JP2008151772A - Method for controlling temperature of microfluidic chip, specimen analysis system, and microfluidic chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To stably detect specimens by preventing reductions and condensation in the quantity of liquid associated with evaporation, the movement of the liquid, and the occurrence of air bubbles when a prescribed quantity of liquid is heated in a channel of a microchip to acquire desired chemical reactions. <P>SOLUTION: In a method for controlling the temperature of a microfluidic chip 200, the temperature of a liquid stored in a fine reaction channel 35 is controlled in the microfluidic chip 200 having the reaction channel 35 formed in a plane plate. The reaction channel 35 is heated to a prescribed reaction temperature, and, simultaneously, a first channel 49a communicating with one end of the reaction channel 35 and a second channel 49b communicating with the other end of the reaction channel are maintained at the same temperatures different from the prescribed reaction temperature by heating or cooling. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a microfluidic chip temperature control method, a specimen analysis system, and a microfluidic chip that are used when performing analysis using various samples, chemical reactions, and the like.

  Recent advances in molecular biology have shown that by analyzing biological substances such as blood, it is possible to predict individual differences due to the effects of drug administration and side effects on the treatment of diseases. There is a growing interest in using this to provide optimal treatment for individuals. For example, if it is known that the effect and side effects of a specific gene and a specific therapeutic drug are strongly correlated, it is necessary to know the nucleotide sequence of the patient's gene in order to use this information for the treatment of a specific patient. is there. Genetic diagnosis for obtaining information on endogenous gene mutation or single nucleotide polymorphism (SNP) can be performed by amplification and detection of a target nucleic acid containing such mutation or single nucleotide polymorphism. Therefore, there is a need for a simple method that can rapidly and accurately amplify and detect a target nucleic acid in a sample.

  In particular, a nucleic acid amplification reaction by PCR (Polymerase Chain Reaction) is a basic technology in the biotechnology field. The PCR method is a method of amplifying a target DNA by adjusting the temperature of a reaction solution in which a template DNA, a primer, a substrate, a heat-resistant polymerase enzyme, etc. are mixed, and sequentially changing the temperature to three predetermined temperatures. It is. Specifically, the temperature of the reaction solution is adjusted to a temperature at which a dynalation reaction for dissociating double-stranded DNA into single-stranded DNA is performed, and then to a temperature at which an annealing reaction is performed to associate a primer with single-stranded DNA. The temperature is adjusted, and then the temperature is adjusted to a temperature at which a double-strand elongation reaction with a thermostable polymerase enzyme is performed. In this way, DNA can be amplified by sequentially adjusting the temperature of the reaction solution to three stages.

  As this type of technology, for example, there is a nucleic acid amplification substrate disclosed in Patent Document 1. In this nucleic acid amplification substrate, a reaction liquid reservoir is formed on a part of the substrate, and the reaction liquid reservoir is filled with a reaction liquid containing components necessary for the nucleic acid amplification reaction, and the temperature of the reaction liquid reservoir is adjusted. A nucleic acid amplification reaction is performed in the reaction reservoir to amplify the nucleic acid. That is, the nucleic acid amplification substrate is obtained by laminating a first layer made of a glass plate and a second layer made of silicon rubber. A gap is formed between the first layer and the second layer, and a void pattern (groove pattern) is formed. ) Form a nucleic acid amplification / separation flow path. The nucleic acid amplification / separation flow path is provided with a reaction liquid reservoir, and the reaction liquid reservoir is connected to a liquid supply path and a liquid discharge path. Then, a predetermined amount of liquid is allowed to flow from the reaction liquid supply path into the reaction liquid reservoir, and this liquid is heated for a predetermined time, so that DNA can be amplified by setting the atmosphere to a predetermined temperature.

JP 2006-115741 A

However, the above-described conventional nucleic acid amplification substrate arranges a certain amount of liquid at a predetermined position (reaction liquid reservoir) in the flow path and heats this liquid for a certain period of time. In this case, the fluid is communicated with the flow path (liquid supply path, liquid discharge path). As the heating progresses, the evaporation proceeds, and the liquid amount may be reduced or concentrated. When such a decrease in liquid volume or concentration occurs, for example, when the reaction liquid reservoir is inspected by fluorescence detection, there is a problem that stable detection cannot be performed.
In addition, the pressure on the upstream and downstream flow paths of the liquid rises due to the evaporation of moisture and the expansion of air, resulting in a difference in the pressure between the upstream and downstream flow paths. That is, an equal amount of heat is transmitted from the heated reaction liquid reservoir to the liquid supply path and the liquid discharge path, but due to the difference in heat capacity due to the difference in the flow path shape, the temperature in both flow paths, that is, the flow path A difference occurs in the internal pressure, causing a phenomenon in which the liquid in the reaction liquid reservoir moves. As a result, as shown in FIG. 11A, the heated liquid Lq moves from the reaction liquid reservoir 1 to the liquid supply path 3 or the liquid discharge path 5, and the fluorescence detection test is performed in the reaction liquid reservoir 1. In the case of performing the above, there arises a problem that stable detection cannot be performed in the same manner as described above.
Furthermore, when the substrate is made of a resin molding material, there is a resin filling defect such as a weld line in the reaction liquid reservoir 1, or when the lid material is adhered to the groove of the substrate, adhesive application unevenness occurs. If a minute space is formed for some reason such as inaccuracy of the mold itself (see weld line WL, chamfer radius R in FIG. 5A), the minute space generated due to the above cause when the reaction channel liquid is introduced. The liquid does not flow into the inside, and a minute wet residue, that is, a minute air pool is formed (see FIG. 5B). Then, if bubbles b are generated by expansion and increase of the air pool due to heating (see FIG. 5C), there is a problem that the accuracy of fluorescence detection is reduced as described above. FIG. 11B shows a state where bubbles b1 are generated when a weld line is present, FIG. 11C shows a state where bubbles b2 are generated when adhesive application unevenness is present, and FIG. A state is shown in which bubbles b3 are generated when the chamfer radius R of the flow path corner in the cross section is large.

  The present invention has been made in view of the above situation. When a predetermined amount of liquid is heated in the flow path of the microfluidic chip to obtain a desired chemical reaction, the amount of liquid is reduced and concentrated due to evaporation. An object of the present invention is to provide a microfluidic chip temperature control method, a sample analysis system, and a microfluidic chip that do not cause movement and generation of bubbles, and to stably detect the reaction state of the sample.

The above object of the present invention is achieved by the following configuration.
(1) A microfluidic chip temperature control method for controlling the temperature of a liquid stored in a reaction channel with respect to a microfluidic chip having a reaction channel formed by fine grooves,
While heating the reaction channel to a predetermined reaction temperature,
The first flow path communicating with one end of the reaction flow path and the second flow path communicating with the other end of the reaction flow path are maintained at the same temperature different from the predetermined reaction temperature by heating or cooling. A temperature control method for a microfluidic chip.

  According to this temperature control method of the microfluidic chip, after filling the reaction channel with the liquid, the central part between the first channel side and the second channel side is heated at a desired temperature necessary for the chemical reaction. However, by adjusting the temperature so that other locations are kept at a temperature different from this temperature, the decrease and concentration of the liquid accompanying evaporation and the movement of the liquid are suppressed, and it proceeds only in the center of the reaction channel. The chemical reaction to be performed can be sequentially examined.

(2) In the temperature control method for a microfluidic chip according to (1),
A temperature control method for a microfluidic chip, wherein the temperature of the first channel and the second channel is adjusted to a temperature lower than the reaction temperature.

  According to this temperature control method of the microfluidic chip, the central part of the reaction channel is heated at a desired temperature necessary for the chemical reaction, and the liquid temperature is maintained at a temperature lower than the reaction temperature in other places. By adjusting the temperature, even if multiple reaction channels are installed side by side, no chemical reaction occurs at both ends of the reaction channel, or the progress is extremely slow. Multiple chemical reactions can proceed independently without being affected. Moreover, evaporation of the liquid in the reaction channel can be prevented.

(3) In the microfluidic chip temperature control method according to (2),
A temperature control method for a microfluidic chip, wherein the temperature of the first channel and the second channel is adjusted to 20 to 30 ° C.

  According to this temperature control method for the microfluidic chip, the temperature difference between the first flow path and the second flow path is adjusted to normal temperature (20 to 30 ° C.), thereby reducing the temperature difference from the reaction section and reacting. It is possible to prevent the temperature of the part from becoming unstable.

(4) In the temperature control method for a microfluidic chip according to any one of (1) to (3),
After liquid is injected from the first channel into the reaction channel,
A method of controlling a temperature of a microfluidic chip, wherein the liquid is removed from the first flow path and the liquid remains only in the reaction flow path before the heating.

  According to this temperature control method for the microfluidic chip, the liquid is removed from the first channel before heating, so that the first channel and the second channel become liquid-free, and the reaction channel Only the liquid remains. Accordingly, the liquid in the reaction channel is sandwiched between the air layers of the first channel and the second channel, and is less susceptible to the reaction from the adjacent channel, so that the independent chemical reaction proceeds. It becomes possible.

(5) In the microfluidic chip temperature control method according to any one of (1) to (4),
Prior to the heating, a temperature adjusting method for a microfluidic chip, wherein an opening communicating with the first channel and the second channel is sealed.

  According to this temperature control method of the microfluidic chip, the opening at both ends of the reaction channel is sealed before the reaction channel is heated, and the reaction is performed in a sealed state. In other words, the first and second flow paths become sealed spaces, and evaporation exceeding the saturated water vapor pressure does not occur, and the effect of suppressing liquid evaporation can be obtained. At the same time, liquid, for example, amplified DNA flows out of the chip. The risk of polluting the environment and causing carryover is thus avoided.

(6) Micro formed with a reaction channel formed by fine grooves, a first channel communicating with one end of the reaction channel, and a second channel communicating with the other end of the reaction channel A fluid chip;
First temperature control means for controlling the temperature of the liquid injected into the reaction channel;
Second temperature control means for controlling the temperature of the first flow path and the second flow path;
A sample analysis system comprising:

  According to this sample analysis system, the liquid in the reaction flow path is heated to the desired reaction heating by the first temperature control means, while the first flow path and the second flow path in contact with the liquid are kept at the same temperature. Even if there is a difference in heat capacity between the first flow path and the second flow path due to a difference in flow path shape or the like, the pressure in the flow path is the same.

(7) The sample analysis system according to (6),
The second temperature control means controls the temperature of the liquid at the end of the reaction channel in contact with the first channel and the liquid at the end of the reaction channel in contact with the second channel. A specimen analysis system characterized by this.

  According to this sample analysis system, the temperature of the liquid in the reaction channel in contact with the first channel and the second channel is adjusted to the same temperature, and the first channel and the second channel from the liquid in the reaction channel. Heat is not transferred to the first flow path, and the internal pressures of the first flow path and the second flow path become the same with higher accuracy. That is, the movement of the liquid in the reaction channel can be more reliably suppressed.

(8) The sample analysis system according to (6) or (7),
A sample analysis system, wherein a detecting means for detecting a reaction state of the liquid injected into the reaction channel is disposed opposite to the reaction channel.

  According to this sample analysis system, the sensor or the like is not exposed in the reaction channel, and the liquid is not contaminated.

(9) The sample analysis system according to (8),
A sample analysis system, wherein the detection means measures fluorescence emitted by exciting a liquid in a reaction channel.

  According to this sample analysis system, the double-stranded DNA amplified by the reaction in the reaction flow channel emits strong fluorescence when intercalated. By measuring the fluorescence intensity, the presence or absence of the target gene sequence can be detected.

(10) A microfluidic chip used for the sample analysis system according to any one of (6) to (9),
A microfluidic chip, wherein a reagent is spotted in advance in a dry state above the reaction channel.

  According to this microfluidic chip, when the reaction channel is heated from the lower surface, the reagent dissolved as the temperature of the liquid rises and flows downward in the channel due to gravity, and the reagent and liquid are mixed by natural convection. Is done.

(11) The microfluidic chip according to (10), wherein the reagent is previously dried in a central portion of the reaction channel between the first channel side and the second channel side. A microfluidic chip that is spotted.

  According to this microfluidic chip, only the central portion of the reaction channel on which the dry reagent is spotted is heated, and there is no dry reagent on the first channel side and the second channel side of the reaction channel, Further, since no heating is performed, no chemical reaction occurs on the first and second flow path sides of the reaction flow path, or the progress is extremely slow. Therefore, the reactions in the plurality of reaction channels do not interfere with each other without draining the liquid in the first channel. The fact that the liquid in the first channel does not need to be drained not only has the effect of simplifying the operation sequence, but also holds the liquid in the reaction channel when the liquid in the first channel is drained. The relationship of (equivalent pipe diameter of the first flow path)> (equivalent pipe diameter of the reaction flow path), which is a condition for the above, can be ignored, compared with when the liquid in the first flow path is drained Thus, the equivalent pipe diameter of the first flow path can be reduced. As a result, the dead volume of the liquid to be inspected can be reduced.

(12) The microfluidic chip according to (10) or (11),
A microfluidic chip comprising a plurality of the reaction channels arranged in parallel.

  According to this microfluidic chip, a plurality of reaction channels are provided, and different types of reagents to be reacted are set in advance in the center of each channel, so that a plurality of chemical reactions can be simultaneously examined.

(13) The microfluidic chip according to any one of (10) to (12),
In the reaction flow path, the flow path height at the central part of the flow path from the first flow path side to the second flow path side is lower than the height other than the central part of the reaction flow path. A microfluidic chip, wherein a gradient is provided on an upper wall surface of the reaction channel.

According to this microfluidic chip, even when bubbles are generated in the channel during heating, the bubbles are prevented from moving or staying in the center of the channel, and the bubbles generated in the center are Move quickly toward both ends. This prevents a decrease in fluorescence detection accuracy.

(14) The microfluidic chip according to (13),
The microfluidic chip, wherein the gradient is formed by a downwardly convex curved surface in which the upper wall surface protrudes into the reaction channel.

  According to this microfluidic chip, by forming a gradient composed of a downwardly convex curved surface in the reaction channel, the bubbles can be smoothly moved to the end portions even when bubbles are generated.

(15) The microfluidic chip according to any one of (10) to (14),
The microfluidic chip, wherein an inner wall surface of the reaction channel is formed of a continuous smooth surface that prevents formation of a minute gap space that is not filled with the liquid when the liquid flows.

  According to this microfluidic chip, the inner wall surface of the flow path is a continuous smooth surface in which a minute gap space is not formed, and bubbles are not generated in the flow path during heating. This prevents a decrease in fluorescence detection accuracy.

(16) The microfluidic chip according to any one of (10) to (15),
A microfluidic chip, wherein a radius of curvature of a bent portion of an inner wall surface of the reaction channel is 20 μm or less.

  According to this microfluidic chip, when the radius of curvature of the bent portion of the inner wall surface is 20 μm or less, the wettability to the liquid channel surface is particularly poor (the contact angle is about 50 ° or more), and the bent portion is wetted. It is difficult for the residue to occur, and air accumulates in the remaining wet portion, and the air does not become bubbles by heating.

  According to the temperature control method for a microfluidic chip according to the present invention, the reaction channel is heated to a predetermined reaction temperature, and at the same time, the first channel communicating with one end of the reaction channel and the other end of the reaction channel are communicated. The second flow path is maintained at the same temperature different from the predetermined reaction temperature by heating or cooling, so that a certain amount of liquid is heated in the flow path of the microchip to obtain a desired chemical reaction. It is possible to suppress the decrease / concentration of the liquid amount accompanying the evaporation and the movement of the liquid. As a result, the chemical reaction that proceeds only in the central portion of the reaction channel can be sequentially inspected in situ (for example, fluorescence detection) while heating. As a result, stable detection of the reaction state of the specimen can be realized.

  According to the sample analysis system of the present invention, the microfluidic chip in which the reaction channel, the first channel, and the second channel are formed, and the temperature of the liquid injected into the reaction channel is controlled. Temperature control means, and second temperature control means for controlling the temperature of the first flow path and the second flow path, so that the liquid in the reaction flow path can be subjected to a desired reaction by the first temperature control means. While heating to a temperature, the first channel and the second channel in contact with the liquid can be set to the same temperature. Thereby, even when there is a difference in heat capacity between the first flow path and the second flow path due to a difference in flow path shape, the temperature in both flow paths, that is, the pressure in the flow path can be made the same, The phenomenon that the liquid in the reaction liquid reservoir moves can be prevented.

According to the microfluidic chip according to the present invention, since the reagent is preliminarily spotted in a dry state on the upper center portion of the reaction channel, the reagent is disposed on the upper side of the reaction channel when the microchip is used. By heating, the reagent dissolved as the temperature of the liquid rises flows downward in the flow path due to gravity, and the reagent / liquid can be well mixed by natural convection.
Further, only the portion where the dry reagent is present is heated, and there is no dry reagent on the first and second flow path sides of the reaction flow path, and no heating is performed. On the channel side, no chemical reaction occurs or the progress is extremely slow. Therefore, the reactions in the plurality of reaction channels do not interfere with each other without draining the liquid in the first channel, and the liquid feeding operation sequence is simplified. Furthermore, the dimensional condition for retaining the liquid in the reaction channel when the liquid in the first channel is drained can be ignored, and the first flow is compared with the case where the liquid in the first channel is drained. The equivalent pipe diameter of the path can be reduced. As a result, the dead volume of the liquid to be inspected can be reduced.

Hereinafter, preferred embodiments of a microfluidic chip temperature control method, a sample analysis system, and a microfluidic chip according to the present invention will be described in detail with reference to the drawings.
FIG. 1 is a block diagram of a sample analysis system according to the present invention, and FIG. 2 is an explanatory view of a main part schematically showing (a) a plan view of a microfluidic chip holding unit and (b) a sectional view taken along the line AA It is.
In the present embodiment, a microfluidic chip (hereinafter also simply referred to as “chip 200”) 200 into which a specimen has been injected is set in the specimen analysis system 100. When the microfluidic chip 200 is set in the specimen analysis system 100, the specimen liquid injected by the physical action force from the outside of the chip is handled, and for example, a single nucleotide polymorphism multiple target gene is examined. This makes it possible to amplify and detect the target nucleic acid specific to the pathogen causing the infection, for example, by amplifying the target nucleic acid and detecting it, and determine the presence or absence of the pathogen in the sample. It becomes.

  In the present embodiment, the physical acting force is a pneumatic action generated by air supply or air suction from a plurality of ports 13, 15, 17 provided at the start point and end point of the flow path 11 shown in FIG. 2 of the chip 200. Force (pneumatic driving force). Therefore, the movement of the liquid supplied to the flow path 11 can be controlled to a desired position in the flow path by air supply or air suction applied to the start point and end point of the flow path.

  As shown in FIGS. 1 and 2, the sample analysis system 100 includes a pump PMP in which air is used as a working fluid, a valve SV, and a pneumatic driving force from the pump PMP via a valve SV, Port connector 19 connected to 15, 17, specimen heating unit (heater) 21 as the first temperature control means, temperature controller 23 as the second temperature control means, and liquid injected into the reaction channel A fluorescence detection unit 25 as a detection means for detecting the reaction state of the sensor, a positioning mechanism 27 for positioning the microfluidic chip 200, and a control unit 29 connected to these to receive a detection signal or send a control signal. It is provided as a basic component.

  The microfluidic chip 200 is a reaction flow that is a minute flow path 11 formed in a flat plate by forming a fine groove on a flat plate surface 31 that is a lower side surface in the drawing and bonding a lid member 33 to the flat plate surface 31. A channel 35, a first channel 37 that communicates with one end of the reaction channel 35, and a second channel 39 that communicates with the other end of the reaction channel 35 are formed.

  The heater 21 controls the temperature of the liquid injected into the reaction channel 35. The heater 21 may be any one such as a resistance heater as long as it does not emit light such as a halogen lamp. This is to prevent disturbance of the fluorescence detection unit 25. The temperature controller 23 controls the temperatures of the first flow path 37 and the second flow path 39. In the present embodiment, the temperature controller 23 is provided so as to surround the heater 21, but may be separate from the left and right in FIG. 2. As the temperature controller 23, for example, a water-cooled heat sink, a Peltier element, or the like can be used.

  The DNA amplification reaction is maintained at a temperature at which the activity of the enzyme used can be maintained constant by an isothermal amplification reaction. Here, “isothermal” refers to a substantially constant temperature at which the enzyme and primer can substantially function. Furthermore, “substantially constant temperature” means that any temperature change that does not impair the substantial functions of the enzyme and the primer is acceptable.

  In the sample analysis system 100, the liquid injected into the reaction channel 35 is heated to a desired reaction temperature by the heater 21, while the first channel 37 and the second channel 39 in contact with the liquid have the same temperature. Set to Thereby, even if there is a difference in heat capacity between the first flow path 37 and the second flow path 39 due to a difference in flow path shape or the like, the pressure in the flow path becomes the same.

  The positioning mechanism 27 can press and hold the microfluidic chip 200 from above. At this time, either one (heater 21 in the present embodiment) is elastically pressed against the chip 200 by the spring mechanism 43 or the like so that the heater 21 and the temperature controller 23 are surely in contact with the microfluidic chip 200. Is good. Further, in order to reliably transfer the heat of the heater 21 and the temperature controller 23 to the chip 200, it is preferable to improve the adhesion (contact area) with the chip 200. It is preferable to dispose an elastic sheet material having good properties.

  In addition, the temperature controller 23 controls the temperatures of the liquid Lq1 at the end of the reaction channel 35 that contacts the first channel 37 and the liquid Lq2 at the end of the reaction channel 35 that contacts the second channel 39. It is preferable that The liquids Lq1 and Lq2 in the reaction channel 35 in contact with the first channel 37 and the second channel 39 are adjusted to the same temperature, so that the first channel from the liquid Lq in the reaction channel 35 37 and the second flow path 39 are biased and heat is not transferred, and the internal pressures of the first flow path 37 and the second flow path 39 become the same with higher accuracy. Thereby, the movement of the liquid in the reaction channel 35 can be more reliably suppressed.

  The fluorescence detection unit 25 is disposed so as to face the reaction channel 35, so that it is not exposed to the reaction channel 35 and does not contaminate the liquid. The fluorescence detection unit 25 measures the fluorescence emitted when the liquid in the reaction channel 35 is excited. That is, the double-stranded DNA amplified by the reaction in the reaction channel 35 emits strong fluorescence when intercalated. By measuring the fluorescence intensity, the presence or absence of the target gene sequence can be detected.

  That is, the reaction channel 35 is excited by the fluorescence detection unit 25 at a wavelength of about 490 nm, and the intercalated cyber green fluorescence of about 520 nm is measured, thereby confirming the amplification of the target DNA. Therefore, when the target nucleic acid sequence is present, an increase in fluorescence intensity is confirmed, and when it is not present, an increase in fluorescence intensity is not confirmed.

  According to this sample analysis system 100, the microfluidic chip 200 in which the reaction channel 35, the first channel 37, and the second channel 39 are formed, and the temperature of the liquid injected into the reaction channel 35 are controlled. Heater 21 and a temperature controller 23 for controlling the temperature of the first flow path 37 and the second flow path 39, the liquid in the reaction flow path 35 is heated to a desired reaction temperature by the heater 21. On the other hand, the first flow path 37 and the second flow path 39 in contact with the liquid can be set to the same temperature. Thereby, even when there is a difference in heat capacity between the first flow path 37 and the second flow path 39 due to a difference in flow path shape, the temperature in both flow paths, that is, the pressure in the flow path is made the same. And the phenomenon that the liquid in the reaction channel 35 moves can be prevented.

Next, the microfluidic chip 200 will be described.
FIG. 3 is an enlarged view of the main part of the microfluidic chip, showing details of the microfluidic chip in the AA cross-sectional view of FIG. 2 and (b) of which is a plan view thereof, and FIG. FIG. 5 is an explanatory diagram of the reagent mixing state in which the cross-sectional view of the reaction channel is shown in (a) and the enlarged view is in (b). FIG. FIG.
The substrate 41 of the microfluidic chip 200 has an outer shape of 55 × 91 mm in length and width, for example, and a thickness t1 of about 2 mm.

  The substrate 41 is manufactured by injection molding of a thermoplastic polymer. The polymer to be used is not particularly limited, but is preferably optically transparent, high in heat resistance, chemically stable, and easily injection-molded. COP (cycloolefin polymer), COC ( Cycloolefin copolymer), PMMA (methyl methacrylate resin) and the like are suitable. Optically transparent means high transparency at the wavelength of excitation light or fluorescence used for detection, small scattering, and little autofluorescence. The chip 200 has translucency that allows fluorescence to be detected, so that, for example, Cyber Green is used as a detection reagent, and fluorescence emitted by intercalating into double-stranded DNA amplified by the reaction can be measured. Become.

  As shown in FIG. 3, the microfluidic chip 200 more specifically has a reaction portion that has a depth t of the digging portion 45 above the reaction flow channel 35 of 0.5 mm and is the central portion of the reaction flow channel 35. The depth h2 of 47 is 1 mm, the depth h1 of the connecting pipe portions 49a and 49b on the left and right sides of the reaction section 47 is 0.5 mm, and the depth h3 of the narrow pipe 51 that connects the connecting pipe portion 49b and the second flow path 39 is 0.1 mm, the length L1 of the reaction channel 35 is 16 mm, the length L2 of one connecting tube portion 49a is 5.65 mm, the length L4 of the reaction portion 47 is 4 mm, and the length L3 of the other connecting tube portion 49b. 5.75 mm, the width W 1 of the first flow path 37 is 0.5 mm, the width W 3 of the connection pipe portions 49 a and 49 b is 0.3 mm, the width W 4 of the narrow pipe 51 is 0.1 mm, and The width W2 is 0.5 mm, and the widening angle α of the reaction part 47 is 60 °.

  In the present embodiment, a plurality of reaction channels 35 are arranged in parallel as shown in FIG. As shown in FIG. 3, each reaction channel 35 is connected to the first channel 37 via a connecting pipe portion 49a, and to the second channel 39 via a narrow tube 51 and a connecting tube portion 49b. It is connected. Different types of reagents to be reacted are set in advance in the reaction sections 47 of the plurality of reaction channels 35. Thereby, a plurality of chemical reactions can be inspected simultaneously.

  As shown in FIG. 4, reagents 53a, 53b, and 53c are preliminarily spotted in a dry state on the upper side of the reaction units 47a, 47b, and 47c. That is, a primer for target DNA and an aqueous solution of gelatin are dried, solidified and fixed after spotting. By heating the reaction channel 35 to 60 ° C., the solidified gelatin is dissolved and dispersed in the reaction unit 47, and an isothermal amplification reaction is performed. Although only the primer aqueous solution can be spotted on the reaction part 47 and dried and fixed, in this case, when the liquid flows into the reaction part, the primer flows in the flow direction, and the reaction in the reaction part Cannot be detected. For this reason, 0.5% gelatin which is difficult to dissolve in an aqueous solution at room temperature is contained and spotted and solidified.

  An aqueous solution of primer and gelatin is spot-fixed on the upper side of the reaction section 47, and is arranged on the upper surface of the flow path as shown in FIG. 4 when the microchip is used. As shown in FIG. 4 (b), the gelatin ge containing the reagent 53 dissolved with the temperature rise of the liquid is heated by the lid 33 side, that is, the lower surface after the liquid flows in. It flows under the reaction part 47 by gravity. Further, the liquid is heated from the lower surface, thereby causing convection 55 in the reaction unit 47. Due to the synergistic effect of the downward flow of the gelatin ge due to gravity and the convection 55 due to the heating of the liquid, the reagent 53 and the gelatin ge are uniformly mixed and diffused in the reaction portion 47 in a short time.

  By the way, in the channel 11 (see FIG. 2), particularly the reaction channel 35, the corner shape of the groove shown in FIG. When a reagent is introduced into the reaction channel 35, if there is a chamfer radius R at the corner shown in FIG. 5A, that portion remains without touching the reagent as shown in FIG. 5B. . The remaining portion is in a state where air 57 is accumulated, and when heated in this state, as shown in FIG. 5C, the air 57 expands and becomes a large bubble b. The bubble b causes a false detection or a problem that the liquid is pushed out.

Therefore, in order to prevent erroneous detection due to the bubbles b, the height of the central portion of the flow path from the first flow path 37 side to the second flow path 39 side in the reaction flow path 35 is the reaction height. It is preferable to provide a gradient that is lower than the central portion of the flow path. A configuration in which a gradient is provided on the upper wall surface in the reaction channel 35 will be described.
FIG. 6 shows an enlarged cross-sectional view of the central portion of the reaction channel 35.
The reaction part 47 that is the central part of the reaction flow path 35 has a flow path height lower than that of the region other than the central part. That is, the upper wall surface of the reaction channel is formed by a downwardly convex curved surface 51 that protrudes into the channel with a radius R1. An interval La between the connection portions 53a and 53b with the flat surface of the upper wall surface at both ends of the curved surface 51 is set to be approximately equal to the length L4 of the reaction portion 47 described above. Since the curved surface 51 is formed in substantially the entire region of the reaction part 47, even if bubbles are generated in the reaction channel 35, the generated bubbles are generated along the curved surface 51 above the channel in the reaction unit 47. Move quickly to. As a result, the bubbles accumulate in the connection portions 53a and 53b, or flow into other regions of the reaction channel 35, and the bubbles move or stay at the observation position of the reaction unit 47 (the central position of the reaction flow detour). There is nothing. Therefore, in the fluorescence detection in the reaction part 47, the malfunctions, such as an erroneous detection by a bubble and pushing a liquid out, are lost, and the fall of fluorescence detection accuracy is prevented.

FIG. 7 shows another configuration example of the gradient provided in the reaction channel 35 shown in FIG.
In this configuration, the shape of the reaction channel 35 shown in FIG. 3A is close, but the upper wall surface of the reaction channel 35 in the reaction unit 47 projects into the channel with the same radius R2 as in FIG. The curved surface 55 has a downwardly convex shape. End portions 57a and 57b at both ends of the curved surface 55 are formed at positions where the substrate 41 is recessed by the inclined surfaces 59a and 59b to increase the flow path height. Since the end portions 57a and 57b are formed with minute curved surfaces and are formed at positions where the flow path height is higher than the center portion of the curved surface 55, they serve as pockets for containing bubbles. According to the above configuration, it is possible to prevent bubbles from moving to or staying at the observation position of the reaction unit 47 while keeping the capacity of the reaction unit 47 large.

  Here, the curved surfaces 51 and 55 in FIGS. 6 and 7 are not limited to curved shapes, and may be any shapes as long as bubbles do not move to the observation position of the reaction unit 47 or do not stay. For example, two inclined surfaces may be formed in a downward convex shape connected at the center of the reaction portion 47, or one inclined surface may be formed over substantially the entire reaction portion 47.

  The problem of the generation of bubbles as described above mainly occurs when the substrate 41 of the chip 200 is manufactured by resin molding. When the mold is directly processed by an end mill or the like, even a small end mill corner has a curved surface with a radius of about 20 μm. Therefore, in this portion of the mold itself, the portion corresponding to the corner on the lid 33 side of the flow channel groove. A curved surface is formed. In addition, depending on molding conditions, the resin does not reach the corners sufficiently, resulting in insufficient filling, and a larger curved surface can be formed in the molded product. Therefore, the radius R is 20 μm or less, preferably 5 μm or less. Generation of bubbles can be prevented by setting the radius R within the above range.

  Further, when the microfluidic chip 200 is manufactured by resin molding, a groove WL called a weld line may be formed on the bottom surface of the flow path depending on how the resin flows during molding. If there is a groove WL by the weld line in the reaction flow path 35, a large bubble b is generated on the same principle as described above. Similarly to the radius R, the depth of the groove WL is 20 μm or less, preferably 5 μm or less.

  As described above, since the radius of curvature of the bent portion of the inner wall surface in the flow channel 11 is 20 μm or less, even when the wettability of the liquid with respect to the flow channel surface is not good (contact angle is about 50 ° or more) It is possible to effectively prevent the wet residue from being generated in the portion, and the air 57 from being accumulated in the wet remaining portion and the air 57 from being heated to become bubbles b. In other words, the inner wall surface of the flow path 11 may be a continuous smooth surface that prevents the formation of a minute gap space that is not filled with the liquid when the liquid flows. Thereby, the bubble b does not generate | occur | produce in a flow path in the case of a heating, and the fall of fluorescence detection accuracy is prevented.

  In the microfluidic chip 200, the flow path 11 is formed by adhering the lid member 33 to the flat plate surface 31. The lid member 33 is joined to the flat plate surface 31 with an adhesive or an adhesive. As the lid member 33, like the substrate 41, a sheet-like polymer polymer that is optically transparent, has high heat resistance, and is chemically stable is used. In this embodiment, a PCR plate seal having a thickness of 100 μm is used (adhesive is applied to a plastic film).

Further, in the case where a problem occurs when the reactant leaks to the outside, the microfluidic chip 200 may block all the ports 13, 15, and 17 with a sealing material such as an adhesive tape in order to seal the inside. preferable. That is, the microfluidic chip 200 seals the chip 200 by removing the port connector 19 from the ports 13, 15, and 17 and applying an adhesive tape when the desired conveyance is completed. That is, before the reaction channel 35 is heated, the opening with respect to the reaction channel 35, that is, the first channel 37 and the second channel 39 are sealed, and the reaction is performed in a sealed state. Therefore, a space between the first flow path 37 and the second flow path 39 becomes a sealed space, and evaporation exceeding the saturated water vapor pressure does not occur, and an effect of suppressing liquid evaporation can be obtained.
When the amplification reaction is performed without sealing the chip 200, the amplified DNA flows out of the chip, contaminating the environment, and there is a risk of carryover. However, the channel of the chip 200 is sealed before the amplification reaction. This can be prevented by setting the state. As a sealing method for sealing the flow path of the chip 200, in addition to the method of attaching the adhesive tape described above, a method of plugging with a cap having a sealing property, or after pouring UV curable resin into the ports 13, 15, 17 A known sealing method such as solidification by irradiation with UV light can be used.

  Therefore, according to the microfluidic chip 200, the reagent 53 is in a dry state on the upper side surface in the central portion of the flow path between the first flow path 37 side and the second flow path 39 side of the reaction flow path 35. Since the reagent 53 is deposited in advance, when the microchip is used, the reagent 53 is arranged on the upper side of the reaction channel 35 and heated from the lower surface, so that the reagent 53 dissolved as the temperature of the liquid Lq rises is reduced by gravity. Flows inward and downward, allowing good mixing of reagents and liquids by natural convection.

Next, a sample analysis method using the sample analysis system 100 and the microfluidic chip 200 will be described.
FIGS. 8A and 8B are operation explanatory views at the time of liquid injection into the reaction channel, where FIG. 8A is a plan view and FIG. 8B is a cross-sectional view of the reaction channel. FIGS. 9A and 9B are explanatory views of the operation at the time of liquid removal from the first flow path. FIG. 9A is a plan view and FIG. 9B is a cross-sectional view of the reaction flow path. FIG. 10 is a graph showing the temperature distribution in the reaction channel and its vicinity.
First, the microfluidic chip 200 is positioned and held in the positioning mechanism 27 of the sample analysis system 100. In this state, the temperature controller 23 is adjusted to normal temperature (for example, 25 ° C.). Next, a predetermined amount of reagent is introduced from the port 13 and introduced into the first flow path 37.

  When the reagent reaches just before the port 15, the conveyance is stopped. Next, the port connector 19 (see FIG. 1) connected to the port 17 is decompressed by the pump PMP, and the reagent is introduced into the reaction channel 35 as shown in FIG. Next, the port 17 is closed, the port connector 19 connected to the port 15 is pressurized by the pump PMP, and the reagent remaining in the first flow path 37 is pushed back to the port 13 as shown in FIG.

  Thereafter, the temperature of the heater 21 is adjusted to a temperature required for the chemical reaction (60 ° C. in the present embodiment), and the reaction is started. The first flow path 37 and the second flow path 39 are held at 25 ° C. by the temperature controller 23 and the reaction flow path 35 is held at 60 ° C., so that the substrate 41 is placed in the Y direction in FIG. The respective positions of the connecting pipe portion 49a of the first flow path 37, the reaction flow path 35, and the connecting pipe section 49b of the second flow path 39 along the temperature distribution shown in FIG. Since both the connecting pipe portion 49a of the first flow path 37 and the connecting pipe section 49b of the second flow path 39 are set to 25 ° C., evaporation from both ends of the reaction flow path 35 is extremely small, and the reaction flow The reagent in the channel 35 is not reduced. In addition, since the air temperature in the first flow path 37 and the second flow path 39 does not increase and the evaporation from the reaction flow path 35 is so small that it can be ignored, the atmospheric pressure in the pipeline does not increase. There is no pressure difference between both ends of the reaction channel 35, and no reagent leaks from the reaction channel 35.

Note that the operation of pushing the reagent remaining in the first flow path 37 back to the port 13 is not necessarily required. Even when different types of chemical reactions are performed in the reaction sections 47 of the plurality of reaction channels 35, the reagent to be reacted is spotted only on the reaction section 47, and only the reaction section 47 is heated, The rate at which the chemical reaction proceeds to both ends is negligibly slow. Therefore, even when the reagent dispensed into the plurality of reaction flow paths 35 is connected via the first flow path 37, each reaction is not affected.
That is, according to this microfluidic chip, the reactions in the plurality of reaction channels 35 do not interfere with each other without draining the liquid in the first channel 37. Moreover, the fact that the liquid in the first flow path 37 does not have to be drained has the effect of simplifying the operation sequence and the liquid in the reaction flow path 35 when the liquid in the first flow path 37 is drained. The relationship of the condition for maintaining (equivalent pipe diameter of the first flow path)> (equivalent pipe diameter of the reaction flow path) can be ignored. Therefore, the equivalent tube diameter of the first flow path 37 can be made smaller than when the liquid in the first flow path 37 is removed, and as a result, the dead volume of the liquid to be inspected can be reduced.

  According to the temperature control method of the microfluidic chip 200, the reaction channel 35 is heated to a predetermined reaction temperature (60 ° C.), and at the same time, the first channel 37 and the reaction channel communicating with one end of the reaction channel 35 The second flow path 39 communicating with the other end of 35 is the same temperature different from the predetermined reaction temperature by heating or cooling (in this embodiment, 25 ° C., 20 or more, which is a temperature lower than the reaction temperature, In a range of 30 ° C. or less), a certain amount of liquid is heated in the flow path of the chip 200 to obtain a desired chemical reaction. The movement of the liquid can be suppressed. Thereby, the state of the chemical reaction that proceeds only in the reaction section 47 of the reaction flow path 35 can be sequentially inspected (for example, fluorescence detection) while heating. As a result, stable detection of the specimen can be realized. If the temperature of the first flow path 37 and the second flow path 39 is controlled to a room temperature lower than 20 ° C., dew is generated on the surface of the microfluidic chip 200, which hinders detection of the reaction flow path 35. When the temperature is adjusted to a temperature higher than ° C., the effect of preventing the movement of the liquid is reduced. In addition, by suppressing the temperature difference between the first flow path 37, the second flow path 39, and the reaction section 47, it is possible to prevent the temperature of the reaction section 47 from becoming unstable.

FIG. 1 is a block diagram of a sample analysis system according to the present invention. It is principal part explanatory drawing which represented the planar view of the microfluidic chip holding | maintenance part to (a) and the AA cross-sectional view schematically to (b). It is the principal part enlarged view of the microfluidic chip which showed the detail of the microfluidic chip in the AA cross-sectional view of FIG. 2 (a), and represented the planar view in (b). It is explanatory drawing of the reagent mixing condition which represented the cross-sectional view of the reaction flow path where the reagent was spotted in (a), and the enlarged view was represented in (b). It is explanatory drawing which represented the bubble generation process in the reaction flow path by (a)-(c). It is an expanded sectional view in the center part of a reaction channel. It is an expanded sectional view which shows the other structural example of the gradient provided in the reaction flow path shown in FIG. FIG. 2 is an explanatory view of operation at the time of liquid injection into a reaction channel, (a) is a plan view, and (b) is a sectional view of the reaction channel. FIG. 5A is a plan view of an operation when liquid is removed from a first flow path, and FIG. 5B is a cross-sectional view of a reaction flow path. It is a graph showing the temperature distribution of the reaction channel and its vicinity. It is explanatory drawing which shows the malfunction (a)-(d) in the conventional flow path.

Explanation of symbols

21 Heater (first temperature control means)
23 Temperature controller (second temperature control means)
25 Fluorescence detection part (detection means)
31 Flat surface 33 Lid 35 Reaction channel 37 First channel 39 Second channel 51,55 Down-convex curved surface 100 Sample analysis system 200 Microfluidic chip Lq Liquid Lq1 Reaction flow in contact with the first channel Liquid at the end of the channel Lq2 Liquid at the end of the reaction channel in contact with the second channel

Claims (16)

A microfluidic chip temperature control method for controlling the temperature of a liquid stored in the reaction channel with respect to a microfluidic chip having a fine reaction channel formed in a flat plate,
While heating the reaction channel to a predetermined reaction temperature,
The first flow path communicating with one end of the reaction flow path and the second flow path communicating with the other end of the reaction flow path are maintained at the same temperature different from the predetermined reaction temperature by heating or cooling. A temperature control method for a microfluidic chip. In the temperature control method of the microfluidic chip according to claim 1,
A temperature control method for a microfluidic chip, wherein the temperature of the first channel and the second channel is adjusted to a temperature lower than the reaction temperature. In the temperature control method of the microfluidic chip according to claim 2,
The temperature adjusting method for a microfluidic chip, wherein the temperature to be adjusted is 20 to 30 ° C. In the temperature control method of the microfluidic chip according to any one of claims 1 to 3,
After liquid is injected from the first channel into the reaction channel,
A method of controlling a temperature of a microfluidic chip, wherein the liquid is removed from the first flow path and the liquid remains only in the reaction flow path before the heating. In the temperature control method of the microfluidic chip according to any one of claims 1 to 4,
Prior to the heating, a temperature adjusting method for a microfluidic chip, wherein an opening communicating with the first channel and the second channel is sealed. A microfluid having a fine reaction channel formed in a flat plate, a first channel communicating with one end of the reaction channel, and a second channel communicating with the other end of the reaction channel Chips,
First temperature control means for controlling the temperature of the liquid injected into the reaction channel;
Second temperature control means for controlling the temperature of the first flow path and the second flow path;
A sample analysis system comprising: The sample analysis system according to claim 6, wherein
The second temperature control means controls the temperature of the liquid at the end of the reaction channel in contact with the first channel and the liquid at the end of the reaction channel in contact with the second channel. A specimen analysis system characterized by this. The sample analysis system according to claim 6 or 7,
A sample analysis system, wherein a detecting means for detecting a reaction state of the liquid injected into the reaction channel is disposed opposite to the reaction channel. The sample analysis system according to claim 8, wherein
A sample analysis system, wherein the detection means measures fluorescence emitted by exciting a liquid in a reaction channel. A microfluidic chip used in the sample analysis system according to any one of claims 6 to 9,
A microfluidic chip, wherein a reagent is spotted in advance in a dry state above the reaction channel. The microfluidic chip according to claim 10, wherein
A microfluidic chip, wherein a reagent is spotted in advance in a dry state at a central portion of a channel between the first channel side and the second channel side in the reaction channel. The microfluidic chip according to claim 10 or 11,
A microfluidic chip comprising a plurality of the reaction channels arranged in parallel. The microfluidic chip according to any one of claims 10 to 12,
In the reaction flow path, the flow path height at the central part of the flow path from the first flow path side to the second flow path side is lower than the height other than the central part of the reaction flow path. A microfluidic chip, wherein a gradient is provided on an upper wall surface of the reaction channel. The microfluidic chip according to claim 13, wherein
The microfluidic chip, wherein the gradient is formed by a downwardly convex curved surface in which the upper wall surface protrudes into the reaction channel. The microfluidic chip according to any one of claims 10 to 14,
The microfluidic chip, wherein an inner wall surface of the reaction channel is formed of a continuous smooth surface that prevents formation of a minute gap space that is not filled with the liquid when the liquid flows. The microfluidic chip according to any one of claims 10 to 15,
A microfluidic chip, wherein a radius of curvature of a bent portion of an inner wall surface of the reaction channel is 20 μm or less.

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