Solution exchange method and digital measurement method

Abstract

Provided are a solution exchange method and a digital measurement method that improve the accuracy of solution exchange in micropores. A solution exchange method for exchanging a solution formed in a plurality of micropores (M) of a substrate (B), the method comprising: a step for replacing, with a gas, a liquid that is immiscible with a first solution and that seals the plurality of micropores (M) filled with the first solution; and a step for filling the plurality of micropores (M) with a second solution by introducing the second solution onto the substrate (B) on which the immiscible liquid has been replaced with a gas.

Description

Technical Field

[0001] The present invention relates to a solution exchange method and a digital assay method. Background Art

[0002] Methods for exchanging solutions in microwells are known. For example, Non-Patent Document 1 describes a method in which the solution in the microwells is exchanged from the lysis buffer to the RT reaction mix by removing the perfluorinated oil used to seal a plurality of microwells filled with a lysis buffer with a detergent-containing buffer.

[0003] Prior art literature

[0004] Patent Literature

[0005] Non-patent literature 1: Scientific Reports volume 6, Article number: 33883 (2016) "An Automated Microwell Platform for Large-Scale Single Cell RNA-Seq" Summary of the invention

[0006] However, in the above-mentioned conventional solution exchange method, the perfluoro oil sealing the micropores may not be sufficiently removed, and the solution exchange in some of the micropores may not be performed.

[0007] An object of the present invention is to provide a technology for improving the accuracy of solution exchange in microwells.

[0008] The solution exchange method of the present invention is a solution exchange method for exchanging solutions in multiple micropores (M) formed on a substrate (B), comprising: a process of replacing an immiscible liquid that is immiscible with the first solution and seals the multiple micropores (M) filled with a first solution with a gas; and a process of filling the multiple micropores (M) with a second solution by introducing a second solution onto the substrate (B) in which the immiscible liquid is replaced with the gas.

[0009] According to the solution exchange method of the present invention, the steps of replacing the immiscible liquid that is immiscible with the first solution and seals the plurality of micropores (M) filled with the first solution with a gas, and filling the plurality of micropores (M) with the second solution by introducing the second solution onto the substrate (B) in which the immiscible liquid is replaced with the gas. Therefore, in the solution exchange method of the present invention, it is possible to suppress the immiscible liquid from remaining around a portion of the micropores M. Therefore, it is possible to suppress the obstruction of the exchange from the first solution to the second solution due to the immiscible liquid remaining around a portion of the micropores M, thereby improving the accuracy of the solution exchange in the micropores.

[0010] The digital measurement method of the present invention is a digital measurement method for performing digital measurement in multiple stages using multiple micropores (M) formed on a substrate (B), comprising the following steps: performing a first measurement in multiple micropores filled with a first measurement reagent and sealed with an immiscible liquid that is immiscible with the first measurement reagent; performing a gas-replacement step for sealing the multiple micropores (M); filling the multiple micropores (M) with the second measurement reagent by introducing a second measurement reagent onto the substrate (B) where the immiscible liquid is replaced with the gas; sealing the multiple micropores (M) filled with the second measurement reagent with an immiscible fluid by introducing an immiscible liquid onto the substrate (B); and performing a second measurement in the multiple micropores (M) filled with the second measurement reagent and sealed with the immiscible liquid.

[0011] According to the digital measurement method of the present invention, the following steps are performed: a step of performing a first measurement in a plurality of micropores filled with a first measurement reagent and sealed with an immiscible liquid that is immiscible with the first measurement reagent; a step of replacing the immiscible liquid that seals the plurality of micropores (M) with a gas; a step of filling the plurality of micropores (M) with a second measurement reagent by introducing a second measurement reagent onto a substrate (B) in which the immiscible liquid is replaced with a gas; a step of sealing the plurality of micropores (M) filled with the second measurement reagent with an immiscible liquid by introducing an immiscible liquid onto the substrate (B); and a step of performing a second measurement in the micropores (M) filled with the second measurement reagent and sealed with the immiscible liquid. Therefore, in the digital measurement method of the present invention, the accuracy of solution exchange in the micropores can be improved. In addition, in the digital measurement method of the present invention, multiple digital measurements including the first digital measurement and the second digital measurement can be continuously performed in a common micropore via at least a solution exchange method.

[0012] According to the present invention, the accuracy of solution exchange in microwells can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 This is a schematic diagram showing the outline of the solution exchange method.

[0014] Figure 2 It is a schematic diagram showing the outline of the digital measurement method and the multi-stage digital measurement method.

[0015] Figure 3 This is a schematic diagram showing the outline of a multi-stage digital measurement method using a solution exchange method.

[0016] Figure 4 It is a diagram showing the overall configuration of a closed-type control system in the first embodiment.

[0017] Figure 5 This is a schematic diagram showing an outline of a method for forming a three-phase plug.

[0018] Figure 6 This is a schematic diagram showing an outline of a method for adding a three-phase plug.

[0019] Figure 7 This is a schematic diagram showing an outline of a method for forming a two-phase plug.

[0020] Figure 8 This is a schematic diagram showing an outline of a method for adding a two-phase plug.

[0021] Fig. 9 This is a diagram showing an example of a cross-sectional view of a closed-type micropore device.

[0022] Fig.10 This is a diagram showing an example of a top view of a closed-type microwell device.

[0023] Fig.11 It is a diagram showing the overall configuration of an open control system in the second embodiment.

[0024] Fig.12 This is a diagram showing an example of a cross-sectional view of an open-type microporous device.

[0025] Fig.13 This is a diagram showing an example of a top view of an open-type microwell device.

[0026] Fig.14 This is a schematic diagram showing an outline of a first example of a method for producing a closed microwell device.

[0027] Fig.15 This is a schematic diagram showing an outline of a second example of a method for producing a closed microwell device.

[0028] Fig.16 It is a schematic diagram showing the flow of the first embodiment.

[0029] Fig.17 It is a schematic diagram showing the flow of a comparative example of the first embodiment.

[0030] Fig.18 It is a graph showing the results of the first embodiment and the comparative example.

[0031] Fig.19 It is a schematic diagram showing the flow of the second embodiment.

[0032] Fig. 20 It is a figure which shows the experimental result of the second example.

[0033] Fig.21 It is a schematic diagram showing the flow of the third embodiment.

[0034] Fig. 22 It is a graph showing the experimental results of the third embodiment.

[0035] Fig.23 It is a schematic diagram showing the flow of the fourth embodiment.

[0036] Fig.24 It is a graph showing the experimental results of the fourth embodiment.

[0037] Fig.25 It is a diagram showing other experimental results of the fourth embodiment.

[0038] Fig.26 It is a diagram showing other experimental results of the fourth embodiment.

[0039] Fig. 27 It is a schematic diagram showing the flow of the fifth embodiment.

[0040] Fig.28 It is a graph showing the first experimental result of the fifth embodiment.

[0041] Fig.29 (a) is a graph showing the fluorescence intensity in the fluorescence image of A647ch. Fig.29 (b) is a graph showing the fluorescence intensity in the fluorescence image of FLch.

[0042] Fig.30 (a) to Fig.30 (c) is a graph showing a regression line of the relationship between fluorescence intensity and impurity concentration.

[0043] Fig.31 It is a graph showing the second experimental result of the fifth embodiment.

[0044] Fig.32 It is a schematic diagram showing the flow of the sixth embodiment.

[0045] Fig.33 (a) means in Fig.32FIG. 4 is a diagram showing an example of a fluorescent image A acquired in step 4 and a fluorescent image B acquired in step 9 . Fig.33 (b) is a graph showing an example of the fluorescence intensity of the microwell where ALP was detected. Fig.33 (c) is a graph showing an example of the fluorescence intensity of a microwell in which β-Gal is detected.

[0046] Fig.34 It is a schematic diagram showing the flow of the seventh embodiment.

[0047] Fig.35 It is a schematic diagram showing the analysis flow of the seventh embodiment.

[0048] Fig.36 (a) and Fig.36 (b) means Fig.34 The diagram shows an example of the detection results when steps 2 to 11 are carried out using a sample prepared without containing exosomes in step 1. Fig.36 (c) and Fig.36 (d) means Fig.34 The diagram shows an example of the detection results when steps 2 to 11 are carried out using a sample prepared so as to contain 16 μg of exosomes per sample in step 1.

[0049] Fig.37 It is a figure which shows other experimental results of the seventh embodiment.

[0050] Description of Reference Numerals

[0051] 1: fluid system, 3: detection system, 5: control system, 11: injection pump, 14: water storage tank, 15: oil management unit, 16A: reagent A management unit, 16B: reagent B management unit, 16C: reagent C management unit, 18: waste liquid tank A, 19: waste liquid tank B, 31: flow cell, 33: microscope, 51: PC, 61: dispenser, 63: tip rack, 65: solution rack, 67: tip waste box, 69: track, 100A: closed control system, 100B: open control system, M: microwell, B: substrate, UG: upper surface glass, DT: double-sided tape, ST: single-sided tape. DETAILED DESCRIPTION

[0052] Hereinafter, preferred embodiments of the present invention will be described with reference to the accompanying drawings. In addition, the same reference numerals are given to the same components, and repeated descriptions are omitted. In addition, unless otherwise specified, positional relationships such as up, down, left, and right are assumed to be based on the positional relationships shown in the accompanying drawings. Furthermore, the dimensional ratios of the accompanying drawings are not limited to the ratios shown in the drawings. In addition, the following embodiments are examples for illustrating the present invention, and the present invention is not limited to these embodiments.

[0053] Figure 1 Schematic diagram showing the outline of an embodiment of the solution exchange method of the present invention. The solution exchange method is implemented using a microporous device MWD. Figure 1 As shown, the microporous device MWD comprises: a substrate B having a plurality of micropores M formed therein, and an upper surface glass UG disposed above the substrate B. The upper surface glass UG is disposed separately from the substrate B so that a space SP can be formed between the substrate B and the upper surface glass UG. The solution exchange method of an embodiment of the present invention is a method for exchanging a solution SA in a plurality of micropores M formed on the substrate B with another solution. In the solution exchange method, for example, a plurality of micropores M filled with a solution SA (a first solution) are sealed with a volatile oil VO (an immiscible liquid) (process 1). In the solution exchange method, air A (gas) is transferred (introduced) into a space SP between the substrate B and the upper surface glass UG (a second substrate) disposed above the substrate B, and the volatile oil VO is replaced with air A (process 2). Next, in the solution exchange method, a solution SB (a second solution) is transferred (introduced) into a space SP on the substrate B where the volatile oil VO is replaced with air A, and a plurality of micropores M are filled with the solution SB (process 3). In such a solution exchange method, it is possible to suppress the volatile oil VO from remaining around a part of the micropores M. Therefore, it is possible to suppress the volatile oil VO remaining around a part of the micropores M from hindering the exchange from the solution SA to the solution SB, thereby improving the accuracy of the solution exchange (exchange from the solution SA to the solution SB) in the micropores M.

[0054] In any of the above-mentioned processes, it can be implemented by an automated device or a manual method. For example, from the viewpoint of reproducibility, it is preferably implemented by an automated device. The automated device, for example, includes a device composed of an air pressure supply unit such as a syringe pump, a pneumatic pump, an air pressure control unit such as a regulator, a flow path such as a pipe, and a fluid control unit such as a pinch valve and a solenoid valve. The automated device, for example, connects a syringe pump through a Teflon (registered trademark) tube, and injects gas into a substrate formed with a plurality of micropores through the syringe pump, or, by attracting an immiscible liquid, replaces the immiscible liquid in a plurality of micropores filled with the first solution with a gas. In addition, the automated device replaces the first solution with the second solution by injecting the second solution into the substrate using a syringe pump. Furthermore, the automated device replaces the immiscible liquid into the substrate using an immiscible fluid, thereby sealing a plurality of micropores exchanged for the second solution with an immiscible fluid.

[0055] In addition, the automated device may be, for example, a device having a pipette connected to a syringe pump. The automated device, for example, sucks out the immiscible liquid on the substrate having a plurality of micropores from above using a pipette, thereby replacing the immiscible liquid in the micropores filled with the first solution with a gas. In addition, the automated device replaces the first solution with the second solution by discharging the second solution from the pipette onto the substrate. Furthermore, the plurality of micropores replaced with the second solution are sealed with the immiscible fluid by discharging the immiscible liquid from the pipette onto the substrate.

[0056] As the case where the solution exchange method of the embodiment of the present invention can be applied, there is no particular limitation, and in detail, various methods can be used. In the solution exchange method, for example, biological particles such as cells and exosomes are introduced into each micropore independently. In the solution exchange method, a solution of a surface antigen detection reagent added with a reagent for detecting antigens present on the surface of particles (biological particles) derived from an organism, a solution added with a reagent for extracting inclusions from biological particles, and a solution added with a reagent for detecting inclusions of the extracted inclusions are sequentially sealed in each micropore using a solution exchange method, so that biological particles can be analyzed particle by particle. In addition, in the solution exchange method, biological particles such as cells and exosomes are introduced into each micropore independently, and a solution added with a biomarker detection reagent is sealed in each micropore to detect biomarkers, and then the solution in each micropore is exchanged for a cell culture fluid, so that single cell screening can be achieved.

[0057] Figure 2 This is a schematic diagram showing the outline of the digital measurement method and the multi-stage digital measurement method. The digital measurement method refers to a measurement method using a plurality of microscopic spaces such as microwells and droplets, such as Figure 2 As shown in the upper part of the text, for example, a method of sealing target particles one by one in tiny spaces, counting tiny spaces that emit fluorescence or other signals due to the presence of tiny particles as 1, and counting tiny spaces that do not emit fluorescence as 0, and determining the tiny particles qualitatively or quantitatively based on the number of tiny spaces counted as 1.

[0058] like Figure 2As shown in the lower section of the multi-stage digital measurement method, for example, a solution concentrated in a manner that increases the concentration of target cells can be introduced into each microwell, cells are sealed into the microwells one by one ("1. Concentration"), surface antigens of the cells are measured ("2. Expression antigen measurement"), extracts and captures the contents (e.g., contained proteins and contained nucleic acids) from the cells using an extracting solution or the like ("3. Extraction / capture"), contained proteins are labeled using a labeling agent ("4. Labeling"), contained proteins are measured ("5. Contained protein measurement"), contained nucleic acids are labeled using a labeling agent ("6. Labeling"), and contained nucleic acids are measured ("7. Contained nucleic acid measurement"). The digital measurement method is not limited to the above-mentioned techniques, and other steps may be appropriately included.

[0059] In addition, in the digital measurement method, a plurality of digital measurements can be continuously performed in the same space by exchanging solutions in a microscopic space (multi-stage digital measurement).

[0060] Figure 3 Schematic diagram showing the outline of the digital measurement method according to the present invention when it is applied to a multi-stage digital measurement method. Figure 3 As shown, in the multi-stage digital measurement method of the embodiment of the present invention, for example, first, after the introduction of biological particles, a plurality of micropores M filled with the solution SA (first measurement reagent) are sealed with a volatile oil VO (immiscible liquid) that is immiscible with the solution SA (step 1). Here, the first digital measurement (step 1) for detecting the surface molecules of the biological particles introduced into the plurality of micropores M is performed. In addition, in order to enable optical measurement, the substrate B formed with the plurality of micropores M is preferably formed of a transparent member.

[0061] In the multi-stage digital measurement method, air A (gas) is introduced into the space SP between the substrate B and the upper surface glass UG disposed above the substrate B, and the volatile oil VO is pushed out, thereby replacing the volatile oil VO with the air A (step 2). Next, in the multi-stage digital measurement method, a solution SB (a second measurement reagent) is injected into the space SP between the substrate B and the upper surface glass UG in which the volatile oil VO is replaced by the air A, and the plurality of micropores M are filled with the solution SB (step 3).

[0062] Next, in the present multi-stage digital measurement method, volatile oil VO is introduced into the space SP between the substrate B and the upper surface glass UG, the solution SB in the space SP is pushed out, and the volatile oil VO (second immiscible liquid) is used to seal the plurality of micropores M filled with the solution SB (process 4). Here, for example, after extracting the inclusions from the biological particles in the plurality of micropores M, a second digital measurement for detecting the inclusions is implemented (process 4). In addition, the volatile oil VO (immiscible liquid) that seals the solution SA in process 1 and the volatile oil VO (second immiscible liquid) that seals the solution SB in process 4 can be either the same type of immiscible liquid or different types of immiscible liquids. In the following description, an example of the same type of immiscible liquids is described.

[0063] Thus, in the multi-stage digital measurement method of the present embodiment, multiple digital measurements including a first digital measurement using solution SA and a second digital measurement using solution SB are continuously performed in a common micropore at least via a solution exchange method. In addition, in the multi-stage digital measurement method, the number of digital measurements performed is arbitrary as long as it is 2 or more times. In addition, the digital measurement may also include digital measurements other than the above-mentioned first digital measurement for detecting surface molecules of biological particles introduced into the micropore M and the second digital measurement for detecting inclusions after extracting the inclusions from the biological particles in the micropore M.

[0064] <First embodiment>

[0065] Reference Figures 4 to 10 The closed control system in the first embodiment is described below. Fig. 9 The micropore device shown is a control system of a micropore device in which, for example, an upper surface glass UG is arranged above the micropore M to form a closed space SP.

[0066] Figure 4 This is a diagram showing the overall structure of a closed control system. Figure 4 As shown, the closed control system 100A illustratively includes a fluid system 1 , a detection system 3 , and a control system 5 .

[0067] The fluid system 1 includes, for example, a syringe pump 11, a water storage tank 14, an oil management unit 15, a reagent A management unit 16A, a reagent B management unit 16B, a reagent C management unit 16C, a waste liquid tank A18, and a waste liquid tank B19. The above-mentioned parts of the fluid system 1 are connected by a plurality of pipes CB. For example, a three-way solenoid valve SV, a pinch valve PV, and a three-point connector C are arranged at the connection part of the pipe CB. For example, at least one of the above-mentioned parts of the fluid system 1, the three-way solenoid valve SV, and the pinch valve PV can be controlled by a control signal from the PC 51 in the control system 5, or by a control signal from a control unit (not shown) provided in the fluid system 1.

[0068] The syringe pump 11 illustratively includes a syringe A12 for delivering air to the tube CB, for example, and a syringe B13 for delivering a washing buffer solution as a washing liquid to the tube CB. The water storage tank 14 is a tank for storing the washing buffer solution, for example.

[0069] The oil management unit 15, for example, includes a container for storing oil. The oil in the oil management unit 15 is operated by the syringe A12 and is sent to the flow cell 31 through the downstream pipe CB by air pressure. The reagent A management unit 16A, the reagent B management unit 16B, and the reagent C management unit 16C are respectively provided with containers for storing reagent A, reagent B, and reagent C. The reagent A, reagent B, and reagent C of the reagent A management unit 16A, the reagent B management unit 16B, and the reagent C management unit 16C are operated by the syringe A12 and are sent to the flow cell 31 through the downstream pipe CB by air pressure. For example, the reagent A, the reagent B, and the reagent C may be different reagents, respectively, or at least two reagents may be common reagents. In the control system 100A, there is no limitation on the types of reagents, and the types may be two or less or four or more.

[0070] The waste liquid tank A18 is a tank for storing waste liquid generated in the fluid system 1. The waste liquid tank B19 is a tank for storing waste liquid from the flow cell 31.

[0071] The detection system 3 includes, for example, a flow cell 31 and a microscope 33. The flow cell 31 is, for example, a measurement container configured to be able to measure a measurement sample sent from a tube CB. The flow cell 31 includes, for example, a plurality of micropores. As the flow cell 31, for example, Figure 3 The microwell device described includes a substrate B and an upper surface glass UG.

[0072] The microscope 33 is, for example, a device for magnifying an object and photographing it. The microscope 33 includes, for example, a microscope unit and a photographing unit for photographing the object magnified by the microscope unit. The microscope unit of the microscope 33 is, for example, arranged at a position opposite to the microwell to obtain information from the microwell filled with the first solution or the microwell filled with the second solution.

[0073] The microscope section, for example, includes optical microscopes such as a stereo microscope, a fluorescence microscope, a laser scanning microscope, and a confocal laser microscope. As the microscope section, for example, electron microscopes such as a transmission electron microscope, a scanning electron microscope, an atomic force microscope, a scanning tunneling microscope, a scanning near-field light microscope, a scanning probe microscope, an X-ray microscope, and an ultrasonic microscope can be cited. The photographing section can be arranged at a position where an observation image of the microscope can be photographed, such as an eyepiece section, a photo tube, or a C-type interface. The photographing section, for example, is not particularly limited as long as it can photograph at least one of a still image and a dynamic image. The photographing section can be, for example, a digital camera, an analog camera, a digital video camera, or an analog video camera.

[0074] The control system 5, for example, performs motion control of each part of the fluid system 1 headed by the injection pump 11. The control system 5, for example, performs motion control of the microscope 33 in the detection system 3. The control system 5, for example, has a PC (Personal Computer) 51. PC 51 is, for example, a device operated by a user of the control system 100A. PC 51, for example, has a CPU and a memory, and the above-mentioned control function of PC 51 can also be realized by the CPU executing a program stored in the memory. In the case where the CPU executes the program, the program can be stored in a storage medium. The storage medium storing the program can also be a non-transitory computer readable medium that can be read by a computer. The non-transitory storage medium is not particularly limited, for example, it can also be a storage medium such as an SSD (Solid State Drive), a USB (Universal Serial Bus) memory, or a CD-ROM (Compact Disc ROM). PC 51 can also have an output unit such as a display for displaying the detection results of the detection system 3. PC 51 can also have an input unit such as a keyboard for the user to input operation instructions.

[0075] <Formation and transfer method of multiphase plug>

[0076] In the present solution exchange method, for example, when performing a process of replacing an immiscible liquid with a gas, before introducing (transferring) the gas onto the substrate, a multiphase plug (multiphase plug) consisting of three phases of gas, a second solution, and an immiscible liquid may be formed in advance in a tube or a pipette tip, and then the multiphase plug is transferred. In this specification, a "multiphase plug" refers to a collection of fluids of different types connected to each other without mixing. The types of fluids in the "multiphase plug" can be two or more, and may be either gas or liquid, or a combination thereof. The "multiphase plug" is preferably formed in a flow path so as to maintain its shape.

[0077] In particular, when it is necessary to seal with an immiscible liquid just after the micropore is exchanged for the second solution, a multiphase plug is preferably used. According to the method, for example, a surfactant solution (second solution) can be introduced into a micropore filled with biological particles such as cells and exosomes and the biological particles are crushed, and then sealed with an immiscible liquid without leaving time, so that the inclusions of the biological particles can be suppressed from diffusing to adjacent micropores. In addition, since it is possible to seal with an immiscible liquid without leaving time after the matrix solution is introduced into the micropore fixed with the enzyme, it is possible to suppress the enzyme reaction from diffusing to adjacent micropores.

[0078] In the case of forming a multiphase plug in a tube, for example, it is formed by controlling a fluid control device such as an electromagnetic valve while injecting an immiscible liquid, a second solution, and air into the tube in sequence. Specifically, after injecting the immiscible liquid into the tube, the fluid control device is switched to inject the second solution, and a portion of the immiscible liquid in front of the flow direction is replaced by the second solution. Thereafter, the fluid control device is switched to inject gas, and a portion of the second solution in front of the flow direction is replaced by gas. In this way, a three-phase plug can be formed, which is arranged in the order of air, second solution, and immiscible liquid from the front of the flow direction. In the case of forming a multiphase fluid in a pipette tip, for example, it is formed by sucking the immiscible liquid, the second liquid, and air in sequence from the front end of the pipette tip.

[0079] Reference Figures 5 to 8 , the formation / addition method of the three-phase / two-phase plug is specifically described. Figure 5 This is a schematic diagram showing an outline of a method for forming a three-phase plug. Figure 6 This is a schematic diagram showing an outline of a method of sending liquid to a three-phase plug of a flow cell. Figure 7 This is a schematic diagram showing an outline of a method for forming a two-phase plug. Figure 8 This is a schematic diagram showing an outline of a method of sending liquid to a two-phase plug of a flow cell. Figures 5 to 8 Yes Figure 4 The dashed line DL is shown as an enlarged view of the part. Figures 5 to 8In the figure, the pinch valve with "x" added indicates a closed state, and the pinch valve without "x" added indicates an open state.

[0080] use Figure 5 , Figure 6 , the formation and transfer method of the three-phase plug is explained. Figure 5 As shown, in the fluid system 1, the pinch valve PV1 is switched to an open state, and the cleaning buffer is transferred into the tube CB. In this way, the inside of the tube CB is cleaned (process 1). In the fluid system 1, after the pinch valve PV1 is switched to a closed state and the pinch valve PV3 is switched to an open state, oil is introduced into the tube CB (process 2). In the fluid system 1, after the pinch valve PV3 is switched to a closed state and the pinch valve PV5 is switched to an open state, the reagent (solution) is introduced into the tube CB (process 3). In the fluid system 1, after the pinch valve PV5 is switched to a closed state and the pinch valve PV7 is switched to an open state, air is introduced into the tube CB (process 4). After such processes, Figure 5 As shown in FIG. 1 , a three-phase plug is formed in the pipe CB portion between the pinch valve PV1 and the pinch valve PV9, in which air, reagent, and oil are arranged in the order from the front in the flow direction. The above three-phase plug is just an example, and other fluids may form three phases. In addition, the order in which each fluid is introduced into the three-phase plug is also arbitrary.

[0081] like Figure 6 As shown, in the fluid system 1, from Figure 5 The state of forming a three-phase bolt in step 4 ( Figure 6 Step 1) starts, after the pinch valve PV9 is switched to the open state, the pinch valve PV1 is switched to the open state, the syringe B13 is operated, the three-phase plug is pushed out from the tube CB, and transferred to the flow cell 31 ( Figure 6 Step 2) is performed by starting from the state of forming a three-phase bolt Figure 6 Step 2 can be performed without stopping the flow of liquid Figure 3 The solution exchange of steps 2 to 4 described in the above.

[0082] use Figure 7 , 8 , the formation and transfer method of the two-phase plug is explained. Figure 7 As shown, in fluid system 1, the pinch valve PV1 is switched to an open state, and the cleaning buffer is transferred into the tube CB (process 1). Thus, the inside of the tube CB is cleaned. In fluid system 1, after the pinch valve PV1 is switched to a closed state and the pinch valve PV7 is switched to an open state, air is introduced into the tube CB (process 2). After such a process, Figure 7As shown, in the pipe CB portion between the pinch valve PV1 and the pinch valve PV9, a two-phase plug is formed in which air and a washing buffer are arranged in the order from the front in the flow direction. In addition, the above-mentioned two-phase plug is only an example, and two phases can also be formed by other fluids. In addition, the order of introducing each fluid into the two-phase plug is also arbitrary.

[0083] like Figure 8 As shown, in the fluid system 1, from Figure 7 The state of forming a two-phase plug in step 2 ( Figure 8 Step 1) starts, after the pinch valve PV9 is switched to the open state, the pinch valve PV1 is switched to the open state, the syringe B13 is operated, the two-phase plug is pushed out from the tube CB, and transferred to the flow cell 31 ( Figure 8 Process 2).

[0084] Reference Fig. 9 as well as Fig.10 , an example of a microporous device in a closed control system 100A ( Figure 4 An example of a flow cell 31 is shown in FIG. Fig. 9 This is a diagram showing an example of a cross-sectional view of a closed-type micropore device. Fig.10 This is a diagram showing an example of a top view of a closed-type microwell device. Fig. 9 yes Fig.10 A cross-sectional view of the EE section shown in the top view.

[0085] like Fig. 9 as well as Fig.10 As shown, the microporous device MWD of the present embodiment is constituted by, for example, configuring an upper surface glass UG on a double-sided tape DT adhered to a substrate B. In the case of optical measurement, it is preferred that the substrate B is formed of a transparent member. An inlet IP disposed near one end E1 of the space SP and an outlet OP disposed near the other end E2 of the space SP are formed on the upper surface glass UG. For example, in the case of replacing oil (immiscible liquid) with gas, the gas can be introduced into the space SP from the inlet IP disposed near one end E1 of the space SP, and the oil can be discharged from the outlet OP disposed near the other end E2 of the space SP. The inlet IP can also introduce liquids other than gas, such as oil and reagents, into the space SP. In addition, the outlet OP can also discharge liquids other than oil, such as reagents, and gases from the space SP.

[0086] The micropores M are arranged on the substrate B at a pitch of, for example, 12 μm. The shape of the micropores is arbitrary, and in the case of optical measurement, the bottom surface is preferably flat. The width of the micropore (for example, when the bottom surface of the micropore is circular, it represents its diameter) is not particularly limited, but is preferably 100 μm or less, more preferably 10 μm or less, and further preferably 5.0 μm or less.

[0087] The depth of the micropores is not particularly limited, but is preferably 100 μm or less, more preferably 10 μm or less, and further preferably 5.0 μm or less. The number of micropores M included in the micropore device MWD is arbitrary and is 10 3 More than 10, preferably 10 4 When digital measurement is performed using micropore M, 10 or more is more preferable from the viewpoint of high-sensitivity detection of the test substance. 5 In addition, the thickness of the double-sided tape DT may be any thickness as long as it is a thickness required for connecting the upper glass UG and the substrate B, but is preferably about 100 μm, for example.

[0088] <Solution>

[0089] The "solution" (reagent) used in the present embodiment is not particularly limited as long as it is a solution that is immiscible with oil. As the "solvent" contained in the solution, water, hydrophilic alcohol, hydrophilic ether, ketone, nitrile solvent, carboxylic acid, amine solvent, dimethyl sulfoxide, etc. can be listed. Specifically, as hydrophilic alcohol, methanol, ethanol, propanol, glycerol, etc. can be listed, as hydrophilic ether, tetrahydrofuran, polyethylene oxide, etc. can be listed, as ketone, acetone, methyl ethyl ketone, etc. can be listed, as nitrile solvent, acetonitrile, etc. can be listed, as carboxylic acid, formic acid, acetic acid, butyric acid, propionic acid, etc. can be listed, as amine solvent, N, N-dimethylformamide, 2-aminoethanol, 2,2',2"-nitrilotriethanol, 2,2'-iminodiethanol (2,2'-Iminodiethanol), etc. can be listed. Among them, water is preferred because it does not hinder the chemical reaction of biological molecules in the micropores, cell culture, etc.

[0090] As the "solute" contained in the solution, proteins, nucleic acids, physiologically active substances, inorganic salts, surfactants, fluorescent substances, low molecular weight compounds, buffer substances, etc. can be listed. Specifically, as proteins, BSA, casein, antigens, antibodies, alkaline phosphatase, β-galactosidase, horseradish peroxidase and streptavidin can be listed. As nucleic acids, DNA, RNA and aptamers can be listed. As physiologically active substances, cell growth factors, differentiation inducing factors, cell adhesion factors, cytokines, hormones, sugar chains and lipids can be listed. As inorganic salts, Na3PO4, Na2HPO4, NaH2PO4, NaCl, KCl, MgCl2 and ZnCl2 can be listed.

[0091] As surfactants, Tween (registered trademark) series, Triton (registered trademark) X-100, SDS and Lipidure (registered trademark) series, etc. can be listed. As fluorescent substances, fluorescein, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, phycoerythrin, Cy (registered trademark) 3, Cy (registered trademark) 5, AlexaFluor (registered trademark) series, FDP, FDG, Amplex red (registered trademark) and Amplex Ultrared (registered trademark) can be listed. As low molecular weight compounds, biotin, etc. can be listed.

[0092] In addition, for example, when chemical reactions such as antigen-antibody reaction and enzyme-substrate reaction are performed in microwells, a solution containing antigen, antibody, enzyme or fluorescent substance can be used. In addition, when cells are cultured in microwells, cell culture fluids such as DMEM can be used as solutions.

[0093] <Immiscible liquids>

[0094] The immiscible liquid used in the present embodiment is a liquid that is immiscible with the above-mentioned solution. The immiscible liquid includes oils such as saturated hydrocarbons, unsaturated hydrocarbons, aromatic hydrocarbons, silicone oils, perfluorocarbons, halogenated solvents, etc. The immiscible liquid is preferably volatile oil, for example. Since volatile oil is easily replaced by gas, the oil that seals the micropores can be reliably removed from the substrate, and the solution is filled into the micropores with good accuracy.

[0095] As saturated hydrocarbons, alkanes or cycloalkanes may be used. As alkanes, hexane or decane may be used. As unsaturated hydrocarbons, squalene may be used. As aromatic hydrocarbons, benzene or toluene may be used. As perfluorocarbons, Fluorinert TM FC-72(3M), Fluorinert TM FC-40 (3M) and Fluorinert TM FC-43 (3M), etc. Examples of the halogenated solvent include chloroform, dichloromethane, chlorobenzene, Asahiklin AE-3000 (AGC), Asahiklin AE-3100 (AGC), Asahiklin AC-6000 (AGC), Asahiklin AC-2000 (AGC), Novec TM 7000(3M), Novec TM 7100(3M), Novec TM 7200(3M), Novec TM7300 (3M) and HFE-7500 (3M), etc. Among them, AE-3100 and Novec-7100 are preferred because of their relatively high vapor pressures.

[0096] The volatility of oil can be expressed by vapor pressure. For example, oil having a vapor pressure of 0.15 kPa or more at 25°C is preferred. The vapor pressure of oil refers to the pressure of the gas in the state of gas-liquid equilibrium at the temperature. The vapor pressure at 25°C can be obtained by adding oil to a vacuum container, maintaining the temperature at 25°C, and measuring the pressure of the gas in the state of gas-liquid equilibrium.

[0097] <Second embodiment>

[0098] Reference Figures 11 to 13 , an open control system in the second embodiment is described. Fig.11 It is a diagram showing the overall configuration of an open control system in the second embodiment. Fig.12 It represents an open microporous device ( Fig.11 FIG. 1 is a diagram of an example of a cross-sectional view of an example of a pool 35 shown. Fig.13 FIG. 1 is a diagram showing an example of a top view of an open-type microporous device. An open-type control system is one having Fig.12 as well as Fig.13 The micropore device shown is a control system for a micropore device in which the top of the micropore M is open. In the following, the differences from the first embodiment will be described in particular.

[0099] like Fig.11 As shown, the fluid system 1 in the control system 100B illustratively includes a dispenser 61 , a tip rack 63 , a solution rack 65 , and a tip disposal box 67 .

[0100] The dispenser 61 takes out one or more pipette tips CP (hereinafter, tips CP) for dispensing a solution or the like from the tip rack 63, and moves the one or more tips CP containing the solution or the like to the reservoir 35. Fig.12 As shown, the dispenser 61 may also use one or more suction heads CP containing a solution, etc., for example, to introduce the solution, etc. into the micro-wells M formed in the pool 35 via a space SP within a range not exceeding the thickness of the single-sided tape ST based on the upper surface of the substrate B. The dispenser 61 may also use one or more suction heads CP that do not contain a solution, etc. (empty), to suck and discharge the solution, etc. formed in the micro-wells M and the space SP of the pool 35. The dispenser 61 is mechanically connected to the movable rail 69 and can move on the movable rail 69.

[0101] The solution rack 65 is a rack for managing one or more solutions. Solutions (reagents) introduced into the reservoir 35, containers containing oil, etc. are placed on the solution rack 65. For example, the dispenser 61 having an empty pipette tip CP sucks the solution or oil from the container placed on the solution rack 65. The pipette tip disposal box 67 is a box for discarding used pipette tips CP.

[0102] like Fig.12 as well as Fig.13 As shown, the micropore device MWD of the present embodiment is formed by, for example, pasting a single-sided tape ST on a substrate B. The micropores M are arranged on the substrate B, for example, at a pitch of 7.0 μm. The shape of the micropores is arbitrary, but in the case of optical measurement, the bottom surface is preferably flat. The width of the micropore (for example, when the bottom surface of the micropore is circular, it represents its diameter.) is not particularly limited, but is preferably 100 μm or less, more preferably 10 μm or less, and further preferably 5.0 μm or less.

[0103] The depth of the micropores is not particularly limited, but is preferably 100 μm or less, more preferably 10 μm or less, and further preferably 5.0 μm or less. The number of micropores M included in the solid line DL of the micropore device MWD is arbitrary and is 10 3 More than 10, preferably 10 4 When digital detection is performed using micropores M, 10 or more is more preferred from the viewpoint of high-sensitivity detection of the substance to be detected. 5 The thickness of the single-sided tape ST is arbitrary, but is preferably about 5 mm, for example.

[0104] <Method for manufacturing a closed microporous device>

[0105] Fig.14 1 is a schematic diagram showing an overview of a first example of a method for producing a closed microporous device. The first example of a method for producing a microporous device is an example in which an antibody or enzyme is not immobilized in the micropore M. Fig.14 As shown, a double-sided tape DT with a predetermined size for demoulding is pasted on the substrate B (process 1). Next, the upper surface glass UG is pressed against the double-sided tape DT for pasting to produce a microporous device (process 2). Afterwards, a washing buffer solution, for example, can be injected into the produced microporous device.

[0106] Fig.15 2 is a schematic diagram showing the outline of a second example of the method for producing a closed micropore device. In the second example of the method for producing a closed micropore device, an antibody or an enzyme is immobilized in the micropore M.

[0107] In the following, an example of modifying micropores M with a biotin-bound PEG linker is described with reference to steps (1) to (10) (process 1). (1) The substrate B formed with the micropores M is placed in an ozone irradiation device, for example, and irradiated with ozone for 20 seconds. (2) The substrate B after (1) is immersed in, for example, ethanol, and ultrasonically cleaned for 5 minutes. (3) In a glass cup, for example, (3-Mercaptopropyl)-trimethoxysilane is added dropwise to 150 mL of a reaction solution (2.5% acetic acid / 90% ethanol) to a final concentration of 2 w / v%, and stirred at 650 rpm for 5 minutes. (4) The substrate B after (2) is immersed in the glass cup of (3), and allowed to stand at room temperature for, for example, more than 12 hours. (5) The substrate B after (4) is washed with ultrapure water and dried with a blower. (6) Place the substrate B after (5) in a constant temperature dryer (for example, set temperature: 90°C) and let it stand for 30 minutes. (7) Take out the substrate B after (6) from the constant temperature dryer and return it to room temperature. (8) Prepare 1mM biotin-conjugated PEG reagent / 25mM MES solution. (9) Add, for example, 400μL of the biotin-conjugated PEG solution of (8) to the substrate B after (7), degas with a degasser for 5 minutes, and let it stand at room temperature for 1 hour. (10) Rinse the substrate B after (9) with ultrapure water and dry it with a blower.

[0108] A double-sided tape DT with a predetermined size for demolding is pasted on the substrate B after step 1 (step 2). Next, the upper surface glass UG is pressed against the double-sided tape DT for pasting to produce a microporous device (step 3). After that, for example, a solution containing avidin, target biotinylated antibody, target enzyme, etc. is injected into the microporous device (step 4). The biotinylated antibody, for example, contains at least one of an anti-PSA monoclonal antibody and an anti-Tau monoclonal antibody.

[0109] <Overview of each embodiment>

[0110] The following is a description of each embodiment. The outline of each embodiment is summarized as follows.

[0111]

[0112] <Micropore devices of the first, second, fourth and fifth embodiments>

[0113] Here, the method for preparing the closed microporous device used in the first, second, fourth and fifth embodiments is specifically described. In this preparation method, a PSA antibody is bound to a biotin-binding PEG linker that modifies the micropores of the microporous device.

[0114] (1) First, prepare to implement Fig.15The microporous device after step 3, i.e., the microporous device modified with a biotin-binding PEG linker. (2) Prepare 0.001 mg / mL NeutrAvidin / Tris solution. The composition of the Tris solution includes Tris-HCl: 0.1 M, NaCl: 150 mM, MgCl2: 1 mM, ZnCl2: 0.1 mM, goat IgG: 1 mg / mL, mouse IgG: 1 mg / mL, BSA: 1% (w / v), casein: 0.5% (w / v), blocking reagent: 30 μg / mL and NaN3: 0.1% (w / v).

[0115] (3) Add 1 μL of 1 mg / mL biotinylated antibody solution to 510 μL of neutravidin / Tris solution (2), and stir at room temperature for 10 minutes. Here, the antibody includes anti-PSA monoclonal antibody (Product No. A45170) manufactured by Biospecix. Biotinylation was performed using Biotin Labeling Kit SH (Product No. LK10) manufactured by DOJINDO.

[0116] (4) Inject 200 μL of HISCL washing solution (Sysmex) into the microporous device of (1). (5) Inject 100 μL of the solution of (3) into the microporous device after (4) and let it stand at room temperature for 30 minutes. (6) Inject 200 μL of HISCL washing solution into the microporous device after (5).

[0117] <Micropore device of the seventh embodiment>

[0118] Next, the method for preparing the closed microporous device used in Example 7 is specifically described. In this preparation method, a Tau antibody is bound to a biotin-binding PEG linker that modifies the microporous device.

[0119] (1) First, prepare to implement Fig.15 The microporous device after step 3, that is, the microporous device modified with a biotin-bound PEG linker. (2) Add 0.001 mg / mL of neutravidin (31000, ThermoFisher) to the Tris solution to a final concentration of 0.001 mg / mL. (3) Add the biotinylated anti-Tau antibody solution to the neutravidin / Tris solution of (2) to a final concentration of 0.002 mg / mL, and stir at room temperature for 10 minutes. Here, the antibody includes an anti-Tau monoclonal antibody manufactured by BioLegend (Product No. 806501). Biotinylation is performed using the Biotin Labeling Kit SH (Product No. LK10) manufactured by DOJINDO.

[0120] (4) Inject 200 μL of HISCL washing solution (Sysmex) into the microporous device after (1). (5) Inject 100 μL of the solution in (3) into the microporous device after (4) and let it stand at room temperature for 30 minutes. (6) Inject 500 μL of HISCL washing solution into the microporous device after (5).

[0121] <Micropore device of the sixth embodiment>

[0122] Next, the method for preparing the closed microporous device used in Example 6 is specifically described. In this preparation method, an enzyme is bound to the biotin-binding PEG linker that modifies the microporous device.

[0123] (1) First, prepare to implement Fig.15 The microporous device after step 3, i.e., the microporous device modified with a biotin-bound PEG linker. (2) Prepare a 12.0 pM streptavidin-labeled β-Gal (S3887-.5MG, Sigma Aldrich) / PBS solution. (3) Prepare an ALP reaction solution (pH = 9.25) using the following composition. The composition includes 2,2'-iminodiethanol (11920-65, Nacalai Tesque): 1.0 M, MgCl2 (133-00161, Wako): 1.0 mM, and Tween 20 (T2530, Tokyo Chemical Industry): 0.10% (v / v).

[0124] (4) Prepare 2.8 pM streptavidin-labeled ALP (11089161001, Roche) / ALP reaction solution. (5) Add 100 μL of the streptavidin-labeled ALP / ALP reaction solution of (4) to 100 μL of the streptavidin-labeled β-Gal / PBS solution of (2). (6) Inject 200 μL of HISCL washing solution (HISCL Washing Solution, Sysmex) into the microporous device after (1). (7) Inject 150 μL of the mixed solution of (5) into the microporous device after (6), and immediately inject 400 μL of HISCL washing solution.

[0125] Here, before describing each example in detail, reagents and samples used in each example and an imaging method of a fluorescence microscope used in an experiment and the like will be described.

[0126] <Reagents>

[0127] The reagents include 1. enzyme solution, 2. antibody solution, 3. enzyme substrate solution, 4. fluorescent pigment solution and 5. cleaning solution.

[0128] 1. The enzyme solution includes an ALP solution. (1) An ALP reaction solution (pH = 9.25) was prepared using the following composition. The composition includes 2,2'-iminodiethanol (11920-65, Nacalai Tesque): 1.0 M, MgCl2 (133-00161, Wako): 1.0 mM, and Tween 20 (T2530, Tokyo Chemical Industry): 0.10% (v / v). (2) A 2.8 pM ALP solution was prepared using the ALP reaction solution of (1) and streptavidin-labeled ALP (11089161001, Roche).

[0129] 2. The antibody solution includes an ALP-labeled anti-Tau antibody solution. The ALP-labeled anti-Tau antibody solution is used in the seventh embodiment. Prepare an anti-Tau antibody / Tris solution labeled with 0.25 nM ALP. The antibody includes an anti-Tau monoclonal antibody (product No. 806501) manufactured by BioLegend. Biotinylation uses the ALP labeling kit SH (product No. LK13) manufactured by DOJINDO.

[0130] 3. The enzyme substrate solution includes the (1) ALP fluorescent substrate solution used in the sixth embodiment, the (2) ALP fluorescent substrate solution (containing pigment) used in the seventh embodiment, the (3) β-Gal fluorescent substrate solution used in the sixth embodiment, and the (4) β-Gal fluorescent substrate / extraction solution (containing pigment) used in the seventh embodiment.

[0131] (1) ALP fluorescent substrate solution was prepared as 2.0 mM FDP / ALP reaction solution using the ALP reaction solution (pH = 9.25) described in "1. Enzyme solution" and 50 mM FDP (11600, AAT Bioquest) aqueous solution. (2) ALP fluorescent substrate solution (containing pigment) was prepared as 1.0 mM FDP (11600, AAT Bioquest) / 0.02 mM A647 / ALP reaction solution using the ALP reaction solution (pH = 9.25) described in "1. Enzyme solution", FDP and A647. (3) β-Gal fluorescent substrate solution was prepared as 250 μM FDG (F2756-5MG, Sigma Aldrich) / PBS solution.

[0132] (4) β-Gal fluorescent matrix / extraction solution (containing pigment) (i) First, prepare 2% (v / v) Triton X-100 (A16046, Alfa Aesar) / specimen diluent (CS617657, Sysmex). (ii) Prepare 2% (w / v) BSA / specimen diluent. (iii) Mix the solutions prepared in (i) and (ii) in equal amounts to prepare an extraction solution. (iv) Prepare 0.25mM FDG (F2756-5MG, Sigma Aldrich) / 0.02mM A647 (A20347, ThermoFisher) / extraction solution.

[0133] 4. The fluorescent pigment solution includes (1) fluorescein / PBS solution used in the first, second and third embodiments, (2) fluorescein / HISCL cleaning solution used in the fifth embodiment, (3) A488 / extraction solution used in the fourth embodiment, (4) A647 / extraction solution used in the fourth and fifth embodiments, and (5) A647 / ALP reaction solution used in the fifth embodiment.

[0134] (1) Fluorescein / PBS solution was prepared by adding PBS (P4417-100TAB, Sigma Aldrich) aqueous solution to a 20 mM fluorescein (F0096, Tokyo Chemical Industry) aqueous solution to a final concentration of 0.20 mM. (2) Fluorescein / HISCL cleaning solution was prepared as follows. (i) A 200 mM fluorescein (F0096, Tokyo Chemical Industry) aqueous solution was prepared. (ii) The fluorescein aqueous solution prepared in (i) was diluted with HISCL cleaning solution to a final concentration of 4 mM.

[0135] (3) Prepare A488 / extraction solution as follows. (i) Prepare 2% (v / v) Triton X-100 (A16046, AlfaAesar) / specimen diluent (CS617657, Sysmex). (ii) Prepare 2% (w / v) BSA / specimen diluent. (iii) Mix equal amounts of the solution prepared in (i) and the solution prepared in (ii) to prepare an extraction solution. (iv) Prepare 10 μM A488 (A10254, ThermoFisher) / extraction solution.

[0136] (4) A647 / extract solution was prepared as follows. (3) Using the extract solution prepared in (iii), 10 μM A647 (A20347, ThermoFisher) / extract solution (the extract solution used in the fourth example) was prepared. In addition, (3) Using the extract solution prepared in (iii), 0.3 mM A647 (A20347, ThermoFisher) / extract solution (the extract solution used in the fifth example) was prepared. (5) A647 / ALP reaction solution was prepared as a 0.3 mM A647 / ALP reaction solution using the aforementioned ALP reaction solution.

[0137] 5. Washing solution used in Example 1 and Comparative Example 1. A washing solution (pH = 7.9) of the following composition was prepared: Tris-HCl (314-90065, NIPPON GENE): 20 mM, Tween 20 (T2530, Tokyo Chemical Industry): 0.1% and NaCl (191-01665, Wako): 50 mM.

[0138] <Sample>

[0139] The immune complex solution containing exosomes, magnetic particles and β-Gal used in the seventh embodiment is described. The immune complex solution is prepared using the following preparation method. (1) Magnetic particles labeled with anti-CD9 antibodies are dispensed into a tube at a rate of approximately 10 million per sample and washed with HISCL washing solution. After washing, all the HISCL washing solution is discarded. The antibodies include anti-CD9 monoclonal antibodies manufactured by BioLegend (Product No. 312102). Magnetic particles include those manufactured by Thermo Fisher Scientific (Product No. 14311D). (2) 80 μL of Tris solution is added to the tube of (1). (3) Using PBS (Sigma Aldrich, P4417-100TAB), a dilution series of the exosome solution is prepared (the amount of exosomes per sample: 0.128 μg, 3.2 μg, 16 μg, 80 μg). Exosomes were recovered from the supernatant of HCT116 cell line culture cells by size exclusion chromatography.

[0140] Next, (4) add 20 μL of each exosome solution of (3) to the solution of (2). In the case of exosome-negative samples, add 20 μL of Tris solution. (5) Shake the sample of (4) on a shaker for 1 hour (1200 rpm, room temperature). (6) Wash the sample of (5) twice with 200 μL of HISCL cleaning solution. After washing, discard all the HISCL cleaning solution. (7) Prepare 0.001 mg / mL biotinylated anti-CD63 antibody / Tris solution. The antibody includes anti-CD63 monoclonal antibody (Product No. 353014) manufactured by BioLegend. Biotinylation is performed using the Biotin Labeling Kit SH (Product No. LK10) manufactured by DOJINDO. (8) Add 100 μL of the solution of (7) to the tube of (6). (9) Shake the sample of (8) on a shaker for 30 minutes (1200 rpm, room temperature).

[0141] Next, (10) wash the sample of (9) twice with 200 μL of HISCL cleaning solution. After washing, discard all the HISCL cleaning solution. (11) prepare a 1.2 pM streptavidin-labeled β-Gal (Sigma, No.: S3887-5MG) / Tris solution. Filter the prepared solution using a 0.22 μm filter. (12) add 100 μL of the solution of (11) to the tube of (10). (13) shake the sample of (12) on a shaker for 30 minutes (1200 rpm, room temperature). (14) wash the sample of (13) three times with 500 μL of HISCL cleaning solution. After washing, discard all the HISCL cleaning solution. (15) add 20 μL of Tris solution to the tube of (14).

[0142] The average number λ of exosomes per magnetic particle is given by the following formula 1.

[0143] (Formula 1)

[0144] λ=N exo / N bead

[0145] (N exo : The number of exosomes in the sample, and N bead : Number of magnetic particles in the sample (= 10 million)

[0146] In addition, N is calculated by formula 2 exo .

[0147] (Formula 2)

[0148] N exo =m exo ×n exo

[0149] (mexo : The quality of exosomes, and n exo : Number of exosomes per 1 mg of exosomes (=1.4×10 8 pieces / mg))

[0150] <Photography method>

[0151] In the photographing method based on fluorescence microscopy of each embodiment, the following fluorescence microscope and photographing conditions are used. The fluorescence microscope includes a microscope: IX-83, Olympus, a camera: Zyla 4.2sCMOS, ANDOR, an objective lens: UCPLFLN20X, Olympus, a light source: X-Cite TURBO, EXCELITAS Technologies, and a fluorescence mirror unit: U-FBNA, Olympus. The photographing conditions include the excitation light intensity: about 0.08 mW, the exposure time: 100 ms, and the number of fields of view: 60 fields of view (the size of one field of view: 685.3 mm×685.3 mm).

[0152] The following describes each embodiment in detail. First, the content of the first embodiment is described.

[0153] <First embodiment>

[0154] Reference Figures 16 to 18 , the first embodiment and the comparative example are described. Regarding the removal of the sealing layer (oil layer), in the first embodiment, the cleaning solution is injected after the air replacement process, and in Comparative Example 1, the cleaning solution is injected without the air replacement process. In the first embodiment and the comparative example, a closed micropore device with an anti-PSA monoclonal antibody fixed thereon is used. In addition, assuming an antigen-antibody reaction in the micropores, the antibody-immobilized micropores are used, but the antigen is not included in the reagents. In addition, in the first embodiment and the comparative example, fluorescein / PBS solution, a cleaning solution, and a volatile oil (FC-43) are used as reagents. The vapor pressure of FC-43 at 25°C is 0.17 kPa, and the boiling point is 174°C.

[0155] Fig.16 Schematic diagram showing the process of the first embodiment. First, as a prerequisite for process 1, 200 μL of fluorescein / PBS solution is introduced into each micropore M as solution SA. Next, 200 μL of oil VO (FC-43) is introduced into the space SP as a sealing layer, and then photographed using a microscope 33 to obtain a fluorescent image A (process 1). 50 mL of air A is introduced into the space SP. As a result, the oil VO in the space SP is replaced by air A (process 2). After the oil VO in the space SP is replaced by air A, a fluorescent image B is obtained by photographing the micropore device using a microscope 33 (process 3).

[0156] Next, a cleaning liquid WL (HISCL cleaning liquid 600 μL) is introduced into the space SP where the oil VO is replaced by the air A. Thus, the solution SA contained in each micropore M is replaced with the cleaning liquid WL. (Step 4). After the space SP and each micropore M are filled with the cleaning liquid WL, a fluorescent image C is acquired by photographing the micropore device using a microscope 33 (Step 5).

[0157] Fig.17 Schematic diagram showing the process of the comparative example of the first embodiment. The comparative example does not have the step of introducing 50 mL of air A in the first embodiment ( Fig.16 Step 2) and the subsequent step of obtaining the fluorescent image B ( Fig.16 Except for step 3), the above steps are the same as those of the first embodiment. Fig.17 As shown, first, as a prerequisite for step 1, 200 μL of fluorescein / PBS solution is introduced as solution SA into each microwell M. Next, 200 μL of oil VO (FC-43) is introduced into the space SP as a sealing layer, and the microwell device is photographed using a microscope 33 to obtain a fluorescent image A' (step 1). Next, a cleaning liquid WL (600 μL of HISCL cleaning liquid) is introduced into the space SP, and the oil VO is pushed out of the space SP (step 2). After the cleaning liquid WL is filled in the space SP, the microwell device is photographed using a microscope 33 to obtain a fluorescent image C' (step 3).

[0158] Fig.18 1 is a diagram showing the results of the first embodiment and the comparative example. A bright spot in each fluorescent image corresponds to a micropore M. When a bright spot exists, it means that a solution SA exists in the micropore M. Fig.18 As shown, in Example 1, according to the fluorescent image C, after the cleaning liquid is added, the bright spots from the micropores M all disappear. That is, in the first embodiment, it is shown that the solution SA in each micropore M is replaced by the cleaning liquid WL. That is, it is shown that the number of micropores M whose solutions are not exchanged is suppressed, and high-precision solution exchange can be performed. This is because, in the case of executing the process of introducing air A into the space SP where the oil VO exists, even if a part of the oil VO is not pushed out by the air A and remains in the space SP, the remaining oil VO is vaporized and mixed in the air A, and it will not hinder the exchange from the solution SA to the cleaning liquid WL in each micropore M. On the other hand, in Comparative Example 1, according to the fluorescent image C' after the cleaning liquid is added, most of the bright spots do not disappear. That is, in Comparative Example 1, it is shown that due to the lack of the process of introducing 50mL of air ( Fig.16Since the oil VO sealing the micropores M is not completely removed by the cleaning liquid WL, the solution remains in some of the micropores M. This is because, when the process of introducing the air A into the space SP where the oil VO exists is not performed, a part of the oil VO is not pushed out by the air A and remains in the space SP, and the remaining oil VO remains in the space SP as a liquid, thereby hindering the exchange of the solution SA to the cleaning liquid WL in each micropore M.

[0159] <Second embodiment>

[0160] Reference Fig.19 as well as Fig. 20 , the second embodiment is described. In the second embodiment, a closed micropore device with an anti-PSA monoclonal antibody fixed thereto is used. In addition, assuming an antigen-antibody reaction within the micropores, an antibody-immobilized micropore is used, but the antigen is not included in the reagents. In addition, in the second embodiment, fluorescein / PBS solution, a cleaning solution, and volatile oils (AE-3000 and FC-43) are used as reagents. For AE-3000, the vapor pressure at 25°C is 31 kPa, the evaporation rate (ratio) is 67 when diethyl ether is set to 100, and the boiling point is 56°C. The vapor pressure of FC-43 at 25°C is 0.17 kPa, and the boiling point is 174°C.

[0161] Fig.19 : is a schematic diagram showing the process of the second embodiment. In the second embodiment, when AE-3000 is used as the volatile oil and when FC-43 is used, the following steps are implemented. First, as a prerequisite for step 1, after 100 μL of HISCL cleaning solution is introduced into the space SP, 200 μL of fluorescein / PBS solution is introduced into the space SP and each micropore M as a solution SA (step 1). Next, 200 μL of oil VO (AE-3000) is introduced into the space SP as a sealing layer (step 2). Thus, the solution SA in the micropore M is sealed with the oil VO. After the solution SA is sealed with the oil VO, a fluorescent image A is obtained by photographing the micropore device using a microscope 33 (step 3). 50 mL of air A is introduced into the space SP. Thus, the oil VO in the space SP is replaced by air A (step 4).

[0162] After the oil VO in the space SP is replaced by the air A, the microporous device is photographed by the microscope 33 to obtain a fluorescent image B (step 5). The cleaning liquid WL (HISCL cleaning liquid 600μL) is introduced into the space SP where the oil VO is replaced by the air A. As a result, the solution SA contained in each micropore M is replaced with the cleaning liquid WL. (step 6). After the space SP and each micropore M are filled with the cleaning liquid WL, the microporous device is photographed by the microscope 33 to obtain a fluorescent image C (step 7). In addition, as described above, FC-43 is used as the oil VO to implement the above steps 1 to 7.

[0163] Fig. 20 It is a figure which shows the experimental result of the second example. Fig. 20 The images A, B, and C shown correspond to the fluorescent image A acquired in step 3, the fluorescent image B acquired in step 5, and the fluorescent image C acquired in step 7 when AE-3000 is used as the volatile oil. Fig. 20 The images A', B', and C' shown correspond to the fluorescent image A acquired in step 3, the fluorescent image B acquired in step 5, and the fluorescent image C acquired in step 7 when FC-43 is used as the volatile oil. Fig. 20 As shown in the fluorescent images C and C' of FIG, the bright spots from the micropores M all disappeared by adding the air A and the cleaning liquid WL. This indicates that the solution SA in each micropore M is replaced by the cleaning liquid WL, and it can be seen that as the oil VO, either AE-3000 having a vapor pressure of 31 kPa at 25°C or FC-43 having a vapor pressure of 0.17 kPa at 25°C can be used. In other words, it can be seen that as the oil VO, an oil having a vapor pressure of 0.15 kPa or more at 25°C can be used.

[0164] <Third embodiment>

[0165] Reference Fig.21 and Fig. 22 , the third embodiment is described. In the third embodiment, unlike other embodiments based on the closed control system 100A, an open control system 100B is implemented. In the third embodiment, as a microporous device, a Fig.12 , Fig.13 The open-type microwell device MWD described above uses fluorescein / PBS solution, cleaning solution, and volatile oil (Novec-7100) as reagents. Novec-7100 has a vapor pressure of 28 kPa at 25°C and a boiling point of 61°C.

[0166] Fig.21: is a schematic diagram showing the process of the third embodiment. First, as a prerequisite for process 1, 150 μL of HISCL cleaning solution is introduced into the space SP within the range not exceeding the thickness of the single-sided tape ST based on the upper surface of the substrate B using the pipette tip CP. Thereafter, 150 μL of HISCL cleaning solution is removed. After 100 μL of fluorescein / PBS solution is introduced into the space SP as the solution SA using the pipette tip CP, 80 μL of the fluorescein / PBS solution is removed from the space SP. Thus, the fluorescein / PBS solution is contained in each microwell M (process 1). Next, 130 μL of oil VO (Novec-7100) is introduced into the space SP using the pipette tip CP, and the solution SA in the microwell M is sealed with the oil VO (process 2). After the solution SA in the microwell M is sealed with the oil VO, a fluorescent image A is acquired by photographing the microwell device using a microscope 33 (process 3).

[0167] The oil VO is sucked and removed from the space SP by the suction head CP. As a result, the oil VO in the space SP is replaced by the air A (step 4). After the oil VO in the space SP is replaced by the air A, the microporous device is photographed by the microscope 33 to obtain a fluorescent image B (step 5). Thereafter, the cleaning liquid WL is introduced from the space SP by the suction head CP, and the solution SA in each micropore M is replaced by the cleaning liquid WL (step 6). After the above-mentioned solution exchange in the micropore is performed, the microporous device is photographed by the microscope 33 to obtain a fluorescent image C (step 7).

[0168] Fig. 22 is a graph showing the experimental results of the third embodiment. Fig. 22 As shown in the fluorescence image C, by removing the oil VO (Novec-7100) and adding the cleaning liquid WL, all the bright spots from the micropores M disappeared. That is, even when using an open micropore device (refer to Fig.12 and Fig.13 ), and also enables high-precision solution exchange.

[0169] <Fourth embodiment>

[0170] Reference Figure 23 to Figure 26 , the fourth embodiment is described. In the fourth embodiment, Figure 4 In the closed control system 100A shown, liquid can be automatically delivered to the closed microporous device. In the fourth embodiment, the solution exchange (exchange between the extracting solutions) of A488 / extracting solution (first solution) and A647 / extracting solution (second solution) is implemented. Figure 4In the closed control system 100A shown, the reagent A management unit 16A stores A488 / extracting solution, the reagent B management unit 16B stores A647 / extracting solution, and the HISCL cleaning solution is stored in the water storage tank 14. In the fourth embodiment, a closed microporous device with anti-PSA monoclonal antibody fixed thereto is used. In addition, assuming an antigen-antibody reaction in the microporous, an antibody-immobilized microporous device is used, but the antigen is not included in the reagents. In addition, in the fourth embodiment, fluorescein / PBS solution, cleaning solution, and volatile oil (AE-3000) are used as reagents.

[0171] Fig.23 : is a schematic diagram showing the process of the fourth embodiment. First, a solution SA is contained in each micropore M, and a micropore device in a state where the solution SA is sealed with oil VO is prepared, and a fluorescent image A is acquired by photographing the micropore device with a microscope 33 (process 1). Next, a three-phase plug (starting from the front of the flow direction, air A, A647 / extracting solution (solution SB) and oil VO) is formed in the tube CB, and it is transferred to the micropore device (flow cell 31). Thus, the oil VO in the space SP is replaced by the air A, and the solution SB is introduced into the space SP after the oil VO is replaced by the air A, so that the solution SA is exchanged with the solution SB in the micropore M, and the solution SB is sealed with oil VO (process 2). After the solution SB is contained in the micropore M and sealed with oil VO, a fluorescent image B is acquired by photographing the micropore device with a microscope 33 (process 3).

[0172] A three-phase plug (air, A488 / extract (solution SA), and oil VO, starting from the front in the flow direction) is formed in the tube CB and transferred to the micropore device (flow cell 31). Thus, the oil VO in the space SP is replaced by air A, and the solution SA is introduced into the space SP after the oil VO is replaced by air A, so that the solution SB is replaced by the solution SA in the micropore M, and the solution SA is sealed by the oil VO (step 4). Then, returning to step 1, after the solution SB is replaced by the solution SA in the micropore M and is sealed by the oil VO, the micropore device is photographed with a microscope 33 to obtain a fluorescent image C. Such a cycle of steps 1 to 4 is repeatedly performed.

[0173] Reference Figure 24 to Figure 26 , the experimental results of the fourth embodiment are described. Fig.24 A diagram showing a fluorescent image acquired by a microscope. Fig.24 A488ch is an image captured by filtering the wavelength of fluorescence emitted from the solution SA, and each bright spot corresponds to the microwell M containing the solution SA. Fig.24A647ch is an image captured by filtering the wavelength of fluorescence emitted from solution SB, and each bright spot corresponds to the microwell M containing solution SB. In addition, fluorescent images A, C, E, G, and I are images captured in process 1, and fluorescent images B, D, F, H, and J are images captured in process 4. As shown in fluorescent images A, C, E, G, and I, it can be seen that in process 1, solution SA is contained in each microwell M, and solution SB is not contained. As shown in fluorescent images B, D, F, H, and J, it can be seen that in process 4, solution SB is contained in each microwell M, and solution SA is not contained. Fig.25 This is a graph showing the number of micropores M where bright spots were detected for the fluorescence image of A488ch and the fluorescence image of A647ch, respectively. The vertical axis shows the number of micropores M where bright spots were detected, and the 1st, 3rd, 5th, 7th, and 9th rounds on the horizontal axis correspond to the fluorescence images taken in process 1, and the 2nd, 4th, 6th, 8th, and 10th rounds correspond to the fluorescence images taken in process 4. As shown in the graph, in the 1st, 3rd, 5th, 7th, and 9th rounds, a plurality of bright spots based on the fluorescence emitted from the solution SA were detected, while on the other hand, no bright spots based on the fluorescence emitted from the solution SB were detected. In addition, in the 2nd, 4th, 6th, 8th, and 10th rounds, a plurality of bright spots based on the fluorescence emitted from the solution SB were detected, while on the other hand, no bright spots based on the fluorescence emitted from the solution SA were detected. Based on this, it can be seen that in process 1, each micropore M contains solution SA and does not contain solution SB, and in process 4, each micropore M contains solution SB and does not contain solution SA. Fig.26 : is a graph showing the fluorescence intensity in the fluorescence image of A488ch and the fluorescence image of A647ch. Fig.26 The graph of the enlarged region of fluorescence intensity 0-300 is also shown in the graph. It can be seen from these graphs that in rounds 1, 3, 5, 7, and 9, the fluorescence intensity emitted from solution SA is strong, and the fluorescence intensity emitted from solution SB is close to 0. In addition, in rounds 2, 4, 6, 8, and 10, the fluorescence intensity emitted from solution SB is strong, and the fluorescence intensity emitted from solution SA is close to 0. That is, it can be seen from the graph that in process 1, each microwell M contains solution SA and does not contain solution SB, and in process 4, each microwell M contains solution SB and does not contain solution SA. As shown in FIG. Figure 24 to Figure 26 As shown, it is shown that the solution SA (A488 / extract solution) and the solution SB (A647 / extract solution) can be continuously exchanged using the three-phase plug formed in the tube CB.

[0174] <Fifth embodiment>

[0175] Reference Figures 27 to 31, the fifth embodiment is described. In the fifth embodiment, the solution exchange using different types of solutions is verified. Specifically, in the fifth embodiment, the solution exchange using a surfactant solution and an organic solvent is implemented. In the fifth embodiment, the exchange of different types of solutions (first experiment) and the verification of the carryover during the solution exchange (second experiment) are implemented.

[0176] In the first experiment, a closed microwell device with anti-PSA monoclonal antibody immobilized was used. In addition, the antibody-immobilized microwell was used, assuming an antigen-antibody reaction within the microwell, but the antigen was not included in the reagents. Figure 4 In the closed control system 100A shown, the reagent A management unit 16A stores A647 / ALP reaction solution, the reagent C management unit 16C stores A647 / extraction solution, and the water tank 14 stores fluorescein / HISCL cleaning solution. AE-3000 was used as the volatile oil.

[0177] Fig. 27 3 is a schematic diagram showing the process of the fifth embodiment. After the fluorescein / HISCL cleaning solution WL is introduced into the space SP and the space SP and each microwell M are filled with the cleaning solution WL, the microwell device is photographed using a microscope 33 to obtain a fluorescent image A (process 1). Figure 5 and Figure 6 As shown, a three-phase plug (starting from the front of the flow direction, air A, A647 / extracting liquid (solution SA) and oil VO) is formed in the tube CB and transferred to the micropore device (flow cell 31). Thus, the cleaning liquid WL in the space SP is replaced by air A, and the solution SA is introduced into the space SP after the cleaning liquid WL is replaced by air A, so that the cleaning liquid WL is replaced by the solution SA in the micropore M, and the solution SA is sealed by the oil VO (process 2). After the solution SA is contained in the micropore M and sealed by the oil VO, the micropore device is photographed by a microscope 33 to obtain a fluorescent image B (process 3).

[0178] Next, if Figure 7 and Figure 8As shown, a two-phase plug (starting from the front of the flow direction, air A, fluorescein / HISCL cleaning solution WL) is formed in the tube CB, and the two-phase plug is transferred to the microporous device (flow cell 31) and introduced into the space SP. Thus, the oil VO in the space SP is replaced by air A, and the cleaning solution WL is introduced into the space SP after the oil VO is replaced by air A, so that the solution SA is replaced by the cleaning solution WL in each micropore M (process 4). After the space SP is filled with the cleaning solution WL and the cleaning solution WL is also contained in the micropore M, the micropore device is photographed using a microscope 33 to obtain a fluorescent image C (process 5). A three-phase plug (starting from the front of the flow direction, air A, A647 / ALP reaction solution (solution SB) and oil VO) is formed in the tube CB, and it is transferred to the micropore device (flow cell 31). Thus, the cleaning solution WL in the space SP is replaced by air A, and the solution SB is introduced into the space SP after the cleaning solution WL is replaced by air A, so that the cleaning solution WL is replaced by the solution SB in the micropore M, and the solution SB is sealed by the oil VO (process 6). After the solution SB is contained in the microwell M and sealed with the oil VO, the microwell device is photographed using the microscope 33 to obtain a fluorescent image D (step 7).

[0179] Reference Fig.28 and Fig.29 , the first experimental results of the fifth embodiment are described. Fig.28 FIG. 1 is a diagram showing an example of fluorescent images A to D. Fig.28 As shown, for images A and C, there are no bright spots in the fluorescence image of A647ch, and the fluorescence intensity is high as a whole in the fluorescence image of FLch. This means that in steps 1 and 5 of taking images A and C, there is no solution SA and solution SB in each microwell M, and it is filled with the cleaning liquid WL. For images B and D, there are bright spots corresponding to each microwell in the fluorescence image of A647ch, and no fluorescence is detected as a whole in the fluorescence image of FLch. This means that in steps 3 and 7 of taking images B and D, each microwell M is filled with solution SA or solution SB, and there is no cleaning liquid WL. Fig.29 : is a graph showing the fluorescence intensity in the fluorescence image of A647ch and the fluorescence image of FLch. Fig.29 As shown, in steps 1 and 5 corresponding to the 1st and 3rd rounds, the fluorescence intensity from solution SA and solution SB is almost 0, and the fluorescence intensity from the cleaning liquid WL is high. In addition, in steps 3 and 7 corresponding to the 2nd and 4th rounds, the fluorescence intensity from solution SA or solution SB is high, and the fluorescence intensity from the cleaning liquid WL is almost 0. It can be seen from this that in steps 1 and 5, there is no solution SA and solution SB in each microwell M, which is filled with the cleaning liquid WL, and in steps 3 and 7, each microwell M is filled with solution SA or solution SB, and there is no cleaning liquid WL. Fig.28 and Fig.29As shown, even solutions with different compositions such as A647 / extraction solution (solution SA) and A647 / reaction solution (solution SB) can be exchanged with high precision.

[0180] In the second experiment, a closed microwell device with anti-PSA monoclonal antibody fixed was used. In addition, the antibody-immobilized microwell was used assuming an antigen-antibody reaction in the microwell, but the reagents did not contain antigens. As reagents, the following mixed solution was prepared assuming the carryover when each solution was exchanged in Experiment 1. In addition, AE-3000 was used as the volatile oil.

[0181] (1) A solution prepared by mixing fluorescein / HISCL cleaning solution with A647 / extraction solution at a ratio of 10% (v / v), 1% (v / v), or 0% (v / v)

[0182] (2) A solution prepared by mixing A647 / extracting solution with fluorescein / HISCL cleaning solution at a ratio of 10% (v / v), 1% (v / v), or 0% (v / v)

[0183] (3) A solution prepared by mixing the fluorescein / HISCL cleaning solution with the A647 / ALP reaction solution at a ratio of 10% (v / v), 1% (v / v), or 0% (v / v)

[0184] The fluorescence intensity of the 10% (v / v), 1% (v / v), and 0% (v / v) solutions prepared in (1) was measured. Fig.30 As shown in (a), a regression line is prepared to show the relationship between fluorescence intensity and impurity concentration. Similarly, the fluorescence intensity of each solution of 10% (v / v), 1% (v / v), and 0% (v / v) prepared in (2) is measured. Fig.30 As shown in (b), a regression line showing the relationship between fluorescence intensity and impurity concentration was prepared, and the fluorescence intensity of each solution of 10% (v / v), 1% (v / v), and 0% (v / v) prepared in (3) was measured. Fig.30 As shown in (c), a regression line showing the relationship between the fluorescence intensity and the impurity concentration is prepared.

[0185] Steps 1 to 7 were carried out in the same manner as in Experiment 1. In Experiment 2, Steps 1 to 7 were carried out three times in order to confirm the reproducibility.

[0186] Reference Fig.31 , the second experimental result of the fifth embodiment is described. Fig.31In the figure, the second round corresponds to process 3, the third round corresponds to process 5, and the fourth round corresponds to process 7. The left vertical axis represents the fluorescence intensity of the fluorescence image of FLch in the second and fourth rounds, and the right vertical axis represents the fluorescence intensity of the fluorescence image of A647ch in the third round. N1 to N3 represent the fluorescence intensities in the first to third trials, respectively. The dotted lines (1) to (3) are used Fig.30 The regression lines shown in (a) to (c) show the fluorescence intensity corresponding to 1% impurity concentration. That is, if the fluorescence intensity of the fluorescence image is the intensity shown by the dotted lines (1) to (3), it means that 1% carryover occurs. Fig.31 As shown, the fluorescence intensity of the 2nd to 4th rounds is lower than the dashed lines (1) to (3), and in the second experiment of the fifth embodiment, it is shown that the solution exchange in the micro space can be performed with less than 1% carryover.

[0187] <Sixth embodiment>

[0188] Reference Fig.32 as well as Fig.33 , the sixth embodiment is described. In the sixth embodiment, solution exchange of substrate solutions using different types of enzymes (enzyme reactions in microwells caused by different enzymes) is performed. Specifically, in the sixth embodiment, continuous single-molecule detection using ALP and β-Gal is implemented. Figure 4 In the closed control system 100A shown, the reagent A management unit 16A stores ALP fluorescent substrate solution, the reagent B management unit 16B stores β-Gal fluorescent substrate solution, and the HISCL cleaning solution is stored in the water tank 14. In the sixth embodiment, a closed microwell device with two enzymes immobilized thereon is used.

[0189] Fig.32 is a schematic diagram showing the process of the sixth embodiment. The space SP in the microporous device including a plurality of microporous pores M immobilized with ALP and another plurality of microporous pores M immobilized with β-Gal and each microporous pore M are filled with a fluorescein / HISCL cleaning solution WL (process 1). Next, as Figure 5 and Figure 6 As shown, a three-phase plug (from the front in the flow direction, air A, ALP fluorescent substrate solution (solution SA) and oil VO) is formed in the tube CB and transferred to the microwell device (flow cell 31) (step 2). Thus, the solution SA is contained in each microwell M and sealed by the oil VO (step 3). After that, the ALP fixed in the microwell M reacts with the ALP fluorescent substrate (FDP: Substrate for ALP) contained in the solution SA contained in the microwell M, and the microwell device after the reaction is photographed by a microscope 33 to obtain a fluorescent image A (step 4).

[0190] like Figure 7 and Figure 8As shown, a two-phase plug (starting from the front of the flow direction, air A and HISCL cleaning solution WL) is formed in the tube CB and transferred to the micropore device (flow cell 31) (step 5). As a result, the space SP and each micropore M are filled with the cleaning solution WL (step 6). A three-phase plug (starting from the front of the flow direction, air A, β-Gal fluorescent substrate solution (solution SB) and oil VO) is formed in the tube CB and transferred to the micropore device (flow cell 31) (step 7). As a result, the solution SB is contained in the micropore M and is sealed by the oil VO (step 8). Thereafter, the β-Gal fixed in the micropore M reacts with the β-Gal fluorescent substrate (FDG: Substrate for β-Gal) contained in the micropore M, and the fluorescent image B is acquired by photographing the micropore device after the reaction using a microscope 33 (step 9). Thereafter, a two-phase plug (starting from the front of the flow direction, air A and HISCL cleaning solution WL) is formed in the tube CB and transferred to the micropore device (flow cell 31) (step 10). This cycle of steps 1 to 10 is repeatedly performed.

[0191] Fig.33 It is a figure which shows the experimental result of the sixth example. Fig.33 (a) means in Fig.32 FIG. 4 is a diagram showing an example of a fluorescent image A acquired in step 4 and a fluorescent image B acquired in step 9. FIG. Fig.33 (b) is a graph showing an example of the fluorescence intensity of a microwell where ALP is detected (each line corresponds to a bright spot in each of a plurality of microwells). Fig.33 (c) is a graph showing an example of the fluorescence intensity of a microwell where β-Gal is detected (each line corresponds to a bright spot in each of a plurality of microwells). Fig.33 As shown in (a) to (c), it is shown that in step 4 of the reaction of ALP with the fluorescent substrate, the fluorescence intensity becomes high in a part of the microwells M, and in step 9 of the reaction of β-Gal with the fluorescent substrate, the fluorescence intensity becomes high in another part of the microwells M. That is, in the sixth embodiment, it is shown that continuous single-molecule detection of ALP and β-Gal can be performed.

[0192] <Seventh embodiment>

[0193] Reference Figure 34 to Figure 37 , the seventh embodiment is described. In the seventh embodiment, the confirmation of single exosome analysis (detection of single particles and quantification of particle inclusions) is implemented. Specifically, in the seventh embodiment, the dependence of exosome addition on the detection of Tau inclusion in exosomes is verified. Figure 4In the closed control system 100A shown, the reagent A management unit 16A stores the ALP fluorescent matrix solution (containing pigment), the reagent B management unit 16B stores the ALP labeled anti-Tau antibody solution, the reagent C management unit 16C stores the β-Gal fluorescent matrix / extraction solution (containing pigment), and the HISCL cleaning solution is stored in the water tank 14. In the seventh embodiment, a closed microporous device with an anti-Tau monoclonal antibody fixed thereto is used. In addition, a prepared sample containing volatile oil (AE-3000) and a magnetic particle-exosome-β-Gal complex is used.

[0194] Fig.34 Schematic diagram showing the process of the seventh embodiment. Fig.34 In the figure, for the convenience of explanation, in a part of the process, one micropore M among the plurality of micropores M is enlarged and illustrated. Fig.34 As shown, a sample (magnetic particles, exosomes, and immune complexes formed by β-Gal labeled antibodies) is prepared and added to the microporous device (step 1). The immune complex containing the magnetic particles is moved toward the direction approaching the magnet by a magnet not shown in the figure approaching the micropore M. That is, a concentration (magnetic collection) step is performed on the sample contained in the micropore M. In addition, in order to keep the immune complex in the micropore M, magnetic collection is continued in each step 2 to 11 except step 4 (step 2). As shown Figure 5 and Figure 6 As shown, a three-phase plug (from the front in the flow direction, air A, β-Gal fluorescent substrate / extract and oil VO) is formed in the tube CB and transferred to the microporous device (flow cell 31). As a result, each microporous M is filled with β-Gal fluorescent substrate / extract and sealed with oil VO. In addition, in the microporous M where the immune complex is magnetically collected, β-Gal reacts with the fluorescent substrate to emit fluorescence (step 3).

[0195] Fluorescent images A and B are obtained by photographing the microporous device using a microscope 33 (step 4). Next, the extraction / capture of Tau contained in the exosomes is performed. That is, the exosome-containing Tau is extracted from the exosomes by the action of the surfactant contained in the β-Gal fluorescent matrix / extraction solution, and the extracted exosome-containing Tau is captured by the anti-Tau monoclonal antibody fixed in the micropore M (step 5). Next, as Figure 7 and Figure 8As shown, a two-phase plug is formed in the tube CB (starting from the front of the flow direction, air A and HISCL cleaning solution WL), and it is transferred to the microporous device (flow cell 31). As a result, the space SP and each micropore M are filled with the cleaning solution WL (step 6). Next, a two-phase plug is formed (starting from the front of the flow direction, air A and ALP-labeled anti-Tau antibody solution), and it is transferred to the microporous device (flow cell 31). As a result, the space SP and each micropore M are filled with the ALP-labeled anti-Tau antibody solution (step 7). In the micropore M where the immune complex is magnetically collected, ALP labeling of Tau encapsulated in exosomes based on the antigen-antibody reaction is implemented (step 8).

[0196] Next, the HISCL cleaning liquid WL is transferred to the micropore device (flow cell 31). As a result, the space SP and each micropore M are filled with the cleaning liquid WL (step 9). Figure 5 and Figure 6 As shown, a three-phase plug (from the front of the flow direction, air A, ALP fluorescent substrate solution and oil VO) is formed in the tube CB and transferred to the microporous device (flow cell 31). As a result, each micropore M is filled with the ALP fluorescent substrate solution and sealed with oil VO. In addition, in the micropore M where the immune complex is magnetically collected, the ALP marker reacts with the fluorescent substrate and emits fluorescence (process 10). The microporous device is photographed using a microscope 33 to obtain a fluorescent image C (process 11).

[0197] Fig.35 Schematic diagram showing the analysis process of the seventh embodiment. Fig.35As shown, the analysis process of the seventh embodiment includes the following four steps. The analysis process of the seventh embodiment includes "beads detection", "exosome detection", "Tau detection" and "data analysis" in the order of implementation. "Beads detection" is a process for determining the micropores M where the immune complex is magnetically collected based on the fluorescent image taken in step 4. "Exosome detection" is a process for obtaining the intensity of fluorescence emitted from each micropore M determined in the "beads detection" based on the fluorescent image taken in step 4. "Tau detection" is a process for obtaining the intensity of fluorescence emitted from each micropore M determined in the "beads detection" based on the fluorescent image taken in step 11. "Data analysis" is a process for plotting on a scatter plot for each micropore M determined in the "beads detection" based on the fluorescence intensity of the fluorescent image of step 4 and the fluorescence intensity of the fluorescent image of step 11. If the scatter plot is divided into four parts, the microwells marked in the upper left area show that exosomes were not detected, but Tau contained in exosomes was detected, the microwells marked in the upper right area show that both exosomes and Tau contained in exosomes were detected, the microwells marked in the lower left area show that neither exosomes nor Tau contained in exosomes were detected, and the microwells marked in the lower right area show that exosomes were detected, but Tau contained in exosomes was not detected.

[0198] Fig.36 as well as Fig.37 It is a figure which shows the experimental result of the seventh example. Fig.36 The horizontal axis of (a) to (d) represents the fluorescence intensity during the detection of exosomes (the fluorescence intensity obtained by "exosome detection"), and the vertical axis represents the fluorescence intensity during the detection of Tau (the fluorescence intensity obtained by "Tau detection"). Fig.36 (a) and (b) are used in Fig.34 The detection results when the sample prepared in step 1 without containing exosomes was subjected to steps 2 to 11, Fig.36 (c) and (d) are used in Fig.34 The detection results are obtained when the samples prepared in step 1 so as to contain 16 μg of exosomes per sample were subjected to steps 2 to 11. Fig.36 (b) is to Fig.36 The enlarged view of the solid line frame L1 part of (a), Fig.36 (d) is to Fig.36 (c) is an enlarged view of the solid line frame L2 part. Fig.36As shown in (b), in the sample prepared without exosomes, almost all the plots are present in the lower left area, and almost no false positives are generated. In addition, it is believed that the plots present in the upper left area are affected by the ALP labeled antibody that is non-specifically adsorbed to the micropores. Fig.36 As shown in (d), in the sample prepared by containing exosomes, a large number of plots exist in the lower right area, and it can be seen that the exosomes contained in the sample can be detected. In addition, there are plots in the upper right area, and it can be seen that Tau contained in exosomes can also be detected. In addition, since Tau contained in exosomes is not contained in all exosomes, the number of plots in the upper right area is less than that in the lower right area.

[0199] Fig.37 The experimental results are shown in which the average number (λ) of exosomes captured by magnetic particles was changed in the sample preparation of step 1, and each step 2 to 11 and the analysis flow of Example 7 were performed. In the experimental results, λ represents the number of Tau ch(+) & Exo ch(+) of about 0.7 (i.e., Fig.35 The number of plots in the upper right area of ​​the scatter plot of "Data Analysis" is 1×10 5 This indicates that Tau contained in exosomes can be detected from the immune complex that captures only one exosome. That is, in the seventh embodiment, it is shown that single exosome analysis can be performed.

[0200] <Other embodiments>

[0201] The above embodiments are for easy understanding of the present invention and are not intended to limit the present invention. The present invention can be modified / improved without departing from its gist (for example, combining the various embodiments, omitting a part of the structure of the various embodiments), and the present invention also includes its equivalents.

Claims

1. A solution exchange method, which is a solution exchange method for exchanging solutions in a plurality of micropores formed in a substrate, comprising: a step of replacing an immiscible liquid that is immiscible with the first solution and seals the plurality of micropores filled with the first solution with a gas; as well as A step of filling the plurality of micropores with the second solution by introducing a second solution onto the substrate in which the immiscible liquid is replaced with the gas.

2. The solution exchange method according to claim 1, wherein: After the step of filling the plurality of micropores with the second solution, the method further includes: introducing a second immiscible liquid that is immiscible with the second solution onto the substrate, thereby sealing the plurality of micropores filled with the second solution with the immiscible liquid.

3. The solution exchange method according to claim 1, wherein: A second substrate is disposed above the plurality of micro-wells, and the immiscible liquid is accommodated in a space between the second substrate and the substrate on which the plurality of micro-wells are formed.

4. The solution exchange method according to claim 3, wherein: The step of replacing the immiscible liquid with the gas includes introducing the gas into the space from an inlet provided near one end of the space, and discharging the immiscible liquid from a discharge port provided near the other end of the space.

5. The solution exchange method according to claim 1, wherein: The upper portion of the substrate on which the plurality of microholes are formed is open.

6. The solution exchange method according to claim 5, wherein: The step of replacing the immiscible liquid with the gas includes: sucking out the immiscible liquid from the upper side with a pipette.

7. The solution exchange method according to claim 1, wherein: The immiscible liquid is oil having a vapor pressure of 0.15 kPa or more at 25°C.

8. The solution exchange method according to claim 1, wherein: The gas is air.

9. A digital measurement method, which is a digital measurement method for performing digital measurement in multiple stages using a plurality of micropores formed on a substrate, comprising: A step of performing a first measurement in the plurality of microwells filled with a first measurement reagent and sealed with an immiscible liquid that is immiscible with the first measurement reagent; a step of replacing the immiscible liquid sealing the plurality of micropores with a gas; a step of introducing a second measuring reagent onto the substrate in which the immiscible liquid is replaced with the gas, thereby filling the plurality of microwells with the second measuring reagent; a step of introducing a second immiscible liquid that is immiscible with the second solution onto the substrate, thereby sealing the plurality of microwells filled with the second measuring reagent with the immiscible liquid; as well as A step of performing a second measurement in the microwell filled with the second measurement reagent and sealed with the immiscible liquid.

10. The digital determination method according to claim 9, wherein: Before the step of performing the first measurement, the method further includes the step of introducing particles derived from a biological organism into the plurality of micropores. The first assay is an assay for detecting surface molecules of the particle, Before the step of performing the second measurement, the method further includes the step of extracting the inclusions from the particles in the plurality of micropores. The second assay is an assay for detecting the inclusions.

11. The digital determination method according to claim 9, wherein: The immiscible liquid is oil having a vapor pressure of 0.15 kPa or more at 25°C.

12. The digital determination method according to claim 9, wherein: The gas is air.

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