Liquid delivery method, flow path apparatus, and liquid delivery device

Abstract

Provided is a liquid feeding method, a flow path apparatus, and a liquid feeding device, which are capable of applying a pressure required to suppress bubbles to a solution when a chemical reaction is performed in a flow path, do not require a preliminary liquid feeding step, and are capable of coping with reciprocating liquid feeding. A liquid feeding method for removing bubbles from a solution introduced into a flow path apparatus, the method comprising: a placement step of placing the solution at a first position on an opposite side of the flow path with respect to a second chamber; a liquid feeding step of pressurizing and feeding the solution to move the solution in the second chamber toward the flow path; and a flow path introduction step of pressurizing and introducing the solution from the second chamber to the flow path.

Description

Technical Field

[0001] This invention relates to a liquid delivery method, a flow path device, and a liquid delivery apparatus. Background Technology

[0002] In recent years, technologies for performing chemical reactions such as nucleic acid amplification and gene detection within flow paths formed on small substrates (flow path devices) have been developing. By setting the flow path to the optimal temperature for the chemical reaction using methods such as placing the flow path device on a heater, the solution for the chemical reaction is delivered to the flow path, thereby realizing the chemical reaction within the flow path. Examples of chemical reactions performed within flow paths include nucleic acid amplification based on PCR (Polymerase Chain Reaction) and gene detection based on microarray methods.

[0003] In PCR-based nucleic acid amplification, microtubes containing reaction solutions are typically placed in a heating block. The temperature of the heating block is varied to repeatedly denature, anneal, and elongate the DNA (thermal cycling), thereby amplifying the target DNA. In this case, the heating and cooling time of the heating block itself becomes the rate limit, rather than the heating and cooling of the solution; the reaction typically lasts about one hour. Alternatively, there are methods that use multiple heaters set to the desired PCR temperature to create flow paths, and PCR is performed by circulating the solution within these paths. It is known that since the flow paths are always set to the desired temperature, thermal cycling can be achieved solely through the heating and cooling of the solution, completing PCR in less than 10 minutes.

[0004] Furthermore, in microarray-based gene detection, specific molecules (probes) that chemically react with the target substance are immobilized on a substrate, and the target substance is then detected. Typically, a solution containing the target molecules flows over a substrate with a relatively wide area relative to the area where the probe is immobilized, resulting in a low frequency of contact between the target substance and the probe, and gene detection taking several hours to tens of hours. On the other hand, methods are known to increase the frequency of contact between the target substance and the probe by immobilizing the probe in a narrow flow path and allowing a solution containing the target substance to flow through it, thus completing gene detection in just a few minutes.

[0005] Another advantage of conducting chemical reactions in the flow path is the reduction in reagent usage. Compared to substrates, the chemical reaction takes place in a small space, thus reducing the amount of reagent solution used.

[0006] Existing technical documents

[0007] Patent documents

[0008] Patent Document 1: Japanese Patent Application Publication No. 2007-85998

[0009] Patent Document 2: U.S. Patent Application Publication No. 2007 / 0087353 Summary of the Invention

[0010] The problem that the invention aims to solve

[0011] If air bubbles are present in the flow path when the flow path is filled with reaction solution for a chemical reaction, in addition to hindering the delivery of the solution, the efficiency of the chemical reaction will also be reduced. For example, during nucleic acid amplification by PCR in the flow path, if air bubbles are present, they may block the flow path, hindering the delivery of the solution for thermal cycling; the air bubbles may also inactivate the nucleic acid amplification enzyme, reducing the reaction efficiency.

[0012] As a method for removing air bubbles from the flow path, Patent Document 1 discloses a method of pressurizing the solution within the flow path. By increasing the pressure applied to the solution, the solubility of the gas in the solution increases, causing the air inside the air bubbles to dissolve in the solution, thereby removing the air bubbles. In this document, a secondary flow path is connected downstream of the main flow path where the chemical reaction takes place, increasing the solution pressure inside the main flow path.

[0013] However, in this literature, the liquid delivery under pressure is limited to one direction. For example, in gene detection such as microarrays within a flow path, it is not suitable for situations where reciprocating liquid delivery is desired, considering the aim of improving the chemical reaction efficiency between the target material and the probe. Furthermore, in the method described in this literature, when the volume of the sample solution is smaller than the volume of the flow path, liquid needs to be delivered first to maintain the pressure of the sample solution. It is argued that delivering liquid first may cause probe dissociation, thus affecting the chemical reaction.

[0014] Therefore, the object of the present invention is to provide a liquid delivery method, flow path device and liquid delivery apparatus that can apply pressure to the solution to suppress bubbles when a chemical reaction is carried out in the flow path, without the need to deliver liquid first, and can also handle reciprocating liquid delivery.

[0015] Methods for solving problems

[0016] One example of the liquid delivery method of the present invention is a liquid delivery method for removing air bubbles from a solution introduced into a flow path device, wherein the flow path device comprises: a flow path of volume v, a first chamber of volume V1 connected to a first side of the flow path, and a second chamber of volume V2 connected to a second side of the flow path different from the first side, wherein V1, V2, and v satisfy equation (1).

[0017] [Number 1]

[0018]

[0019] in,

[0020] [Number 2]

[0021]

[0022] α is the solubility of the gas in the solution at the temperature of the second chamber, β is the solubility of the gas in the solution at the temperature of the flow path, P0 is the pressure of the surrounding environment of the flow path device, ΔP is the pressure rise inside the bubble, ΔP=4σ / d, σ is the surface tension of the solution, and d is the diameter of the flow path.

[0023] The above methods have the following advantages:

[0024] In the preparation process, a solution is prepared at a first position on the opposite side of the flow path, relative to the second chamber described above.

[0025] In the liquid delivery process, the solution is pressurized and delivered to move it toward the flow path in the second chamber; and

[0026] In the flow path introduction process, the solution is introduced into the flow path under pressure from the second chamber.

[0027] An example of the flow path device of the present invention is a flow path device for introducing and pressurizing a solution. The flow path device includes: a flow path of volume v, a first chamber of volume V1 connected to a first side of the flow path, and a second chamber of volume V2 connected to a second side of the flow path that is different from the first side. V1, V2, and v satisfy equation (1).

[0028] [Number 3]

[0029]

[0030] in,

[0031] [Number 4]

[0032]

[0033] α is the solubility of the gas in the solution at the temperature of the second chamber, β is the solubility of the gas in the solution at the temperature of the flow path, P0 is the pressure of the surrounding environment of the flow path device, ΔP is the pressure rise inside the bubble, ΔP=4σ / d, σ is the surface tension of the solution, and d is the diameter of the flow path.

[0034] An example of the liquid delivery device of the present invention is a liquid delivery device for delivering the solution in the aforementioned flow path device. The liquid delivery device includes a pressurizing unit connected to the aforementioned solution inlet, and the pressurizing unit delivers the solution by applying pressure via the aforementioned solution inlet.

[0035] Invention Effects

[0036] According to the liquid delivery method, flow path device, and liquid delivery apparatus of the present invention, when a chemical reaction is carried out in the flow path, a pressure required to suppress bubbles can be applied to the solution. Therefore, the liquid delivery obstruction caused by bubbles and the reduction in chemical reaction efficiency are improved, enabling the chemical reaction to be carried out more stably or efficiently within the flow path.

[0037] Furthermore, the liquid delivery method, flow path device, and liquid delivery apparatus according to the present invention can suppress air bubbles in the flow path and can deliver liquid reciprocally. Moreover, even when the solution volume is smaller than the flow path volume, it is not necessary to deliver the solution first, and air bubbles can be suppressed.

[0038] Other issues, structures, and effects not described above will become clear through the following examples. Attached Figure Description

[0039] Figure 1 This is a diagram of the device configuration in Embodiment 1.

[0040] Figure 2 This is a cross-sectional view of the device in Example 1.

[0041] Figure 3 This is the workflow of Example 1.

[0042] Figure 4 yes Figure 3 A schematic diagram of the flow path device in step (a).

[0043] Figure 5 yes Figure 3 A schematic diagram of the flow path device in step (d).

[0044] Figure 6 This is a diagram of the device configuration in Example 2.

[0045] Figure 7 These are cross-sectional views of the apparatus in Embodiment 2 and its variations.

[0046] Figure 8 This is the workflow of Example 2.

[0047] Figure 9 yes Figure 8 A schematic diagram of the flow path device in step (b).

[0048] Figure 10 yes Figure 8 A schematic diagram of the flow path device in step (f).

[0049] Figure 11 This is a diagram of the device configuration in Example 3.

[0050] Figure 12 This is a cross-sectional view of the device in Example 3.

[0051] Figure 13 This is the workflow of Example 3.

[0052] Figure 14 This is a diagram of the device configuration in Example 4.

[0053] Figure 15 It is a graph showing the relationship between the chamber volume ratio and the probability of bubble formation.

[0054] Symbol Explanation

[0055] 101…Flow path device, 102…Substrate, 103…Flow path, 104…First chamber, 105…Second chamber, 106…Solution inlet, 107…Pressure control device (pressurization unit), 108…Tube, 109…Sealing material, 110…Solution, 120…Probe, 121…Bead, 201…Valve, 202…Pressure control device connection, 203…Valve, 301…First external heat source (heating mechanism), 302…Second external heat source (heating mechanism), 303…Equipment holding part, 304…First temperature zone, 305…Second temperature zone, 401…Liquid delivery device, 402…Cover, 403…External heat source (heating mechanism), 404…Temperature control part, 405…Sensor, 406…Signal detection part. Detailed Implementation

[0056] Hereinafter, embodiments of the present invention will be described with reference to the accompanying drawings.

[0057] [Example 1]

[0058] The flow path device according to Embodiment 1 of the present invention will be described below. This flow path device is used for performing PCR-based nucleic acid amplification, microarray-based gene detection, etc., and is particularly configured to introduce a solution and pressurize it to move the introduced solution.

[0059] Figure 1 This is a top view of the flow path device 101. Figure 2 Indicates along Figure 1 A cross-sectional view of the AA line. The flow path device 101 includes a substrate 102 and a sealing material 109.

[0060] The substrate 102 is preferably made of a material that is stable against temperature and pressure changes, is not easily corroded by solutions used in chemical reactions, and has good formability. Examples of such materials include cyclic olefin polymers (COP), polycarbonate (PC), and acrylic resins (PMMA).

[0061] The substrate 102 includes a flow path 103, a first chamber 104, a second chamber 105, and a solution inlet 106. The first chamber 104 is connected to one side (the first side) of the flow path 103. The second chamber 105 is connected to the other side (the second side, which is different from the first side) of the flow path 103.

[0062] The solution inlet 106 is machined into a through hole on the substrate 102. The solution inlet 106 opens on the upper surface of the flow path device 101 (for example, the surface opposite to the surface where the sealing material 109 is pressed). The flow path 103, the first chamber 104, and the second chamber 105 are formed by machining the substrate 102 and sealing it with the sealing material 109.

[0063] The solution inlet 106 is disposed on the side opposite to the flow path 103 relative to the second chamber 105. By providing the solution inlet 106 at this position, solution can be easily introduced into the second chamber 105 without interfering with the flow path 103.

[0064] Flow path 103 is not limited to Figure 1 as well as Figure 2 The straight line shape shown could also be, for example, a winding shape. In Figure 1 In the example, flow path 103 branches into multiple branches (3 branches), but it can also be a single flow path without branches. By branching flow path 103 into multiple branches, the flow rate can be increased without increasing the cross-sectional area of ​​each flow path 103. The cross-sectional shape of flow path 103 can be designed arbitrarily, for example, it can be square, rectangular or circular.

[0065] Specific molecules (probes 120) that can chemically react with specific target substances can also be immobilized in flow path 103. Examples of target substances include nucleic acids, antigens, antibodies, and peptides.

[0066] More than one bead 121 can be disposed within the flow path 103, for example, multiple beads 121 can be filled within the flow path 103. Specific molecules (probes) that chemically react with a specific target substance are fixed on the surface of the beads 121, for example.

[0067] Specific target substances can be detected by using probe 120 and / or bead 121. Alternatively, probe 120 and / or bead 121 can be omitted.

[0068] First chamber 104 Figure 1 as well as Figure 2 The example shown is a cuboid shape, but it can also be other shapes, such as a cylinder. Additionally, the first chamber 104 can also be... Figure 1 as well as Figure 2 The flow path shape is as shown in the second chamber 105.

[0069] The second chamber 105 is in Figure 1 as well as Figure 2 The example shown is a flow path shape, but it can also be other shapes. For example, it could be... Figure 1 and Figure 2 The first chamber 104 shown can be a cuboid shape, or it can be a cylindrical shape.

[0070] The definition of "flow path shape" can be appropriately determined by those skilled in the art; for example, it can be described as a shape having a defined axial direction and not having portions (height difference portions) in the whole including both ends of the axial direction whose cross-sectional shape changes discontinuously along the axial direction. "Axial direction" does not need to be a straight line direction, and can also be as follows: Figure 1 The second chamber 105 contains a curved section. The axial direction can be referred to as the length direction or the direction of solution movement. In other words, the flow path shape can be described as a shape in which the cross-sectional shape does not change with the axial position or changes only continuously. With such a shape, since there are no parts where the cross-sectional shape changes discontinuously (parts with height differences), the solution will not stagnate in such parts, and more appropriate liquid delivery can be achieved.

[0071] The sealing material 109 has the function of sealing with the substrate 102. The surface of the sealing material 109 that seals with the substrate 102 can be adhesive or pressure-sensitive. The sealing material 109 is preferably a film with a thickness of about 0.1 mm made of polyolefin (PO), polypropylene (PP), or other resins.

[0072] A pressure-reducing device 107 is configured in association with the flow path device 101. The pressure-reducing device 107 is connected to the solution inlet 106 of the flow path device 101 via a pipe 108. The pressure-reducing device 107 is an example of a pressurizing unit, which moves the solution within the flow path device 101 by pressurizing and / or depressurizing. Thus, the pressure-reducing device 107 functions as a liquid delivery control mechanism. The pressure-reducing device 107 can be, for example, a syringe pump, a diaphragm pump, a miniature blower, etc.

[0073] Let the volume of flow path 103 be v, the volume of the first chamber 104 be V1, and the volume of the second chamber 105 be V2. V1, V2, and v are designed to satisfy equation (1).

[0074] [Number 5]

[0075]

[0076] in,

[0077] [Number 6]

[0078]

[0079] α is the solubility of the gas in the solution at the temperature of the second chamber 105. The value of α varies depending on the composition of the solution introduced into the flow path device 101 and the composition of the gas dissolved in the solution. As an example, α is the value at room temperature. Alternatively, as an example, α is the solubility of air in the solution.

[0080] β is the solubility of the gas in the solution at the temperature of flow path 103. That is, when the temperature of the second chamber 105 is different from the temperature of flow path 103, there is a possibility that α≠β.

[0081] P0 is the pressure of the surrounding environment of the flow path device 101, which is atmospheric pressure in a typical indoor environment.

[0082] ΔP is the pressure rise inside the bubble, ΔP = 4σ / d. σ is the surface tension of the solution, and d is the diameter of the flow path 103. It should be noted that d is the value assuming the cross-section of the flow path 103 is circular. However, if the cross-section of the flow path 103 is not circular, those skilled in the art can appropriately determine the value of d based on its shape using known techniques. For example, it can also be set to the diameter of a circular plate with an area equal to the cross-section of the flow path 103.

[0083] Figure 3 This describes the reciprocating liquid delivery process from the solution inlet 106 to the pressurized flow path. This process describes a liquid delivery method for removing air bubbles from the solution introduced into the flow path device 101. Hereinafter, according to... Figure 3 The steps of each action are explained.

[0084] First, a solution for the chemical reaction in the flow path is introduced through the solution inlet 106. Figure 3 (a)). The space formed by the second chamber 105 connected to the solution inlet 106, the flow path 103, and the first chamber 104 is sealed outside the solution inlet 106, so the solution stays near the solution inlet 106 in this step.

[0085] Figure 4 This indicates the position of solution 110 at that moment. This position is designated as the first position. That is, the liquid delivery method of this embodiment may include a step (placement step) of placing solution 110 at a first position opposite to flow path 103 relative to the second chamber 105. In one example, this first position is not included in the second chamber 105. That is, when solution 110 is located at this first position, the volume V2 of the second chamber 105 does not contain any part of solution 110, or substantially does not contain solution 110.

[0086] Next, the pressure-reducing device 107 is connected to the solution inlet 106 via pipe 108. Figure 3(b)). Next, pressurization of the flow path device 101 is initiated via the pressure increasing / depressurizing device 107. Figure 3 (c)). By applying pressure, the solution moves in the second chamber 105, and soon the solution reaches the front of the flow path 103 within the second chamber 105. Figure 3 (d) Thus, the liquid delivery method of this embodiment includes a step (liquid delivery step) in which the solution is pressurized and delivered by means of a pressure-reducing device 107 connected to the second chamber 105 to move the solution toward the flow path 103 in the second chamber 105.

[0087] Figure 5 This indicates the position of solution 110 at the end of step (d). At this moment, the solution is pressurized to a sufficient pressure to remove air bubbles from the flow path 103 (the calculation of the pressure will be described later).

[0088] Next, in order to introduce the solution into the flow path 103 and carry out the chemical reaction, the flow path device 101 is further pressurized using the pressure reducing device 107. Figure 3 (e)). Thus, the solution moves from the second chamber 105 to the flow path 103 and is introduced into the flow path 103 ( Figure 3 (f) Thus, the liquid delivery method of this embodiment includes a step of pressurizing and introducing a solution from the second chamber 105 into the flow path 103 (flow path introduction step).

[0089] When or after a solution is introduced into flow path 103, bubbles may sometimes be generated within flow path 103. However, if sufficient pressure is applied to the solution to pressurize and remove the bubbles, the bubbles generated within flow path 103 will soon disappear.

[0090] Next, part or all of the solution flows through flow path 103 into the first chamber 104. Figure 3 (g)). Then, to allow the solution to circulate, the pressure-reducing device 107 is switched to pressure reduction (g). Figure 3 (h)). Thus, the solution moves through flow path 103 into the second chamber 105. Figure 3 (i) When part or all of the solution passes through flow path 103, one reciprocating flow of the solution is completed. This process is repeated when multiple reciprocating flows of the solution are performed. Figure 3 (e)~ Figure 3 (i) process.

[0091] It should be noted that the solution may also be conveyed in one direction instead of reciprocating. In this case, it is advantageous to provide a valve in the first chamber 104 for discharging the solution to the outside of the flow path device 101. During the liquid delivery process... Figure 3After step (g), the valve in the first chamber 104 is switched to open the first chamber 104 to the outside, and the solution is discharged out of the first chamber 104, thereby realizing one-way liquid delivery.

[0092] Next, the reason for applying the pressure required to suppress bubbles to the solution during liquid delivery when Equation (1) is satisfied will be explained.

[0093] When the solution moves to flow path 103, which is set to a higher temperature for the chemical reaction than room temperature or the temperature of the second chamber 105, the solubility of the gas in the solution decreases, and the gas expelled from the solution becomes bubbles. In this embodiment, the gas inside the bubbles thus generated is dissolved in the solution by applying pressure, thereby suppressing the bubbles.

[0094] Before the solution is introduced into flow path 103, the gas dissolves to its saturation concentration. By introducing the solution into flow path 103, the solubility decreases, and therefore the saturation concentration also decreases. If the amount of gas exceeding the saturation concentration does not escape from the solution, the gas pressure ratio within the solution is α / β. Therefore, if the solution is introduced immediately after entering flow path device 101 ( Figure 3 (a)) is set to the initial pressure P0, then before the solution is introduced into the flow path ( Figure 3 (d) If a pressure exceeding (α / β)·P0 is applied to the solution, no bubbles will be generated in the solution due to the increase in temperature.

[0095] Furthermore, since this type of flow path device is typically used at atmospheric pressure, P0 is atmospheric pressure. On the other hand, if the flow path device is used in a pressure chamber for some reason, the pressure inside the pressure chamber becomes P0.

[0096] However, since the gas inside the bubble is effectively pressurized by the surface tension at the gas-liquid interface, the pressure applied from the outside can be reduced accordingly. Regarding the increase in internal pressure of the bubble, given σ as the surface tension of the solution and d as the diameter of the bubble, ΔP = 4σ / d can be calculated. Therefore, if a pressure of (α / β)·P0 - ΔP is applied to the solution, the bubble can be removed. The value of d can be appropriately determined based on the size of the bubble to be removed.

[0097] Therefore, in reality, just before the solution is introduced into the flow path ( Figure 3 (d) If the solution is subjected to a pressure of (α / β)·P0-ΔP, then the pressure required to suppress bubbles can be applied at all times during the delivery of the solution.

[0098] It should be noted that d can also be the diameter of the flow path. Bubbles are formed by the growth of tiny bubble nuclei. The purpose of this embodiment is to suppress bubbles before they grow too largely as their diameter exceeds the flow path diameter; the bubble diameter is assumed to be less than the flow path diameter. The larger the bubble diameter, the smaller the increase in internal pressure (ΔP), and the greater the pressure required to suppress the bubbles. Therefore, the applied pressure can be specified by assuming that the bubble diameter is equal to the flow path diameter.

[0099] Here, the liquid delivery is carried out very slowly, and the pressurization of the solution is an isothermal process. According to Boyle's law, before the solution is introduced into flow path 103 ( Figure 3 (d) The pressure P applied to the solution 3d as follows.

[0100] [Number 7]

[0101] P0(V1+v+V2)=P 3d (V1+v)

[0102]

[0103] If the P 3d If the value is greater than or equal to (α / β)·P0-ΔP, then there will be no gas release (bubble generation) due to the increase in temperature. That is, the condition becomes P 3d ≥(α / β)·P0-ΔP. Rearranging the above two equations, bubbles within flow path 103 can be suppressed when the following conditions are met.

[0104] [Number 8]

[0105]

[0106]

[0107]

[0108] It should be noted that Patent Document 2 discloses a flow path device with a chamber. This document describes a method of utilizing compressed air within a chamber provided at one end of the flow path during reciprocating liquid delivery. However, the device described in this document has an air chamber connected to one side of the flow path (corresponding to the first chamber 104), but does not have a chamber connected to the opposite end of the flow path (corresponding to the second chamber 105). That is, the volume corresponding to V2 is extremely small compared to the volumes corresponding to V1 and v, failing to satisfy equation (1), and therefore cannot suppress air bubbles in the flow path.

[0109] Thus far, the suppression of bubbles caused by the decrease in solubility accompanying the setting of the flow path to a higher temperature has been explained. However, the ways in which bubble generation can be suppressed are not limited to this.

[0110] For example, when introducing a solution into a flow path, there may be areas where the solution is not fully filled due to bends in the flow path, resulting in air bubbles. Even if air bubbles are generated in this way, they can be removed by dissolving the gas in the solution using pressure. The pressure required in this case will be explained below.

[0111] Let V be the total volume of bubbles generated when the solution is introduced into the flow path 103. b Let the volume of the solution be V. s Before the solution is introduced into flow path 103, the gas is dissolved to a saturated concentration.

[0112] In this case, besides the gas αV dissolved in the solution before the solution is introduced into flow path 103, s In addition, it is also necessary to make the volume V b The gas dissolves in the solution. The required pressure relative to the initial pressure P0 is shown below.

[0113] [Number 9]

[0114]

[0115] As mentioned above, when considering the effect of pressure increase inside the bubble, if the solution is about to be introduced into the flow path 103 ( Figure 3 (d) Applying the pressure shown below to the solution can suppress bubbles.

[0116] [Number 10]

[0117]

[0118] As before, before the solution is introduced into flow path 103 ( Figure 3 (d) The pressure P applied to the solution 3d′ for:

[0119] [Number 11]

[0120] P0(V1+v+V2)=P 3d′ (V1+v)

[0121]

[0122] If the P 3d′ If the pressure is above the value shown below, bubbles can be suppressed.

[0123] [Number 12]

[0124]

[0125] Right now,

[0126] [Number 13]

[0127]

[0128] If it is tidied up, bubbles in the flow path can be suppressed when the following formula is satisfied.

[0129] [Number 14]

[0130]

[0131]

[0132] Here, the volume ratio of the bubbles to the solution is set as R = V. b / V s In chemical reactions using flow paths, if R ≥ 0.05, issues arise due to impediment to liquid delivery and reduced chemical reaction efficiency caused by air bubbles. Therefore, as a threshold, R = 0.05 is set, and the volumes of flow path 103, first chamber 104, and second chamber 105 are designed to satisfy the following formula, thereby eliminating air bubbles that cause the aforementioned problems.

[0133] [Number 15]

[0134]

[0135] Thus, this embodiment is also effective in suppressing bubbles generated during solution introduction.

[0136] [Example 2]

[0137] Hereinafter, Embodiment 2 of the present invention will be described. It should be noted that the description of parts that are common to Embodiment 1 is sometimes omitted.

[0138] Figure 6 This is a top view of the flow path device 101. Figure 7 (a) indicates along Figure 6 A cross-sectional view of line AA. This embodiment is relative to... Figure 1 The configuration shown differs from Embodiment 1 in that it includes a valve 201 that can open to the atmosphere. The valve 201 is located on the side opposite to the flow path 103 for the first chamber 104. Furthermore, this embodiment differs from Embodiment 1 in that the solution inlet 106 is located at the connection point between the second chamber 105 and the second side of the flow path 103 (i.e., the side different from the first side connecting the first chamber 104).

[0139] In this embodiment, the solution inlet 106 may be part of the flow path 103 or part of the second chamber 105. That is, if the solution inlet 106 has a volume that cannot be ignored, the volume may be configured to be included in the volume v of the flow path 103 or in the volume V2 of the second chamber 105.

[0140] exist Figure 6 In the middle, flow path 103 is not branched, but flow path 103 can also be connected to... Figure 1 Similarly, branches. Additionally, although the illustrations of probe 120 and bead 121 are omitted, either one or both can be present.

[0141] Valve 201 is, for example, an on / off valve. Figure 6 and Figure 7 As shown, valve 201 can be a miniature valve that can be installed in the flow path. Alternatively, valve 201 can also be a directional switching valve, or a directional switching valve can be installed on the pipe connected to the first chamber 104.

[0142] Figure 8 This describes the flow chart of solution introduction from the solution inlet to the pressurized flow path, involving reciprocating liquid delivery. The following is a description of the process. Figure 8 The steps of each action are explained.

[0143] In this embodiment, valve 201 is operated from a state where it is open to the atmosphere. Figure 8 (a)). First, the solution used for the chemical reaction in flow path 103 is introduced into flow path device 101 through solution inlet 106. Figure 8 (b)). Figure 9 This indicates the position of solution 110 at that moment.

[0144] After the solution is introduced, to prevent contamination of the solution, the opening of the solution inlet 106 is sealed with sealing material 109. Figure 8 (c)). This operation also helps to maintain the airtightness of the flow path device 101 when the solution is subsequently pressurized and delivered.

[0145] Next, the pressure is reduced in the flow path device 101 by the pressure reducing device 107. Figure 8 (d)). By reducing pressure, the solution moves towards the pressure-reducing device connection 202 inside the second chamber 105. Figure 8 (e)). Soon, all or almost all of the solution passes through the second chamber 105, and the depressurization stops at the moment it reaches the depressurization device connection 202. Figure 8 (f)). Figure 10 The position of solution 110 at this moment is schematically shown.

[0146] It should be noted that, in Figure 9 and Figure 10 In, also with Figure 6 Similarly, flow path 103 is not branched, but flow path 103 can also be connected to... Figure 1 Similarly, branches. Additionally, although the illustrations of probe 120 and bead 121 are omitted, either one or both can be present.

[0147] exist Figure 8 At time (f), valve 201 is opened, thereby maintaining the pressure inside flow path 103, first chamber 104, second chamber 105, and solution inlet 106 at the same level as the pressure of the surrounding environment (e.g., atmospheric pressure).

[0148] Figure 10 The position of solution 110 shown is relative to Figure 4 The position of the solution 110 shown is the same as the first position. Thus, the liquid delivery method of this embodiment may include: a step of moving the solution from the solution inlet 106 toward the first position with the valve 201 open; and then a step of placing the solution 110 at the first position on the side opposite to the flow path 103 relative to the second chamber 105 (placement step).

[0149] Next, close valve 201 to ensure the airtightness of flow path device 101. Figure 8 (g)). Then, pressurization of the flow path device 101 is initiated via the pressure-reducing device 107. Figure 8 (h)). By applying pressure, the solution moves in the second chamber 105, and soon the solution reaches the front of the flow path 103 within the second chamber 105. Figure 8 (i)). At this point, the solution is pressurized to a sufficient pressure in order to remove air bubbles in the flow path 103.

[0150] Next, in order to introduce the solution into the flow path 103 and allow for a chemical reaction, the flow path device 101 is further pressurized using the pressure reducing device 107. As a result, the solution moves from the second chamber 105 into the flow path 103 and is introduced into the flow path 103. Figure 8 (j)).

[0151] When or after a solution is introduced into flow path 103, bubbles may sometimes be generated within flow path 103. However, sufficient pressure is applied to the solution to pressurize and remove the bubbles, and the bubbles generated within flow path 103 will soon disappear.

[0152] Next, part or all of the solution flows through flow path 103 into the first chamber 104. Figure 8 (k)). Then, to allow the solution to circulate, the pressure-reducing device 107 is switched to pressure reduction (k). Figure 8(l)). Thus, the solution moves through flow path 103 into the second chamber 105. Figure 8 (m)). When part or all of the solution passes through flow path 103, one reciprocating flow of the solution is completed. In the case of multiple reciprocating flows of the solution, the process is repeated. Figure 8 (h)~ Figure 8 (m) process.

[0153] It should be noted that, as described in Example 1, the liquid delivery may not be reciprocating but can be unidirectional. In the case of liquid delivery in one direction, simply opening valve 201 to discharge the solution outside the first chamber 104 is sufficient.

[0154] Thus, in Example 2, just as in Example 1, the generation of bubbles can be suppressed. Furthermore, the freedom to position the solution inlet 106 is increased.

[0155] Figure 7 (b) shows a cross-sectional view of the flow path device 101 of a modified example of Embodiment 2. Figure 7 (b) is related to Figure 7 (a) Corresponding cross-sectional view. In this modified example, the flow path device 101 has a valve 203 in the solution inlet 106.

[0156] replace Figure 8 (c) involves sealing the opening of the solution inlet 106 with the sealing material 109, and then closing the opening of the solution inlet 106 with the valve 203. This allows for multiple opening and closing of the solution inlet 106, increasing the degree of operational freedom.

[0157] [Example 3]

[0158] Hereinafter, Embodiment 3 of the present invention will be described. This embodiment is a flow path device for performing PCR. It should be noted that descriptions of parts common to Embodiment 1 or Embodiment 2 are sometimes omitted.

[0159] Figure 11 This is a top view of the flow path device 101. Figure 12 express Figure 11 A cross-sectional view of AA. The flow path device 101 includes a substrate 102, a sealing material 109, a first external heat source 301, a second external heat source 302, and a device holding part 303. The first external heat source 301 and the second external heat source 302 each have a built-in thermocouple and a heater, functioning as a temperature control mechanism for forming a temperature band for PCR. The device holding part 303 is preferably made of a material with heat resistance and low thermal conductivity. For example, PEEK (polyetheretherketone) and PC (polycarbonate) are preferred.

[0160] exist Figure 11 In, also with Figure 6 Similarly, the flow path is not branched, but the flow path can also be... Figure 1 Similarly, branches. Additionally, although the illustrations of probe 120 and bead 121 are omitted, either one or both can be present.

[0161] In this embodiment, the flow path has multiple temperature regions, for example, it is divided into a first temperature region 304 and a second temperature region 305. The first temperature region 304 is heated to a first temperature by contacting a first external heat source 301, and the second temperature region 305 is heated to a second temperature (or a temperature different from the first temperature) by contacting a second external heat source 302.

[0162] Thus, the flow path device 101 of this embodiment includes a first external heat source 301 and a second external heat source 302 as a heating mechanism for heating the flow path.

[0163] In this embodiment, to accelerate PCR, a two-step PCR method is used, which involves repeatedly performing denaturation, annealing, and elongation in two temperature zones. The target temperature of the first temperature zone 304 is set as the annealing and elongation temperature band (e.g., 60°C), and the target temperature of the second temperature zone 305 is set as the denaturation temperature band (e.g., 95°C). Alternatively, the first temperature zone 304 can be set as the denaturation temperature band, and the second temperature zone 305 as the annealing and elongation temperature band.

[0164] The solution used in this embodiment can be, for example, a solution obtained by mixing one or more primers, a thermostable enzyme, and four deoxyribonucleoside triphosphates (dATP, dCTP, dGTP, dTTP) into a mixture containing one or more DNAs as the amplification target.

[0165] Figure 13 This describes the operational flow from the introduction of solution into the solution inlet 106 until the reciprocating flow within the pressurized flow path. It covers the operation from solution introduction until the solution passes through the second chamber and moves to the edge of the flow path. Figure 13 (a)~(d)) and Example 1 Figure 3 (a)~(d)) Similarly.

[0166] Then, the pressure-reducing device 107 is used to pressurize the flow path device 101, causing the solution to move to the first temperature region 304. Figure 13 (e)). The solution is left idle for a certain period of time in the first temperature region 304, thereby carrying out the annealing and elongation reaction. Figure 13 (f)). Next, the pressure is reduced by the pressure-reducing device 107, causing the solution to move to the second temperature region 305. Figure 13(g)). The solution is left to standby in the second temperature region of 305 for a certain period of time, thereby undergoing a denaturing reaction. Figure 13 (h)). By Figure 13 The steps (e) to (h) are repeated a specified number of times to amplify the DNA of the target.

[0167] Thus, since the flow path device of Example 3 has a heating mechanism, it can suppress the generation of bubbles in PCR in the same way as in Example 1.

[0168] It should be noted that, as a variation of Example 3, the heating mechanism can be separate, and the heating temperature can also be other temperatures. In particular, if the heating mechanism heats the flow path or at least a portion thereof to above 30°C, it may be applicable to chemical reactions that substantially require heating.

[0169] [Example 4]

[0170] Hereinafter, Embodiment 4 of the present invention will be described. This embodiment relates to a liquid delivery device capable of being equipped with any of the flow path devices of Embodiments 1 to 3 or their variations.

[0171] As an example of this embodiment, in Figure 14 The image shows that it is equipped with Figure 1 The flow path device 101 described herein includes a liquid delivery device 401. The liquid delivery device 401 is used to deliver a solution within the flow path device. The flow path device 101 and the liquid delivery device 401 constitute a liquid delivery device assembly.

[0172] The liquid delivery device 401 can be used in conjunction with the flow path device 101. The liquid delivery device 401 includes a pressure reducing device 107, a device holding part 303, a cover 402, an external heat source 403, a temperature control part 404, a sensor 405, and a signal detection part 406.

[0173] The pressure-reducing device 107 is an example of a pressurizing unit, connected to the solution inlet 106 via a pipe 108. The pressure-reducing device 107 delivers the solution by applying pressure via the solution inlet 106. By using the pressure-reducing device 107, the bubble suppression described in Examples 1 to 4 can be achieved.

[0174] The cover 402 serves to fix the flow path device 101, which is provided in the device holding part 303, in a predetermined position. The material of the cover 402 is preferably a material with heat resistance and low thermal conductivity, such as PC (polycarbonate).

[0175] External heat source 403 is an example of a heating mechanism. A thermocouple and a heater are built into the external heat source 403, and the temperature is maintained at the desired temperature by a temperature control unit 404.

[0176] Sensor 405 acquires information to observe the flow path in a chemical reaction. The signal acquired by sensor 405 is sent to signal detection unit 406. Sensor 405 can be at least one of a pressure sensor, a liquid level sensor, or a bubble detection sensor. For example, if a pressure sensor is used, the pressure increase / decrease of the pressure regulating device 107 can be controlled more appropriately; if a liquid level sensor is used, the position of the solution can be controlled more appropriately; and if a bubble detection sensor is used, the presence or absence of bubbles in the flow path can be confirmed.

[0177] Thus, the liquid delivery device according to Embodiment 4 can deliver the solution more appropriately within the flow path device.

[0178] [Example 5]

[0179] This embodiment is an experimental example of Embodiment 1, which is an experimental example that studies Equation (1) related to the chamber volume ratio in the flow path device required to suppress bubbles.

[0180] <Experiment>

[0181] A groove measuring 0.12 mm wide, 0.12 mm deep, and 10 mm long is machined in the center of a 10 mm × 40 mm × 2 mm thick COP substrate. A first chamber of flow path type with volume V1 is connected to one end of the flow path, and a second chamber of flow path type with volume V2 is connected to the other end. Ten beads with a diameter of 100 μm are filled into the groove, and a 0.1 mm thick PO sealing material is pressed into it to form the flow path (volume v = 0.14 mm). 3 ).

[0182] By varying the volume V1 of the first chamber and the volume V2 of the second chamber, multiple flow path devices with different values ​​of the chamber volume ratio V2 / (V1+v) are created.

[0183] Considering typical hybridization conditions, the flow path device is mounted on a heater to heat the flow path to 60°C and secured with a PC cover. On the opposite side of the flow path connection to the second chamber, 25 μL of a 4×SSC-0.1% SDS solution, serving as the standard hybridization solution, is introduced. The syringe pump is connected to the second chamber via tubing.

[0184] The flow path device is pressurized using a syringe pump, and the solution is introduced into the flow path through the second chamber. A digital microscope is used to confirm the presence of air bubbles in the flow path during the delivery process, and the probability of air bubble generation is calculated for the chamber volume ratio of each flow path device.

[0185] <Results>

[0186] Figure 15This indicates the relationship between the chamber volume ratio and the probability of bubble formation. Bubbles are generated when the chamber volume ratio V2 / (V1+v) is below approximately 0.4, while bubbles are suppressed when the chamber volume ratio V2 / (V1+v) exceeds approximately 0.5.

[0187] Next, the explanation Figure 15 The validity of the experimental results shown. In this embodiment, the solution was heated from room temperature (set at 20°C) to 60°C, which is the set temperature for the flow path. The solubility of air in water at 20°C is α = 0.019 cm⁻¹. 3 / cm 3 At 60℃, β = 0.012 cm. 3 / cm 3 Furthermore, the surface tension of water σ = 72.8 mN / m, and according to the reasons described in Example 1, if the bubble diameter d is equal to the flow path diameter, then d = 100 μm. Substituting these values ​​into equations (1) and (2), we get:

[0188] [Number 16]

[0189]

[0190] (exist Figure 15 (The approximate location of the value of 'a' is indicated by a dashed line). Therefore, Figure 15 The experimental results shown are appropriate, indicating that the chamber volume ratio conditions shown in Equations (1) and (2) can suppress bubbles in the flow path.

[0191] Example 5 is an experimental example of Example 1, but it is believed that the same experimental results can be obtained for Examples 2 to 4.

Claims

1. A method for delivering a solution, removing air bubbles from a solution introduced into a flow path device. The flow path device includes: The flow path of volume v; A first chamber of volume V1, which is connected to the first side of the flow path; and A second chamber of volume V2, which is connected to a second side of the flow path that is different from the first side. V1, V2, and v satisfy equation (1). [Number 1] in, [Number 2] α is the solubility of the gas in the solution at the temperature of the second chamber. β is the solubility of the gas in the solution at the temperature of the flow path. P0 is the pressure of the surrounding environment of the flow path device. ΔP is the pressure rise inside the bubble, ΔP = 4σ / d, where σ is the surface tension of the solution and d is the diameter of the flow path; The temperature of the flow path is set to be higher than the temperature of the second chamber. The method comprises: In the configuration process, a solution is configured at a first position on the opposite side of the flow path, relative to the second chamber; In the liquid delivery process, the solution is pressurized and delivered so that the solution moves toward the flow path in the second chamber; as well as In the flow path introduction process, a solution is introduced into the flow path under pressure from the second chamber.

2. The liquid delivery method according to claim 1, wherein, The flow path device includes: A solution inlet, disposed at the connection portion between the second chamber and the second side of the flow path; and A valve, which is positioned on the side opposite to the flow path relative to the first chamber, The method includes, prior to the configuration step: The process of introducing the solution into the flow path device at the solution inlet; and The process of moving the solution from the solution inlet toward the first position while the valve is open.

3. According to the liquid delivery method of claim 1, further, V1, V2 and v satisfy equation (3). [Number 3] in, [Number 4] 。 4. A flow path device that introduces and pressurizes a solution. The flow path device includes: The flow path of volume v; A first chamber of volume V1, which is connected to the first side of the flow path; and A second chamber of volume V2, which is connected to a second side of the flow path that is different from the first side. V1, V2, and v satisfy equation (1). [Number 5] in, [Number 6] α is the solubility of the gas in the solution at the temperature of the second chamber. β is the solubility of the gas in the solution at the temperature of the flow path. P0 is the pressure of the surrounding environment of the flow path device. ΔP is the pressure rise inside the bubble, ΔP = 4σ / d, where σ is the surface tension of the solution and d is the diameter of the flow path. The temperature of the flow path is set to be higher than the temperature of the second chamber.

5. The flow path device according to claim 4, wherein, The flow path device includes a solution inlet, which is disposed on the side opposite to the flow path relative to the second chamber.

6. The flow path device according to claim 4, wherein, The flow path device includes a solution inlet, which is disposed at the connection portion between the second chamber and the second side of the flow path.

7. The flow path device according to claim 4, wherein, The flow path device includes a heating mechanism for heating the flow path.

8. The flow path device according to claim 7, wherein, The heating mechanism heats the flow path to above 30°C.

9. The flow path device according to claim 4, wherein, The flow path device includes a valve that is positioned on the side opposite to the flow path relative to the first chamber.

10. The flow path device according to claim 4, wherein, The flow path has multiple branches.

11. The flow path device according to claim 6, wherein, The solution inlet is equipped with a valve.

12. The flow path device according to claim 4, wherein, At least one of the first chamber or the second chamber has a flow path shape.

13. The flow path device according to claim 4, wherein, Specific molecules that react chemically with specific substances are fixed in the flow path.

14. The flow path device according to claim 4, wherein, The flow path device includes beads disposed within the flow path, on which specific molecules that chemically react with a specific substance are fixed.

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