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WO2025142891A1 - Système d'analyse d'échantillon liquide et micropuce - Google Patents

Système d'analyse d'échantillon liquide et micropuce Download PDF

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Publication number
WO2025142891A1
WO2025142891A1 PCT/JP2024/045590 JP2024045590W WO2025142891A1 WO 2025142891 A1 WO2025142891 A1 WO 2025142891A1 JP 2024045590 W JP2024045590 W JP 2024045590W WO 2025142891 A1 WO2025142891 A1 WO 2025142891A1
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WIPO (PCT)
Prior art keywords
liquid sample
pipette
flow path
dispensing
pipette tip
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Pending
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PCT/JP2024/045590
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English (en)
Japanese (ja)
Inventor
一雄 酒居
蒔人 萱野
朋子 和田
和也 細川
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Zacros
Zacros Corp
Original Assignee
Zacros
Zacros Corp
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Publication date
Application filed by Zacros, Zacros Corp filed Critical Zacros
Publication of WO2025142891A1 publication Critical patent/WO2025142891A1/fr
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
    • G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N37/00—Details not covered by any other group of this subclass

Definitions

  • the present invention relates to an analytical system and a microchip for liquid samples.
  • liquid samples such as reagents and specimens are poured into microfluidic chips and reacted with each other (see, for example, Patent Document 1).
  • the liquid sample used in the reaction is introduced into the microfluidic chip using, for example, a dispensing pipette (also simply called a pipette) with a pipette tip attached to the end for aspirating and discharging the liquid sample.
  • a dispensing pipette also simply called a pipette
  • a pipette tip attached to the end for aspirating and discharging the liquid sample.
  • the present invention was made in consideration of these circumstances, and its purpose is to provide a technology that ensures the stability of the delivery of the liquid sample introduced through a pipette tip.
  • the liquid sample analysis system includes a pipette tip that aspirates and ejects a liquid sample, a dispensing pipette with the pipette tip attached to its tip and having a space for accommodating the liquid sample aspirated through the pipette tip, a connection section that holds the dispensing pipette with the pipette tip attached, a microchip having a first flow path formed therein that allows the liquid sample introduced from the dispensing pipette to flow into a measurement section for measuring the characteristics of the liquid sample, and a control unit that is connected to the dispensing pipette and controls the flow rate of the liquid sample aspirated and ejected through the pipette tip, and controls the delivery of the liquid sample introduced through the first flow path to the measurement section.
  • This feature ensures stable delivery of the liquid sample introduced through the pipette tip.
  • the microchip may be provided with a filter that maintains the ventilation pressure of the liquid sample delivered to the measurement unit through the first flow path within a certain range.
  • the microchip may also be provided with an air hole connected to the measurement unit through the second flow path, and a sensor that detects that the delivered liquid sample has reached a predetermined area between the measurement unit and the air hole, and the control unit may control a mechanism for discharging the liquid sample from the dispensing pipette so as to stop discharging the liquid sample contained in the dispensing pipette based on a detection signal output from the sensor indicating that the delivered liquid sample has reached the predetermined area.
  • Another aspect of the present invention is a microchip that includes a connection section that holds a dispensing pipette with a pipette tip attached to its tip that aspirates and ejects a liquid sample and has a space for accommodating the liquid sample aspirated through the pipette tip, a flow path that directs the liquid sample introduced from the dispensing pipette held in the connection section into a measurement section for measuring the characteristics of the liquid sample, and a filter that maintains within a certain range the air pressure of the liquid sample delivered to the measurement section through the flow path.
  • Another aspect of the present invention is a microchip that includes a connection section that holds a dispensing pipette with a pipette tip attached to its tip for aspirating and dispensing a liquid sample and having a space for accommodating the liquid sample aspirated through the pipette tip, a first flow path that directs the liquid sample introduced from the dispensing pipette held in the connection section into a measurement section for measuring the characteristics of the liquid sample, an air hole that connects the measurement section to the second flow path, and a sensor that detects when the delivered liquid sample reaches a predetermined area between the measurement section and the air hole.
  • FIG. 1 is a diagram showing a schematic configuration of an analysis system according to an embodiment.
  • FIG. 2 is a diagram illustrating a usage form of the pipette tip according to the embodiment.
  • FIG. 3 is a diagram illustrating a microfluidic chip according to an embodiment.
  • FIG. 4 is a diagram illustrating an air bubble removal structure provided in a flow channel of the microfluidic chip according to the embodiment.
  • FIG. 5 is a diagram for explaining a form that enables tilted and vertical use of the microfluidic chip according to the embodiment.
  • FIG. 6 is a diagram illustrating an air bubble removal structure in a form that allows the microfluidic chip according to the embodiment to be used in an inclined or vertical position.
  • FIG. 1 is a diagram showing a schematic configuration of an analysis system according to an embodiment.
  • FIG. 2 is a diagram illustrating a usage form of the pipette tip according to the embodiment.
  • FIG. 3 is a diagram illustrating a microfluidic chip according to an embodiment
  • FIG. 7 is a table showing an example of a control sequence for a dispensing pipette during testing of the analysis system according to the embodiment.
  • FIG. 8 is an example of a graph showing the results of absorbance measurements.
  • FIG. 9 is a diagram illustrating a schematic configuration of an analysis system according to the second embodiment.
  • FIG. 10 is a top view illustrating the microfluidic chip according to the second embodiment.
  • FIG. 11 is a table showing an example of a control sequence for the dispensing pipette in the second embodiment.
  • FIG. 12 is a top view illustrating an example of the microfluidic chip according to the fourth embodiment.
  • FIG. 13 is a top view illustrating another example of the microfluidic chip according to the fourth embodiment.
  • FIG. 14 is a diagram illustrating a microfluidic chip according to a modified example.
  • drawings referred to in the following description merely show the shapes, sizes, and positional relationships in a schematic manner to enable the contents of the present invention to be understood.
  • the present invention is not limited to the shapes, sizes, and positional relationships illustrated in each drawing.
  • Fig. 1 is a diagram showing a schematic configuration of an analysis system 1 according to this embodiment.
  • the analysis system 1 illustrated in Fig. 1 is a system that measures and analyzes the reaction of a small amount of liquid sample, such as a reagent or specimen, introduced into a microfluidic chip 30 by a dispensing pipette 20.
  • the analysis system 1 includes a control unit 10, a dispensing pipette (also simply called a pipette) 20, and a microfluidic chip 30.
  • the control unit 10 and the dispensing pipette 20 are connected via a predetermined interface.
  • the memory stores, for example, an operating system (OS), various programs, various tables, and the like.
  • OS operating system
  • the delivery of the liquid sample in the microfluidic chip 30 is described as being controlled by the control unit 10 connected to the dispensing pipette 20, but the control unit 10 and the dispensing pipette 20 may be configured as an integrated unit. That is, the dispensing pipette 20 includes a microcomputer chip or the like that executes the functions of the control unit 10 within its housing.
  • the dispensing pipette 20 may be configured to control the delivery of liquid samples according to a program prestored in the memory of the microcomputer chip.
  • the control unit 10 controls various parameters related to the delivery of the liquid sample, such as flow rate, time, and flow rate, in response to the suction and discharge operations of the dispensing pipette 20.
  • various parameters related to the delivery of the liquid sample such as flow rate, time, and flow rate, in response to the suction and discharge operations of the dispensing pipette 20.
  • the microfluidic chip 30 into which the liquid sample has been introduced it becomes possible to stabilize the delivery of the liquid through the flow channel 32 to the measurement section 34, which is an area for analyzing and evaluating the components, properties, etc. of the liquid sample.
  • stable delivery of the liquid can be provided even if the amount of liquid sample used for testing is small (e.g., 5 to 20 ⁇ L).
  • the dispensing pipette 20 has a pipette body 21 that contains a liquid sample, and a pipette tip 22 that is attached to the tip of the pipette body 21.
  • the pipette tip 22 has a fixing part 23 that fixes the outer periphery of the inscribed pipette body 21, and a fitting part 24.
  • the fitting part 24 is formed with a convex structure, such as a barbed structure that protrudes outward from the circumferential surface, and is fitted into a fitting structure 31a, such as a fitting groove, formed on the inner circumferential surface of the connection part 31 on the end side where the pipette tip 22 is attached.
  • the dispensing pipette 20, with the pipette tip 22 attached to the tip of the pipette body 21, is held in the microfluidic chip 30 via a cylindrical connection part 31 provided on the microfluidic chip 30.
  • the connection part 31 is a structure for mounting and holding the dispensing pipette 20 containing a liquid sample on the microfluidic chip 30.
  • the inner circumferential surface of the connection part 31 on the end side where the pipette tip 22 is mounted is provided with a fitting structure 31a, such as a fitting groove, that fits with a return structure (convex structure) that protrudes outward from the circumferential surface of the fitting part 24.
  • the tip part of the fitting part 24 of the pipette tip 22 held in the microfluidic chip 30 through the connection part 31 extends through the cylindrical internal space and into the area where the liquid sample is introduced.
  • FIG. 2 is a diagram explaining how the pipette tip 22 is used.
  • FIG. 2 illustrates an example of how the microfluidic chip 30 is used with a sample Z1 collected through the pipette tip 22.
  • the pipette tip 22 has an anti-contamination filter 25 provided in the internal space that contains the aspirated sample Z1.
  • the pipette body 21 of the dispensing pipette 20 is attached to the pipette tip 22.
  • the outer periphery of the pipette body 21 is fixed to the pipette tip 22 by the fixing part 23 of the pipette tip 22, which is inscribed in the fixing part 23.
  • the dispensing pipette 20 to which the pipette tip 22 is attached aspirates a predetermined amount (e.g., about 5 to 20 ⁇ L) of sample Z1 contained in a container by a suction operation.
  • the aspirated sample Z1 is contained, for example, in the internal space at the tip of the pipette tip 22, where the tip and anti-contamination filter 25 are provided (FIG. 2(b)).
  • pipette body 21 of dispensing pipette 20 is detached from attached pipette tip 22.
  • Pipette body 21 is inserted and removed from fixing part 23 that fixes the inscribed outer periphery, and pipette tip 22 fitted to connection part 31 via fitting part 24 maintains an engagement with microfluidic chip 30.
  • the pipette body 21 When the pipette body 21 is inserted or removed from the fixed part 23, there may be specimen Z1 remaining in the internal space at the tip of the pipette tip 22 without being introduced into the microfluidic chip 30.
  • the specimen Z1 remaining in the internal space at the tip may backflow or scatter to the outside due to the negative pressure generated when the pipette body 21 is inserted or removed, which may cause environmental contamination of the surroundings.
  • the pipette tip 22 is provided with a contamination prevention filter (contamination prevention filter 25), which can prevent environmental contamination of the specimen Z1 caused by the negative pressure generated when the pipette body 21 is inserted or removed.
  • the microfluidic chip 30 is discarded together with the pipette tip 22 fitted into the connection part 31.
  • the dispensing pipette 20 equipped with the pipette tip 22, for example, as a pretreatment of the specimen Z1, it is possible to dilute the specimen Z1 and mix it with a diluent, extract nucleic acid, mix it with a reagent, etc.
  • these pretreatments can be appropriately combined.
  • the dispensing pipette 20 aspirates 10 ⁇ L (microlot) of specimen Z1 through the pipette tip 22, and then further aspirates 90 ⁇ L of diluent, thereby promoting the mixing of the specimen Z1 and the diluent by the aspirating operation, and enabling the specimen Z1 to be diluted 10 times by the diluent.
  • the pipette tip 22 may have, for example, a silica membrane that can specifically bind to nucleic acid in the presence of a high salt concentration.
  • the pipette tip 22 including the silica membrane By attaching the pipette tip 22 including the silica membrane to the dispensing pipette 20, the nucleic acid purified from the aspirated measurement specimen Z1 can be introduced into the microfluidic chip 30.
  • the sample Z1 to be measured include saliva, blood, and nasal swabs that have been treated with a lysis buffer used for nucleic acid extraction.
  • the dispensing pipette 20 After dispensing the sample into the microfluidic chip 30, the dispensing pipette 20 again aspirates and dispenses a cleaning solution containing ethanol, thereby washing the nucleic acids adsorbed to the silica membrane.
  • the liquid sample introduced into the microfluidic chip 30 through the pipette tip 22 includes liquid samples such as reagents and specimens, reactants to react with the liquid samples, cleaning solutions, etc.
  • the liquid sample is not particularly limited as long as it is a sample that can be passed through the microfluidic chip 30. Examples include liquid samples obtained from living organisms such as blood and urine or diluted solutions thereof, extracts from living organisms such as plants and animals, naturally occurring water such as rivers, oceans, and rainfall, cosmetics, cleaning solutions, waste liquids, etc.
  • the components in the sample are also not particularly limited, and examples include proteins, nucleic acids, low molecular weight compounds, sugars, etc.
  • the microfluidic chip 30 is a device in which a groove for a flow path 32 is formed for sending a liquid sample introduced into a specified area of the connection part 31 to a measurement part 34.
  • a device is produced, for example, by bonding a base material having a groove formed on its surface that serves as the flow path to a film material or base material using an adhesive or the like.
  • FIG. 3 is a diagram illustrating the microfluidic chip 30 according to this embodiment.
  • a top view of the approximately rectangular microfluidic chip 30 is illustrated.
  • a recessed region 31b which is a recess for placing a liquid sample introduced in the form of a droplet, is formed in a portion of the microfluidic chip 30 that faces the tip of the fitting portion 24 of the dispensing pipette 20 held by the connection portion 31.
  • a substantially circular measuring portion 34 is formed on the longitudinal end side of the microfluidic chip 30 that faces the recessed region 31b, and a groove of the flow path 32 that connects the recessed region 31b and the measuring portion 34 is formed between the recessed region 31b and the measuring portion 34.
  • the cross-sectional shape of the groove that constitutes the flow path 32 may be any of a concave, U-shaped, and V-shaped.
  • a reaction substrate that reacts with the liquid sample introduced into the recessed region 31b by the dispensing pipette 20 is applied to the flow path 32.
  • the flow path 32 connecting the recessed region 31b and the measurement section 34 is a long and narrow flow path in which multiple roughly U-shaped flow paths 32 are connected when viewed from above, and constitutes a heating section 33 in which heating is performed to accelerate the reaction of a liquid sample, for example.
  • a heating mechanism such as a heater that heats the introduced liquid sample is provided below the flow path 32 in which the heating section 33 is formed.
  • the liquid sample introduced into the flow path 32 via the heating section 33 can be heated in multiple stages depending on the evaluation of its characteristics and properties.
  • the flow path 32 constituting the heating section 33 and the measurement section 34 are provided with bubble removal structures 32a, 34a for capturing bubbles that occur during the processing of the liquid sample introduced into the microfluidic chip 30.
  • the measurement section 34 is, for example, an area for analyzing and evaluating the components and properties of the liquid sample that reacts with a reaction substrate, etc.
  • the measurement section 34 may be provided with, for example, a stirrer for stirring the liquid sample sent through the heating section 33. The operation of the stirrer is controlled, for example, by a stirring mechanism that utilizes magnetic force.
  • the microfluidic chip 30 by providing an air bubble removal structure 32a in the flow path 32 that constitutes the heating section 33, it is possible to suppress the introduction of air bubbles that occur during the processing of the liquid sample sent to the measurement section 34. Also, by providing an air bubble removal structure 34a in the measurement section 34, it is possible to suppress interference with the inspection and evaluation of the liquid sample, for example, caused by air bubbles that occur during the stirring process of the liquid sample flowing into the measurement section 34. In the microfluidic chip 30, by suppressing the introduction of air bubbles, it is possible to increase the stability of the delivery of the introduced liquid sample.
  • the liquid sample introduced into the flow path 32 is heated through the heating unit 33 to a temperature range of 90 to 98 degrees.
  • the liquid sample introduced into the flow path 32 is heated through the heating unit 33 to a temperature range of 60 to 70 degrees. In either case, it is desirable to generate sufficient bubbles by allowing the heated liquid sample to remain in the area where the heating unit 33 is provided for about 3 to 10 minutes. Bubbles generated during the processing of the liquid sample are captured through the air bubble removal structure 32a formed in the flow path 32, and the liquid sample from which the bubbles have been removed is introduced into the measurement unit 34.
  • the microfluidic chip 30 is composed of a long and thin channel that is connected to multiple meandering, roughly U-shaped channels 32, which relatively increases the contact area between the channel wall surface in the heating section 33 and the introduced liquid sample, thereby increasing the degree of bubble generation. Furthermore, by providing multiple repeating structures for trapping bubbles in the region of the heating section 33, such as a meandering channel 32, a bubble removal structure 32a, a meandering channel 32, and a bubble removal structure 32a, the bubble capture rate can be improved.
  • FIG. 4 is a diagram illustrating the air bubble removal structure 32a provided in the flow path 32.
  • FIG. 4(a) illustrates an enlarged top view of a partial region of the flow path 32 in which the air bubble removal structure 32a is formed.
  • FIG. 4(b) illustrates a cross-sectional view taken along line B-B# in FIG. 4(a)
  • FIG. 4(c) illustrates a cross-sectional view taken along line C-C# in FIG. 4(a).
  • the air bubble removal structure 32a is formed in a straight flow path 32 that connects the flow paths that meander in an approximately U-shape.
  • the air bubble removal structure 32a is formed as a convex structure that protrudes from the surface side of the microfluidic chip 30 (the surface side where the connection part 31 is provided) in the region of the heating part 33.
  • the protruding height of the convex structure that protrudes from the surface side of the air bubble removal structure 32a is linearly inclined so that the protruding height is relatively high in the flow direction from the upstream side to the downstream side of the flow channel 32.
  • bubble Z4 illustrates a cross-sectional view of the measurement unit 34 taken along line A-A#.
  • the air bubble removal structure 34a is formed on the periphery of the measurement unit 34 as a convex structure that protrudes toward the surface side of the microfluidic chip 30.
  • a flat portion 34b is formed, which is a measurement area for measuring the turbidity, absorbance, and other characteristics of the introduced liquid sample.
  • the air bubble removal structure 34a provided in the measurement unit 34 is formed on the periphery, surrounding the flat portion 34b and protruding toward the surface side.
  • the microfluidic chip 30, in which the flow path 32 for introducing the liquid sample is formed may be used in a manner in which it is inclined with respect to the horizontal plane, or in a manner in which it is inverted vertically.
  • the short side of the microfluidic chip 30 on which the connection part 31 is provided is positioned vertically downward, and the short side on which the measurement part 34 is provided is positioned upward, in a manner in which it is inclined with respect to the horizontal plane, or in a manner in which it is inverted vertically.
  • the liquid sample introduced into the microfluidic chip 30 is transferred from the lower side to the upper side in the vertical direction through the flow path 32.
  • the structure for trapping air bubbles is different from the form in which the microfluidic chip 30 is placed horizontally, as described in Figures 3 and 4.
  • FIG. 5 is a diagram for explaining a microfluidic chip 30 in a form that allows the above-mentioned inclined use and vertical use.
  • a form of a microfluidic chip 30 that allows reaction measurement of multiple channels (3 channels x 4 blocks) is exemplified.
  • a flow path 32 is formed from the recessed region 31b to each measurement unit 34 provided in the multiple channels, and heating units 33a and 33b are provided in two stages on the lower side (recessed region 31b side) and upper side (measurement unit 34 side) of the flow path 32.
  • Each of the heating units 33a and 33b enables heating of the liquid sample introduced into the flow path 32 at an individual temperature.
  • the flow path 32 connecting the heating unit 33a and the heating unit 33b, and the flow path 32 connecting the heating unit 33b and the measurement unit 34 of the multiple channels are each provided with an air bubble removal structure 32b for capturing air bubbles generated during the processing of the introduced liquid sample.
  • FIG. 6 is a diagram illustrating the bubble removal structure provided in the microfluidic chip 30 in a form that allows for tilted and vertical use.
  • FIG. 6(a) is an enlarged view of the circled area Z3 in FIG. 5, illustrating the bubble removal structure 34c provided in the upper region of each measurement section of the three channels.
  • FIG. 6(b) is an enlarged view of the circled area Z2 in FIG. 5, illustrating the bubble removal structures 32b and 32c formed in the flow path 32 of the heating section 33b.
  • air bubbles accumulate on the upper side of the flow path 32 that meanders in an approximately U-shape, and on the upper side of the measurement unit 34 in the vertical direction.
  • air bubble removal structure 32c that expands the flow path space between the upper and lower flow path walls in the vertical direction can be provided to increase the efficiency of air bubble accumulation.
  • the air bubble removal structure 32b provided in the flow path 32 that connects the heating unit 33a and the heating unit 33b.
  • the air bubble removal structure 32b can accumulate air bubbles on the upper flow path wall side by making the flow path wall on the upper side of the vertical direction of the flow path 32 into an approximately triangular shape that expands upward.
  • an air bubble removal structure 34c can be formed that collects air bubbles on the upper side of the flow channel wall in the vertical direction.
  • the measurement unit 34 is provided with a filter 35 that maintains the air pressure of the flow path 32 from the recessed area 31b to the measurement unit 34 within a certain range of pressure.
  • the filter 35 is provided, for example, in the measurement unit 34 at a position opposite the inlet through which the liquid sample flows in after passing through the heating unit 33.
  • the filter 35 allows air to pass but does not allow the liquid sample to pass through, so that the air pressure between the recessed area 31b and the measurement unit 34 is maintained within a certain range. In other words, when the liquid sample reaches the filter 35 provided in the measurement unit 34 at a position opposite the inlet through which the liquid sample flows, the flow of liquid through the flow path 32 from the recessed area 31b to the measurement unit 34 is stopped.
  • the liquid sample is uniformly introduced into each of the multiple measurement sections.
  • the liquid sample is uniformly introduced into the three measurement sections 34 of each block arranged on the upper side in the vertical direction and the three measurement sections 34 of each block arranged on the lower side in the vertical direction.
  • the measurement section 34 With a filter 35, the liquid sample can be stopped at the point where the liquid sample reaches the measurement section 34.
  • the liquid sample can be continuously introduced in sequence to each channel connected to the branched path that has not yet reached the liquid sample.
  • the liquid sample can be uniformly introduced into each measurement section 34.
  • the filter 35 is provided, but instead of the filter 35, a sensor that detects when the delivered liquid sample reaches a predetermined area (e.g., the measurement unit 34) may be provided.
  • a sensor that detects when the delivered liquid sample reaches a predetermined area (e.g., the measurement unit 34) may be provided.
  • sensors include a liquid sensor or electrode sensor that detects the liquid level, and a pressure sensor that detects the air pressure in the flow path 32 from the recessed area 31b to the measurement unit 34.
  • the control unit 10 of the analysis system 1 detects, for example, using a liquid sensor or an electrode sensor, that the liquid sample delivered through the flow path 32 has reached a predetermined area.
  • the control unit 10 may perform control to stop the delivery of the liquid sample based on the arrival state indicated by a binary status signal indicating an active status or inactive status output from the liquid sensor or electrode sensor.
  • the control unit 10 controls the dispensing pipette 20 to continue delivery when the status signal output from the electrode sensor is an inactive status indicating a not yet reached state, and to stop delivery otherwise.
  • a liquid sensor or an electrode sensor it becomes possible to stop the delivery of the delivered liquid sample at the target location (the desired location of the flow path 32 in the microfluidic chip 30).
  • the control unit 10 of the analysis system 1 can, for example, use a pressure sensor to detect the air pressure in the flow path 32 leading from the recessed region 31b to the measurement unit 34, and control the liquid transfer to stop when the detected pressure reaches a certain pressure (threshold value).
  • a pressure sensor by using a pressure sensor, it becomes possible to detect a constant increase in the air pressure in the flow path 32 leading from the recessed region 31b to the measurement unit 34 and stop the liquid transfer to the microfluidic chip 30.
  • Fig. 7 is a table showing an example of a control sequence for the dispensing pipette 20 during the test
  • Fig. 8 is an example of a graph showing the measurement results of absorbance.
  • plasma and a diluent were used as liquid samples, and the liquid samples were introduced into the microfluidic chip 30 using the dispensing pipette 20 equipped with a pipette tip 22. Thereafter, the delivery of the liquid sample was controlled, and the change in absorbance of the liquid sample delivered to the measurement unit 34 was measured.
  • plasma was aspirated using a dispensing pipette 20 equipped with a pipette tip 22.
  • the plasma was aspirated at a flow rate of 3 ⁇ L/sec for 1 second, resulting in aspirating a flow rate of 3 ⁇ L (step 1).
  • the diluent was aspirated at a flow rate of 50 ⁇ L/sec for 3 seconds, resulting in aspirating a flow rate of 150 ⁇ L (step 2).
  • the dispensing pipette 20 equipped with the pipette tip 22 was then fixed to the connection part 31 of the microfluidic chip 30, and measurement was started.
  • the diluted plasma sample was introduced into the recessed area 31b through the pipette tip 22 of the dispensing pipette 20 fixed to the connection part 31, and the liquid was sent to the measurement part 34 through the flow path 32 formed in the microfluidic chip 30.
  • the diluted plasma sample was discharged at a flow rate of 50 ⁇ L/sec, and the total amount (153 ⁇ L) was discharged (step 3).
  • the diluted plasma sample is sent from the recessed area 31b through the flow path 32, reaches the heating unit 33, where the heating temperature is set to 37°C, and is heated.
  • the heating temperature is set to 37°C, and is heated.
  • the air in the dispensing pipette is discharged through the dispensing pipette 20.
  • the air is discharged at a flow rate of 50 ⁇ L/sec and a flow rate of 153 ⁇ L (steps 4 and 5).
  • the plasma sample heated by the heating unit 33 provided in the flow path 32 of the microfluidic chip 30 is sent and injected into the measurement unit 34.
  • Figure 8 shows an absorbance curve, where the change in absorbance is the result of binding between the plasma sample and the anti-D-dimer antibody immobilized on the latex beads, and is exemplified as a pseudo-reaction curve Z10.
  • the vertical axis of the graph shown in Figure 8 represents absorbance
  • the horizontal axis represents time (minutes).
  • the absorbance (transmittance) of the plasma sample by the latex beads changes from approximately 1.1 to approximately 1.43 over a period of 5 minutes from the start of measurement. Over the 5-minute period from 5 to 10 minutes, the change in absorbance becomes gradual, shifting from approximately 1.43 to approximately 1.5. After 10 minutes, the change in absorbance was confirmed to be a slight increase, shifting around 1.5.
  • the microfluidic chip 30 of the first embodiment is configured to maintain the air pressure of the flow path 32 from the recessed region 31b to the measurement unit 34 within a certain range by the filter 35 provided at a portion facing the inlet of the measurement unit 34 through which the liquid sample flows.
  • the microfluidic chip 30 is provided with an air vent hole 36, which is an air vent hole, instead of the filter 35, and a sensor 37 is installed in the flow path 38 between the measurement unit 34 and the air vent hole 36.
  • the sensor 37 is, for example, a liquid sensor or an electrode sensor that detects that the delivered liquid sample has reached a predetermined region.
  • control unit 10 of the analysis system 1 can control the dispensing pipette 20 to stop discharging the liquid sample based on a binary status signal (detection signal) output from the sensor 37.
  • a binary status signal detection signal
  • the analysis system 1 can perform sample measurement with improved accuracy.
  • FIG. 9 is a diagram showing a schematic configuration of the analysis system 1 according to the second embodiment
  • FIG. 10 is a top view illustrating the microfluidic chip 30 according to the second embodiment.
  • the microfluidic chip 30 is provided with an air vent hole 36 instead of the filter 35, and a sensor 37 is installed in the flow path 38 between the measurement unit 34 and the air vent hole 36.
  • the control unit 10 of the analysis system 1 according to the second embodiment is different in that it controls the delivery and stopping of the liquid sample discharged from the dispensing pipette 20 based on a binary status signal output from the sensor 37.
  • the analysis system 1 according to the second embodiment will be described below, focusing mainly on the differences.
  • the flow path 32 is an example of the "first flow path”
  • the flow path 38 is an example of the "second flow path”.
  • the first flow path may have a storage section, a pre-treatment section, an observation section, etc.
  • the second flow path may have a waste liquid storage section, etc.
  • the control unit 10 receives a binary status signal output from the sensor 37. For example, when the status signal output from the sensor 37 is an inactive status indicating that the flow path 38 between the measurement unit 34 and the air vent hole 36 has not yet been reached, the control unit 10 controls the dispensing pipette 20 to continue discharging the liquid sample. On the other hand, when the status signal output from the sensor 37 is an active status indicating that the flow path 38 between the measurement unit 34 and the air vent hole 36 has been reached, the control unit 10 controls the dispensing pipette 20 to stop discharging the liquid sample and stops liquid transfer in the microfluidic chip 30.
  • the air vent hole 36 is formed on the longitudinal end side facing the recessed area 31b of the approximately rectangular microfluidic chip 30.
  • the sensor 37 is installed in the flow path 38 between the measurement unit 34 and the air vent hole 36.
  • the liquid sample sent from the dispensing pipette 20 to the microfluidic chip 30 reaches the measurement unit 34 while pressing the air in the flow path 32. Gas such as air present in the flow path 32 pressed by the liquid sample is released to the outside of the microfluidic chip 30 through the air vent hole 36.
  • the liquid sample that has reached the measurement unit 34 reaches the installation position of the sensor 37 installed in the flow path 38 between the measurement unit 34 and the air vent hole 36.
  • the sensor 37 detects the liquid sample that has reached the installation position and transitions the status signal from an inactive status to an active status.
  • the control unit 10 controls the dispensing pipette 20 to stop discharging the liquid sample.
  • the microfluidic chip 30 according to Example 2 the flow of the liquid sample from the recessed region 31b through the flow channel 32 to the measurement unit 34 is stopped.
  • FIG. 11 is a table showing an example of a control sequence for the dispensing pipette 20 in Example 2.
  • latex particles 50 ⁇ L
  • the latex particles sealed in the heating unit 33 are sealed in the elongated flow channel 32, so they remain stably in the flow channel without moving or scattering during storage and transportation of the microfluidic chip.
  • the processing of steps 1 to 3 is the same as in Example 1. That is, the dispensing pipette 20 equipped with the pipette tip 22 was used to aspirate plasma (3 ⁇ L), then the diluent (150 ⁇ L) was aspirated, the dispensing pipette 20 equipped with the pipette tip 22 was fixed to the connection part 31 of the microfluidic chip 30, and the measurement was started. After the measurement was started, the dispensing pipette 20 introduced the diluted plasma sample into the recessed area 31b, and started to send the diluted plasma sample to the measurement part 34 through the flow path 32 formed in the microfluidic chip 30. The diluted plasma sample was discharged at a flow rate of 50 ⁇ L/sec for approximately 3 seconds, discharging a flow rate of 153 ⁇ L.
  • the diluted plasma sample is sent from the recessed region 31b through the flow path 32, reaches the heating section 33, whose heating temperature is set to 37°C, and is heated (step 4). At this time, the latex particles sealed in the heating section 33 move to the measurement section 34 together with the plasma sample. Then, three minutes after the liquid supply is stopped in step 3, the diluted plasma sample (153 ⁇ L) is discharged through the dispensing pipette 20 (step 5). The plasma sample heated in the heating section 33 is injected into the measurement section 34 and mixed with the latex particles, starting a reaction.
  • the sensor 37 installed in the flow path 32 between the measurement section 34 and the air vent hole 36 detects the plasma sample that has reached the region, and the liquid supply of the plasma sample in the microfluidic chip 30 is stopped (step 6).
  • the liquid supply of the liquid sample in the microfluidic chip 30 may be controlled in combination with a pressure sensor, a valve, etc.
  • the transfer of liquid from the dispensing pipette 20 equipped with the pipette tip 22 to the microfluidic chip 30 can be controlled using parameters such as flow rate, time, and flow rate.
  • the transfer of the liquid sample that has reached the measurement unit 34 through the flow path 32 formed in the microfluidic chip 30 can be stopped using a binary output signal output from the filter 35 or a sensor 37 such as a liquid sensor or an electrode sensor.
  • a binary output signal output from the filter 35 or a sensor 37 such as a liquid sensor or an electrode sensor.
  • RNA extraction solution was prepared from the nasal swab using an RNA extraction kit (Loopamp Virus RNA Extraction Kit; Eiken Chemical).
  • the dispensing pipette 20 aspirated the RNA extraction solution (specimen) through the pipette tip 22, and introduced the aspirated RNA extraction solution into the recessed area 31b of the microfluidic chip 30 in which it was fitted.
  • the specimen was then sent to the heating unit 33 through the flow path 32, and stopped for three minutes.
  • the heating unit 33 was heated to 65 degrees by a heater. After three minutes had passed, the dispensing pipette 20 was controlled to start sending the liquid in the microfluidic chip 30.
  • reaction unit A (measurement unit 34, channel A)
  • reaction unit B reaction unit 34, channel B
  • Lamp method influenza test reagents Liken Chemical: A is the measurement, B is the positive control, and C is the negative control.
  • reaction zones A, B, and C are heated to 60 degrees. Downstream of reaction zones A, B, and C continues flow path 32, which is connected to filter 35. Filter 35 allows air to pass through but not liquid. Reaction zones A, B, and C are filled with the sample by liquid delivery via dispensing pipette 20, which reacts with the reagent applied to each reaction zone. Reaction zones A, B, and C were evaluated for turbidity using a semiconductor laser with a wavelength of 600 nm. In the case of a positive result, an increase in turbidity is detected for reaction zones A and B.
  • Example 4 A DNA extract was prepared from a blood sample using a DNA extraction kit (DNeasy Blood & Tissue Kit: Qiagen). As in Example 3, the dispensing pipette 20 aspirated the DNA extract (specimen) through the pipette tip 22, and introduced the aspirated DNA extract into the recessed area 31b of the fitted microfluidic chip 30.
  • the microfluidic chip 30 used in Example 4 has a form shown in FIG. 12, for example.
  • the specimen was sent to the heating section 33c through the flow path 32, and stopped for three minutes. The heating section 33c was heated to 95 degrees by a heater.
  • the dispensing pipette 20 was controlled to start sending the liquid in the microfluidic chip 30.
  • the specimen heated to a predetermined temperature (95 degrees) through the heating section 33c passed through the air bubble removal structure 32a provided in the flow path 32, and the specimen from which air bubbles had been removed was stopped at the heating section A (33d).
  • the sample is sent to the heating unit B (33e) and stopped for 30 seconds.
  • the heating unit B (33e) is heated to 55 degrees by a heater.
  • the sample is sent to the heating unit C (33f) and stopped.
  • the heating unit B (33e) is heated to 72 degrees by a heater.
  • the suction operation of the dispensing pipette 20 is controlled, and the sample that has reached the heating unit C (33f) is drawn back to the flow path 32 of the heating unit A (33d).
  • the suction and discharge operations of the dispensing pipette 20 are appropriately controlled, and the above cycle is repeated about 20 to 30 times to advance the PCR reaction.
  • the PCR amplification by each cycle can be monitored as fluorescence intensity, for example, by the fluorescence detection unit 80 provided in the flow path 32 between the heating unit A (33d) and the heating unit B (33e).
  • PCR amplification can be detected visually by lateral flow by using a microfluidic chip 30 incorporating a test slip 83 for nucleic acid chromatography, as shown in FIG. 13.
  • the sample is sent to the reagent mixing section (measurement section 34) by the dispensing pipette 20 and stopped for a certain period of time in the reagent mixing section (measurement section 34).
  • the sample may be stirred using an inserted stirrer 81, or may be stopped for several minutes to allow the sample to diffuse naturally.
  • the sample is mixed with the reagent and then further sent to reach the test slip reagent reaction section 82. After that, the sample reaches the test slip 83 for nucleic acid chromatography, and the test results can be evaluated using nucleic acid chromatography.
  • the microfluidic chip 30 is described as an instrument having a substantially rectangular shape and a groove for the flow channel 32 for sending a liquid sample from the recessed region 31b of the connection portion 31 for fixing the dispensing pipette 20 to which the pipette tip 22 is attached to the measurement portion 34.
  • a disk-shaped microchip is used in PCR, immunoassays, etc.
  • a disk shape is applied to the shape of the microfluidic chip 30 to which the dispensing pipette 20 sends liquid.
  • FIG. 14 is a diagram illustrating a microfluidic chip 50 according to a modified example.
  • FIG. 14(a) shows a top view of the microfluidic chip 50 according to the modified example
  • FIG. 14(b) shows a side view of the microfluidic chip 50.
  • the microfluidic chip 50 is disk-shaped, and a connection part 51 is formed in the center for fixing a dispensing pipette 20 to which a pipette tip 22 is attached.
  • a reagent coating that reacts with the liquid sample, a plasma separation filter, a nucleic acid extraction filter, etc. can be installed below the connection part 51.
  • the grooves of the flow paths 52 are formed radially, extending linearly from the center where the connection part 51 is provided in the circumferential direction, and the grooves of the flow paths 52 are connected to the measurement part 54 formed on the periphery. Note that the configuration illustrated in FIG. 14(a) is an example of a configuration in which four grooves of the flow paths 52 are formed so as to intersect at right angles in a cross shape at the center.
  • the dispensing pipette 20 fitted with the pipette tip 22 is attached to a connection part 51 provided at the center of a disk-shaped microfluidic chip 50 in the same manner as in Examples 1 and 2, and the liquid sample is injected into the flow path 52 and the liquid is delivered stepwise to the measurement part 54.
  • the attached dispensing pipette 20 is then detached from the connection part 51, and liquid delivery by centrifugation is continued, enabling two-stage liquid delivery. That is, in the modified embodiment, by employing a disk-shaped microfluidic chip 50, it becomes possible to perform two-stage liquid delivery: delivery by the dispensing pipette 20 with controlled flow rate, time, flow rate, etc., and delivery by centrifugation. In the modified embodiment, precise control by multiple stages of liquid delivery becomes possible.
  • the analysis system 1 can be combined with valves, liquid sensors, electrode sensors, pressure sensors, etc. to enable complex liquid transfer control.
  • liquid transfer can be performed, for example, to mix a specimen with a reagent, separate plasma from a blood specimen, or extract nucleic acid from a specimen as a pre-processing step for PCR.
  • the reagent is applied to the flow path 52, and the specimen is injected up to the site where the reagent is applied. Then, mixing can be achieved by repeatedly aspirating and discharging the specimen injected into the flow path with the dispensing pipette 20.
  • pre-processing such as separating plasma from whole blood as a pre-processing step, and then perform liquid transfer by centrifugation.

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  • Life Sciences & Earth Sciences (AREA)
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Abstract

La présente invention concerne un système d'analyse d'échantillon liquide comprenant : une pointe de pipette qui aspire et évacue un échantillon liquide ; et une pipette de distribution dont l'extrémité avant reçoit la pointe de pipette, et disposant d'un espace pour recevoir l'échantillon liquide aspiré à travers la pointe de pipette. Le système comprend une micropuce dans laquelle est formé un premier canal d'écoulement qui achemine le flux de l'échantillon liquide introduit par la pipette de distribution vers une section de mesure pour mesurer les caractéristiques de l'échantillon liquide, et une section de liaison qui maintient la liaison entre la pipette de distribution et la pointe de pipette attachée. Le système comprend une unité de commande qui est reliée à la pipette de distribution, qui régule le débit de l'échantillon liquide aspiré et évacué par la pointe de pipette, et qui commande le transfert de l'échantillon liquide introduit par le premier canal d'écoulement vers la section de mesure.
PCT/JP2024/045590 2023-12-28 2024-12-24 Système d'analyse d'échantillon liquide et micropuce Pending WO2025142891A1 (fr)

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JP2023222269A JP2025104454A (ja) 2023-12-28 2023-12-28 液体試料の分析システムおよびマイクロチップ
JP2023-222269 2023-12-28

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011027851A1 (fr) * 2009-09-07 2011-03-10 コニカミノルタホールディングス株式会社 Système d'alimentation en liquide pour micropuce, dispositif de détection d'échantillons et procédé d'alimentation en liquide pour système d'alimentation en liquide d'une micropuce
JP2015190855A (ja) * 2014-03-28 2015-11-02 凸版印刷株式会社 マイクロチップへの試薬注入量制御装置及び方法
WO2017094674A1 (fr) * 2015-12-01 2017-06-08 日本板硝子株式会社 Récipient pour amplification par pcr, dispositif de pcr et procédé de pcr
WO2017130359A1 (fr) * 2016-01-28 2017-08-03 コニカミノルタ株式会社 Procédé d'approvisionnement en liquide, et système de détection et dispositif de détection pour la mise en œuvre dudit procédé
JP2020520634A (ja) * 2017-04-20 2020-07-16 バイオフルイディカ、インコーポレイテッド 液体サンプルからバイオマーカーを単離するための流体密封性フローシステム

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011027851A1 (fr) * 2009-09-07 2011-03-10 コニカミノルタホールディングス株式会社 Système d'alimentation en liquide pour micropuce, dispositif de détection d'échantillons et procédé d'alimentation en liquide pour système d'alimentation en liquide d'une micropuce
JP2015190855A (ja) * 2014-03-28 2015-11-02 凸版印刷株式会社 マイクロチップへの試薬注入量制御装置及び方法
WO2017094674A1 (fr) * 2015-12-01 2017-06-08 日本板硝子株式会社 Récipient pour amplification par pcr, dispositif de pcr et procédé de pcr
WO2017130359A1 (fr) * 2016-01-28 2017-08-03 コニカミノルタ株式会社 Procédé d'approvisionnement en liquide, et système de détection et dispositif de détection pour la mise en œuvre dudit procédé
JP2020520634A (ja) * 2017-04-20 2020-07-16 バイオフルイディカ、インコーポレイテッド 液体サンプルからバイオマーカーを単離するための流体密封性フローシステム

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