Filling method and filling system

Abstract

To provide a filling method capable of highly efficiently filling a target capturing substance into a microwell.SOLUTION: A filling method for filling a target-capturing substance into a microwell, the method comprising: a capture substance information acquisition step of acquiring capture substance information on a target-capturing substance to be used; a rate determination step of determining, based on the capture substance information acquired in the capture substance information acquisition step, an introduction rate at which a dispersion medium in which the target-capturing substance is dispersed is introduced into a region provided with a plurality of the microwells on a substrate provided with the microwells; A capture substance filling step of filling the one or more microwells with the target-capturing substance.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present disclosure relates to a filling method and a filling system for use in microwell-based detection. [Background technology]

[0002] Rapid and accurate detection of a target substance in a sample is important in fields such as medical diagnosis and academic research. For example, in the medical field, a target substance is detected from the amount of signal emitted by a reporter in an antibody-based assay, and the concentration is measured. In recent years, a highly sensitive measurement method has been developed, as shown in Patent Document 1, in which a detection reagent containing a reporter is delivered to a microwell, the microwell is subsequently isolated with a hydrophobic solvent, and the reaction is then detected using a fluorescence microscope or the like. Furthermore, a measurement method using a target capture substance has been developed as a method for more efficiently filling a target substance into a microwell. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent Application No. 2012-556164 Summary of the Invention [Problem to be solved by the invention]

[0004] According to the investigations of the present inventors, in conventional detection methods, when filling the microwells with the target capture substance, the filling rate is low or the uniformly micro-compartmented area is narrow, which can reduce the number of detectable target substances and decrease the detection sensitivity. Also, uneven filling of the microwells with the target capture substance can occur, which can prevent appropriate detection results from being obtained. Therefore, the present disclosure aims to provide a filling method and a filling system that enable highly efficient filling of target capture substances into microwells. [Means for solving the problem]

[0005] A method of filling according to one aspect of the present disclosure includes: 1. A method for filling microwells with a target capture substance, comprising: a capture substance information acquisition step of acquiring capture substance information regarding the target capture substance to be used; a rate determination step of determining an introduction rate for introducing a dispersion medium in which the target capture substance is dispersed into a region on the substrate on which the plurality of microwells are provided, based on the capture substance information acquired in the capture substance information acquisition step; and a capture substance filling step of filling one or more of the microwells with the target capture substance by introducing the dispersion medium into the substrate at the introduction speed; It has.

[0006] Furthermore, a filling system according to another aspect of the present disclosure includes: 1. A filling system for filling microwells with a target capture substance, comprising: a capture substance information acquisition unit that acquires capture substance information regarding the target capture substance to be used; a rate determination unit that determines an introduction rate for introducing a dispersion medium in which the target capture substance is dispersed into a region on the substrate on which the plurality of microwells are provided, based on the capture substance information acquired by the capture substance information acquisition unit; and a capture substance filling section that fills one or more of the microwells with the target capture substance by introducing the dispersion medium into the substrate at the introduction speed; It has. [Effects of the Invention]

[0007] According to the present disclosure, a filling method and a filling system are provided that enable highly efficient filling of target capture substances into microwells. [Brief explanation of the drawings]

[0008] [Figure 1] FIG. 2 is a functional block diagram of a filling system according to a first embodiment of the present disclosure. [Figure 2] 1 is a flowchart showing an outline of a filling method according to a first embodiment of the present disclosure. [Figure 3] 1 is a block diagram illustrating an example of a hardware configuration of a filling system according to a first embodiment of the present disclosure. [Figure 4] FIG. 1 is a cross-sectional view showing an example of a well plate. [Figure 5] FIG. 10 is a functional block diagram of a filling system according to a modified example of the first embodiment of the present disclosure. [Figure 6] 10 is a flowchart showing an outline of a filling method according to a modified example of the first embodiment of the present disclosure. [Figure 7] FIG. 10 is a functional block diagram of a filling system according to a modified example of the first embodiment of the present disclosure. [Figure 8] 10 is a flowchart showing an outline of a filling method according to a modified example of the first embodiment of the present disclosure. [Figure 9] FIG. 10 is a functional block diagram of a detection system according to a second embodiment of the present disclosure. [Figure 10] 10 is a flowchart showing an outline of a detection method according to a second embodiment of the present disclosure. [Figure 11] FIG. 10 is a diagram showing an example of an image obtained by photographing the result of filling a microwell provided in a substrate with a target capture substance. DETAILED DESCRIPTION OF THE INVENTION

[0009] In a detection method for detecting a target substance by encapsulating the target substance in a microwell using a target capture substance, a dispersion medium in which the target capture substance is dispersed is introduced onto a substrate having microwells formed therein. Subsequently, as the dispersion medium moves across the substrate, the target capture substance is sequentially filled into the microwells formed on the substrate.

[0010] FIG. 11 shows an example of an image obtained by photographing the result of filling a microwell provided in a substrate with a target capture substance. When the target capture substance is properly filled into the microwells, the target capture substance dispersed in the dispersion medium spreads over the substrate together with the dispersion medium, and is filled in an almost uniformly dispersed manner into each of the multiple microwells provided on the substrate, as shown in Figure 11.

[0011] According to the investigations of the present inventors, in conventional detection methods using microwells, when a dispersion medium containing dispersed target capture substances is introduced onto a substrate, the target capture substances sometimes aggregate and accumulate outside the microwells. Aggregation of the target capture substances and accumulation outside the microwells can reduce the number of target capture substances filled into the microwells and hinder the flow of the dispersion medium. As a result of further investigation, the inventors of the present application found that by appropriately controlling the speed at which the dispersion medium is introduced onto the substrate, it is possible to improve the filling rate of the target capture substance into the microwells, achieve uniform microcompartmentalization over a wider area, and suppress unevenness in the filling state.

[0012] Hereinafter, embodiments of the present disclosure will be described in detail with reference to the drawings. However, the components described in the embodiments are merely examples, and the technical scope of the present disclosure is determined by the claims and is not limited to the individual embodiments described below. In the drawings, similar or corresponding elements are denoted by the same reference numerals, and their description may be omitted or simplified.

[0013] [First embodiment] 1 shows a functional block diagram of a filling system according to a first embodiment of the present disclosure. The filling system 10 according to this embodiment is a filling system for filling microwells with a target capture substance. The filling system 10 includes a capture substance information acquisition unit 101, a rate determination unit 102, a capture substance filling unit 103, an input unit 104, an output unit 105, and a memory unit 106.

[0014] A description will be given of a filling method according to a first embodiment of the present disclosure, which is carried out using the filling system 10 shown in Fig. 1. Fig. 2 is a flowchart showing an outline of the filling method according to the first embodiment of the present disclosure.

[0015] First, in step S201, a capture agent information acquisition step, the capture agent information acquisition unit 101 acquires capture agent information relating to the target capture agent to be used. The capture agent information includes information related to factors that may affect the filling of the microwells. The capture agent information may also include information about the dispersion medium in which the target capture agent is dispersed.

[0016] As described above, the target capture substance dispersed in the dispersion medium spreads over the substrate together with the dispersion medium and fills each microwell provided on the substrate. The behavior of the target capture substance in the dispersion medium varies depending on the type of target capture substance and dispersion medium used. In the present disclosure, information on factors affecting the behavior of the target capture substance in the dispersion medium is obtained as capture substance information.

[0017] The capture substance information includes, for example, material property information relating to the properties of at least one of the target capture substance and the dispersion medium. The material property information may be a physical property value of at least one of the target capture substance and the dispersion medium that affects the sedimentation velocity of the target capture substance in the dispersion medium.

[0018] Specific examples of the properties of the target capture substance include values ​​related to the size of the target capture substance, such as its volume and diameter, its mass, and its concentration in the dispersion medium. Specific examples of the properties of the dispersion medium include its viscosity.

[0019] The capture substance information may also include specific information for identifying at least one of the target capture substance and the dispersion medium, such as a product name, a generic name of the substance, a chemical substance name, a code for identifying the chemical substance, a structural formula, and a composition or analytical data indicating the composition.

[0020] There are two types of methods for acquiring capture substance information: automatic and manual. In the automatic method, the filling system 10 may further include a capture substance image acquisition unit that acquires images of the target capture substance and an image analysis unit that analyzes the images acquired by the capture substance image acquisition unit, and the capture substance information is automatically acquired based on the results obtained by the image analysis unit. In the manual method, the filling system 10 presents specific capture substance information options displayed on the output unit 105, such as "target capture substance A, target capture substance B, target capture substance C," and the user selects the option from the input unit 104.

[0021] The capture substance information acquisition unit 101 may acquire the capture substance information from the storage unit 106, or from an external server, etc. Furthermore, the capture substance information acquisition unit 101 may acquire the capture substance information as an analysis result directly from a functional site that performs a specific analysis, as in the example described above.

[0022] Next, in the rate determination step of step S202, the rate determination unit 102 determines the introduction rate for introducing the dispersion medium into the region on the substrate where the microwells are provided, based on the capture substance information acquired in the capture substance information acquisition step.

[0023] In the present disclosure, highly efficient filling of the target capture substance into the microwells is possible by determining the rate at which the dispersion medium is introduced onto the substrate to an appropriate value in accordance with the capture substance information.

[0024] An example of the relationship between material property information on the characteristics of a target capture substance (capture substance information) and control of the rate at which a dispersion medium is introduced onto a substrate is shown in Table 1. Also, an example of the relationship between material property information on the characteristics of a dispersion medium (capture substance information) and control of the rate at which a dispersion medium is introduced onto a substrate is shown in Table 2.

[0025] [Table 1]

[0026] [Table 2]

[0027] For example, if the diameter of the target capture substance is relatively large, it is considered difficult for the target capture substance to enter the microwell, and therefore a relatively slow introduction rate is determined in the rate determination step. Also, for example, if the mass of the target capture substance is relatively small, the rate at which the target capture substance settles in the dispersion medium is slow, and therefore a relatively slow introduction rate is determined in the rate determination step. Also, for example, if the concentration of the target capture substance in the dispersion medium is relatively high, the target capture substance is in a high-density state in the dispersion medium and therefore prone to aggregation, and therefore a relatively fast introduction rate is determined in the rate determination step. Also, for example, if the viscosity of the dispersion medium is relatively high, the dispersion medium is difficult to flow, and therefore a relatively fast introduction rate is determined in the rate determination step.

[0028] In the rate determination step, the specific method for determining the introduction rate based on the capture substance information is not particularly limited. When determining the specific introduction rate, for example, a value calculated from the capture substance information using the following formula (Stokes' equation) may be referenced.

[0029]

number

[0030] For example, if the terminal velocity of the target capture substance calculated using the above formula is fast, it can be determined that the target capture substance can be efficiently filled into the microwell by increasing the introduction rate.

[0031] Furthermore, for example, if an appropriate introduction rate is known depending on the type of target capture substance, the combination of the target capture substance and the dispersion medium, etc., the appropriate introduction rate may be determined in the rate determination process depending on the specific information obtained as the capture substance information.

[0032] The method for determining the introduction rate may be either automatic or manual. In the automatic case, for example, the filling system 10 automatically determines the introduction rate. When the introduction rate is determined manually, there may be multiple methods for determining the introduction rate. For example, the user may select from speed conditions such as "fast, medium, or slow" presented by the filling system 10 via the output unit 105 by inputting the speed conditions via the input unit 104, and the optimal introduction rate for each target capture substance may be appropriately determined based on the filling results. Alternatively, the filling system 10 may determine the optimal introduction rate based on the capture substance information input by the user to the filling system 10.

[0033] Next, in the capture substance filling step of step S203, the capture substance filling unit 103 fills one or more microwells with the target capture substance by introducing the dispersion medium into the substrate at the introduction speed determined in the speed determination step. Specific examples of methods for introducing the dispersion medium into the substrate using the capture substance loading unit 103 include using a liquid delivery device such as a micropipette or syringe to directly pour the dispersion medium into an inlet port of a channel provided in the substrate. Loading can also be achieved by dripping the dispersion medium into the inlet port and then aspirating it through an outlet port, or by applying centrifugal force to the substrate. In this case, the dispersion medium containing the dispersed target capture substance is preferably introduced so as to cover the entire microwell. Next, the distribution of target capture substances into the microwells will be explained.

[0034] When distributing the target capture substance into the microwells, it is preferable to leave the substrate with the microwells under reduced pressure and degas the space above the area on the substrate where the microwells are provided. Specifically, for example, a method can be used in which the substrate is left in a vacuum desiccator at 0.1 atmospheres for a predetermined period of time. By degassing, the air in the microwells is removed, allowing the dispersion medium in which the target capture substance is dispersed to be efficiently filled into the microwells. The degassing time is not particularly limited and can be set as desired. Note that the method of filling the dispersion medium into the microwells is not limited to degassing.

[0035] Next, a sealing medium is introduced into the space above the region of the substrate where the microwells are provided, thereby sealing the space, i.e., replacing the dispersion medium present in the space above the microwells with the sealing medium.

[0036] Next, an example of the hardware configuration of the filling system according to the first embodiment will be described. FIG. 3 is a block diagram showing an example of the hardware configuration of the filling system according to the first embodiment. The filling system 10 includes an information processing device 30 and a filling device 31 .

[0037] The information processing device 30 has computer functions. For example, the information processing device 30 may be integrated with a desktop personal computer (PC), a laptop PC, a tablet PC, a smartphone, or the like.

[0038] The information processing device 30 includes a central processing unit (CPU) 301, a random access memory (RAM) 302, a read only memory (ROM) 303, and a hard disk drive (HDD) 304 to function as a computer that performs calculations and storage. The information processing device 30 also includes a communication interface (I / F) 305, an output device 306, and an input device 307. The CPU 301, RAM 302, ROM 303, HDD 304, communication I / F 305, output device 306, and input device 307 are interconnected via a bus. The output device 306 and input device 307 may be connected to the bus via a drive device (not shown) for driving these devices.

[0039] 3, the components constituting the information processing device 30 are illustrated as an integrated device, but some of these functions may be configured by external devices. For example, the output device 306 and the input device 307 may be external devices separate from the components constituting the computer functions including the CPU 301, etc.

[0040] The CPU 301 performs predetermined operations in accordance with programs stored in the RAM 302, HDD 304, etc., and also has the function of controlling each unit of the information processing device 30. The processing performed by the CPU may include obtaining capture substance information, determining the introduction speed, image analysis, etc.

[0041] The RAM 302 is made up of a volatile storage medium and provides a temporary memory area necessary for the operation of the CPU 301. The ROM 303 is made up of a non-volatile storage medium and stores necessary information such as programs used for the operation of the information processing device 30.

[0042] The HDD 304 is a storage device that is made up of a nonvolatile storage medium and stores information about the capture substance and the like.

[0043] The communication I / F 305 is a communication interface based on standards such as Wi-Fi (registered trademark) and 4G, and is a module for communicating with other devices including the filling device. The output device 306 may be a display device such as a liquid crystal display or an OLED (Organic Light Emitting Diode) display, and may be used to display moving images, still images, characters, etc. The input device 307 is a button, touch panel, keyboard, pointing device, etc., and is used by the user to operate the information processing device 30. The output device 306 and the input device 307 may be integrally formed as a touch panel.

[0044] The filling device 31 is a device for introducing a dispersion medium onto a substrate to fill the microwells with a target capture substance. The filling device 31 may include a member used for liquid delivery, such as a micropipette or syringe, and a mechanism for supplying power for delivering the dispersion medium from the member used for liquid delivery. The filling device 31 may also include, for example, a mechanism for degassing the space above the substrate.

[0045] The hardware configuration shown in FIG. 3 is an example, and other devices may be added, or some devices may not be provided. Also, some devices may be replaced with other devices having similar functions. Furthermore, some functions may be provided by other devices via a network, and the functions constituting this embodiment may be distributed and realized among multiple devices. For example, the HDD 304 may be replaced with an SSD (Solid State Drive) using semiconductor elements such as flash memory, or may be replaced with cloud storage. Next, the materials used in the filling method according to the first embodiment will be described in more detail.

[0046] <Microwell> Microwells are independently separated compartments. The volume of each compartment is preferably very small, preferably between 0.1 fL and 1000 fL. A preferred example of a microwell is a minute hole provided on the upper surface of a substrate.

[0047] <Well plate> A preferred example of a member having a substrate with microwells is a well plate, an example of which is shown in Figure 4.

[0048] 4 shows a cross-sectional view of a well plate 400. The well plate 400 has a base material made up of a lower substrate 401 and a partition wall 403, and an upper substrate 402, with microwells 404 provided on the upper surface of the base material.

[0049] The microwells 404 are recesses that contain a dispersion medium and are separated from each other by partition walls 403. The bottom surface of the microwells 404 can be the lower substrate 401, and the shape of the area surrounded by the bottom surface and side surfaces of the microwells 404 may be, for example, cylindrical or prismatic. When the microwells 404 are cylindrical with a circular bottom surface, the diameter of the bottom surface of the microwells 404 is preferably 0.5 μm to 12 μm and the depth of the microwells 404 is preferably 0.5 μm to 12 μm. Furthermore, the diameter of the bottom surface of the microwells 404 is more preferably 1 μm to 9 μm and the depth of the microwells 404 is more preferably 1 μm to 9 μm. The upper substrate 402 preferably faces the opening of the microwells 404 and the upper surface of the partition wall 403 across a space 405. The space 405 serves as a flow path for fluids such as a dispersion medium and a sealing medium, and the fluids such as the dispersion medium and the sealing medium can flow from an inlet (not shown) toward an outlet (not shown). A commercially available well plate such as SIMOA (registered trademark) DISC by Quanterix may be used as the well plate 400, or a well plate may be fabricated.

[0050] <Target capture substance> Examples of target capture substances include particles. Antibodies or the like may be bound to the particles in order to capture the target. Specific examples of particles include polymer resin (styrene resin, acrylic resin, etc.) particles, silica particles, agarose carrier resin particles, metal particles, latex particles, magnetic particles, etc. Use of a target capture substance makes it possible to separate the target by centrifugation or using magnetism. As the target capture substance, particles with a particle size of 1 μm or more and 10 μm or less can be preferably used.

[0051] <Dispersion medium> The dispersion medium can be appropriately selected from various media depending on the target capture substance used. Among these, hydrophilic liquid media are often suitable for use as dispersion media. Examples of hydrophilic liquid media that can be used as dispersion media include at least one selected from the group consisting of water, hydrophilic alcohols, hydrophilic ethers, ketones, nitrile-based solvents, dimethyl sulfoxide, and N,N-dimethylformamide, or mixtures containing these. Examples of hydrophilic alcohols include ethanol, methanol, propanol, and glycerin. Examples of hydrophilic ethers include tetrahydrofuran, polyethylene oxide, and 1,4-dioxane. Examples of ketones include acetone and methyl ethyl ketone. Examples of nitrile-based solvents include acetonitrile. The dispersion medium may further contain a buffer, a surfactant, and the like.

[0052] <Sealing medium> In many cases, a hydrophobic liquid medium can be suitably used as the sealing medium. Specific examples of the sealing medium include fluorine-based oils, saturated aliphatic hydrocarbons, unsaturated aliphatic hydrocarbons, aromatic hydrocarbons, and silicone oils. Examples of fluorine-based oils include Fluorinert, Asahiklin AE-3000 (manufactured by AGC), and Fomblin (manufactured by Solvay). Examples of saturated hydrocarbons include Isopar (manufactured by ExxonMobil) and mineral oil.

[0053] [Modification 1 of the First Embodiment] Next, a first modification of the first embodiment of the present disclosure described above will be described. 5 shows a functional block diagram of a filling system according to Modification 1 of the first embodiment. The filling system 10 according to this embodiment further includes a correspondence information acquisition unit 501 in addition to the components described in the first embodiment.

[0054] FIG. 6 is a flowchart showing an outline of a filling method according to the first modification of the first embodiment. In the filling method according to this embodiment, in the correspondence information acquisition process of step S601, the correspondence information acquisition unit 501 acquires correspondence information relating to the correspondence relationship between capture substance information and rate index information. Here, the capture substance information relating to the correspondence relationship is capture substance information for one or more target capture substances, including the target capture substance to be used. Furthermore, the rate index information is information relating to an index for determining the introduction rate at which a dispersion medium containing a dispersed target capture substance is introduced into a region on a substrate having a plurality of microwells, where the microwells are provided.

[0055] The rate index information may be information indicating only a preferred direction of change from a predetermined initial value, such as increasing or decreasing the introduction rate from the predetermined initial value. Alternatively, the rate index information may be information including a recommended value for the degree of change in addition to the direction of change from the predetermined initial value for the introduction rate. Alternatively, the rate index information may be information including a recommended value for a specific introduction rate corresponding to the capture substance information.

[0056] In the capture substance filling step of step S203, if there are multiple ways to introduce the dispersion medium onto the substrate, the degree of influence of each element of the capture substance information on the filling of the target capture substance into the microwells may differ depending on the way the dispersion medium is introduced onto the substrate. Therefore, the above-mentioned correspondence information may be prepared for each introduction way.

[0057] Subsequently, in a speed determination step of step S202, the speed determination unit 102 refers to the correspondence information acquired by the correspondence information acquisition unit 501 when determining the introduction speed. As in the present first modification, by referring to the correspondence information in the rate determination step, the introduction rate can be determined more easily.

[0058] [Modification 2 of the First Embodiment] Next, a second modification of the first embodiment of the present disclosure described above will be described. 7 shows a functional block diagram of a filling system according to Modification 2 of the first embodiment. The filling system 10 according to this embodiment further includes a model acquisition unit 701 in addition to the components described in the first embodiment.

[0059] FIG. 8 is a flowchart showing an outline of a filling method according to the second modification of the first embodiment. In the filling method according to this embodiment, in the model acquisition step of step S801, the model acquisition unit 701 acquires a trained model. Here, the trained model acquired by the model acquisition unit 701 is a machine learning model that has been trained to output a recommended value for the introduction speed of the dispersion medium onto the substrate so as to obtain a predetermined filling efficiency when capture substance information is input. There are no particular limitations on the type of machine learning model used for learning or the learning method, and any known machine learning model or learning method can be used. For example, past filling results may be image-analyzed and fed back to update the trained model so that the optimal introduction speed can be output each time through machine learning.

[0060] In this embodiment, if there are multiple ways to introduce the dispersion medium onto the substrate in the capture substance filling step of step S203, the trained model may be prepared for each introduction method. Alternatively, the trained model may be trained to input the introduction method of the dispersion medium in addition to the capture substance information and output a recommended introduction rate that will achieve a predetermined filling efficiency.

[0061] Next, in the rate determination process of step S202, the rate determination unit 102 determines the introduction rate by referring to the recommended introduction rate value output from the trained model acquired by the model acquisition unit 701 using the capture substance information acquired in step S201 as input. As in the present modification 2, the recommended value of the introduction speed is output using a trained model in the speed determination process, and by referring to this, the introduction speed can be determined more easily.

[0062] [Second embodiment] As a second embodiment, an example of a method for detecting target molecules that utilizes the filling method described in the first embodiment will be described.

[0063] 9 shows a functional block diagram of a detection system according to a second embodiment of the present disclosure. A detection system 90 according to this embodiment includes a target capture unit 901, a target loading unit 902, a detection unit 903, an input unit 104, an output unit 105, and a memory unit 106. The detection unit 903 also includes an image acquisition unit 903a. A detection method according to a second embodiment will be described, which is carried out using the detection system 90 shown in Fig. 9. Fig. 10 is a flowchart showing an outline of the detection method according to the second embodiment of the present disclosure.

[0064] The detection method according to this embodiment relates to a method for distributing a sample containing a target molecule into a plurality of microwells and then detecting the target molecule in the microwells. By distributing a sample containing a target molecule into microwells, the sample appears concentrated, making it possible to detect the target molecule without an amplification step and shortening the time it takes for the signal to saturate. The volume per microwell compartment can be made sufficiently small so that each compartment contains one or fewer target molecules. Counting the number of compartments from which a signal is obtained enables a digital assay that calculates the concentration of the target molecule in the sample. In the detection method according to this embodiment, first, in a target capture step in step S1001, the target capture unit 901 performs an operation for capturing a target molecule in a dispersion medium using a target capture substance. Specifically, for example, the target capture step includes a step for mixing a dispersion medium in which the target capture substance is dispersed with a sample containing the target molecule to be detected.

[0065] The target molecule can be any substance without limitation, and specific examples of the target molecule include proteins, nucleic acids, lipids, sugars, low-molecular-weight compounds, enzymes, ligands, receptors, antibodies, antigens, cytokines, hormones, and membrane proteins. The target capture step can include, in addition to mixing the dispersion medium and the sample, a step for promoting the association between the target capture substance and the target molecule. Examples of such steps include heating and shaking. The target capture step may also include a step for adding an additive to the mixture (mixture) of the dispersion medium and the sample to initiate or promote the association between the target capture substance and the target molecule. The target capture step may also include a step for adding a detection reagent (detection reagent) to the mixture (mixture) of the dispersion medium and the sample. It should be noted that the detection system 90 does not necessarily have to have the target capture unit 901. In other words, the operation in the target capture step is not limited to being performed automatically, but may be performed manually by the user.

[0066] Next, in the target filling step of step S1002, the target capture substance is filled into the microwells by the filling method described in embodiment 1. The detection system 90 may include, for example, a mounting unit for placing a substrate having microwells formed therein, and the dispersion medium may be introduced into the substrate placed on the mounting unit according to the filling method described in embodiment 1.

[0067] The microwells may contain a detection reagent, or the sample containing the target molecule may contain a detection reagent, which preferably generates a signal upon interaction with the target molecule. Any known detection reagent can be used without limitation. For example, when the target molecule is a nucleic acid and the CRISPR / Cas system is used, the detection reagent can include an effector protein, a crRNA that binds to the target nucleic acid, and a reporter molecule. That is, the crRNA binds to the target nucleic acid, activates the effector protein, and the activated effector protein modifies the reporter molecule, generating fluorescence as a signal. When the target molecule is a nucleic acid, the detection reagent is not limited to the above system, and various probes that emit signals depending on the presence or amount of the target nucleic acid can be used.

[0068] Examples of detection reagents when the target molecule is a protein include antibodies, modified antibodies, ligands, and aptamers, which can be labeled with a fluorescent dye, enzyme, or the like to generate a signal. When the target molecule is an enzyme, an enzyme substrate can be used as the detection reagent. The enzyme substrate may be labeled with a fluorescent dye and modified by the substrate to generate fluorescence as a signal.

[0069] Effective digital assays can be performed by limiting the number of target molecules per microwell to one or less. Alternatively, when using a target capture substance, limiting the number of target molecules per target capture substance to one or less can achieve the same effect, making digital assays possible.

[0070] Subsequently, in the detection step of step S1003, the detection unit 903 detects the target molecule based on a signal emitted by the target capture substance that has captured the target molecule.

[0071] Signal Generation: In the microwells into which the sample containing the target molecule is dispensed, a reaction can be carried out to generate a signal to be used in detecting the target molecule in the detection step. For example, in the example using the CRISPR / Cas system described above, the substrate with the microwells is incubated at 37°C. This incubation allows the CRISPR-Cas trans-cleavage reaction to proceed, resulting in the generation of fluorescence from the fluorescent substance possessed by the reporter molecule. In the example where the target molecule is an enzyme, signal generation can be achieved by proceeding with the reaction at the optimal temperature for that enzyme.

[0072] A method for detecting a target molecule based on a signal resulting from a reaction between the target molecule and a target capture substance filled in a microwell will be described. In this embodiment, a method using image capture to acquire signal information will be described.

[0073] In this embodiment, the detection unit 903 has an image acquisition unit 903a, which acquires images of the plurality of microwells in an image acquisition step included in the detection step. The image acquisition unit 903a may acquire images of the microwells using an imaging device separate from the detection system 90, or the image acquisition unit 903a may be equipped with an imaging device.

[0074] The image includes all information contained in the image, such as brightness, color, and shade for each coordinate, and the image acquisition means can acquire necessary information from all of this information. The image may be an extracted image. The image may be of the entire substrate or the entire well plate, or may be of a part of the substrate or the well plate. The image acquired by the image acquisition unit 903a may be at least one of a bright field image and a fluorescent image.

[0075] An example of signal detection in the detection step when the image acquired by the image acquisition unit 903a is, for example, a fluorescent image will be described. For example, in the example where the target molecule is an enzyme, an enzyme reaction generates fluorescence, which accumulates in the microwell. The image acquisition unit 903a acquires an image of this using an imaging device such as a microscope or CCD camera. Next, the image is analyzed to detect a signal resulting from the reaction between the target molecule and the target capture substance filled in the microwell.

[0076] In this embodiment, the detection unit 903 has the image acquisition unit 903a, and the target molecule is detected using an image of the microwell acquired by the image acquisition unit 903a. However, the detection method in the detection step is not limited to this. That is, the detection unit 903 does not have to have the image acquisition unit 903a, and an appropriate detection device can be used for the detection unit 903 depending on the mechanism used to detect the target molecule.

[0077] The present disclosure can also be realized by executing the following process. That is, software (programs) for causing a computer (or a CPU, MPU, etc.) to execute the functions of the above-described embodiments are supplied to a system or device via a network or various storage media. The computer (or a CPU, MPU, etc.) of the system or device then reads and executes the programs. The present disclosure may also be realized, for example, by a circuit (e.g., an ASIC) that implements one or more functions.

[0078] It should be noted that the above-described embodiments merely illustrate specific examples of implementing the filling method and detection method according to the present disclosure, and the technical scope of the present disclosure should not be interpreted as being limited by these embodiments. In other words, the present disclosure can be implemented in various forms without departing from its technical concept or main features. For example, it should be understood that embodiments in which some components of any of the embodiments are added to or replaced with some components of other embodiments are also embodiments to which the present disclosure can be applied.

[0079] Embodiments of the present disclosure include the following configurations and methods. (Method 1) 1. A method for filling microwells with a target capture substance, comprising: a capture substance information acquisition step of acquiring capture substance information regarding the target capture substance to be used; a rate determination step of determining an introduction rate for introducing a dispersion medium in which the target capture substance is dispersed into a region on the substrate on which the plurality of microwells are provided, based on the capture substance information acquired in the capture substance information acquisition step; and a capture substance filling step of filling one or more of the microwells with the target capture substance by introducing the dispersion medium into the substrate at the introduction speed; A filling method comprising: (Method 2) The loading method of method 1, wherein the capture material information includes material property information relating to properties that affect the introduction rate. (Method 3) The capture substance information includes identification information for identifying at least one of the target capture substance and the dispersion medium. The filling method according to method 1 or 2. (Method 4) a correspondence information acquisition step of acquiring correspondence information relating to a correspondence relationship between the capture substance information for one or more target capture substances including the target capture substance used and rate index information relating to an index for determining the introduction rate, 4. The filling method according to any one of Methods 1 to 3, wherein the rate determining step includes determining the introduction rate using the correspondence information. (Method 5) 5. The method of claim 4, wherein the rate indicator information includes a recommended value for the introduction rate. (Method 6) 6. The loading method according to any one of Methods 1 to 5, wherein the target capture substance is a particle having a particle size of 1 μm or more and 10 μm or less. (Method 7) 7. The filling method according to any one of Methods 1 to 6, wherein the volume of the microwell is 0.1 fL or more and 1000 fL or less. (Method 8) a target capturing step of capturing target molecules in the dispersion medium by the target capturing substance; a target filling step of filling the target capture substance into the microwells by the filling method according to any one of methods 1 to 7; a detection step of detecting the target molecule based on a signal emitted by the target capture substance that has captured the target molecule; A detection method comprising: (Method 9) The detection method described in Method 8, wherein the detection step includes an image acquisition step of acquiring images of a plurality of the microwells. (Method 10) The detection method described in Method 9, wherein the image is at least one of a bright field image and a fluorescent image. (Configuration 1) 1. A filling system for filling microwells with a target capture substance, comprising: a capture substance information acquisition unit that acquires capture substance information regarding the target capture substance to be used; a rate determination unit that determines an introduction rate for introducing a dispersion medium in which the target capture substance is dispersed into a region on the substrate on which the plurality of microwells are provided, based on the capture substance information acquired by the capture substance information acquisition unit; and a capture substance filling section that fills one or more of the microwells with the target capture substance by introducing the dispersion medium into the substrate at the introduction speed; A filling system having (Configuration 2) A filling system according to configuration 1; a detection unit that detects the target molecule based on a signal emitted by the target capture substance that has captured the target molecule; A detection system comprising: (Configuration 3) 3. The detection system according to configuration 2, wherein the detection unit has an image acquisition unit that acquires images obtained by imaging the plurality of microwells. (Configuration 4) 4. The detection system of configuration 3, wherein the image is at least one of a bright-field image and a fluorescent image. [Explanation of symbols]

[0080] 10 Filling System 30 Information processing equipment 31 Filling equipment 90 Detection System 101 Captured Substance Information Acquisition Department 102 Speed ​​determining section 103 Captured substance filling section 400-well plate 401 Lower board 402 Upper board 403 Bulkhead 404 Microwell 405 Space 501 Correspondence information acquisition unit 701 Model Acquisition Department 901 Target Acquisition Unit 902 Target filling part 903 Detector 903a Image acquisition unit

Claims

1. 1. A method for filling microwells with a target capture substance, comprising: a capture substance information acquisition step of acquiring capture substance information regarding the target capture substance to be used; a rate determination step of determining an introduction rate for introducing a dispersion medium in which the target capture substance is dispersed into a region on the substrate on which the plurality of microwells are provided, based on the capture substance information acquired in the capture substance information acquisition step; and a capture substance filling step of filling one or more of the microwells with the target capture substance by introducing the dispersion medium into the substrate at the introduction speed; A filling method comprising:

2. The method of claim 1 , wherein the capture material information includes material property information relating to properties that affect the introduction rate.

3. The capture substance information includes identification information for identifying at least one of the target capture substance and the dispersion medium. The method of claim 1.

4. a correspondence information acquisition step of acquiring correspondence information relating to a correspondence relationship between the capture substance information for one or more target capture substances including the target capture substance used and rate index information relating to an index for determining the introduction rate, The method of claim 1 , wherein the rate determining step includes determining the rate of introduction using the correspondence information.

5. The method of claim 4 , wherein the rate indicator information includes a recommended value for the introduction rate.

6. 2. The method according to claim 1, wherein the target capture substance is a particle having a particle size of 1 μm or more and 10 μm or less.

7. The filling method according to claim 1, wherein the volume of the microwell is 0.1 fL or more and 1000 fL or less.

8. a target capturing step of capturing target molecules in the dispersion medium by the target capturing substance; a target filling step of filling the target capture substance into the microwells by the filling method according to claim 1; a detection step of detecting the target molecule based on a signal emitted by the target capture substance that has captured the target molecule; A detection method comprising:

9. The detection method according to claim 8 , wherein the detection step includes an image acquisition step of acquiring images of the plurality of microwells.

10. The detection method according to claim 9 , wherein the image is at least one of a bright-field image and a fluorescent image.

11. 1. A filling system for filling microwells with a target capture substance, comprising: a capture substance information acquisition unit that acquires capture substance information regarding the target capture substance to be used; a rate determination unit that determines an introduction rate for introducing a dispersion medium in which the target capture substance is dispersed into a region on the substrate on which the plurality of microwells are provided, based on the capture substance information acquired by the capture substance information acquisition unit; and a capture substance filling section that fills one or more of the microwells with the target capture substance by introducing the dispersion medium into the substrate at the introduction speed; A filling system having

12. A filling system according to claim 11; a detection unit that detects the target molecule based on a signal emitted by the target capture substance that has captured the target molecule; A detection system comprising:

13. The detection system according to claim 12 , wherein the detection unit has an image acquisition unit that acquires images obtained by imaging the plurality of microwells.

14. The detection system of claim 13 , wherein the image is at least one of a bright-field image and a fluorescent image.

Images