark formed on the resin sheet can be used even if it is not necessarily a physically concave shape. The light reflection characteristics of the formed shape mark change when pressed against the tip of the nozzle 109, preventing the light irradiated from the optical system from traveling linearly and being reflected back to the imaging device. As a result, the linearly reflected light in this area is attenuated, making it appear darker than the surrounding area, and it can be recognized as a dark shape mark in the captured image ( Figure 27C ). In fact, when the height distribution of the shape mark formed on the surface of a transparent resin sheet was measured using a white light interference microscope, it was confirmed that the shape mark was only deformed to a degree that was not significantly recognizable as a physical concave, but was recognized as a contrast change in the coaxial epi-illumination system.

[0181] The resin sheet 125 may be made of the same material as the micro-reaction chambers, or may be provided separately as a separate material. When made of the same material as the micro-reaction chambers, a nozzle-shaped trace can be formed at a position away from the area where the micro-reaction chambers are provided. The advantage of this method is that the shape of the nozzle-shaped trace is highly reproducible and high correction accuracy can be ensured. In addition, there is no need to prepare separate components for correcting the position coordinates of the tip of the nozzle 109; the components used in the microparticle filling method can be used as is, making it possible to correct the position coordinates of the tip of the nozzle 109 easily, at low cost, and in a short time. Furthermore, due to the above characteristics, it can be easily incorporated into a continuous loop sequence in the microparticle filling method.

[0182] Since this method applies a mechanical load to the tip of the nozzle 109, damage to the tip of the nozzle 109 can be prevented by detecting and controlling the mechanical load applied to the tip of the nozzle 109 using an appropriate threshold. For example, a strain signal obtained from a sensor system 116 using a leaf spring and a strain sensor can be converted into a load value applied to the tip of the nozzle 109. Therefore, by setting an appropriate threshold for the signal from the sensor system 116, it is possible to achieve a mechanical load with high reproducibility without damaging the tip of the nozzle 109, and to form a highly reproducible shape imprint.

[0183] Various materials can be used as the material for the resin sheet 125, and examples thereof include various resin materials (polycarbonate-based, polycycloolefin-based, polyolefin-based, polypropylene-based, polyethylene-based, acrylic-based, acrylonitrile-based, polystyrene-based, polydimethylsiloxane-based resins, etc.).

[0184] <Technical Effects> As described above, in the fifth embodiment, a resin sheet 125 having mechanical plasticity is used, and the tip of the nozzle 109 is brought into contact with the resin sheet 125 and a mechanical load is applied, thereby forming a nozzle-shaped mark 111 on the resin sheet 125 that is identical to the tip of the nozzle 109. This nozzle-shaped mark 111 can be used to correct the "horizontal differential distance from the reference position in the captured image to the horizontal center position of the tip of the nozzle" that is necessary for alignment.

[0185] <Verification of Effects of Fifth Embodiment> The effects of the fifth embodiment will be verified below. The same device configuration as in the second to fourth embodiments was used. Specifically, in the microparticle filling method shown in FIG. 17 , during the correction step after cleaning the tip of the nozzle 109 in step S2-0, the formation of a nozzle-shaped mark was carried out multiple times. The nozzle-shaped mark was formed by pressing the tip of the nozzle (circular outer diameter 66 μm, circular inner diameter 42 μm) against a resin sheet made of the same material as the micro-reaction chamber and having the mechanical plasticity of a transparent body with a mechanical load of 3.2 mN for 3 seconds.

[0186] FIG. 28A is a photographed image showing an example of multiple nozzle-shaped marks (formed at 100 μm intervals). For the nozzle-shaped marks photographed in this way, the pixel deviation from the reference position (center position) of the photographed image was detected by image processing. Image processing was performed using a circle edge detection method to detect both the outer and inner circles, and the average value of the center positions of each circle on the image was calculated. The average value of the center positions of each circle on the image was calculated to determine the number of pixels that deviated from the center position of the image, and this was converted into the XY coordinate deviation on the first drive stage 106 using a conversion coefficient (1 pixel = 1.1625 μm) that converts this to XY coordinate values ​​on the first drive stage 106. In this way, the corrected "horizontal differential distance from the reference position of the photographed image to the horizontal center position of the nozzle tip" was calculated using Equation (3).

[0187] 28B is a graph showing the results of the correction amount for the "difference in horizontal distance from the reference position in the photographed image to the horizontal center position of the nozzle tip." In this way, it was confirmed that accurate alignment could be achieved by periodically correcting the deviation amount of several to 15 μm for the "difference in horizontal distance from the reference position in the photographed image to the horizontal center position of the nozzle tip" even during continuous particle filling operation.

[0188] Sixth Embodiment In the second embodiment, an example was described in which the micro-reaction chamber 101 has a simple cylindrical shape. In the sixth embodiment, a technique will be described in which the shape of the micro-reaction chamber can be changed to relax the allowable alignment accuracy when aligning the tip of the nozzle 109 with the micro-reaction chamber.

[0189] 29A is a schematic cross-sectional view of a planar substrate 102 according to the sixth embodiment. As shown in FIG. 29A , the shape of the micro-reaction chambers 101 may be such that the size of the upper opening 103a is larger than the size of the bottom 103b. For example, the micro-reaction chambers 101 may have an inverted truncated cone shape in which the diameter of the upper opening 103a is larger than the diameter of the bottom 103b. By using an inverted truncated cone shape in this manner, the diameter of the upper opening 103a can be made larger than in the case of a simple cylindrical shape. In particular, when the volume of the micro-reaction chambers 101 is constant, the diameter of the upper opening 103a can be made larger in the inverted truncated cone shape than in the case of a simple cylindrical shape.

[0190] Fig. 29B is a cross-sectional schematic diagram showing the state immediately before the tip of the nozzle 109 is lowered into the interior of the micro-reaction chamber 101. As shown in Fig. 29B, when filling the same volume of microparticles, using a micro-reaction chamber in the shape of an inverted truncated cone leads to a higher probability of successfully lowering the tip of the nozzle 109 into the interior of the micro-reaction chamber 101 compared to using a micro-reaction chamber in the shape of a cylindrical cone.

[0191] In this case, the tip of the nozzle 109 can be provided with a degree of freedom so that it can move slightly horizontally. By allowing the tip of the nozzle 109 to move, even if the tip of the nozzle 109 descends slightly off-center from the center position of the upper opening 103a of the inverted truncated cone-shaped micro-reaction chamber 101, the tip of the nozzle 109 can reach the bottom 103b while contacting the inclined side wall surface 103c. In this case, to prevent the tip of the nozzle 109 from stopping on the side wall surface 103c during its descent and not reaching the bottom 103b, the diameter of the tip of the nozzle 109 can be made equal to or smaller than the circular diameter of the bottom 103b.

[0192] The cross-sectional shape of such a structure in which the size of the upper opening 103a is larger than the size of the bottom is not limited to an inverted truncated cone shape, but may be other elliptical or polygonal shapes. The important point is that the size of the upper opening 103a is larger than when the side wall of the micro-reaction chamber is vertical, and the likelihood that the tip of the nozzle 109 can descend into the inside of the micro-reaction chamber increases, and any other shape may be used as long as such dimensional relationship can be realized.

[0193] As described above, the important driving force is the process in which the tip of the nozzle 109 contacts the bottom 103b of the micro-reaction chamber 101, and when the tip of the nozzle 109 is pulled up, the high-concentration microparticle solution 113 contacts the side wall surface 103c of the micro-reaction chamber 101 due to its wettability, causing the liquid to move forward. Therefore, if the tip of the nozzle 109 were linear, for example, if the shape of the micro-reaction chamber 101 were an inverted truncated cone, the gap between the tip of the nozzle 109 and the side wall surface 103c of the micro-reaction chamber would be large near the opening of the micro-reaction chamber 101. This would reduce the Laplace pressure as the high-concentration microparticle solution 113 moves forward, potentially resulting in insufficient filling. Therefore, it is important to note that if the shape of the micro-reaction chamber 101 is changed, it is desirable to change the shape of the tip of the nozzle 109 to a similar shape.

[0194] Fig. 29C is a cross-sectional schematic diagram showing an example in which the shape of the tip of the nozzle 109 is changed. When the micro-reaction chamber is shaped like an inverted truncated cone as shown in Fig. 29C, the tip of the nozzle 109 can also be shaped like an inverted truncated cone with the same proportions.

[0195] <Technical Effects> As described above, in the sixth embodiment, by changing the shape of the micro-reaction chambers, it is possible to relax the allowable alignment accuracy when aligning the tip of the nozzle 109 with the micro-reaction chambers.

[0196] <Verification of Effects of the Sixth Embodiment> The effects of the sixth embodiment will be verified below. The same device configuration as in the second to fifth embodiments was used. In addition, the technology for improving alignment accuracy described in the second to fifth embodiments was used.

[0197] Fig. 30A is a photographed image of a chip having micro-reaction chambers in the shape of an inverted truncated cone. The chip shown in Fig. 30A has 20 x 20 micro-reaction chambers 101 (400 in total). Each micro-reaction chamber has an inverted truncated cone shape (taper angle 8°) with an upper opening 103a having a circular diameter of 83 µm, a bottom 103b having a circular diameter of 66.7 µm, and a depth of 65 µm.

[0198] Figure 30B is a photographed image of the side of a nozzle whose tip is shaped like an inverted truncated cone. The tip of the nozzle is shaped like an inverted truncated cone (taper angle 8°), and a glass capillary was used, with the tip of the nozzle 109 having an outer diameter of 64 μm and an inner diameter of 42 μm. Figure 30B shows the state when attempting to fill microparticles. In the case of such a tip of the nozzle 109 and micro-reaction chamber 101, the difference between the diameter of the upper opening 103a and the outer diameter of the tip of the nozzle 109 is 19 μm, and this difference is the amount of deviation in alignment accuracy that can be tolerated. 16A and 16B is used, and a glass capillary with a cylindrical tip having an outer diameter of 71 μm and an inner diameter of 54 μm is used as the nozzle, the difference between the diameter of the upper opening 103a and the outer diameter of the tip of the nozzle 109 is 4 μm, and the tolerance for deviation in alignment accuracy is small. Therefore, by adopting a structure in which the size of the upper opening 103a is larger than the size of the bottom 103b as the micro-reaction chamber 101, the tolerance for alignment accuracy can be relaxed.

[0199] [Seventh embodiment] In the seventh embodiment, a technology is described that can increase the probability of filling the inside of the micro-reaction chamber 101 with a high-concentration microparticle solution 113 by adjusting the difference between the size of the micro-reaction chamber 101 and the size of the tip of the nozzle 109.

[0200] As described in the sixth embodiment, it has been shown that an inverted truncated cone shape in which the diameter of the upper opening is wider than the diameter of the bottom is suitable for the alignment method in the micro-reaction chamber 101. This embodiment shows that in an example in which the tip shape of the nozzle 109 is a hollow inverted truncated cone and the micro-reaction chamber 101 is also inverted truncated cone shape, the difference between the size of the bottom diameter of the micro-reaction chamber 101 and the size of the tip of the nozzle 109 affects the probability of filling the high-concentration microparticle solution 113 inside the micro-reaction chamber 101, and that it is possible to increase the yield by adjusting this difference to 1 μm or less.

[0201] As described above, when the tip of the nozzle 109 comes into contact with the bottom 103b of the micro-reaction chamber 101 and the tip of the nozzle 109 is pulled up, the high-concentration micro-particle solution 113 comes into contact with the side wall surface 103c of the micro-reaction chamber 101 due to its wettability, which is an important driving force in the process of advancing the high-concentration micro-particle solution 113. Therefore, when the bottom diameter of the micro-reaction chamber 101 having an inverted truncated cone shape and the circular outer diameter of the tip of the nozzle 109 are as similar as possible, the probability that the high-concentration micro-particle solution 113 will come into contact with the side wall surface 103c of the micro-reaction chamber 101 increases, and the success rate of filling increases.

[0202] When adjusting the size of the micro-reaction chamber 101 and the size of the tip of the nozzle 109, the size of the smallest part of the tip of the nozzle 109 (for example, the outer diameter of a circle in the case of the hollow inverted truncated cone shape described above) can be made equal to or smaller than the size of the smallest part of the micro-reaction chamber 101 (for example, the bottom diameter in the case of the inverted truncated cone shape described above). However, when the material of the micro-reaction chamber 101 is elastically deformable, even if the size of the smallest part of the tip of the nozzle 109 is larger than the size of the smallest part of the micro-reaction chamber 101, it is possible for the nozzle 109 to deform and come into contact with the bottom 103b of the micro-reaction chamber 101 by applying an appropriate load.

[0203] <Technical Effects> As described above, in the seventh embodiment, by adjusting the difference between the size of the micro-reaction chambers 101 and the size of the tip of the nozzle 109, it is possible to further increase the yield.

[0204] <Verification of the Effects of the Seventh Embodiment> The effects of the seventh embodiment will be verified below. The same device configuration as in the second to sixth embodiments was used. Furthermore, the technique for improving alignment accuracy described in the second to sixth embodiments was employed. A planar substrate-like resin sheet with eight chips, as shown in FIG. 30A, was used. Each chip was provided with 400 micro-reaction chambers. Each micro-reaction chamber had an inverted truncated cone shape (taper angle 8°) with an upper opening 103a having a diameter of 83 μm, a bottom opening 103b having a diameter of 66.7 μm, and a depth of 65 μm. A glass capillary with a tip having an inverted truncated cone shape (taper angle 8°) was used as the nozzle. The outer diameter of the tip of the nozzle 109 was varied from 55 μm to 66.7 μm, and the filling success rate was calculated when the difference between the diameter of the bottom 103b of the micro-reaction chamber 101 and the outer diameter of the tip of the nozzle 109 was varied.

[0205] FIG. 31A is a table showing the results of the filling success rate when the difference between the diameter of the bottom 103b of the micro-reaction chamber 101 and the outer diameter of the tip of the nozzle 109 was varied. Eight chips, each with 20 x 20 micro-reaction chambers 101 (400 in total), were provided on a resin sheet. Multiple filling trials were performed on these micro-reaction chambers 101. As a result, as shown in FIG. 31A , the filling success rate was significantly improved by reducing the difference. In particular, it was confirmed that the filling success rate could be increased to 99.2% by reducing the difference to 1 μm or less. On the other hand, it was confirmed that the number of filling failures increased when the difference between the diameter of the bottom 103b of the micro-reaction chamber 101 and the outer diameter of the tip of the nozzle 109 was large.

[0206] Fig. 31B is a captured image of a chip in which the filling of particles has failed. As shown in Fig. 31B, a plurality of micro-reaction chambers have been formed in which the particles have not been filled entirely.

[0207] Therefore, the filling success rate can be increased by reducing the difference between the circular diameter of the bottom 103b of the micro-reaction chamber and the circular outer diameter of the tip of the nozzle 109. When the micro-reaction chamber has a simple cylindrical shape, the circular diameter of the bottom 103b and the diameter of the upper opening 103a are equal, so reducing this difference increases the required accuracy for alignment, resulting in a trade-off between successful filling and successful alignment. Even in such a case, if the micro-reaction chamber has an inverted truncated cone shape as in the sixth embodiment, it is possible to both increase the filling success rate by reducing the difference between the circular diameter of the bottom 103b of the micro-reaction chamber and the circular outer diameter of the tip of the nozzle 109, and increase the diameter of the upper opening 103a to increase the likelihood of alignment and thereby increase the success rate of alignment.

[0208] Eighth Embodiment In the eighth embodiment, a technique will be described that can increase the probability of filling the high-concentration microparticle solution 113 into the micro-reaction chamber 101 by increasing the pressure inside the tip of the nozzle 109.

[0209] As described above, when the tip of the nozzle 109 comes into contact with the bottom 103b of the micro-reaction chamber, and the tip of the nozzle 109 is pulled up, the high-concentration micro-particle solution 113 comes into contact with the side wall surface 103c of the micro-reaction chamber 101 due to its wettability, and this is an important driving force in the process of advancing the high-concentration micro-particle solution 113. Therefore, the Laplace pressure due to the surface tension determined among the three components of the micro-reaction chamber 101, the nozzle 109, and the high-concentration micro-particle solution 113 is the driving force that advances the high-concentration micro-particle solution 113 into the inside of the micro-reaction chamber 101. Therefore, by further applying pressure to the inside of the tip of the nozzle 109 in addition to this Laplace pressure, the driving force can be increased.

[0210] Specifically, in the particle filling system 100 described in the second embodiment, the electromagnetic valve is closed, and the entire system from the tip of the nozzle 109 to the syringe pump is closed, and the internal pressure of the entire system is increased using the syringe pump. This allows the high-concentration particle solution 113 inside the tip of the nozzle 109 to be pressurized.

[0211] FIG. 32A is a cross-sectional schematic diagram showing a state in which the high-concentration microparticle solution 113 inside the nozzle 109 is pressurized. As shown in FIG. 32A , pressurization slightly pushes the high-concentration microparticle solution 113 out of the tip of the nozzle 109, wetting the tip of the nozzle 109. This also assists the process of the high-concentration microparticle solution 113 advancing from the tip of the nozzle 109 to the micro-reaction chamber 101. In this way, the Laplace pressure plus the auxiliary pressure can assist wetting inside the micro-reaction chamber 101, thereby improving the probability that the solution will wet and contact the side walls of the micro-reaction chamber 101. At the same time, this pressurization must be controlled to a value that does not exceed the Laplace pressure at the tip of the nozzle 109 so that the high-concentration microparticle solution 113 remains at the tip of the nozzle 109. If the Laplace pressure is exceeded, the high-concentration microparticle solution 113 will be released from the tip of the nozzle 109 as large droplets, making it difficult to transition to an appropriate filling state.

[0212] Appropriate pressurization control can be performed immediately before the tip of the nozzle 109 is lowered into the inside of the micro-reaction chamber 101. Specifically, it can be performed between step (c) and step (d) described in the first embodiment. By applying pressurization control between these steps, it is possible to assist the driving force that advances the high-concentration microparticle solution 113 into the inside of the micro-reaction chamber 101 at an appropriate timing.

[0213] <Technical Effects> As described above, in the eighth embodiment, by increasing the pressure inside the tip of the nozzle 109, it is possible to increase the probability that the high-concentration microparticle solution 113 will be filled inside the micro-reaction chamber 101.

[0214] <Verification of the Effects of the Eighth Embodiment> The effects of the eighth embodiment will be verified below. The same device configuration as in the second to seventh embodiments was used. The technique for improving alignment accuracy described in the second to sixth embodiments was also used. Furthermore, the same resin sheet and glass capillary as in the seventh embodiment were used. The pressure inside the tip of the nozzle 109 was changed using a syringe pump, and increased by Δ0.5 kPa from atmospheric pressure. Under these conditions, the filling success rate was determined when the pressure inside the tip of the nozzle 109 was increased.

[0215] 32B is a table showing the results of the filling success rate when the pressure inside the tip of the nozzle 109 is increased. As shown in FIG. 32B, it was confirmed that by applying pressure to the tip of the nozzle 109, the filling success rate increased from 87% (without pressure) to 98% (with pressure).

[0216] [Ninth Embodiment] In the seventh embodiment, a technique was described in which the success rate of filling the high-concentration microparticle solution 113 into the micro-reaction chamber 101 is increased by reducing the difference (gap) between the bottom diameter of the micro-reaction chamber 101 and the circular outer diameter of the tip of the nozzle 109. In this embodiment, a technique will be described in which the probability of filling the high-concentration microparticle solution 113 into the micro-reaction chamber 101 is increased by reducing this gap.

[0217] An elastically deformable resin material is used as the material for the micro-reaction chamber 101. When filling the microparticles, the tip of the nozzle 109 is brought into contact with the inside of the micro-reaction chamber 101, a load is applied to deform the micro-reaction chamber, and the gap between the side wall surface 103c of the micro-reaction chamber 101 and the tip of the nozzle 109 is reduced.

[0218] FIG. 33A is a cross-sectional schematic diagram showing the elastic deformation of the micro-reaction chamber 101 according to the ninth embodiment. As shown in FIG. 33A, the micro-reaction chamber 101 can be deformed in a contracting direction by applying a mechanical load to the bottom 103b of the micro-reaction chamber 101 using the tip of the nozzle 109. This example illustrates an example using a micro-reaction chamber with an inverted truncated cone shape and a nozzle 109 with an inverted truncated cone shape, as in the sixth embodiment. In this case, a space with no components installed directly below the micro-reaction chamber, for example, a space of about 100 μm, can achieve this deformation state. Providing such a micro-gap directly below the micro-reaction chamber can also be achieved by installing a magnet 121 directly below the micro-reaction chamber as described in the second embodiment, thereby accumulating the high-concentration microparticle solution 113 at the tip of the nozzle 109 in a short time.

[0219] <Technical Effects> As described above, in the ninth embodiment, an elastically deformable resin material is used as the material for the micro-reaction chamber 101. Then, the tip of the nozzle 109 is brought into contact with and applied with a load to the inside of the micro-reaction chamber 101 to deform the micro-reaction chamber, thereby reducing the gap between the micro-reaction chamber and the tip of the nozzle 109. This increases the probability that the high-concentration micro-particle solution 113 will be filled inside the micro-reaction chamber 101.

[0220] <Verification of the Effects of the Ninth Embodiment> The effects of the ninth embodiment will be verified below. The same device configuration as in the second to seventh embodiments was used. Furthermore, the technology for improving the alignment accuracy described in the second to sixth embodiments was used. Furthermore, the same resin sheet and glass capillary as in the seventh embodiment were used. This resin sheet has elastic deformation properties. A 100 μm gap was provided directly below the micro-reaction chamber, and the shape of the micro-reaction chamber 101 elastically deformed by pressing the tip of the nozzle 109 into the bottom 103b of the micro-reaction chamber. Under these conditions, an attempt was made to fill the microparticles.

[0221] Fig. 33B is a table showing the results of the success rate of filling with microparticles when elastically deforming the micro-reaction chambers 101. As shown in Fig. 33B, it was confirmed that the success rate of filling increased from 87% (without elastic deformation) to 100% (with elastic deformation) by reducing the gap between the micro-reaction chambers 101 and the tip of the nozzle 109 through elastic deformation.

[0222] [Tenth Embodiment] Even when the above-described first or second embodiment and the third to ninth embodiments are utilized, there will be a low probability of 1 to 2% or less of micro-reaction chambers that are "unfilled" with microparticles. In the tenth embodiment, a technology will be described that automatically determines and detects the filling state of microparticles in a micro-reaction chamber, and further attempts to automatically refill the detected "unfilled" micro-reaction chamber.

[0223] 34A to 34C are schematic diagrams for explaining a particle filling method according to a tenth embodiment. Figures 34A to 34C show steps that are additionally performed after the series of flows explained in the first or second embodiment. The method of this embodiment includes a step of processing images of a plurality of micro-reaction chambers taken using a first drive stage 106 and a first image capture device 107.

[0224] The processing of this embodiment is initiated after attempting to fill all micro-reaction chambers with microparticles while referring to a list of micro-reaction chambers 101 of predetermined numbers to be filled, which are paired with pre-registered micro-particle solutions 110 of predetermined numbers. The processing of this embodiment can be broken down into the following steps (I) to (III). (I) The first drive stage 106 is moved to position the micro-reaction chambers 101 after multiple filling trials directly below the first imaging device 107 and photograph them. (II) "Unfilled" micro-reaction chambers are identified and detected from the photographed images by image processing. (III) An attempt is made to refill the detected list of multiple "unfilled" micro-reaction chambers with the micro-particle solution of the same predetermined number.

[0225] In step (I), first, all of the target micro-reaction chambers after the filling trial are photographed in a bird's-eye view. At this time, the first moving stage 106 is used to move a predetermined group of multiple micro-reaction chambers to a position where they can be photographed by the first imaging device 107, at XY coordinates on the first moving stage. At this time, if multiple micro-reaction chambers cannot fit within a single field of view of the first imaging device 107, they are divided into multiple fields of view, and the information is combined and processed in a subsequent process. This processing is essentially similar to the technology described in the third embodiment and can be performed in a similar manner. In particular, since the XY coordinates on the first moving stage of the multiple micro-reaction chambers acquired in advance can be referenced, the coordinate positions where all of the micro-reaction chambers can be photographed in a bird's-eye view can be calculated from this coordinate information.

[0226] Step (II) of determining "unfilled" micro-reaction chambers from the captured image by image processing is performed as follows: First, because the technology described in the third embodiment already holds the XY coordinate information on the first drive stage 106 of the micro-reaction chambers 101 that "should be filled," this coordinate information is used as reference information.

[0227] FIG. 34A shows image information registered in advance as the "filled" state. FIG. 34B is a diagram for explaining image processing for detecting micro-reaction vessels in the "filled" state from a captured image. Next, using the image information registered as the "filled" state as shown in FIG. 34A from the captured image, a group of micro-reaction vessels similar to the image information of the "filled" state as shown in FIG. 34B is detected by a pattern matching method. The same method as in the third embodiment can be used as the pattern matching method. In this case, the micro-reaction vessels detected by pattern matching and determined to be in the "filled" state can be acquired as XY coordinate information on the first drive stage 106, as in the third embodiment.

[0228] FIG. 34C is a diagram illustrating a method for determining the filling state of a micro-reaction chamber that "should be filled." As shown in FIG. 34C, (c-1) the XY coordinate position information of the micro-reaction chamber that "should be filled" is compared with (c-2) the XY coordinate position information of the micro-reaction chamber detected as being in a "filled" state. If the position information (c-2) is included within a predetermined threshold from the position information (c-1), the micro-reaction chamber that "should be filled" (c-1) can be determined to be "filled." On the other hand, if the position information (c-2) is not included within a predetermined threshold from the position information (c-1), the micro-reaction chamber that "should be filled" (c-1) can be determined to be "unfilled." In this way, by using so-called negative detection using a pattern matching method, it is possible to detect a micro-reaction chamber that is in an "unfilled" state.

[0229] 35 is a diagram showing a workflow for detecting unfilled micro-reaction chambers according to the tenth embodiment. The image processing technology of this embodiment is executed by the control device 120. In step S201, coordinate positions at which all of the micro-reaction chambers can be photographed in a bird's-eye view are calculated from previously acquired XY coordinate information on the first drive stage of the multiple micro-reaction chambers. In step S202, the area for photographing the chip is divided into N x M parts, and the chip is moved to the photographing area where its position is detected by a pattern matching method (repeated N x M times).

[0230] In step S203, the setting values ​​(analog gain value, etc.) of the first imaging device are switched. In step S204, a pattern matching method is performed on the captured image. In step S205, the detected positions of the micro-reaction chambers on the image are converted into XY coordinates on the first drive stage. In step S206, it is determined whether processing for all setting values ​​has been completed. If YES in step S206, the process returns to step S202. If NO in step S206, the process returns to step S203.

[0231] In step S207, it is determined whether or not the detection of the positions of the micro-reaction chambers for all divided regions has been completed. If the answer is YES in step S207, the process proceeds to step S208. If the answer is NO in step S207, the process returns to step S202.

[0232] In step S208, all XY coordinate information of the micro-reaction chambers detected as being in the "filled" state is merged, and overlapping information is reduced to a single point. In step S209, (c-1) the XY coordinate position information of the micro-reaction chamber numbered i "to be filled" is compared with (c-2) the XY coordinate position information of the micro-reaction chamber detected as being in the "filled" state. In step S210, it is determined whether the position information of (c-2) is included within a predetermined threshold from the position information of (c-1). If the answer is YES in step S210, the process proceeds to step S211. If the answer is NO in step S210, the process proceeds to step S212.

[0233] In step S211, a "filled" determination is made. That is, the micro-reaction vessel numbered i "to be filled" is determined to be in a "filled" state. In step S212, an "unfilled" determination is made. That is, the micro-reaction vessel numbered i "to be filled" is determined to be "unfilled."

[0234] The advantages of this method are described below. The first advantage is that by increasing the judgment threshold in pattern matching with images of "filled" micro-reaction vessels, the risk of misidentifying "filled" micro-reaction vessels as "unfilled" (so-called false negative detection) can be reduced. The second advantage is that the risk of overlooking "unfilled" micro-reaction vessels can be reduced. To use so-called positive detection, which directly detects similar images of "unfilled" micro-reaction vessels using pattern matching, multiple reference images must be prepared. However, as shown in Figure 2B, there are multiple unfilled states for "unfilled" micro-reaction vessels, and various image patterns exist. Therefore, to address these various image patterns, it is necessary to prepare a large number of training images using machine learning or deep learning. Furthermore, it is not necessarily possible to cover all image patterns. On the other hand, by detecting "unfilled" micro-reaction vessels using negative detection, as in this method, these issues can be avoided at low cost.

[0235] By referring to the list of multiple "unfilled" micro-reaction chambers detected by this method, an attempt is made to refill a micro-reaction chamber with the same micro-particle solution. In this way, even the unfilled micro-reaction chambers can be automatically refilled again, thereby further increasing the yield.

[0236] During automatic refilling, it is also possible to reset the device control variables using log information about the non-filling that occurred in the previous attempt and then attempt refilling. As an example, if a predetermined condition was not met in the previous attempt (for example, if the volume of the high-concentration microparticle solution 113 does not fall within the target threshold, i.e., if the volume of the high-concentration microparticle solution 113 is too small or too large and the refilling attempt is skipped), feedback control can be performed by referring to the conditions in the previous attempt to increase or decrease the volume of the microparticle solution 110 to be sucked into the nozzle 109, thereby increasing the success rate of refilling.

[0237] <Technical Effects> As described above, in the tenth embodiment, the yield can be further increased by automatically detecting unfilled micro-reaction chambers and attempting to automatically refill them.

[0238] <Verification of the Effects of the Tenth Embodiment> The effects of the tenth embodiment will be verified below. The same device configuration as in the second to eighth embodiments was used. The same resin sheet and glass capillary as in the seventh embodiment were also used. The technique for improving the alignment accuracy described in the first to sixth embodiments and the technique for improving the filling success rate described in the seventh and eighth embodiments were also used simultaneously. Under these conditions, an attempt was made to fill micro-reaction chambers with microparticles, and those that were detected as "unfilled" were retried to fill with microparticles.

[0239] 36A is a table showing the results of the particle filling success rate when particle refilling was performed in the tenth embodiment. As shown in FIG. 36A, it was confirmed that the filling success rate increased from 99.6% to 99.94% after adding the automatic refilling function.

[0240] FIG. 36B is an overhead observation image of a resin sheet provided with well-filled micro-reaction chambers in this embodiment.

[0241] [Eleventh Embodiment] In the eleventh embodiment, a technique for a continuous filling method that can shorten the time required to fill a micro-reaction chamber with a microparticle solution will be described. This method is a modified example of the workflow described in the second embodiment.

[0242] In the first and second embodiments, as shown in Fig. 13, the intention is to fill the entire amount of the high-concentration microparticle solution 113 formed at the tip of the nozzle 109 into the micro-reaction chamber 101. That is, the total volume of all microparticles contained in the high-concentration microparticle solution 113 is adjusted to be equal to or less than the total volume of the micro-reaction chamber 101. In the case of such an operation process, it is preferable to perform an operation of aspirating the microparticle solution again every time the high-concentration microparticle solution 113 is filled into the micro-reaction chamber 101, as in the workflow shown in Fig. 18. This workflow is suitable for the purpose of precisely controlling the volume of the high-concentration microparticle solution 113 filled into the micro-reaction chamber 101.

[0243] On the other hand, a filling method using a different operation process is also possible using the configuration of the present disclosure. A continuous filling method can be used as a variation of such a workflow. The continuous filling method is a method in which, where L is a natural number, a microparticle solution 110 sufficient for L fillings is previously aspirated into the nozzle 109, and a high-concentration microparticle solution 113 sufficient for L fillings is concentrated at the tip of the nozzle 109, and then L micro-reaction chambers are continuously filled with the microparticle solution 110.

[0244] Fig. 37 is a schematic diagram for explaining a continuous filling method for microparticles according to the 11th embodiment. The basic flow is similar to that of Fig. 13, but differs in that microparticles are continuously filled into a plurality of micro-reaction chambers until filling is performed L times.

[0245] 38 is a diagram showing the workflow of the continuous filling method according to the eleventh embodiment. In the workflow, as shown in FIG. 38, steps S2-1, S2-2, S2-3, and S3 are performed only at the first filling or after L fillings of the microparticle solution. In other cases, continuous filling can be achieved by skipping steps S2-1, S2-2, S2-3, and S3. The determination of "first filling or after L fillings" can be processed by, for example, counting the number of fillings in the control program and making a determination using this number of fillings.

[0246] In the continuous filling method described above, when the nozzle is pulled up at a very low speed of approximately 1 to 130 μm / s in step (e) of FIG. 37 , the volume of microparticles filled into the micro-reaction chamber can be controlled by controlling the amount of pulling. Upon completion of filling in step (f) of FIG. 13 or step (f) of FIG. 37 , the nozzle is pulled up from the micro-reaction chamber at a speed significantly faster than that in step (e) of FIG. 37 , for example, a speed of 1 to several tens of mm / s. This transition occurs when the nozzle 109 and the high-concentration microparticle solution 113 transition from a liquid-connected state to a separated state. Therefore, by appropriately controlling the amount of pulling in step (e) of FIG. 37 , the volume of the high-concentration microparticle solution 113 desired to remain inside the micro-reaction chamber 101 can be controlled. This control effect is not limited to the continuous filling method, but can also be implemented in the first embodiment and the filling method in the workflow of FIG. 18 .

[0247] Since the amount of microparticle filling can be controlled as described above, even if "at the first filling or after L fillings" is YES, the step of waiting until the microparticles are concentrated in step S3 is not essential and can be skipped. Since a large amount of microparticle solution is aspirated, when magnetically accelerated sedimentation is performed, a sufficient amount of high-concentration microparticle solution for L fillings is typically concentrated at the tip of the nozzle 109 in 1 to 2 seconds. Therefore, since a sufficient amount of high-concentration microparticle solution is supplied to the tip of the nozzle 109 while step S4 is being performed, step S3 can always be skipped.

[0248] In this embodiment, when performing step S2-3 in Fig. 38, not only is the coordinate detection and correction of one micro-reaction vessel performed, but multiple micro-reaction vessels photographed in the same image are simultaneously processed by image recognition to detect and correct the coordinates, thereby making it possible to skip the coordinate correction process of step S2-3. This image recognition method can be realized by expanding and applying the third or fourth image processing technique to multiple micro-reaction vessels.

[0249] The effect of this continuous filling method is that it is possible to shorten the time required to fill the micro-reaction chamber with the microparticle solution by skipping steps S2-1, S2-2, S2-3, and S3 in Fig. 38. In particular, this method leads to a significant reduction in time when the purpose is to fill multiple micro-reaction chambers with the same type of microparticle solution.

[0250] In this embodiment, a large amount of high-concentration microparticle solution capable of filling L times is accumulated at the tip of the nozzle 109, so that the viscous resistance of the high-concentration microparticle solution as a fluid at the tip of the nozzle 109 is greater than that of a high-concentration microparticle solution capable of only one filling, and the pressure required to spread the high-concentration microparticle solution into the inside of the micro-reaction chamber 101 increases due to the wetting principle described with reference to Fig. 12. Therefore, by simultaneously using the seventh and eighth embodiments, it is possible to increase the probability that the high-concentration microparticle solution 113 will be filled into the inside of the micro-reaction chamber 101.

[0251] <Technical Effects> As described above, in the eleventh embodiment, by using the continuous filling method, it is possible to shorten the time required to fill the micro-reaction chamber with the microparticle solution.

[0252] <Verification of Effects of Eleventh Embodiment> The effects of the eleventh embodiment will be verified below. The same device configuration as in the second to eighth embodiments was used. The same resin sheet and glass capillary as in the seventh embodiment were also used. The workflow in FIG. 38 was adopted as the continuous filling method. The number of continuous fillings was set to four, and a total of 12 filling trials were performed under the condition that step S3 was always skipped.

[0253] Fig. 39A is a graph showing the filling time per micro-reaction chamber when the workflow of Fig. 18 in the second embodiment is used. In Fig. 39A, the typical filling time per micro-reaction chamber was about 40 seconds.

[0254] 39B is a graph showing the filling time per micro-reaction chamber when using the continuous filling method of Embodiment 11. As shown in FIG. 39B, it was confirmed that when the continuous filling method is used, the filling time per micro-reaction chamber can be typically shortened to about 6 seconds when no solution is being aspirated.

[0255] [Modifications] The present disclosure is not limited to the above-described embodiments and includes various modifications. For example, the above-described embodiments have been described in detail to clearly explain the present disclosure, and it is not necessary to include all of the described configurations. Furthermore, a part of one embodiment can be replaced with a configuration of another embodiment. Furthermore, a configuration of another embodiment can be added to a configuration of one embodiment. Furthermore, a part of the configuration of each embodiment can be added to, deleted from, or substituted for a part of the configuration of another embodiment.

[0256] 100 Microparticle filling system 101 Microreaction chamber 102 Planar substrate 103a Upper opening 103b Bottom 103c Side wall surface 104 Bottom surface of planar substrate 105 Through-hole 106 First driving stage 107 First imaging device 108 Second driving stage 109 Nozzle 109a Outer wall of nozzle 110 Microparticle solution 111 Nozzle shape trace 112 Planar substrate for shape trace 113 High-concentration microparticle solution 114 Second imaging device 115 Driving mechanism 116 Sensor system 117 Dispensing mechanism 118 Storage container 119 Stirring mechanism 120 Control device 121 Magnet 122 Single-cell analysis chip 123 Microreaction chamber of single-cell analysis chip 124 Through-hole for cell capture 125 Resin sheet

Claims

1. A particle filling method for filling a container with particle, comprising: aligning a nozzle and a container using an image captured by an imaging device; and filling the container with particle from the nozzle, wherein the aligning includes aligning the tip of the nozzle with the horizontal center position of an opening of the container using (a) a horizontal differential distance from a reference position of the image captured by the imaging device to the horizontal center position of the tip of the nozzle, and (b) coordinates that position the horizontal center position of the opening of the container at the reference position.

2. A method for filling fine particles as described in claim 1, wherein the horizontal position of said imaging device and the horizontal position of said nozzle are fixed, and said container is configured to be movable in said horizontal direction.

3. The microparticle filling method of claim 1, wherein the aligning step further includes: forming a trace of the nozzle on a substrate horizontal to the nozzle; photographing the trace of the nozzle with the imaging device; and (a) correcting a horizontal differential distance from a reference position of the image photographed by the imaging device to a horizontal center position of the tip of the nozzle by analyzing the image of the trace of the nozzle photographed by the imaging device.

4. The microparticle filling method of claim 1, wherein the alignment step includes analyzing the image of the container captured by the imaging device and, using a pattern matching method, (b) obtaining coordinates where the horizontal center position of the opening of the container is located at the reference position.

5. The microparticle filling method of claim 1, wherein the alignment step includes analyzing the image of the container captured by the imaging device and, using an edge detection method, (b) obtaining coordinates where the horizontal center position of the opening of the container is located at the reference position.

6. A method for filling fine particles as described in claim 1, wherein the fine particles are magnetic, and filling the fine particles includes placing a magnet below the container, thereby accelerating the settling of the fine particles at the tip of the nozzle by magnetic force and shortening the time required to form a high-concentration suspension having a predetermined fine particle concentration.

7. The method for filling fine particles according to claim 1, wherein the container has an inverted truncated cone shape with the diameter of the opening being larger than the diameter of the bottom.

8. A method for filling fine particles as described in claim 7, wherein the tip of the nozzle is smaller than the opening of the container, and the difference between the diameter of the bottom of the container and the diameter of the tip of the nozzle is 1 μm or less.

9. The method of claim 1, wherein filling the microparticles includes pressurizing the inside of the nozzle immediately before lowering the tip of the nozzle into the interior of the container, thereby wetting the tip of the nozzle with a high-concentration microparticle solution.

10. A method for filling fine particles as described in claim 1, wherein the container has elastic deformation properties, a space is provided below the container, and when the nozzle is lowered, the container elastically deforms, thereby reducing the gap between the nozzle and the container.

11. The method for filling microparticles as described in claim 1, further comprising: detecting whether filling of the microparticles into the container is complete or incomplete from an image captured by the imaging device; and re-filling the microparticles into the container that is incomplete.

12. A particle filling system for filling one or more containers with particles, comprising: a nozzle; a dispensing mechanism for controlling the suction of a suspension of the particles into the nozzle; a first drive mechanism for moving a stage supporting the container in a horizontal direction; a second drive mechanism for moving the nozzle vertically; a control device for controlling the dispensing mechanism, the first drive mechanism, and the second drive mechanism; and an imaging device for photographing the container from above, wherein the control device performs the following steps: causes the nozzle to suction the suspension using the dispensing mechanism; concentrates the particles in the suspension to form a high-concentration suspension having a predetermined particle concentration at the tip of the nozzle; processes the image captured by the imaging device, and drives the first drive mechanism to align the tip of the nozzle with the horizontal center position of the opening of the container using (a) a horizontal differential distance from a reference position of the image captured by the imaging device to the horizontal center position of the tip of the nozzle, and (b) coordinates at which the horizontal center position of the opening of the container is located at the reference position; and driving the second driving mechanism to fill the high-concentration suspension into the container.

13. A particulate filling system as described in claim 12, wherein the horizontal position of said imaging device and the horizontal position of said nozzle are fixed, and said stage is fixed in position in the vertical direction.

14. The particulate filling system according to claim 12, further comprising a substrate horizontal to the nozzle, and the control device executes a process for forming a trace of a shape identical to the tip shape of the nozzle on the horizontal substrate.

15. The fine particle filling system described in claim 14, wherein the horizontal substrate has mechanical plasticity, and the control device drives the first driving mechanism and the second driving mechanism to perform a process of forming a trace of the nozzle shape by pressing the tip of the nozzle into the horizontal substrate.

16. The fine particle filling system of claim 12, wherein the fine particles are magnetic, the fine particle filling system further comprises a magnet arranged below the container, and in the process of forming the highly concentrated suspension, the magnetic force of the magnet accelerates the sedimentation of the fine particles at the tip of the nozzle, thereby shortening the time required to form the highly concentrated suspension.

17. The particulate filling system of claim 12, wherein the container has an inverted truncated cone shape with the diameter of the opening being greater than the diameter of the bottom.

18. A fine particle filling system as described in claim 17, wherein the tip of the nozzle is smaller than the opening of the container, and the difference between the diameter of the bottom of the container and the diameter of the tip of the nozzle is 1 μm or less.

19. The fine particle filling system described in claim 12, wherein the container has elastic deformation properties, a space is provided below the container, and in the process of filling the container with the high concentration suspension, the container elastically deforms when the nozzle is lowered, thereby reducing the gap between the nozzle and the container.

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