Liquid sample analysis system and microchip

Abstract

The present invention provides a liquid sample analysis system comprising: a pipette tip that aspirates and discharges a liquid sample; and a dispensing pipette on the leading end of which the pipette tip is attached, and which has a space for accommodating the liquid sample aspirated through the pipette tip. The system comprises a microchip in which is formed a first flow channel that carries a flow of the liquid sample introduced from the dispending pipette to a measuring section for measuring the characteristics of the liquid sample, and a connecting section that holds the dispending pipette with the attached pipette tip. The system comprises a control unit which is connected to the dispensing pipette, controls the flow rate of the liquid sample aspirated and discharged through the pipette tip, and controls the transfer of the liquid sample introduced via the first flow channel to the measuring section.

Description

Liquid sample analysis system and microchip

[0001] The present invention relates to a liquid sample analysis system and a microchip.

[0002] Conventionally, in the fields of medicine, biochemistry, and the like, tests and experiments have been conducted in which a small amount of liquid sample, such as a reagent or specimen, is introduced into a microfluidic chip or the like to cause a reaction (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 referred to as a pipette) having a pipette tip attached to the tip for aspirating and discharging the liquid sample.

[0003] International Publication No. 2009 / 069656

[0004] However, when a small amount of liquid sample is used for testing, etc., the stability of the delivery of the liquid sample introduced in droplet form through a pipette tip becomes an issue. The present invention has been made in consideration of such 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.

[0005] In order to achieve the above object, one aspect of the present invention employs the following configuration: That is, a liquid sample analysis system according to one embodiment includes a pipette tip that aspirates and discharges a liquid sample, a dispensing pipette with the pipette tip attached to its tip and having a space for containing the liquid sample aspirated through the pipette tip, a connection unit that holds the dispensing pipette with the pipette tip attached, a microchip formed with a first flow path that allows the liquid sample introduced from the dispensing pipette to flow into a measurement unit that measures the characteristics of the liquid sample, and a control unit that is connected to the dispensing pipette, controls the flow rate of the liquid sample aspirated and discharged through the pipette tip, and controls the delivery of the liquid sample introduced through the first flow path to the measurement unit.

[0006] According to this feature, the stability of the delivery of the liquid sample introduced through the pipette tip can be ensured.

[0007] Here, the microchip may include 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 include an air vent connected to the measurement unit through the second flow path, and a sensor that detects when the delivered liquid sample reaches a predetermined region between the measurement unit and the air vent, and the control unit may control a mechanism for dispensing the liquid sample from the dispensing pipette to stop dispensing 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 region.

[0008] Another aspect of the present invention is a microchip comprising: a connection part for holding a dispensing pipette having 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 flow path for flowing the liquid sample introduced from the dispensing pipette held in the connection part into a measurement part for measuring the characteristics of the liquid sample; and a filter for maintaining the air pressure of the liquid sample sent through the flow path to the measurement part within a certain range.

[0009] Even in this configuration, the stability of the delivery of the liquid sample introduced through the pipette tip can be ensured.

[0010] Another aspect of the present invention is a microchip comprising: a connection part for holding a dispensing pipette having 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 for directing the liquid sample introduced from the dispensing pipette held in the connection part into a measurement part for measuring the characteristics of the liquid sample; an air vent connecting the measurement part to the second flow path; and a sensor for detecting when the transported liquid sample has reached a predetermined region between the measurement part and the air vent.

[0011] Even in this configuration, the stability of the delivery of the liquid sample introduced through the pipette tip can be ensured.

[0012] 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 pattern of a pipette tip according to an embodiment. FIG. 3 is a diagram illustrating a microfluidic chip according to an embodiment. FIG. 4 is a diagram illustrating a bubble removal structure provided in a flow channel of a microfluidic chip according to an embodiment. FIG. 5 is a diagram illustrating a configuration that allows tilted and vertical use of a microfluidic chip according to an embodiment. FIG. 6 is a diagram illustrating a bubble removal structure in a configuration that allows tilted and vertical use of 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 an analysis system according to an embodiment. FIG. 8 is an example of a graph showing absorbance measurement results. FIG. 9 is a diagram showing a schematic configuration of an analysis system according to Example 2. FIG. 10 is a top view illustrating a microfluidic chip according to Example 2. FIG. 11 is a table showing an example of a control sequence for a dispensing pipette in Example 2. FIG. 12 is a top view illustrating an example of a microfluidic chip according to Example 4. FIG. 13 is a top view illustrating another example of a microfluidic chip according to Example 4. FIG. 14 is a diagram illustrating a microfluidic chip according to a modified example.

[0013] Hereinafter, one mode for carrying out the invention (hereinafter also referred to as one embodiment or embodiment) will be described with reference to the drawings. The configurations of the following embodiment are examples, and the configurations of the microfluidic chip and analysis system disclosed in this embodiment can be appropriately modified depending on various conditions. Unless otherwise specified, the configurations disclosed in this embodiment are not intended to limit the technical scope of the invention to only those, and can be combined as much as possible.

[0014] Furthermore, the drawings referred to in the following description merely show the shapes, sizes, and positional relationships in a schematic manner to the extent that the contents of the present invention can be understood. That is, the present invention is not limited to the shapes, sizes, and positional relationships exemplified in each drawing. Furthermore, there may be parts in which the dimensional relationships and ratios differ between the drawings.

[0015] [Embodiment 1] Fig. 1 is a diagram showing the 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. As shown in Fig. 1, the analysis system 1 is configured to include a control unit 10, a dispensing pipette (also simply referred to as a pipette) 20, and a microfluidic chip 30. The control unit 10 and the dispensing pipette 20 are connected via a predetermined interface.

[0016] The control unit 10 controls the intake and discharge of small amounts of liquid samples, such as reagents and specimens, contained in the dispensing pipette 20, and also controls the delivery of liquid samples introduced into the microfluidic chip 30 in droplet form. The introduction of reagents and the like into the microfluidic chip 30 using the dispensing pipette 20 is also referred to as "droplet application." The control unit 10 is a microcomputer unit including, for example, a processor such as a central processing unit (CPU), memory such as random access memory (RAM), read-only memory (ROM), and flash memory, and a communication interface with external devices. The memory stores, for example, an operating system (OS), various programs, various tables, and the like. In this embodiment, the delivery of liquid samples in the microfluidic chip 30 is described as being controlled by the control unit 10 connected to the dispensing pipette 20; however, 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 the liquid sample according to a program stored in advance in a memory or the like within the microcomputer chip.

[0017] The control unit 10 according to this embodiment 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. In 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 unit 34, which is an area for analyzing and evaluating the components, properties, and the like of the liquid sample. The analysis system 1 according to this embodiment can provide stable delivery of the liquid sample even when the amount of liquid sample used for testing, etc. is small (for example, 5 to 20 μL).

[0018] The dispensing pipette 20 includes a pipette body 21 that contains a liquid sample and a pipette tip 22 attached to the tip of the pipette body 21. The pipette tip 22 includes a fixing portion 23 that fixes the outer periphery of the inscribed pipette body 21 and a fitting portion 24. The fitting portion 24 is formed with a convex structure, such as a barbed structure, that protrudes outward from the periphery. The fitting portion 24 fits into a fitting structure 31a, such as a fitting groove, formed on the inner periphery of the connecting portion 31 at the end where the pipette tip 22 is attached. The dispensing pipette 20 aspirates and dispenses a predetermined amount (e.g., 5 to 20 μL) of the liquid sample contained in the pipette body 21 through the pipette tip 22. The connecting portion 31 may be integral with the microfluidic chip 30 or may be a separate component from the microfluidic chip 30. The connecting portion 31 may be detachable from the microfluidic chip 30.

[0019] 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 connecting portion 31 provided on the microfluidic chip 30. The connecting portion 31 is a structure for mounting and holding the dispensing pipette 20 containing a liquid sample in the microfluidic chip 30. The inner surface of the connecting portion 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 into a barbed structure (convex structure) that protrudes outward from the peripheral surface of the fitting portion 24. As shown in FIG. 1 , the tip portion of the fitting portion 24 of the pipette tip 22 held in the microfluidic chip 30 via the connecting portion 31 passes through the cylindrical internal space and extends to a region where the liquid sample is introduced.

[0020] The relative positioning of the tip of the pipette tip 22 with respect to the microfluidic chip 30 is appropriately adjusted by the engagement of the convex structure on the engagement portion 24 with an engagement structure 31a, such as an engagement groove, on the inner circumferential surface of the connection portion 31. Furthermore, when the dispensing pipette 20 is attached to the microfluidic chip 30 via the connection portion 31, as shown in FIG. 1 , the outer circumferential surface of the connection portion 31, on which the engagement structure 31a is provided, closely contacts the inner circumferential surface of the pipette tip 22 at the portion where the convex structure of the engagement portion 24 is provided. The engagement of the convex structure on the engagement portion 24 with the engagement structure 31a on the inner circumferential surface of the connection portion 31 enables a tight seal to be formed to prevent leakage of air or the like when a liquid sample or the like is introduced into the microfluidic chip 30 through the attached dispensing pipette 20. A contamination prevention filter 25 may be provided in the pipette tip 22 between the portion of the engagement portion 24 where the convex structure is provided and the tip. The contamination prevention filter 25 is made of, for example, a material that allows air to pass through but does not allow the liquid sample to pass through.

[0021] Fig. 2 is a diagram illustrating 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. As shown in Fig. 2, the pipette tip 22 has an anti-contamination filter 25 provided in the internal space that accommodates the aspirated sample Z1.

[0022] As shown in Figure 2(a), before collecting 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 with the pipette tip 22 attached aspirates a predetermined amount (e.g., approximately 5 to 20 µL) 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, which is provided with the tip and an anti-contamination filter 25 (Figure 2(b)).

[0023] 2(c), the dispensing pipette 20 containing the sample Z1 in the internal space at the tip of the pipette tip 22 is attached to the microfluidic chip 30 via the connecting portion 31. The tip of the pipette tip 22 is positioned at a predetermined position relative to the microfluidic chip 30 by fitting a convex structure provided on the fitting portion 24 with a fitting structure 31a, such as a fitting groove, provided on the inner circumferential surface of the connecting portion 31. The outer circumferential surface of the connecting portion 31, on which the fitting structure 31a is provided, tightly contacts the inner circumferential surface of the pipette tip 22 at the portion where the convex structure of the fitting portion 24 is provided, and the joint between the connecting portion 31 and the pipette tip 22 is sealed.

[0024] 2(d), when testing or measurement of sample Z1 using microfluidic chip 30 is completed, pipette body 21 of dispensing pipette 20 is detached from attached pipette tip 22. Pipette body 21 is inserted into and removed from fixing portion 23 that fixes the inscribed outer periphery, and pipette tip 22, which is fitted into connecting portion 31 via fitting portion 24, maintains its engagement with microfluidic chip 30.

[0025] When the pipette body 21 is inserted or removed from the fixing portion 23, the sample Z1 may remain in the internal space at the tip of the pipette tip 22 without being introduced into the microfluidic chip 30. The sample 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. The pipette tip 22 is provided with an anti-contamination filter (anti-contamination filter 25), which can prevent environmental contamination of the sample 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 connecting portion 31.

[0026] The dispensing pipette 20 equipped with the pipette tip 22 can be operated to pretreat the sample Z1, for example, by diluting the sample Z1 and mixing it with a diluent, extracting nucleic acids, or mixing it with a reagent. These pretreatments can be combined as appropriate. For example, the dispensing pipette 20 can aspirate 10 μL (microliters) of sample Z1 through the pipette tip 22, followed by an additional 90 μL of diluent. This aspirating action promotes mixing of the sample Z1 with the diluent, enabling a 10-fold dilution of the sample Z1 with the diluent. The pipette tip 22 may also have a silica membrane capable of specifically binding nucleic acids in the presence of high salt concentrations. By attaching a pipette tip 22 including a silica membrane to the dispensing pipette 20, purified nucleic acids from the aspirated sample Z1 can be introduced into the microfluidic chip 30. Examples of the sample Z1 to be measured include saliva, blood, and nasal swabs after treatment with a lysis buffer used for nucleic acid extraction. After dispensing the sample into the microfluidic chip 30, the dispensing pipette 20 again aspirates and dispenses a cleaning solution containing ethanol or the like, thereby washing the nucleic acid adsorbed to the silica membrane.

[0027] The pipette tip 22 may also be configured to include, for example, a sponge coated with a reagent. During the inhalation and discharge operations of the dispensing pipette 20 equipped with the pipette tip 22, the reagent coated on the sponge can be mixed with the sample Z1. For example, a sponge coated with calcium chloride is loaded into the tip of the pipette tip 22, blood or plasma anticoagulated with sodium citrate is aspirated, and the dispensing pipette 20 is then attached to the microfluidic chip 30. The dispensing pipette 20 can then discharge the blood or plasma aspirated into the pipette tip 22, thereby introducing calcium-added blood or plasma into the microfluidic chip 30. Coating the microfluidic chip 30 with a blood coagulation activating reagent, such as tissue factor or a contact factor activating reagent (kaolin, celite, etc.), enables evaluation of blood clotting time within the microfluidic chip 30.

[0028] As described above, 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 sample, cleaning solutions, etc. The liquid sample is not particularly limited as long as it can be passed through the microfluidic chip 30. Examples of the liquid sample 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.

[0029] 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 predetermined region of a connection part 31 to a measurement part 34. Such a device is produced, for example, by bonding a substrate having a groove formed on its surface that serves as the flow path to a film material or substrate using an adhesive or the like.

[0030] FIG. 3 is a diagram illustrating a microfluidic chip 30 according to this embodiment. FIG. 3 illustrates a top view of the generally rectangular microfluidic chip 30. As shown in FIG. 3 , a recessed region 31b, which is a depression for placing a liquid sample introduced in droplet form, is formed in a portion of the microfluidic chip 30 facing the tip of the fitting portion 24 of the dispensing pipette 20 held by the connecting portion 31. A generally circular measuring portion 34 is formed at the longitudinal end of the microfluidic chip 30 facing the recessed region 31b. A groove for a flow channel 32 connecting 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 constituting the flow channel 32 may be concave, U-shaped, or V-shaped. For example, a reaction substrate or the like that reacts with the liquid sample introduced into the recessed region 31b by the dispensing pipette 20 is applied to the flow channel 32.

[0031] The flow channel 32 connecting the recessed region 31b and the measurement unit 34 is a long and narrow flow channel in which a plurality of substantially U-shaped flow channels 32 are connected when viewed from above, and constitutes, for example, a heating unit 33 in which heating is performed to accelerate the reaction of a liquid sample. A heating mechanism such as a heater for heating the introduced liquid sample is provided below the flow channel 32 in which the heating unit 33 is formed. The liquid sample introduced into the flow channel 32 via the heating unit 33 can be heated in multiple stages depending on the evaluation of its characteristics and properties.

[0032] The flow path 32 constituting the heating unit 33 and the measurement unit 34 are provided with bubble removal structures 32a, 34a for capturing bubbles generated during the processing of the liquid sample introduced into the microfluidic chip 30. The measurement unit 34 is an area for analyzing and evaluating, for example, the components and properties of the liquid sample that reacts with a reaction substrate or the like. The measurement unit 34 may be provided with, for example, a stirrer for stirring the liquid sample sent through the heating unit 33. The operation of the stirrer is controlled, for example, by a stirring mechanism that utilizes magnetic force.

[0033] In the microfluidic chip 30, by providing an air bubble removal structure 32a in the flow path 32 that constitutes the heating unit 33, it is possible to suppress the intrusion of air bubbles that occur during the processing of the liquid sample sent to the measurement unit 34. Furthermore, by providing an air bubble removal structure 34a in the measurement unit 34, it is possible to suppress interference with the testing and evaluation of the liquid sample, for example, caused by air bubbles that occur during the stirring process of the liquid sample that has flowed into the measurement unit 34. In the microfluidic chip 30, suppressing the intrusion of air bubbles can increase the stability of the delivery of the introduced liquid sample.

[0034] For example, when PCR is performed using the microfluidic chip 30, the liquid sample introduced into the flow channel 32 is heated to a temperature range of 90 to 98 degrees Celsius via the heating unit 33. Similarly, when the Lamp method is applied, the liquid sample introduced into the flow channel 32 is heated to a temperature range of 60 to 70 degrees Celsius via the heating unit 33. 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 approximately 3 to 10 minutes. Bubbles generated during the liquid sample processing are captured via the bubble vent structure 32a formed in the flow channel 32, and the liquid sample from which the bubbles have been removed is introduced into the measurement unit 34. Furthermore, by providing the bubble vent structure 34a in the measurement unit 34, bubbles introduced via the flow channel 32 can be captured. 3, the microfluidic chip 30 is configured with a long, narrow channel formed by connecting a plurality of meandering, approximately 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 rate of bubble generation. Furthermore, by providing a plurality of repeating structures for trapping bubbles in the region of the heating section 33, such as a meandering channel 32, a bubble vent structure 32a, another meandering channel 32, and a bubble vent structure 32a, the bubble capture rate can be improved.

[0035] 4A and 4B are diagrams illustrating the bubble release structure 32a provided in the flow path 32. FIG. 4A illustrates an enlarged top view of a partial region of the flow path 32 in which the bubble release structure 32a is formed. FIG. 4B illustrates a cross-sectional view taken along line B-B# in FIG. 4A, and FIG. 4C illustrates a cross-sectional view taken along line C-C# in FIG. 4A. As shown in FIG. 4A, in the region of the heating section 33, the bubble release structure 32a is formed in the linear flow path 32 connecting the flow paths that meander in a generally U-shape.

[0036] 4(b) and 4(c), the bubble release structure 32a is formed as a convex structure that protrudes from the surface side of the microfluidic chip 30 (the surface side on which 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 bubble release structure 32a is linearly inclined so that the protruding height is relatively high with respect to the flow direction from the upstream side to the downstream side of the flow channel 32. By providing such a structure, it is possible to increase the collection efficiency of bubbles generated during the heating process.

[0037] Returning to Figure 3, bubble Z4 illustrates a cross-sectional view of the measurement unit 34 taken along line A-A#. As shown in bubble Z4, the bubble release structure 34a is formed on the periphery of the measurement unit 34 as a convex structure that protrudes toward the surface of the microfluidic chip 30. Inside the bubble release structure 34a, 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 bubble release structure 34a provided in the measurement unit 34 is formed on the periphery, surrounding the flat portion 34b and protruding toward the surface.

[0038] Regarding the removal of air bubbles generated during the processing of a liquid sample introduced into the microfluidic chip 30, the microfluidic chip 30, which has the flow channel 32 for introducing the liquid sample, may be used in an inclined position relative to the horizontal plane or in an inverted position. For example, the short side of the microfluidic chip 30, on which the connection unit 31 is provided, is positioned vertically downward, and the short side on which the measurement unit 34 is provided is positioned vertically upward. In such a configuration, the liquid sample introduced into the microfluidic chip 30 is transported from the lower side to the upper side through the flow channel 32 in the vertical direction. The structure for trapping air bubbles is different from that of the microfluidic chip 30, which is positioned horizontally, as described in FIGS. 3 and 4 .

[0039] FIG. 5 illustrates a microfluidic chip 30 configured to enable both tilted and vertical use. FIG. 5 illustrates a microfluidic chip 30 capable of multi-channel (3 channels x 4 blocks) reaction measurement. The microfluidic chip 30 of FIG. 5 has flow paths 32 extending from recessed regions 31b to the respective measurement units 34 of the multi-channels. Two heating units 33a and 33b are provided on the lower side (recessed region 31b side) and upper side (measurement unit 34 side) of the flow paths 32, respectively. Each of the heating units 33a and 33b heats a liquid sample introduced into the flow paths 32 at a different temperature. The flow paths 32 connecting the heating units 33a and 33b and the flow paths 32 connecting the heating units 33b and the multi-channel measurement units 34 are each provided with a bubble vent structure 32b for capturing bubbles generated during the processing of the introduced liquid sample.

[0040] 6A and 6B are diagrams illustrating the bubble removal structure provided in the microfluidic chip 30 configured to allow tilted and vertical use. Fig. 6A 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 unit for three channels. Fig. 6B 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 unit 33b.

[0041] When the microfluidic chip 30 is tilted or vertically positioned, bubbles accumulate on the vertically upper side of the flow path 32, which meanders in a substantially U-shape, and on the vertically upper side of the measurement unit 34. Therefore, as shown in FIG. 6B , in the flow path 32, which meanders in a substantially U-shape, the bubble collection efficiency can be improved by providing a bubble release structure 32c that widens the flow path space between the vertically upper and lower flow path walls. The same applies to the bubble release structure 32b provided in the flow path 32 connecting the heating unit 33a and the heating unit 33b. For example, the bubble release structure 32b can accumulate bubbles on the upper flow path wall side by forming the vertically upper flow path wall of the flow path 32 into a substantially triangular shape that expands upward. Furthermore, in the measuring section 34, by configuring the shape of the measuring section 34 as being approximately drop-shaped when viewed from the surface side, it is possible to form an air bubble removal structure 34c that collects air bubbles on the upper side of the flow path wall in the vertical direction.

[0042] The measurement unit 34 is provided with a filter 35 that maintains the ventilation pressure of the flow path 32 from the recessed region 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 to the inlet through which the liquid sample flows 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 ventilation pressure between the recessed region 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 to the inlet through which the liquid sample flows, the flow of liquid through the flow path 32 from the recessed region 31b to the measurement unit 34 is stopped.

[0043] In the microfluidic chip 30 shown in FIG. 6 , which is capable of multi-channel inspection and testing, it is necessary to ensure that the liquid sample flows uniformly into each of the multiple measurement units. For example, it is necessary to ensure that the liquid sample flows uniformly into the three measurement units 34 in each block arranged vertically upward and the three measurement units 34 in each block arranged vertically downward. By providing the measurement units 34 with filters 35, the delivery of the liquid sample can be stopped when the liquid sample reaches the measurement unit 34. In other words, since the delivery of the liquid sample to the measurement unit 34 arranged vertically downward can be stopped when the liquid sample reaches the measurement unit 34, the liquid sample can be delivered sequentially to each of the unreached channels connected to the branched paths. As a result, even in a microfluidic chip 30 having branched flow paths to multiple (e.g., 10 or more) measurement units 34, the liquid sample can be delivered uniformly to each measurement unit 34.

[0044] 3 and other figures show a configuration including the filter 35, but a sensor that detects that the delivered liquid sample has reached a predetermined region (e.g., the measurement unit 34) may be provided instead of the filter 35. Examples of such a sensor 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 region 31b to the measurement unit 34.

[0045] 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 region. The control unit 10 may control the liquid sample delivery to stop based on the arrival state indicated by a binary status signal representing an active status or an inactive status output from the liquid sensor or electrode sensor. For example, 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 an incomplete delivery state, and to stop delivery otherwise. In this embodiment, the use of a liquid sensor or an electrode sensor makes it possible to stop delivery of the delivered liquid sample at the target location (a desired location in the flow path 32 in the microfluidic chip 30).

[0046] Furthermore, the control unit 10 of the analysis system 1 can use, for example, a pressure sensor to detect the ventilation pressure in the flow path 32 leading from the recessed region 31b to the measurement unit 34, and can perform control so as to stop the liquid transfer when the detected pressure reaches a certain pressure (threshold value). In this embodiment, by using a pressure sensor, it is possible to detect a certain increase in the ventilation 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.

[0047] Example 1 Next, the results of a liquid delivery test performed by the analysis system 1 according to this embodiment will be described. Fig. 7 is a table showing an example of a control sequence for the dispensing pipette 20 during the test, and Fig. 8 is an example of a graph showing the results of absorbance measurement. In the liquid delivery test, 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.

[0048] As shown in Table Tb1 of Fig. 7, plasma was aspirated using the dispensing pipette 20 equipped with the pipette tip 22. The plasma was aspirated at a flow rate of 3 µL / sec for 1 second, resulting in aspirating a flow rate of 3 µL (step 1). The diluent was aspirated at a flow rate of 50 µL / sec for 3 seconds, resulting in aspirating a flow rate of 150 µL (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.

[0049] After the start of the measurement, the diluted plasma sample was introduced into the recessed region 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 channel 32 formed in the microfluidic chip 30. The diluted plasma sample was ejected at a flow rate of 50 μL / sec, and the entire volume (153 μL) was ejected (step 3).

[0050] The diluted plasma sample was transported from the recessed region 31b through the flow path 32, reached the heating unit 33, whose heating temperature was set at 37°C, and heated. Three minutes after the liquid transport was stopped in step 3, the air in the dispensing pipette was ejected through the dispensing pipette 20. The air was ejected at a flow rate of 50 μL / sec and a flow rate of 153 μL (steps 4 and 5). By the ejection operation in step 5, the plasma sample heated by the heating unit 33 provided in the flow path 32 of the microfluidic chip 30 was transported and injected into the measurement unit 34.

[0051] The measurement unit 34 is equipped with latex agglutination assay beads and a stirrer (0.5 mm diameter x 1 mm length). The plasma sample injected into the measurement unit 34 is stirred at 500 rpm by the stirrer. After stirring, the change in absorbance at a wavelength of 600 nm is measured. The change in absorbance is measured as a change in transmittance using, for example, anti-D-dimer antibody-immobilized latex beads. The capacity of the measurement unit is 203 μL.

[0052] FIG. 8 shows an absorbance curve, in which 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 FIG. 8 represents absorbance, and the horizontal axis represents time (minutes). As shown in the pseudo-reaction curve Z10, the absorbance (transmittance) of the plasma sample by the latex beads changes from approximately 1.1 to approximately 1.43 over the five minutes from the start of measurement. Over the five minutes from the fifth to the tenth minute, 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, remaining near 1.5.

[0053] Example 2 The microfluidic chip 30 of Example 1 is configured to maintain the air pressure in the flow path 32 from the recessed region 31b to the measurement unit 34 within a certain range using a filter 35 provided at a location facing the inlet of the measurement unit 34 through which the liquid sample flows. In Example 2, the microfluidic chip 30 is provided with an air vent hole 36, which is an air vent, 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 when the delivered liquid sample reaches a predetermined area. Even with this configuration, the control unit 10 of the analysis system 1 can control the dispensing pipette 20 to stop dispensing the liquid sample based on a binary status signal (detection signal) output from the sensor 37. As a result, Example 2 ensures stable liquid delivery in the microfluidic chip 30, reduces the cost of components on the chip, and achieves accurate liquid volume control. The analysis system 1 of Example 2 enables sample measurement with improved accuracy.

[0054] FIG. 9 is a diagram showing a schematic configuration of an analysis system 1 according to Example 2, and FIG. 10 is a top view illustrating a microfluidic chip 30 according to Example 2. As described above, Example 2 differs in that, instead of the filter 35, the microfluidic chip 30 is provided with an air vent hole 36, 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 Example 2 also differs in that it controls the delivery and stopping of the liquid sample dispensed from the dispensing pipette 20 based on a binary status signal output from the sensor 37. Below, the analysis system 1 according to Example 2 will be described, focusing mainly on the differences. The flow path 32 is an example of a "first flow path," and the flow path 38 is an example of a "second flow path." The first flow path may include a storage section, a pre-processing section, an observation section, etc. The second flow path may include a waste liquid storage section, etc.

[0055] 9 , a binary status signal output from the sensor 37 is input to the control unit 10. For example, when the status signal output from the sensor 37 is an inactive status indicating that the liquid sample has not yet reached the flow path 38 between the measurement unit 34 and the air vent hole 36, the control unit 10 controls the dispensing pipette 20 to continue dispensing the liquid sample. On the other hand, when the status signal output from the sensor 37 is an active status indicating that the liquid sample has reached the flow path 38 between the measurement unit 34 and the air vent hole 36, the control unit 10 controls the dispensing pipette 20 to stop dispensing the liquid sample and to stop liquid transfer in the microfluidic chip 30.

[0056] As shown in FIGS. 9 and 10 , the air vent hole 36 is formed on the longitudinal end side of the substantially rectangular microfluidic chip 30, facing the recessed region 31b. The sensor 37 is installed in the flow path 38 between the measurement unit 34 and the air vent hole 36. The liquid sample delivered from the dispensing pipette 20 to the microfluidic chip 30 reaches the measurement unit 34 while pressing against 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. When the liquid sample continues to be discharged from the dispensing pipette 20, 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 changes the status signal from inactive to active. When the status signal changes to active, the control unit 10 controls the dispensing pipette 20 to stop dispensing the liquid sample. In the microfluidic chip 30 according to the second embodiment, the transfer of the liquid sample from the recessed region 31b through the flow channel 32 to the measurement unit 34 is stopped.

[0057] In the configuration of Example 2, a liquid transfer test was conducted in the same manner as in Example 1. Fig. 11 is a table showing an example of a control sequence for the dispensing pipette 20 in Example 2. In Example 2, in order to provide a sensor 37 that detects when a liquid sample has reached a predetermined region, latex particles (50 µL) were previously sealed in the heating unit 33. The latex particles sealed in the heating unit 33 are sealed in the elongated flow channel 32, and therefore remain stably within the flow channel without moving or scattering during storage or transportation of the microfluidic chip.

[0058] 11 , the processes of steps 1 to 3 are the same as those in Example 1. Specifically, plasma (3 μL) was aspirated using the dispensing pipette 20 equipped with the pipette tip 22, followed by aspirating a diluent (150 μL). The dispensing pipette 20 equipped with the pipette tip 22 was then secured to the connector 31 of the microfluidic chip 30, and measurement was initiated. After measurement was initiated, the dispensing pipette 20 introduced the diluted plasma sample into the recessed region 31b and began delivering the diluted plasma sample to the measurement unit 34 through the flow path 32 formed in the microfluidic chip 30. The diluted plasma sample was discharged at a flow rate of 50 μL / sec for approximately 3 seconds, dispensing 153 μL.

[0059] The diluted plasma sample was transferred from the recessed region 31b through the flow path 32 and reached the heating unit 33, whose heating temperature was set at 37°C, where it was heated (step 4). At this time, the latex particles encapsulated in the heating unit 33 moved to the measurement unit 34 along with the plasma sample. Three minutes after the liquid transfer was stopped in step 3, the diluted plasma sample (153 μL) was ejected through the dispensing pipette 20 (step 5). The plasma sample heated by the heating unit 33 was injected into the measurement unit 34 and mixed with the latex particles, initiating a reaction. The sensor 37 installed in the flow path 32 between the measurement unit 34 and the air vent hole 36 detected the plasma sample reaching this region, and the transfer of the plasma sample in the microfluidic chip 30 was stopped (step 6). In Example 2, the transfer of the liquid sample in the microfluidic chip 30 may be controlled in combination with a pressure sensor, a valve, or the like.

[0060] As described above, in the analysis system 1 according to this embodiment, 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. Furthermore, 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 the sensor 37, such as a liquid sensor or an electrode sensor. In this embodiment, even if the amount of liquid sample used for testing or the like is small, stable transfer of the liquid sample introduced through the flow path formed in the microfluidic chip to a predetermined region can be provided.

[0061] Example 3: An RNA extract was prepared from a nasal swab using an RNA extraction kit (LooPamp Viral RNA Extraction Kit; Eiken Chemical). The dispensing pipette 20 aspirated the RNA extract (sample) through the pipette tip 22 and introduced it into the recessed region 31b of the fitted microfluidic chip 30. The sample was then delivered to the heating unit 33 through the flow path 32 and stopped for three minutes. The heating unit 33 was heated to 65°C by a heater. After three minutes, the dispensing pipette 20 was controlled to start delivery of the sample to the microfluidic chip 30. The sample heated to a predetermined temperature (65°C) via the heating unit 33 passed through the bubble removal structure 32a provided in the flow path 32, and the sample, from which air bubbles had been removed, was introduced into reaction unit A (measurement unit 34, channel A) and reaction unit B (measurement unit 34, channel B). Reaction sections A, B, and C (measurement section 34, channel C) were coated with Lamp method influenza test reagents (Loopamp Influenza B Virus Detection Reagent Kit; Eiken Chemical): A for measurement, B for positive control, and C for negative control.

[0062] Each of reaction zones A, B, and C is heated to 60 degrees. A flow path 32 connected to a filter 35 continues downstream of reaction zones A, B, and C. Air passes through filter 35, but liquid does not. Reaction zones A, B, and C are filled with the sample by liquid transfer via a dispensing pipette 20, and the sample 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. If the result is positive, an increase in turbidity is detected for reaction zones A and B.

[0063] 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 region 31b of the fitted microfluidic chip 30. The microfluidic chip 30 used in Example 4 had the configuration shown in FIG. 12, for example. By controlling the dispensing operation of the dispensing pipette 20, the specimen was delivered to the heating unit 33c through the flow path 32 and stopped for three minutes. The heating unit 33c was heated to 95°C by a heater. After three minutes, the dispensing pipette 20 was controlled to start delivery of the specimen through the microfluidic chip 30. The specimen, heated to a predetermined temperature (95°C) through the heating unit 33c, passed through the 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 unit A (33d). After one minute has passed, the sample is sent to heating unit B (33e) and stopped for 30 seconds. Heating unit B (33e) is heated to 55°C by a heater. After 30 seconds, the sample is sent to heating unit C (33f) and stopped. Heating unit B (33e) is heated to 72°C by a heater. The inhalation operation of the dispensing pipette 20 is then controlled, and the sample that has reached heating unit C (33f) is drawn back into the flow path 32 of heating unit A (33d). The inhalation and discharge operations of the dispensing pipette 20 are appropriately controlled, and the above cycle is repeated approximately 20 to 30 times to allow the PCR reaction to proceed. PCR amplification during each cycle can be monitored as fluorescence intensity, for example, by a fluorescence detection unit 80 provided in the flow path 32 between heating unit A (33d) and heating unit B (33e).

[0064] Furthermore, PCR amplification can be detected visually by lateral flow using a microfluidic chip 30 incorporating a nucleic acid chromatography test slip 83, as shown in FIG. 13 . In this case, after performing PCR nucleic acid amplification using the dispensing pipette 20 as described above, the dispensing pipette 20 delivers the sample to the reagent mixing section (measurement section 34) and stops the process there for a certain period of time. The sample may be stirred in the reagent mixing section (measurement section 34) using an inserted stirrer 81, or the process may be stopped for several minutes to allow the sample to naturally diffuse. In the reagent mixing section (measurement section 34), the sample is mixed with the reagent and then further delivered to the test slip reagent reaction section 82. The sample then reaches the nucleic acid chromatography test slip 83, allowing the test results to be evaluated using nucleic acid chromatography.

[0065] (Modification) In the first embodiment, the microfluidic chip 30 has been described as a device having a substantially rectangular shape and a groove for the flow path 32 for delivering 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. For example, as disclosed in Japanese Patent Application Laid-Open Nos. 2021-159011, 2019-088332, and 2018-014966, disk-shaped microchips are used in PCR, immunoassays, and the like. In the analysis system 1 of the modification, the shape of the microfluidic chip 30 to which the dispensing pipette 20 delivers liquid is disk-shaped.

[0066] 14A and 14B are diagrams illustrating a microfluidic chip 50 according to a modified example. FIG. 14A shows a top view of the microfluidic chip 50 according to the modified example, and FIG. 14B shows a side view of the microfluidic chip 50. As shown in FIGS. 14A and 14B, the microfluidic chip 50 is disk-shaped, and a connection part 51 is formed in the center for fixing a dispensing pipette 20 equipped with a pipette tip 22. Below the connection part 51, for example, a reagent coating that reacts with a liquid sample, a plasma separation filter, a nucleic acid extraction filter, or the like can be installed.

[0067] Grooves of flow channels 52 are formed radially, extending linearly in the circumferential direction from the center where connection section 51 is provided, and the grooves of flow channels 52 are connected to measurement section 54 formed on the periphery. Note that the configuration illustrated in Figure 14(a) is an example of a configuration in which four grooves of flow channels 52 are formed that intersect at right angles in a cross shape at the center.

[0068] The dispensing pipette 20 equipped with the pipette tip 22 is attached to a connection part 51 provided at the center of a disk-shaped microfluidic chip 50, as in Examples 1 and 2, and a liquid sample is injected into a flow channel 52 and transferred stepwise to a measurement part 54. The attached dispensing pipette 20 is then detached from the connection part 51, and liquid transfer by centrifugation is carried out, thereby enabling two-stage liquid transfer. That is, in the modified embodiment, by employing a disk-shaped microfluidic chip 50, it is possible to perform two-stage liquid transfer: liquid transfer by the dispensing pipette 20 with controlled flow rate, time, flow rate, etc., and liquid transfer by centrifugation. In the modified embodiment, precise control of liquid transfer in multiple stages is possible.

[0069] As in the first and second embodiments, the analysis system 1 in the modified example also enables complex liquid transfer control by combining it with valves, liquid sensors, electrode sensors, pressure sensors, etc. Liquid transfer in the modified example can be performed, for example, to mix a specimen with a reagent, separate plasma from a blood specimen, or extract nucleic acids from a specimen as a pre-processing step for PCR. When mixing a specimen with a reagent, for example, the reagent is applied to the flow path 52, and the specimen is then injected up to the area where the reagent is applied. Mixing can then be achieved by repeatedly aspirating and discharging the specimen injected into the flow path using the dispensing pipette 20. It is also possible to perform pre-processing, such as separating plasma from whole blood, before transferring the specimen by centrifugation.

[0070] 1. Analysis system, 10. Control unit, 20. Dispensing pipette, 21. Pipette body, 22. Pipette tip, 23. Fixing part, 24. Fitting part, 25. Contamination prevention filter, 30, 50. Microfluidic chip, 31, 51. Connection part, 31a. Fitting structure, 31b. Recessed area, 32, 38, 52. Flow path, 32a, 32b, 34a, 34c. Air vent structure, 33, 33a, 33b, 33c, 33d, 33e, 33f. Heating part, 34, 54. Measurement part, 34b. Flat part, 35. Filter, 36. Air vent hole, 37. Sensor

Claims

1. A liquid sample analysis system comprising: a pipette tip for sucking and discharging a liquid sample; a dispensing pipette having a space for accommodating the liquid sample sucked through the pipette tip, the dispensing pipette being attached to the tip of the pipette tip; a connection part for holding the dispensing pipette to which the pipette tip is attached; a microchip in which a first flow path is formed for allowing the liquid sample introduced from the dispensing pipette to flow into a measurement part for measuring the characteristics of the liquid sample; and a control unit connected to the dispensing pipette for controlling the flow rate of the liquid sample sucked and discharged through the pipette tip and for controlling the feeding of the introduced liquid sample to the measurement part through the first flow path.

2. The liquid sample analysis system according to claim 1, wherein the microchip includes a filter for maintaining the ventilation pressure of the liquid sample fed to the measurement part through the first flow path within a certain range.

3. The liquid sample analysis system according to claim 1, wherein the microchip includes a ventilation hole connected to the measurement part by a second flow path and a sensor for detecting that the fed liquid sample has reached a predetermined region between the measurement part and the ventilation hole, and the control unit controls the dispensing pipette to stop discharging the liquid sample accommodated therein based on a detection signal output from the sensor indicating that the fed liquid sample has reached the predetermined region.

4. A microchip comprising: a connection part for holding a dispensing pipette having a space for accommodating the liquid sample sucked through the pipette tip, the pipette tip for sucking and discharging the liquid sample being attached to the tip; a flow path for allowing the liquid sample introduced from the dispensing pipette held by the connection part to flow into a measurement part for measuring the characteristics of the liquid sample; and a filter for maintaining the ventilation pressure of the liquid sample fed to the measurement part through the flow path within a certain range.

5. A connection part that holds a dispensing pipette with a pipette tip for sucking and discharging a liquid sample attached to the tip and having a space for accommodating the liquid sample sucked through the pipette tip; a first flow path for flowing the liquid sample introduced from the dispensing pipette held by the connection part into a measurement part for measuring the characteristics of the liquid sample; a vent hole connected to the measurement part by a second flow path; and a sensor for detecting that the fed liquid sample has reached a predetermined region between the measurement part and the vent hole. A microchip comprising these components.

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