Liquid delivery method, digital measurement method, and digital measurement device

Abstract

The present invention provides a liquid delivery method, a digital measurement method, and a digital measurement apparatus capable of easily controlling the transfer of a solution and an immiscible liquid to a device having a plurality of micropores. A liquid feeding method for feeding liquid to a device (MWD) in which a plurality of micropores (M) are formed on a substrate (B), the liquid feeding method including: feeding a liquid to the device (MWD) at a position different from the position of the device (MWD); a step in which a multiphase plug in which a solution filling a plurality of micropores (M) and an immiscible liquid that is immiscible with the solution and that seals the plurality of micropores (M) filled with the solution are arranged in this order from the front in the direction of flow is formed in advance; and a step for transferring the multiphase plug to the device (MWD).

Description

Technical Field

[0001] The invention relates to a liquid feeding method, a digital measuring method and a digital measuring device. Background Art

[0002] Methods related to liquid delivery to a microwell array are known. For example, Patent Document 1 describes a liquid delivery method using a digital microfluidics (DMF) device, which is a liquid delivery method in which an oil droplet is arranged at a predetermined position of the DMF device, and a DMF electrode is used to move the aqueous droplet to the opening of a hole arranged on the DMF device via the arrangement position of the oil droplet. In this liquid delivery method, since the oil droplet moves with the movement of the aqueous droplet and becomes a two-phase droplet, the hole filled with the aqueous droplet is sealed by the oil droplet.

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent Document 1: U.S. Patent Publication No. 2020-0001300 Summary of the invention

[0006] In the above-mentioned conventional liquid feeding method, the oil droplets need to be moved along with the movement of the aqueous droplets. Therefore, there is a problem that the liquid feeding conditions such as the amount and flow rate of the aqueous droplets and oil droplets have low freedom and it is difficult to control the liquid feeding.

[0007] An object of the present invention is to provide a liquid feeding method, a digital measurement method, and a digital measurement device capable of easily controlling the transfer of a solution and an immiscible liquid to a device having a plurality of micropores.

[0008] The liquid delivery method of the present invention is a liquid delivery method for delivering liquid to a device (MWD) having a plurality of micropores (M) formed on a substrate (B), comprising: a process of pre-forming, at a position different from the device (MWD), a multiphase plug having a solution filled with a plurality of micropores (M) and an immiscible liquid that is immiscible with the solution and is used to seal the plurality of micropores (M) filled with the solution, arranged in sequence from the front in the flow direction; and a process of transferring the multiphase plug to the device (MWD).

[0009] According to the liquid delivery method of the present invention, the following steps are performed: a step of pre-forming a multiphase plug in which a solution filling a plurality of micropores (M) and an immiscible liquid that is immiscible with the solution and is used to seal the plurality of micropores (M) filled with the solution are arranged in sequence from the front in the flow direction at a position different from the device (MWD); and a step of transferring the multiphase plug to the device (MWD). Therefore, there is no need to form a multiphase plug on the device (MWD), and the degree of freedom of liquid delivery conditions such as the amount of the solution and the immiscible liquid, the flow rate, etc. is high. Therefore, it is possible to easily control the delivery of the solution and the immiscible liquid to the device (MWD).

[0010] The digital measurement method of the present invention is a digital measurement method for performing digital measurement using a device (MWD) having a plurality of micropores (M) formed on a substrate (B), comprising: a step of pre-forming, at a position different from the device (MWD), a multiphase plug having a solution filled with a plurality of micropores (M) and an immiscible liquid that is immiscible with the solution and is used to seal the plurality of micropores (M) filled with the solution, arranged in sequence from the front in the flow direction; a step of transferring the multiphase plug to the device (MWD); and a step of performing digital measurement in a plurality of micropores (M) containing a solution and sealed with an immiscible liquid by transferring the multiphase plug.

[0011] According to the digital measurement method of the present invention, the following steps are performed: a process of pre-forming a multiphase plug in which a solution filling a plurality of micropores (M) and an immiscible liquid that is immiscible with the solution and is used to seal the plurality of micropores (M) filled with the solution are arranged in sequence from the front in the flow direction at a position different from the device (MWD); a process of transferring the multiphase plug to the device (MWD); and a process of performing digital measurement in a plurality of micropores (M) containing the solution and sealed with the immiscible liquid by transferring the multiphase plug. In the digital measurement method of the present invention, it is not necessary to form a multiphase plug on the device (MWD), and the degree of freedom of the liquid delivery conditions such as the amount and flow rate of the solution and the immiscible liquid is high. Therefore, it is possible to easily control the transfer of the solution and the immiscible liquid to the device (MWD). Therefore, for example, it is possible to control the time from the accommodating of the solution in the micropores (M) to the sealing with the immiscible liquid. Thus, it is possible to immediately seal the micropores (M) with the immiscible liquid after the solution is contained in the micropores (M). Thus, the outflow of the reactant from the micropore (M) caused by the solution can be suppressed, and the signal obtained from the micropore (M) can be increased in the digital measurement. More specifically, for example, after introducing a surfactant solution into a micropore (M) filled with biological particles such as cells and exosomes and crushing the biological particles, it is possible to seal with an immiscible liquid without vacating time. In addition, for example, after introducing a matrix solution into a micropore (M) filled with an enzyme-labeled test substance and starting an enzyme-matrix reaction, it is possible to seal with an immiscible liquid without vacating time.

[0012] The digital measuring device of the present invention is a digital measuring device for performing digital measurements, comprising: a device (MWD) having a plurality of micropores (M) formed on a substrate; a fluid management unit connected to a liquid receiving port of the device (MWD), forming a multiphase plug having a solution filling the plurality of micropores (M) and an immiscible liquid that is immiscible with the solution and is used to seal the plurality of micropores (M) filled with the solution, arranged in sequence from the front in the flow direction, at a position different from the device (MWD), and transferring the multiphase plug to the device (MWD); and a detection unit that detects a measurement object in the plurality of micropores (M) containing the solution and sealed with the immiscible liquid through the transfer of the multiphase plug.

[0013] According to the digital measuring device of the present invention, it includes: a device (MWD) having a plurality of micropores (M) formed on a substrate; and a fluid management unit connected to the liquid receiving port of the device (MWD), and a multiphase plug is formed at a position different from the device (MWD), in which a solution filling a plurality of micropores (M) and an immiscible liquid that is immiscible with the solution and is used to seal the plurality of micropores (M) filled with the solution are arranged in sequence from the front in the flow direction, and the multiphase plug is transferred to the device (MWD). In the digital measuring device of the present invention, it is not necessary to form a multiphase plug on the device (MWD), and the degree of freedom of the liquid delivery conditions such as the amount and flow rate of the solution and the immiscible liquid is high. Therefore, it is possible to easily control the transfer of the solution and the immiscible liquid to the device (MWD). Therefore, for example, it is possible to control the time from the accommodating of the solution in the micropores (M) to the sealing with the immiscible liquid. As a result, it is possible to immediately seal the micropores (M) with the immiscible liquid after the solution is accommodated in the micropores (M). Thus, the outflow of the reactant from the micropore (M) caused by the solution can be suppressed, and the signal obtained from the micropore (M) can be increased in the digital measurement. More specifically, for example, after introducing a surfactant solution into a micropore (M) filled with biological particles such as cells and exosomes and crushing the biological particles, it is possible to seal with an immiscible liquid without vacating time. In addition, for example, after introducing a matrix solution into a micropore (M) filled with an enzyme-labeled test substance and starting an enzyme-matrix reaction, it is possible to seal with an immiscible liquid without vacating time.

[0014] According to the present invention, it is possible to provide a liquid feeding method, a digital measurement method, and a digital measurement device capable of easily controlling the transfer of a solution and an immiscible liquid to a device having a plurality of micropores. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is a schematic diagram showing the outline of the liquid feeding method.

[0016] Figure 2 It is a schematic diagram showing the outline of the digital measurement method and the multi-stage digital measurement method.

[0017] Figure 3 This is a schematic diagram showing an outline of a digital measurement method using a liquid feeding method.

[0018] Figure 4 It is a diagram showing the overall configuration of a closed control system in the embodiment.

[0019] Figure 5 This is a schematic diagram showing an outline of a method for forming a three-phase plug.

[0020] Figure 6 This is a schematic diagram showing an outline of a method for transferring a three-phase plug.

[0021] Figure 7This is a schematic diagram showing an outline of a method for forming a two-phase plug.

[0022] Figure 8 This is a schematic diagram showing an outline of a method for transferring a two-phase plug.

[0023] Fig. 9 This is a diagram showing an example of a cross-sectional view of a closed-type micropore device.

[0024] Fig.10 This is a diagram showing an example of a top view of a closed-type microwell device.

[0025] Fig.11 This is a schematic diagram showing an outline of a first example of a method for producing a closed microwell device.

[0026] Fig.12 This is a schematic diagram showing an outline of a second example of a method for producing a closed microwell device.

[0027] Fig.13 It is a schematic diagram showing the flow of the first embodiment.

[0028] Fig.14 It is a figure which shows the experimental result of the first example.

[0029] Fig.15 It is a figure which shows other experimental results of the first embodiment.

[0030] Fig.16 It is a schematic diagram showing the flow of the second embodiment.

[0031] Fig.17 It is a figure which shows the experimental result of the second example.

[0032] Fig.18 (a) is a graph showing the fluorescence intensity in the fluorescence image of A647ch. Fig.18 (b) is a graph showing the fluorescence intensity in the fluorescence image of FLch.

[0033] Fig.19 It is a schematic diagram showing the flow of the third embodiment.

[0034] Fig. 20 (a) means Fig.19 FIG. 1 is a diagram showing an example of a fluorescent image A acquired in step 1 and a fluorescent image B acquired in step 3. Fig. 20 (b) is a graph showing an example of the fluorescence intensity of the microwell where ALP was detected. Fig. 20 (c) is a graph showing an example of the fluorescence intensity of a microwell in which β-Gal is detected.

[0035] Fig.21It is a schematic diagram showing the flow of the fourth embodiment.

[0036] Fig. 22 It is a schematic diagram showing the analysis flow of the fourth embodiment.

[0037] Fig.23 (a) and Fig.23 (b) indicates the use of Fig.21 The diagram shows an example of the detection results when the sample prepared in step 1 without containing exosomes is subjected to steps 2 to 11. Fig.23 (c) and Fig.23 (d) indicates the use of Fig.21 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.

[0038] Fig.24 It is a diagram showing other experimental results of the fourth embodiment.

[0039] Fig.25 It is a schematic diagram showing the flow of the fifth embodiment.

[0040] Fig.26 It is a schematic diagram showing a flow of a comparative example of the fifth embodiment.

[0041] Fig. 27 It is a diagram showing the comparison results between the fifth embodiment and the comparative example.

[0042] Fig.28 (a) is a diagram showing an example of the inside of the micropore device in the fifth embodiment. Fig.28 (b) is a diagram showing an example of the state inside a micropore device in a comparative example.

[0043] Description of Reference Numerals

[0044] 1: Fluid system, 3: Detection system, 5: Control system, 11: Syringe 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, M: Microwell, B: Substrate, UG: Upper surface glass, DT: Double-sided tape, ST: Single-sided tape. DETAILED DESCRIPTION

[0045] 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.

[0046] Figure 1 Schematic diagram showing the outline of an embodiment of the liquid feeding method of the present invention. The liquid feeding method is a liquid feeding method for feeding liquid to a microporous device MWD (device). Figure 1 As shown, the microporous device MWD includes a substrate B having a plurality of micropores M formed therein, and an upper surface glass UG arranged above the substrate B. The upper surface glass UG is arranged separately from the substrate B so that a space SP can be formed between the substrate B and the upper surface glass UG. The liquid feeding method of an embodiment of the present invention is a method of transferring a multiphase plug (multiphase plug) described later to the microporous device MWD. ​​In the liquid feeding method, for example, a multiphase plug is pre-formed at a position different from the microporous device MWD, in which at least a solution SA (for example, a reagent solution) filled with a plurality of micropores M and an immiscible liquid (for example, a volatile oil VO) that is immiscible with the solution SA and is used to seal the plurality of micropores M filled with the solution SA, which are arranged in sequence from the front in the flow direction (process 1). In Figure 1 In the example, air A is also included in the multiphase plug, and the multiphase plug is formed by arranging air A, solution SA and immiscible liquid in sequence from the front in the flow direction. Here, the position different from the microporous device MWD refers to, for example, a specified position in the tube CB connected to the liquid delivery port IP of the microporous device MWD. ​​In this specification, a "multiphase plug" refers to a collection of fluids in which fluids of different types are connected to each other without mixing. The types of fluids in the "multiphase plug" can be more than two, and can be either gas or liquid, or a combination thereof. The "multiphase plug" is preferably formed in the flow path to maintain its shape.

[0047] When the pinch valve PV is controlled to be in an open state, the transfer of the multiphase plug to the space SP of the microporous device MWD begins (process 2). Initially, the space SP is filled with, for example, a cleaning liquid WL, and by transferring the multiphase plug, air A, solution SA, and volatile oil VO slowly enter in sequence from the front in the flow direction. Afterwards, after a plurality of micropores M are respectively filled with the solution SA, the volatile oil VO is used to seal the plurality of micropores M respectively (process 3). In such a liquid delivery method, a multiphase plug is pre-formed at a position different from the microporous device MWD. ​​Therefore, there is no need to form a multiphase plug on the microporous device MWD, and the liquid delivery conditions such as the amount of solution and immiscible liquid, flow rate, etc. have a high degree of freedom. Therefore, the transfer of solution and immiscible liquid to the microporous device (MWD) can be easily controlled.

[0048] Here, for example, the volume of the solution SA in the multiphase plug is set based on the time from when a predetermined micropore M among the plurality of micropores M is filled with the solution SA to when it is sealed with the volatile oil VO. The volume (V) of the solution SA is actually set by the following formula.

[0049] V=S×v×t

[0050] (S: cross-sectional area in the space SP with respect to the liquid feeding direction, v: flow rate of the multiphase plug in the space SP, and t: time from when the micropores M are filled with the solution SA until they are sealed with the volatile oil VO)

[0051] The time t from when the micropores M are filled with the solution SA to when they are sealed with the volatile oil VO is arbitrary, and can be appropriately set, for example, according to the capacity of the solution SA. When the capacity of the solution SA is relatively large, the time t is preferably less than 4 seconds. When the capacity of the solution SA is relatively small, the time t is preferably less than 1 second.

[0052] The volume of the volatile oil VO in the multiphase plug is equal to or larger than the volume of the space SP of the micropore device MWD. ​​According to this structure, since the volatile oil VO fully fills the space SP, the solution SA contained in the plurality of micropores M can be appropriately sealed.

[0053] In any of the above-mentioned processes, it is possible to implement by an automation device or a manual method. For example, from the viewpoint of reproducibility, it is preferably implemented by an automation device. The automation device, for example, includes a device composed of an air pressure supply unit such as a syringe pump and 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 automation device, for example, connects a syringe pump to a pipe such as a Teflon (registered trademark) pipe, forms a multiphase plug in the pipe by the syringe pump, and transfers it to a microporous device, so that the immiscible liquid of a plurality of micropores filled with a solution can be sealed with gas replacement.

[0054] As the situation that this liquid delivery method can be applied, there is no particular limitation, and in detail, it can be various ways. In the liquid delivery method, for example, biological particles such as cells and exosomes are independently introduced into each micropore. In the liquid delivery 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 of a reagent for extracting an inner substance from biological particles, and a solution of an inner substance detection reagent added with a reagent for detecting the extracted inner substance are sequentially sealed in each micropore by a liquid delivery method, so that biological particles can be analyzed particle by particle. In addition, in the liquid delivery method, biological particles such as cells and exosomes are independently introduced into each micropore, 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.

[0055] 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.

[0056] like Figure 2 As 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 particles derived from a biological body can be introduced into each micropore, cells can be sealed into the micropores one by one ("1. Concentration"), surface antigens of the cells can be measured ("2. Expression antigen measurement"), extraction liquid can be used to extract and capture the contents (for example, contained proteins and contained nucleic acids) from the cells ("3. Extraction / capture"), contained proteins can be labeled with a labeling agent ("4. Labeling"), contained proteins can be measured ("5. Contained protein measurement"), contained nucleic acids can be labeled with a labeling agent ("6. Labeling"), and contained nucleic acids can be measured ("7. Contained nucleic acid measurement"). In the digital measurement method, it is not limited to the above-mentioned techniques, and other steps can also be appropriately included.

[0057] 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).

[0058] Figure 3Schematic diagram showing an outline of an embodiment of a digital measurement method using a liquid delivery method of the present invention. The digital measurement method of an embodiment of the present invention is a digital measurement method using a microporous device MWD for digital measurement. Figure 3 As shown, in the digital measurement method of the embodiment of the present invention, for example, a multiphase plug is pre-formed in the tube CB connected to the liquid delivery port IP of the microporous device MWD, which contains at least a solution SA (for example, a reagent solution) filled with a plurality of micropores M and an immiscible liquid (for example, volatile oil VO) that is immiscible with the solution SA and is used to seal the plurality of micropores M filled with the solution SA (process 1). Figure 3 In the example of , air A is also included in the multiphase plug, and the multiphase plug is formed by arranging air A, solution SA, and immiscible liquid in this order from the front in the flow direction.

[0059] When the pinch valve PV is controlled to be in an open state, the transfer of the multiphase plug to the space SP of the microporous device MWD begins (process 2). Initially, the space SP is filled with, for example, a cleaning liquid WL, and by transferring the multiphase plug, air A, solution SA, and volatile oil VO are slowly introduced in sequence from the front of the flow direction. Afterwards, by transferring the multiphase plug, digital measurement is performed in a plurality of micropores M containing solution SA and sealed with volatile oil VO (process 3). In addition, the digital measurement method is not limited to the above-mentioned process. For example, before the process of transferring the multiphase plug to the microporous device MWD, a process of introducing particles (biological particles) derived from a biological body into a plurality of micropores M may also be included. In the case of this method, the solution contains a substance for detecting surface molecules of the particles (for example, the fluorescent matrix (FDG) shown in process 3), and the digital measurement implemented in process 3 is a measurement for detecting surface molecules of the particles. In addition, in order to enable optical measurement, the substrate B having a plurality of micropores M is preferably formed by a transparent component.

[0060] In the digital measurement method using the present liquid delivery method, there is no need to form a multiphase plug on the microporous device MWD, and the liquid delivery conditions such as the amount and flow rate of the solution and the immiscible liquid are highly free. Therefore, it is possible to easily control the transfer of the solution and the immiscible liquid to the microporous device (MWD). Thus, it is possible to seal with an immiscible liquid immediately after the reagent solution is accommodated in the micropore. Thus, it is possible to suppress the outflow of the reactant from the micropore caused by the reagent solution, and in the digital measurement, it is possible to increase the signal obtained from the micropore M. More specifically, for example, it is possible to introduce a surfactant solution into a micropore (M) filled with biological particles such as cells and exosomes and crush the biological particles, and then seal with an immiscible liquid without leaving time. In addition, for example, it is possible to introduce a matrix solution into a micropore (M) filled with an enzyme-labeled test substance and start an enzyme-matrix reaction, and then seal with an immiscible liquid without leaving time.

[0061] Reference Figures 4 to 10 , the closed control system in the embodiment is described. The closed control system refers to a control system having the following 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.

[0062] Figure 4 This is a diagram showing the overall structure of a closed control system. Figure 4 As shown, the closed control system 100A exemplarily includes a fluid system 1, a detection system 3, and a control system 5. The control system 100A is an example of a digital measurement device that performs digital measurement.

[0063] The fluid system 1 (fluid management unit) includes, for example, a syringe pump (pump unit) 11, a water storage tank 14, an oil management unit (immiscible fluid management unit) 15, a reagent A management unit (first solution management unit) 16A, a reagent B management unit (second solution management unit) 16B, a reagent C management unit (third solution management unit) 16C, a waste liquid tank A 18, and a waste liquid tank B 19. The above-mentioned parts of the fluid system 1 are connected through 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 portion 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 can be controlled by a control signal from a control unit (not shown) provided in the fluid system 1.

[0064] The syringe pump 11 illustratively includes, for example, a syringe A12 for delivering air to the tube CB and a syringe B13 for delivering, for example, a washing buffer solution as a washing liquid to the tube CB. The water storage tank 14 is, for example, a tank for storing the washing buffer solution.

[0065] 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 waste liquid tank A18 or 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 waste liquid tank A18 or the flow cell 31 through the downstream pipe CB by air pressure. For example, the reagent A, the reagent B, and the reagent C can be different reagents, respectively, and at least two reagents can also be common reagents. In the control system 100A, there is no restriction on the types of reagents, and it can be less than two or more than four.

[0066] 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.

[0067] The detection system 3 includes, for example, a flow cell 31 and a microscope (detector) 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.

[0068] 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 facing the microwell to obtain information from the microwell filled with a solution.

[0069] 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.

[0070] 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 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.

[0071] <Formation and transfer method of multiphase plug>

[0072] In the present liquid delivery method, for example, when performing a process of replacing an immiscible liquid with a gas, a multiphase plug consisting of three phases of gas, solution, and immiscible liquid can be pre-formed in a tube or a pipette tip before the gas is transferred to the substrate, and then the multiphase plug can be transferred.

[0073] In particular, in the case where it is necessary to seal with an immiscible liquid just after the micropore is exchanged for a solution, it is preferred to use a multiphase plug. According to the method, for example, a surfactant solution (solution) can be introduced into a micropore filled with biological particles such as cells and exosomes and the biological particles are crushed, and then the immiscible liquid is sealed without leaving time, so that the inclusions of the biological particles can be suppressed from flowing out of the micropore, or the inclusions of the biological particles flowing out diffuse into 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 an enzyme, it is possible to suppress the enzyme reaction from flowing out of the micropore, or the enzyme reaction flowing out diffuses into adjacent micropores.

[0074] When a multiphase plug is formed 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 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 solution, and the immiscible liquid in front of the flow direction is replaced by the solution. Thereafter, the fluid control device is switched to inject the gas, and the solution in front of the flow direction is replaced by the gas. In this way, a three-phase plug can be formed in which air, solution, and immiscible liquid are arranged in the order of the front of the flow direction.

[0075] Reference Figures 5 to 8 , the formation / transfer method of the three-phase / two-phase plug is specifically explained. 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 overview of a method for transferring the three-phase plug to the 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 overview of a method for transferring a two-phase plug to 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 8 In the figure, the pinch valve with "x" added indicates a closed state, and the pinch valve without "x" added indicates an open state.

[0076] use Figure 5 , 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 solution is transferred into the tube CB. Thus, 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. Thus, the oil in front of the flow direction is replaced by the reagent (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. Thus, the reagent in front of the flow direction is replaced by air (process 4). After such a process, 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.

[0077] 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) Start 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 liquid delivery of steps 2 to 4 described in the above.

[0078] 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 clamp 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 clamp valve PV1 is switched to a closed state and the clamp valve PV7 is switched to an open state, air is introduced into the tube CB. Thus, the cleaning buffer in the front of the flow direction is replaced by air (process 2). After such a process, Figure 7 As shown, in the tube CB portion between the pinch valve PV1 and the pinch valve PV9, a two-phase plug is formed in which air and 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 introduction order of each fluid of the two-phase plug is also arbitrary. For example, oil can be introduced into the tube CB instead of the washing buffer in process 1, and reagent can be introduced instead of air in process 2. Thus, a two-phase plug is formed in which reagent (solution) and oil (immiscible liquid) are arranged in the order from the front in the flow direction.

[0079] 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).

[0080] 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 a top view.

[0081] 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 (liquid supply port) 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.

[0082] 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.

[0083] 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.

[0084] <Solution>

[0085] 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.

[0086] 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.

[0087] 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.

[0088] In addition, for example, when a chemical reaction such as an antigen-antibody reaction or an enzyme-substrate reaction is performed in the microwell, a solution containing an antigen, an antibody, an enzyme or a fluorescent substance can be used. In addition, when cells are cultured in the microwell, a cell culture fluid such as DMEM can be used as a solution.

[0089] <Immiscible liquids>

[0090] 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.

[0091] Examples of saturated hydrocarbons include alkanes and cycloalkanes. Examples of alkanes include hexane and decane. Examples of unsaturated hydrocarbons include squalene. Examples of aromatic hydrocarbons include benzene and toluene. Examples of perfluorocarbons include 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 TM 7300 (3M) and HFE-7500 (3M), etc. Among them, AE-3100 and Novec-7100 are preferred because of their relatively high vapor pressure.

[0092] 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.

[0093] <Method for producing a closed microporous device>

[0094] Fig.11 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.11As 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.

[0095] Fig.12 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.

[0096] 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.

[0097] 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.

[0098] <Overview of each embodiment>

[0099] The following is a description of each embodiment. The outline of each embodiment is summarized as follows.

[0100]

[0101] <Micropore Devices of the First and Second Embodiments>

[0102] Here, the method for preparing the closed microporous device used in the first and second 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.

[0103] (1) First, prepare to implement Fig.12 The 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).

[0104] (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.

[0105] (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).

[0106] <Micropore device of the fourth and fifth embodiments>

[0107] Next, the method for preparing the closed microporous device used in the fourth and fifth embodiments is specifically described. In this preparation method, a Tau antibody is bound to a biotin-binding PEG linker that modifies the microporous device.

[0108] (1) First, prepare to implement Fig.12The 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.

[0109] (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).

[0110] <Micropore device of the third embodiment>

[0111] Next, the method for preparing the closed microporous device used in the third embodiment is specifically described. In this preparation method, an enzyme is bound to the biotin-binding PEG linker that modifies the microporous device.

[0112] (1) First, prepare to implement Fig.12 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).

[0113] (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.

[0114] 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.

[0115] <Reagents>

[0116] The reagents include, for example, 1. an enzyme solution, 2. an antibody solution, 3. an enzyme substrate solution, and 4. a fluorescent dye solution.

[0117] 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).

[0118] 2. The antibody solution includes an ALP-labeled anti-Tau antibody solution. The ALP-labeled anti-Tau antibody solution is used in the fourth and fifth embodiments. 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 is performed using the ALP labeling kit SH (product No. LK13) manufactured by DOJINDO.

[0119] 3. The enzyme substrate solution includes the (1) ALP fluorescent substrate solution used in the third embodiment, the (2) ALP fluorescent substrate solution (containing pigment) used in the fourth and fifth embodiments, the (3) β-Gal fluorescent substrate solution used in the third embodiment, and the (4) β-Gal fluorescent substrate / extraction solution (containing pigment) used in the fourth and fifth embodiments.

[0120] (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.

[0121] (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.

[0122] 4. The fluorescent pigment solution includes (1) fluorescein / HISCL cleaning solution used in the second embodiment, (2) A488 / extraction solution used in the first embodiment, (3) A647 / extraction solution used in the first and second embodiments, and (4) A647 / ALP reaction solution used in the second embodiment.

[0123] (1) Prepare a fluorescein / HISCL cleaning solution as follows. (i) Prepare a 200 mM fluorescein (F0096, Tokyo Chemical Industry) aqueous solution. (ii) Dilute the fluorescein aqueous solution prepared in (i) with a HISCL cleaning solution to prepare a final concentration of 4 mM.

[0124] (2) 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.

[0125] (3) Prepare A647 / extract solution as follows. (3) Use the extract solution prepared in (iii) to prepare 10 μM A647 (A20347, ThermoFisher) / extract solution (the extract solution used in the first example). In addition, (2) Use the extract solution prepared in (iii) to prepare 0.3 mM A647 (A20347, ThermoFisher) / extract solution (the extract solution used in the second example). (5) A647 / ALP reaction solution Use the aforementioned ALP reaction solution to prepare a 0.3 mM A647 / ALP reaction solution.

[0126] <Sample>

[0127] The immune complex solution containing exosomes, magnetic particles and β-Gal used in the fourth and fifth embodiments 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 in an amount of about 10 million per sample and washed with HISCL washing solution. After washing, all the HISCL washing solution is discarded. The antibody includes an anti-CD9 monoclonal antibody (product No. 312102) manufactured by BioLegend. The 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.

[0128] 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).

[0129] 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).

[0130] The average number λ of exosomes per magnetic particle is given by the following formula 1.

[0131] (Formula 1)

[0132] λ=N exo / N bead

[0133] (N exo : The number of exosomes in the sample, and N bead : Number of magnetic particles in the sample (= 10 million)

[0134] In addition, N is calculated by formula 2 exo .

[0135] (Formula 2)

[0136] N exo =m exo ×n exo

[0137] (mexo : The quality of exosomes, and n exo : Number of exosomes per 1 mg of exosomes (=1.4×10 8 pieces / mg))

[0138] <Photography method>

[0139] 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).

[0140] The following describes each embodiment in detail. First, the content of the first embodiment is described.

[0141] <First embodiment>

[0142] Reference Figures 13 to 15 , the first embodiment is described. In the first embodiment, Figure 4 In the closed control system 100A shown, liquid can be automatically delivered to the closed microporous device. In the first embodiment, the solution exchange (exchange between the extracting solutions) of A488 / extracting solution (first solution) and A647 / extracting solution (second solution) is implemented. Figure 4 In 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 tank 14. In the first embodiment, a closed microporous device with anti-PSA monoclonal antibodies fixed thereto is used. In addition, assuming an antigen-antibody reaction in the micropores, antibody-immobilized micropores are used, but the antigen is not included in the reagents. In addition, in the first embodiment, fluorescein / PBS solution, cleaning solution, and volatile oil (AE-3000) are used as reagents. The vapor pressure of AE-3000 at 25°C is 31 kPa, and the evaporation rate (ratio) is 67 when ether is set to 100.

[0143] Fig.13: is a schematic diagram showing the process of the first embodiment. First, a solution SA is contained in each micropore M, and a micropore device MWD 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 MWD using 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 MWD. ​​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 MWD using a microscope 33 (process 3).

[0144] 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 MWD. ​​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 with 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 sealed with the oil VO, the micropore device MWD is photographed with a microscope 33 to obtain a fluorescent image C. Such a cycle of steps 1 to 4 is repeatedly performed.

[0145] As described above, the volume of the solution SA in the three-phase plug formed in the tube CB is set based on the time from when a predetermined micropore M among the plurality of micropores M is filled with the solution SA to when it is sealed with the volatile oil VO. The volume (V) of the solution SA is actually set by the following formula.

[0146] V=S×v×t

[0147] (S: cross-sectional area in the space SP with respect to the liquid feeding direction, v: flow rate of the multiphase plug in the space SP, and t: time from when the micropores M are filled with the solution SA until they are sealed with the volatile oil VO)

[0148] In this embodiment, V is set to 30 mm. 3 , S=0.28mm 2 , v=27mm / sec, t=4sec.

[0149] Reference Fig.14 as well as Fig.15 , the experimental results of the first embodiment are described. Fig.14 A diagram showing a fluorescent image acquired by a microscope. Fig.14A488ch 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.14 A647ch 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.

[0150] Fig.15 It is a graph showing the fluorescence intensity in the fluorescence image of A488ch and the fluorescence image of A647ch. From the graph, it can be seen that in the odd-numbered rounds (Rounds) of the three-phase plug for transferring air / A488 solution / oil VO, namely the 1st, 3rd, 5th, 7th, and 9th rounds, the fluorescence intensity emitted from solution SA is strong, and the fluorescence intensity emitted from solution SB is almost 0. In addition, it can be seen that in the even-numbered rounds of the three-phase plug for transferring air / A647 solution / oil VO, namely the 2nd, 4th, 6th, 8th, and 10th rounds, the fluorescence intensity emitted from solution SB is strong, and the fluorescence intensity emitted from solution SA is almost 0. That is, from the graph, 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. As Fig.14 as well as Fig.15 As shown, it is shown that the exchange of solution SA (A488 / extract) and solution SB (A647 / extract) can be continuously performed using the three-phase plug formed in the tube CB. In addition, in this example, AE-3000 is used as the volatile oil, but the same result can be obtained by using FC-43. The vapor pressure of FC-43 at 25°C is 0.17 kPa and the boiling point is 174°C.

[0151] <Second embodiment>

[0152] Reference Figures 16 to 18 , the second embodiment is described. In the second embodiment, the solution exchange using different types of solutions is verified. Specifically, in the second embodiment, the solution exchange using a surfactant solution and an organic solvent is implemented. In the second embodiment, the exchange of different types of solutions is implemented.

[0153] In the second embodiment, a closed microwell device with an anti-Tau monoclonal antibody fixed thereto is used. In addition, assuming an antigen-antibody reaction in the microwell, an antibody-immobilized microwell is used, but the reagents do not contain an antigen. Figure 4In 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.

[0154] Fig.16 : is a schematic diagram showing the process of the second embodiment. Fluorescein / HISCL cleaning solution WL is introduced into the space SP. After the space SP and each micropore M are filled with the cleaning solution WL, a fluorescent image A is acquired by photographing the micropore device MWD using a microscope 33 (process 1). A three-phase plug (starting from the front of the flow direction, air A, A647 / extracting solution (solution SA) and oil VO) is formed in the tube CB and transferred to the micropore device MWD. ​​Thus, the cleaning solution WL in the space SP is replaced by air A, and the solution SA is introduced into the space SP after the cleaning solution WL is replaced by air A, so that the cleaning solution WL is exchanged with the solution SA in the micropore M, and the solution SA is sealed with oil VO (process 2). After the solution SA is contained in the micropore M and sealed with oil VO, a fluorescent image B is acquired by photographing the micropore device MWD using a microscope 33 (process 3).

[0155] Next, a two-phase plug (from the front in the flow direction, air A and fluorescein / HISCL cleaning solution WL) is formed in the tube CB, and the two-phase plug is transferred to the microwell device MWD and introduced into the space SP. Thus, the oil VO in the space SP is replaced by the air A, and the cleaning solution WL is introduced into the space SP after the oil VO is replaced by the air A, so that the solution SA in each microwell M is replaced by the cleaning solution WL (step 4).

[0156] Next, for the solution, instead of A647 / extraction solution, A647 / ALP reaction solution (solution SB) is used to implement steps 1 to 4. After the space SP is filled with the cleaning liquid WL and the cleaning liquid WL is also contained in the micropore M, a fluorescent image C is acquired by photographing the micropore device MWD using a microscope 33 (step 1). 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 transferred to the micropore device MWD. ​​Thus, the cleaning liquid WL in the space SP is replaced by air A, and the solution SB is introduced into the space SP after the cleaning liquid WL is replaced by air A, so that the cleaning liquid WL is exchanged with the solution SB in the micropore M, and the solution SB is sealed with oil VO (steps 2 and 3). After the solution SB is contained in the micropore M and sealed with oil VO, a fluorescent image D is acquired by photographing the micropore device MWD using a microscope 33 (step 4).

[0157] Reference Fig.17 as well as Fig.18 , the experimental results of the second embodiment are described. Fig.17The figure shows an example of fluorescence images A to D. Fig.17 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 indicates that in process 1 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 indicates that in process 3 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.18 : is a graph showing the fluorescence intensity in the fluorescence image of A647ch and the fluorescence image of FLch. Fig.18 As shown, in process 1 corresponding to the 1st and 3rd rounds, the fluorescence intensity from solution SA or solution SB is almost 0, and the fluorescence intensity from cleaning liquid WL is high. In addition, in process 3 corresponding to the 2nd and 4th rounds, the fluorescence intensity from solution SA or solution SB is high, and the fluorescence intensity from cleaning liquid WL is almost 0. It can be seen from this that in process 1, there is no solution SA or solution SB in each microwell M, which is filled with cleaning liquid WL, and in processes 3 and 7, each microwell M is filled with solution SA or solution SB, and there is no cleaning liquid WL. Fig.17 and Fig.18 As shown, even when the solutions have different compositions such as A647 / extraction solution (solution SA) and A647 / reaction solution (solution SB), solution exchange can be performed with high precision.

[0158] <Third embodiment>

[0159] Reference Fig.19 as well as Fig. 20 , the third embodiment is described. In the third embodiment, solution exchange of substrate solutions using different types of enzymes (enzyme reactions in microwells caused by different enzymes) is performed. Specifically, in the third 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 third embodiment, a closed microwell device with two enzymes immobilized thereon is used.

[0160] Fig.19: is a schematic diagram showing the process of the third embodiment. The space SP in the micropore device MWD including a plurality of micropores M fixed with ALP and another plurality of micropores M fixed with β-Gal and each micropore M are filled with fluorescein / HISCL cleaning solution WL. A three-phase plug (starting from the front of the flow direction, air A, ALP fluorescent substrate solution (solution SA) and oil VO) is formed in the tube CB (process 1). The clamp valve PV is controlled to be in an open state, and the three-phase plug formed in process 1 is transferred to the micropore device MWD (process 2). As a result, the solution SA is contained in each micropore M and is sealed by the oil VO (process 3). Thereafter, the ALP fixed in the micropore M reacts with the ALP fluorescent substrate (FDP: Substrate for ALP) contained in the solution SA contained in the micropore M, and a fluorescent image A is acquired by photographing the micropore device MWD after the reaction using a microscope 33 (process 3).

[0161] After a two-phase plug is formed in the tube CB (starting from the front of the flow direction, air A and HISCL cleaning liquid WL), the pinch valve PV is controlled to be in an open state, and the formed two-phase plug is transferred to the micropore device MWD (process 4). As a result, the space SP and each micropore M are filled with the cleaning liquid WL (process 1). Next, a three-phase plug is formed in the tube CB (starting from the front of the flow direction, air A, β-Gal fluorescent substrate solution (solution SB) and oil VO), and when the pinch valve PV is controlled to be in an open state, the three-phase plug is transferred to the micropore device MWD (process 2). As a result, the solution SB is contained in the micropore M and is sealed by the oil VO (process 3). 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 (process 3). After that, a two-phase plug (air A and HISCL cleaning liquid WL from the front in the flow direction) is formed in the pipe CB and transferred to the microwell device MWD (step 4). This cycle of steps 1 to 4 is repeatedly performed.

[0162] Fig. 20 It is a graph showing the experimental results of the third embodiment. Fig. 20 (a) means Fig.19 FIG. 1 is a diagram showing an example of a fluorescent image A acquired in step 1 and a fluorescent image B acquired in step 3. Fig. 22 (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. 22 (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. 20As shown in (a) to (c), in step 1 of the reaction of ALP with the fluorescent substrate, the fluorescence intensity increases in a part of the microwells M, and in step 3 of the reaction of β-Gal with the fluorescent substrate, the fluorescence intensity increases in another part of the microwells M. That is, in the third embodiment, it is shown that continuous single-molecule detection of ALP and β-Gal can be performed.

[0163] <Fourth embodiment>

[0164] Reference Figure 21 to Figure 24 , the fourth embodiment is described. In the fourth embodiment, the confirmation of single exosome analysis (detection of single particles and quantification of particle inclusions) is implemented. Specifically, in the fourth embodiment, the dependence of exosome addition amount on the detection of Tau inclusion in exosomes is verified. Figure 4 In 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 fourth 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.

[0165] Fig.21 Schematic diagram showing the process of the fourth embodiment. Fig.21 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.21 As shown, the sample (immune complex formed by magnetic particles, exosomes and β-Gal labeled antibodies) is prepared and added to the micropore device MWD (process 1). The immune complex containing magnetic particles moves in the direction close to the magnet by approaching the micropore M through a magnet not shown in the figure. That is, the sample contained in the micropore M is subjected to a concentration (magnetic collection) process. In addition, in order to keep the immune complex in the micropore M, magnetic collection is continued in each process 2 to 11 except process 4 (process 2). A three-phase plug is formed in the tube CB (starting from the front of the flow direction, air A, β-Gal fluorescent matrix / extract and oil VO) and transferred to the micropore device MWD. ​​As a result, each micropore M is filled with β-Gal fluorescent matrix / extract and sealed with oil VO. Here, the time t from adding the solution to the micropore M until it is sealed with oil VO is, for example, about 4 seconds. In addition, in the micropore M after the immune complex is magnetically collected, β-Gal reacts with the fluorescent matrix to emit fluorescence (process 3).

[0166] Fluorescent images A and B are acquired by photographing the microporous device using a microscope 33 (step 4). Next, the extraction / capture of Tau contained in exosomes is performed. That is, Tau contained in exosomes is extracted from exosomes by the action of the surfactant contained in the β-Gal fluorescent matrix / extraction solution, and the extracted Tau contained in exosomes is captured by the anti-Tau monoclonal antibody fixed in the micropore M (step 5). 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 MWD (space crushing). 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 MWD. ​​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 contained in exosomes based on the antigen-antibody reaction is implemented (step 8).

[0167] Next, the HISCL cleaning solution WL is transferred to the microporous device MWD. ​​As a result, the space SP and each micropore M are filled with the cleaning solution WL (process 9). A three-phase plug is formed in the tube CB (starting from the front of the flow direction, air A, ALP fluorescent matrix solution and oil VO), and it is transferred to the microporous device MWD. ​​As a result, each micropore M is filled with the ALP fluorescent matrix 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 matrix and emits fluorescence (process 10). The fluorescent image C is obtained by photographing the microporous device MWD using a microscope 33 (process 11).

[0168] Fig. 22 Schematic diagram showing the analysis process of the fourth embodiment. Fig. 22As shown, the analysis process of the fourth embodiment includes the following four steps. The analysis process of the fourth embodiment includes "bead detection", "exosome detection", "Tau detection" and "data analysis" in the order of implementation. "Bead 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 "bead 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 "bead detection" based on the fluorescent image taken in step 11. "Data analysis" is a process for plotting (plot) on a scatter plot for each micropore M determined in the "bead 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.

[0169] Fig.23 as well as Fig.24 It is a figure which shows the experimental result of the fourth example. Fig.23 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.23 (a) and (b) are used in Fig.21 The detection results when the sample prepared in step 1 without containing exosomes was subjected to steps 2 to 11, Fig.23 (c) and (d) are used in Fig.21 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.23 (b) is to Fig.23 The enlarged view of the solid line frame L1 part of (a), Fig.23 (d) is to Fig.23 (c) is an enlarged view of the solid line frame L2 part. Fig.23As 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.23 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.

[0170] Fig.24 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 4 were performed. In the experimental results, λ represents the number of Tau ch(+) & Exo ch(+) of about 0.7 (i.e., Fig. 22 The number of plots in the upper right area of ​​the scatter plot of "Data Analysis" is 1×10 5 This means that Tau contained in exosomes can be detected from the immune complex that captures only one exosome. In other words, in the fourth example, it was shown that single exosome analysis can be achieved.

[0171] <Fifth Example, Comparative Example>

[0172] Reference Figure 25 to Figure 28 , the fifth embodiment and its comparative example are described. In the fifth embodiment, a multiphase plug is formed, and an exosome-encapsulated Tau measurement based on a multiphase plug liquid delivery method in which the formed multiphase plug is transferred is implemented. On the other hand, in the comparative example, a multiphase plug is not formed, and an exosome-encapsulated Tau measurement based on a liquid delivery method in which each fluid is transferred one by one in sequence is implemented. Specifically, in the fifth embodiment, the dependence of the detection of exosome-encapsulated Tau on the amount of exosomes added is verified. In Figure 4 In the closed control system 100A shown, the reagent A management unit 16A stores the ALP fluorescent substrate 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 substrate / extraction solution (containing pigment), and the HISCL cleaning solution is stored in the water tank 14. In the fifth 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.

[0173] Fig.25 FIG. 5 is a schematic diagram showing the process of the fifth embodiment. Fig.21In step 4 of the fourth embodiment shown in FIG. 1 , the fluorescent image B is obtained by photographing the microporous device using a microscope 33, but in Fig.25 The process of the fifth embodiment shown is different in that the fluorescent image is not acquired in step 4, and the other parts are substantially the same, so the detailed description of each step of the process of the fifth embodiment is omitted. In steps 3 and 10 of the fifth embodiment, the time t from the microwell M being filled with the β-Gal fluorescent matrix / extraction solution to being sealed with the oil VO is about 4 seconds.

[0174] Fig.26 FIG. 1 is a schematic diagram showing a flow chart of a comparative example of the fifth embodiment. Fig.25 In step 3 and step 10 of the fifth embodiment shown in FIG. 1 , a multiphase plug including air / FDG (FDP) solution / oil is formed and transferred, but in Fig.26 The flow of the comparative example shown is the same except that a multiphase plug is not formed in step 3 and step 10, and air, FDG (FDP) solution, and oil are sequentially transferred. The differences from the fifth embodiment will be specifically described below.

[0175] exist Fig.26 In step 3, air A is first transferred to the microporous device MWD, then the β-Gal fluorescent matrix / extracting solution is transferred to the microporous device MWD, and finally the oil VO is transferred to the microporous device MWD. ​​Thus, each micropore M is filled with the β-Gal fluorescent matrix / extracting solution and sealed with the oil VO. Here, the time t from the filling of the micropore M with the β-Gal fluorescent matrix / extracting solution to the sealing with the oil VO is about 1 minute. In addition, Fig.26 In step 10, air A is first transferred to the microporous device MWD, then the ALP fluorescent matrix solution is transferred to the microporous device MWD, and finally the oil VO is transferred to the microporous device MWD. ​​Thus, each micropore M is filled with the ALP fluorescent matrix solution and sealed with the oil VO. Here, the time t from the micropore M being filled with the ALP fluorescent matrix solution to being sealed with the oil VO is about 1 minute.

[0176] Fig. 27 It is a diagram showing the comparison results between the fifth embodiment and the comparative example. Fig. 27 is Fig.25 Step 1 of the fifth embodiment and Fig.26 The detection results when steps 2 to 11 were performed using the sample prepared so as to contain 16 μg of exosomes per sample in the sample preparation in step 1 of the comparative example. Here, the average exosome capture amount λ per magnetic particle was "112". Fig. 27The vertical axis represents the ratio of the number of exosome-positive (Exo ch(+)) and Tau-positive pores (Tau ch(+)) to the number of detected magnetic particles. In the fifth embodiment, it can be seen that the number of exosome-positive (Exo ch(+)) and Tau-positive pores (Tau ch(+)) increased compared with the comparative example. In other words, after the micropores M are filled with FDG solution, the oil VO is used to seal for a short time, so that Tau extracted from exosomes can be captured in the micropores.

[0177] Fig.28 It is a diagram showing the contents of the investigation of the comparison results between the fifth embodiment and the comparative example. Fig.28 (a) is a diagram showing an example of the inside of the micropore device in the fifth embodiment. Fig.28 (b) is a diagram showing an example of the appearance of the microporous device in the comparative example. Fig.28 As shown in (a), in the fifth embodiment, it can be seen that by setting the time from filling the micropores M with the FDG solution to sealing to less than 4 seconds, Tau can be captured in the same micropores M as exosomes. Fig.28 As shown in (b), in the comparative example, it can be seen that by setting the time from filling the micropores M with the FDG solution to sealing the micropores M to about 1 minute, the number of cases where Tau cannot be captured in the same micropores M as exosomes increases.

[0178] <Other embodiments>

[0179] 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 liquid delivery method, which is a liquid delivery method for delivering liquid to a device having a plurality of micropores formed on a substrate, comprising: A step of pre-forming, at a position different from the device, a multiphase plug having a solution filling the plurality of micropores and an immiscible liquid immiscible with the solution and used to seal the plurality of micropores filled with the solution, arranged in sequence from the front in the flow direction; as well as and transferring the multi-phase plug to the device.

2. The liquid delivery method according to claim 1, wherein: In the device, a space is formed between the substrate on which the plurality of micropores are formed and a second substrate disposed above the substrate. The step of transferring the multi-phase plug to the device includes the step of transferring the multi-phase plug to the space of the device.

3. The liquid delivery method according to claim 2, wherein: The multiphase plug is transferred to the space of the device, so that after the plurality of microwells are filled with the solution, the plurality of microwells are respectively sealed with the immiscible liquid.

4. The liquid delivery method according to claim 1, wherein: The volume of the solution in the multiphase plug is set based on the time from when a prescribed microwell among the plurality of microwells is filled with the solution until it is sealed with the immiscible liquid.

5. The liquid delivery method according to claim 4, wherein: The volume (V) of the solution is actually set by the following formula, V=S×v×t Wherein, S is the cross-sectional area of ​​the space relative to the liquid delivery direction, v is the flow rate of the multiphase plug, and t is the time from when the plurality of micropores are filled with the solution until they are sealed with the immiscible liquid.

6. The liquid delivery method according to claim 5, wherein: The t is less than 4 seconds.

7. The liquid delivery method according to claim 5, wherein: The t is less than 1 second.

8. The liquid delivery method according to claim 2, wherein: The volume of the immiscible liquid in the multiphase plug is greater than or equal to the volume of the space of the device.

9. The liquid delivery method according to claim 1, wherein: The multiphase plug includes air, the solution, and the immiscible liquid arranged in sequence from the front in the flow direction.

10. The liquid delivery method according to claim 1, wherein: The position different from the device includes a predetermined position in a tube connected to the liquid delivery port of the device.

11. The liquid delivery method according to claim 10, wherein: The step of forming the multiphase plug includes the steps of introducing the immiscible liquid into the tube and replacing the immiscible liquid at the front of the flow direction of the introduced immiscible liquid with the solution.

12. The liquid delivery method according to claim 11, wherein: The step of forming the multiphase plug further includes the step of replacing the solution at the front of the flow direction of the solution introduced by replacing the immiscible liquid with air.

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

14. A digital measurement method, comprising: A step of pre-forming, at a position different from the device, a multiphase plug having a solution filling the plurality of micropores and an immiscible liquid immiscible with the solution and used to seal the plurality of micropores filled with the solution, arranged in sequence from the front in the flow direction; The step of transferring the multi-phase plug to the device; as well as A step of performing digital measurement in the plurality of microwells containing the solution and sealed with the immiscible liquid by transferring the multiphase plug.

15. The digital determination method according to claim 14, wherein: Before the step of transferring the multiphase plug to the device, the method further includes the step of introducing biologically derived particles into the micropores. The solution includes a substance for detecting surface molecules of the particles, The digital assay is an assay for detecting surface molecules of the particle.

16. The digital determination method according to claim 14, further comprising: before the step of transferring the multiphase plug to the device, introducing particles derived from a biological organism into the micropores; A step of pre-forming, at a location different from the device, a first multiphase plug in which the solution contains a substance for detecting surface molecules of the particle and a substance for destroying the particle; The step of transferring the first multi-phase plug to the device; A step of performing a first measurement in the microwell to detect surface molecules of the particle; A step of pre-forming, at a location different from the device, a second multiphase plug in which the solution contains a substance for detecting inclusions of the particle; a step of transferring the second multi-phase plug to the device; and A step of performing a second measurement for detecting contents of the particle in the microwell.

17. The digital determination method according to claim 14, wherein: The multiphase plug includes air, the solution, and the immiscible liquid arranged in sequence from the front in the flow direction.

18. The digital determination method according to claim 14, wherein: The position different from the device includes a predetermined position in a tube connected to the liquid delivery port of the device.

19. The digital determination method according to claim 14, wherein: The immiscible liquid is oil having a vapor pressure of 0.15 kPa or more at 25°C.

20. A digital measuring device, which is a digital measuring device for performing digital measurements, comprising: A device having a plurality of micro-holes formed on a substrate; a fluid management unit connected to the liquid delivery port of the device, forming a multiphase plug having a solution filling the plurality of micropores and an immiscible liquid immiscible with the solution and used for sealing the plurality of micropores filled with the solution in sequence arranged from the front in the flow direction, and transferring the multiphase plug to the device; as well as A detector detects the object to be measured in the plurality of microwells that contain the solution and are sealed with the immiscible liquid by the transfer of the multiphase plug.

21. The digital measuring device according to claim 20, wherein: The fluid management unit includes a tube into which the immiscible liquid is introduced, and a portion of the immiscible liquid at the front in the flow direction of the introduced immiscible liquid is replaced with the solution.

22. The digital measuring device according to claim 21, wherein: The fluid management unit further replaces the solution at the front of the flow direction of the solution introduced into the tube by replacing the immiscible liquid with air.

23. The digital measuring device according to claim 20, wherein: The fluid management unit includes: a solution management unit for managing the solution; an immiscible liquid management unit for managing the immiscible liquid; a tube connected to the solution management unit and the immiscible liquid management unit; and a pump unit for injecting the solution in the solution management unit and the immiscible liquid in the immiscible liquid management unit into the tube.

Images