JP4595457B2 - Microfluidic device having a polymerase chain reaction channel - Google Patents

Description

  The present invention relates to a microfluidic device having a polymerase chain reaction channel for amplifying a DNA fragment having a target base sequence.

The polymerase chain reaction (hereinafter referred to as PCR) method is an excellent method for obtaining a large amount of a DNA fragment having a target base sequence from a small amount of DNA-containing sample by a simple method. . In general, the PCR method is a simple operation method in which a PCR reaction solution is placed in a plastic container and set in a PCR temperature controller consisting of a heat block, but it takes a long time of 1-2 hours. . This is because it takes a lot of time to accurately control the temperature suitable for annealing, elongation and denaturation of DNA strands. As a method for performing PCR in a shorter time, for example, a flow-through PCR apparatus for performing PCR in a capillary tube is disclosed (for example, see Patent Document 1).

Furthermore, a method of performing PCR using a so-called microfluidic device in which a capillary tube is formed on a substrate has been reported (for example, see Non-Patent Document 1). This microfluidic device has a concave portion that becomes a flow path formed on a glass substrate and is bonded to a flat plate, and has a concave portion that becomes a flow path by photolithography using an active energy ray-curable resin. It is disclosed that a microfluidic device that performs PCR can be obtained by forming a member and bonding the member and a flat plate together (see, for example, Patent Document 2).

Japanese Patent Laid-Open No. 07-075544 JP 2002-58470 A Martin U Cop, Andrew J. DeMello, Andreas Manz, “SCIENCE”, 1998, 280, P.A. 1046-1048

Silicon and glass microfluidic devices include a silicon substrate, a glass substrate cutting or etching process, and a process of bonding glass over a long period of time under high temperature and high pressure, which is troublesome and costly. In addition, brittle materials such as silicon and glass are easily broken by impact, so that they require problems in handling and cannot be incinerated at the time of disposal, and are not suitable for use in large quantities. In contrast, resin microfluidic devices have high impact resistance, can be discarded by incineration, and are advantageous in terms of cost.

  A microfluidic device in which a polymerase chain reaction channel (hereinafter referred to as a PCR channel) is provided on a microfluidic device, the liquid flows in the capillary tube, and the liquid flows on the heat block controlled to a predetermined temperature. By passing through, heat is exchanged quickly. Nucleic acid amplification is expected in a short amount of time because there is no time required for temperature rise and fall of the heat block, but the target is due to non-nucleic acid amplification or non-specific amplification occurring in resin microfluidic devices. In many cases, a DNA fragment having the base sequence as described above cannot be efficiently obtained.

Further, in the conventional method described above, since it is necessary to provide a plurality of regions having different temperatures in a plane parallel to the surface of the member, a region having a large temperature difference may be provided when the microfluidic device is very small. It was difficult.

  The problem to be solved by the present invention is a microfluidic device that is lightweight, can be discarded by incineration, etc., is resistant to impact, is a resin-made microfluidic device, and can perform highly accurate nucleic acid amplification. Is to provide.

As a result of intensive studies to solve the above problems, the present inventors have found that it is difficult for a resin microfluidic device to perform highly accurate nucleic acid amplification.
1) Thermal conductivity is low compared to silicon or glass microfluidic devices, and temperature control is difficult to be strictly controlled.
2) In addition, PCR inhibitors may leak from raw materials and inhibit PCR.
It is found that a resin-made microfluidic device capable of performing high-precision nucleic acid amplification can be obtained by optimizing the structure, material, etc. of the microfluidic device. Solved.

That is, the present invention
The substrate resin film (A) and the injection molded resin member (B) having a recess on the surface were bonded together.
Or
The substrate resin film (A), the spacer resin film (C), and the other substrate resin film (D) were bonded together in this order.
A microfluidic device having a polymerase chain reaction channel is provided.

  An accurate microfluidic device that can perform high-precision nucleic acid amplification in a short time, is lightweight, is resistant to impact, and can be discarded by incineration or the like has been obtained.

(Microfluidic device)
The microfluidic device of the present invention is a so-called microfluidic device, microfabricated device, lab-on-chip, or micro total analytical system (μ-TAS). . In other words, a microfluidic device has a fine flow path inside, and the flow path is made of a resin-based material, and the mechanism in which the fluid undergoes temperature changes and the concentration are adjusted. Reaction equipment for chemical and biochemical reactions having a mechanism for receiving a chemical reaction, a mechanism for receiving a chemical reaction, a mechanism for controlling flow velocity, flow branching, mixing or separation, or a mechanism for receiving electrical or optical measurements. Point to.

Most of the microfluidic device of the present invention is made of a resin material,
1) A substrate resin film (A) and an injection molded resin member (B) having a recess on the surface are bonded together.
Or
2) A flow path is formed by laminating the substrate resin film (A), the spacer resin film (C), and the other substrate resin film (D) in this order.
It can be manufactured by an extremely simple method.

The resin material generally has a lower thermal conductivity than silicon or glass, but can form a thin resin film.
Precise temperature control can be performed by bonding an injection molded resin member (B) having a recess to the surface of a thin substrate resin film (A) and heating from the thin substrate resin film (A) side.

Since the thermal conductivity is a value per unit length, when the thickness of the substrate resin film (A) becomes 1/10, a thermal conductivity effect of 10 times can be obtained. When a thin substrate resin film (A) is used, the thickness of the substrate resin film (A) can be set very small with respect to the interval between the temperature setting regions where the temperatures of the adjacent PCR flow paths are different. Interference with a suitable temperature setting region can be reduced. For this reason, precise temperature setting becomes possible, and highly accurate nucleic acid amplification reaction becomes possible.
Moreover, since the thermal conductivity can be further improved by reducing the depth of the concave portion of the injection molded resin member (B) and the thickness of the spacer resin film (C), highly accurate PCR can be performed. .

Furthermore, an optimal flow path design can be performed by selecting the type of resin and the filler. For example, in order to form a thermal conductive anisotropy in which heat is easily transmitted in the depth direction of the flow path and heat is not easily transmitted in the flow direction of the flow path, the substrate resin film (A) is formed as a thin film and Optimization can be achieved by reducing the thermal conductivity (increasing the heat capacity) of the injection-molded resin member (B) and the spacer resin film (C) at the same time as setting the high thermal conductivity. When this design concept is followed, the thermal conductivity of the substrate resin film (A) is 0.1 W / m · K or more, and the thermal conductivity of the injection molded resin member (B) and the spacer resin film (C) is 2 W. / M · K or less is preferable.

(PCR channel)
The PCR channel of the present invention can be of any shape as long as the PCR reaction solution is amplified by passing through this channel. It is a circuit of 20 to 50 cycles corresponding to annealing, extension and denaturation, and is preferably a flow path in which nucleic acid is amplified by moving the liquid in the in-plane direction because temperature control is easy.

(PCR inhibition)
Since the microfluidic device has a very large contact area with the wall surface with respect to the liquid per unit volume, there is a possibility that PCR may be inhibited even by a very small amount of material contained in the wall material. In fact, it has been found that microfluidic devices composed of photocurable resins are particularly prone to this inhibition. Although this inhibitor has not been identified at present, it is assumed that it will be an uncured product of a photocurable resin or a decomposed product upon light irradiation. A resin used for injection molding or a resin used as a resin film has a relatively high molecular weight, and it is considered that there is almost no leakage from the liquid in the flow path. By using the injection molded resin member (B), more accurate PCR could be performed. Details will be described below.

(Substrate resin film (A))
As the substrate resin film (A) to be attached to the injection-molded resin member (B) having a concave portion on the surface used in the present invention, any resin film that does not inhibit PCR can be used. For example, polyester film, nylon film, polypropylene film A biaxially stretched film such as a polystyrene film is preferably used. The thickness of the substrate resin film (A) is preferably 200 μm or less, and more preferably 100 μm or less. When the thickness exceeds 200 μm, the thermal conductivity from the heater for performing PCR is poor and the efficiency of PCR is lowered. The substrate resin film (A) is more preferable if it is thinned, and the thermal conductivity is more preferable. However, if the substrate resin film (A) is too thin, the strength cannot be maintained. It is preferable to do. For this reason, the thickness of the substrate resin film (A) is preferably 1 μm to 200 μm, and more preferably 10 μm to 200 μm.

(Thermal conductivity of substrate resin film (A))
The substrate resin film (A) preferably has a thermal conductivity of 0.01 W / m · K or more. Furthermore, 0.05 W / m · K or more is preferable, and 0.1 W / m · K or more is particularly preferable.
If it is less than 0.01 W / m · K, the thermal conductivity from the heater for performing PCR is poor, and the efficiency of PCR is reduced. As the substrate resin film (A) used in the present invention, a commercially available resin film that satisfies the aforementioned thermal conductivity can also be used. Resin films with improved thermal conductivity, such as those containing a thermally conductive filler in the resin film, or those having a thermally conductive substance fixed on the surface of the resin film, are preferable if they do not inhibit PCR. Can be used.

(Thermal conductive particles in the substrate resin film (A))
The thermal conductivity can be increased by incorporating thermally conductive particles into the substrate resin film (A) used in the present invention. Although it will not specifically limit if it is a filler with high heat conductivity, For example, at least 1 sort (s) chosen from the group of a metal oxide, a metal nitride, silicon carbide, and a hydrated metal compound can be mentioned. Examples of the metal oxide include aluminum oxide. Examples of the metal nitride include boron nitride and aluminum nitride. Examples of the hydrated metal compound include aluminum hydroxide and magnesium hydroxide. Although it can be contained in an amount of 50 to 500 parts by mass with respect to 100 parts by mass of the polymer, since the flexibility of the sheet is impaired when the amount exceeds 500 parts by mass, the followability to the heat block is lowered and the thermal conductivity is lowered.

In this invention, a board | substrate resin film (A) is used for the part which touches a heat block. Compared to a conventional microfluidic device using glass as a substrate, the substrate resin film (A) can change the thermal conductivity depending on the material used, and therefore, a suitable material can be selected. Recently, resin films having a high thermal conductivity of several W / m · K or more have been developed by imparting high orientation and adding thermal conductive particles. Of course, these resin films can also be preferably used. it can.

(Heat conduction efficiency)
The microfluidic device of the present invention includes: λ: thermal conductivity of substrate resin film (A) (W / m · K, l: thickness (μm) of substrate resin film (A), d: depth of flow path ( μm), it is preferable to satisfy the following formula 1.
(L + d) / λ ≦ 2000 (Formula 1)
When (l + d) / λ exceeds 2000, the heat conduction to the PCR reaction solution is reduced, and the PCR efficiency is lowered. That is, although depending on the flow path depth described later, when the substrate resin film (A) is thick, it is preferable to use the substrate resin film (A) having higher thermal conductivity.

(First form)
The first embodiment of the present invention is a microfluidic device having a PCR flow path characterized in that a substrate resin film (A) is bonded to an injection-molded resin member (B) having a recess on the surface. In the present invention, the PCR flow path refers to a flow path portion installed on a microfluidic device for performing PCR, and a PCR reaction that requires heat-resistant DNA polymerase, primer, template DNA, nucleotide, and buffer. As the liquid passes through the PCR flow path of the microfluidic device, PCR proceeds and a DNA fragment having the target base sequence is obtained.

(Injection molded resin member (B))
The injection-molded resin member (B) having a recess on the surface of the present invention is a resin member having a recess corresponding to a PCR flow path as an essential component on a smooth surface. As the resin member, any polymer material can be used as long as it can withstand heat during PCR, and for example, a resin such as polymethyl methacrylate, polycarbonate, polymethylpentene, or the like can be used. By injection molding these resins, an injection molded resin member (B) having a recess on the surface of the present invention can be obtained. A port for introducing a PCR reaction solution into the flow channel or taking out a PCR reaction solution amplified by nucleic acid in the flow channel in accordance with the through port provided in a part of the recess The mold can be designed so that the portion is provided on the surface opposite to the surface having the recess, and a member having both the recess and the port portion can be injection-molded at once.

(Thermal conductivity of injection molded resin member (B))
At least the PCR flow path part of the injection-molded resin member (B) is preferably a thin plate, and the thickness is preferably 2 mm or less, and more preferably 1 mm or less. When the thickness exceeds 2 mm, the heat flux in the horizontal direction of the microfluidic device increases, making it difficult to control the temperature independently.
Further, the appropriate value of the thermal conductivity of the injection molded resin member (B) varies depending on the usage of the microfluidic device of the present invention.
When heating only from the substrate resin film (A) side of the microfluidic device of the present invention, the thermal conductivity of the injection molded resin member (B) is preferably 2 W / m · K or less, preferably 1 W / m / K. The following is more preferable. If the thermal conductivity of the injection molded resin member (B) is higher than 2 W / m · K, the heat flux in the horizontal direction of the microfluidic device increases and it becomes difficult to control the temperature independently.
On the other hand, when the microfluidic device of the present invention is heated by a heat block from both sides of the substrate resin film (A) and the injection molded resin member (B), the thermal conductivity of the injection molded resin member (B) is approximately the substrate resin. It is preferable that it is comparable as a film (A).

(Depth of injection molded resin member (B))
The depth of the concave portion of the injection molded resin member (B) having a concave portion on the surface of the present invention is arbitrary, but the depth of the concave portion corresponding to the PCR flow path portion is preferably 300 μm or less, and 200 μm or less. It is more preferable that If it exceeds 300 μm, the thermal conductivity from the heater for performing PCR is poor and the efficiency of PCR is reduced. When the recess is deep, the efficiency of PCR can be improved by lowering the flow rate of the microfluidic device, but the PCR time is lengthened and the effect of using the microfluidic device is diminished. In addition, if the concave portion becomes too shallow, the pressure loss when the liquid flows is too large, or when the substrate resin film (A) is pasted, the depth of the flow path is difficult to be uniform, and a constant flow rate cannot be maintained. Therefore, the thickness is preferably 1 μm or more, and more preferably 10 μm or more. From the above, it is most preferable that the depth of the concave portion of the injection molded resin member (B) is 10 μm to 200 μm.

(Channel depth and width of PCR channel)
Further, the channel depth of the PCR channel part of the present invention is preferably 300 μm or less, and more preferably 200 μm or less. If it exceeds 300 μm, the thermal conductivity from the heater for performing PCR is poor and the efficiency of PCR is reduced. When the channel portion is deep, the efficiency of PCR can be improved by lowering the flow rate of the microfluidic device, but the PCR time becomes longer and the effect of using the microfluidic device is diminished. In addition, if the flow path becomes too shallow, the pressure loss at the time of flowing the liquid becomes too large, or the depth of the flow path tends to be uneven and a constant flow rate cannot be maintained. Preferably, it is preferably 10 μm or more. From the above, it is most preferable that the depth of the concave portion of the injection molded resin member (B) is 10 μm to 200 μm.
The channel width is preferably 500 μm or less, and preferably 200 μm. From the viewpoint of manufacturing accuracy, it is preferably 1 μm or more, more preferably 10 μm or more. From the above, the channel width is most preferably 10 to 200 μm.

(Bonding of substrate resin film (A) and injection-molded resin member (B) having a recess on the surface)
In order to bond the substrate resin film (A) and the injection-molded resin member (B) having a concave portion on the surface, bonding may be performed by a known method that does not use an adhesive such as heat fusion or ultrasonic fusion. preferable. If an adhesive is not used, leakage of a substance that inhibits PCR can be suppressed. Of course, it is possible to bond using an adhesive, and it is of course possible to bond the substrate resin film (A) coated with adhesive on the entire surface with an injection molded resin member (B) having a recess on the surface. It is more preferable to bond so that the agent or the like does not face the recess. For example, an adhesive is applied to the surface other than the recess of the injection molded resin member (B) having a recess on the surface, and is bonded to the substrate resin film (A). Such a method is preferably used. The adhesive preferably has thermosetting property or active energy ray curable property. By heating or irradiating light after bonding, the adhesive can be prevented from coming into contact with the liquid flowing in the flow path and inhibiting PCR.

(Method of using the microfluidic device of the first form)
Next, a method for using the microfluidic device according to the first embodiment of the present invention will be described. This microfluidic device is used by bringing the substrate resin film (A) side of the microfluidic device into contact with a heat block adjusted to at least two temperatures of annealing and denaturation. As the PCR reaction solution flows through the PCR flow path, nucleic acid amplification is performed through 20 to 50 cycles through three temperature regions suitable for annealing, denaturation, and extension reactions existing between the two temperatures. . Of course, a third heat block adjusted to a temperature suitable for the extension reaction may be used as the heat block. The PCR flow path part of the present invention is disposed close to the thin substrate resin film (A) with a flow path for performing PCR, so that the heat conductivity from the heat block is good, annealing, stretching, The temperature can be accurately controlled to a temperature suitable for denaturation. Further, since the microfluidic device has a thin PCR channel, the heat flux between regions controlled at different temperatures can be suppressed, and independent temperature control can be performed. The heat block may be provided on the injection molded resin member (B) side as an auxiliary.

  The microfluidic device of the present invention is obtained by bonding a substrate resin film (A) to an injection molded resin member (B) having a concave portion on the surface. For convenience, the injection molded resin member (B) having the ceiling and the substrate resin film (A) are referred to as the bottom, the vertical distance of the flow path is referred to as depth, and the horizontal distance is referred to as width. Therefore, it does not define how to use the substrate resin film (A) side as a bottom surface, but uses an injection-molded resin member (B) having a concave portion on the surface as a bottom surface, or injection having a concave portion on the surface. Arbitrary usage is possible, such as using the molded resin member (B) and the substrate resin film (A) upright so as to be side surfaces.

(Second form)
The second form of the present invention is a microfluid having a PCR flow path in which a substrate resin film (A), a spacer resin film (C), and another substrate resin film (D) are laminated in this order. It is a device. In the present invention, the PCR flow path refers to a flow path portion installed on a microfluidic device for performing PCR, and a PCR reaction that requires heat-resistant DNA polymerase, primer, template DNA, nucleotide, and buffer. As the liquid passes through the PCR flow path of the microfluidic device, PCR proceeds and a DNA fragment having the target base sequence is obtained. That is, the spacer resin film (C) is sandwiched between the two substrate resin films (A) and the substrate resin film (D) so that the flow channel equivalent portion constituting the PCR flow channel is essential. In other words, the microfluidic device is characterized in that the portions corresponding to the flow channels constituting the PCR flow channel are stacked so as to be voids, and the voids are used as the flow channels.

  The microfluidic device in which the resin films of the present invention are laminated does not show a manufacturing method in which three films of a substrate resin film (A), a spacer resin film (C), and a substrate resin film (D) are essential. The substrate resin film (A) and the substrate resin film (D) may be made as a single resin film (A ′), and the resin film (A ′) may be folded so as to sandwich the spacer resin film (C). The substrate resin film (A), the spacer resin film (C), and the substrate resin film (D) may have a laminated structure. In addition, many resin films may be laminated and used as long as PCR is not suppressed.

In this second form, either one of the substrate resin film (A) or the substrate resin film (D), or both of the resin films, on the portion that becomes the ceiling or bottom of the flow path when laminated. It is preferable to provide a through hole in advance. When a microfluidic device was produced by laminating a substrate resin film (A), a spacer resin film (C), and a substrate resin film (D), a PCR reaction solution was introduced from this through-hole or a nucleic acid was amplified. It can be used as an outlet for taking out a PCR reaction solution. Moreover, a port member can also be adhere | attached and used according to this through-hole as needed.

(Spacer resin film (C))
As the spacer resin film (C) used in the present invention, any resin film that does not inhibit PCR can be used. For example, a biaxially stretched film such as a polyester film, a nylon film, a polypropylene film, or a polystyrene film is preferably used. .
In order to form the flow path equivalent part used in the present invention, the spacer resin film (C) laminated between the two substrate resin films forms a flow path between the resin films cut into strips. And a mode in which the flow path portion is a resin film cut out. The spacer resin film (C) from which the flow path portion is cut out may be formed by cutting through the resin film through the portion that becomes the flow path by a known and usual method. The spacer resin film (C) is approximately the thickness of the flow path of the microfluidic device. The thickness of the resin film is preferably 300 μm or less, and more preferably 200 μm or less. If it exceeds 300 μm, the thermal conductivity from the heater for performing PCR is poor and the efficiency of PCR is reduced. Further, the thickness of the resin film is preferably 1 μm or more, and more preferably 10 μm or more. When the resin film is thinner than 1 μm, it becomes difficult to handle the resin film, the pressure loss when flowing the liquid becomes too large, or when applying the resin film, the depth of the flow path is difficult to be uniform, and a constant flow rate is maintained. Problems such as inability to keep. For this reason, the thickness of the spacer resin film (C) is most preferably 10 to 200 μm.
In either case of forming the flow path between the resin films cut into strips, and in the case of forming the flow path by the resin film from which a part of the film is cut, the flow path width is preferably 500 μm or less, 200 μm is preferred. From the viewpoint of manufacturing accuracy, it is preferably 1 μm or more, more preferably 10 μm or more. From the above, the channel width is most preferably 10 to 200 μm.

(Thermal conductivity of spacer resin film (C))
The thermal conductivity of the spacer resin film (C) is preferably 2 W / m · K or less, and more preferably 1 W / m / K or less. When the thermal conductivity of the spacer resin film (C) is higher than 2 W / m · K, the heat flux in the horizontal direction of the microfluidic device becomes large, and it becomes difficult to control the temperature to an independent temperature.

(Substrate resin film (D))
As the substrate resin film (D) used in the present invention, any resin film that does not inhibit PCR can be used. For example, a biaxially stretched film such as a polyester film, a nylon film, a polypropylene film, or a polystyrene film is preferably used. . The thickness of the substrate resin film (D) is not particularly limited, but when heated from the substrate resin film (D) side by an auxiliary heater for performing PCR, the thickness of the substrate resin film (D) is 200 μm or less. Is preferable, and it is more preferable that it is 100 micrometers or less. When the thickness exceeds 200 μm, the thermal conductivity from the heater for performing PCR is poor and the efficiency of PCR is lowered. In addition, the substrate resin film (D), like the substrate resin film (A), is preferably thinner, more preferable for thermal conductivity. However, if the substrate resin film (D) is too thin, the strength cannot be maintained. When using, it is preferable to make it thin as long as the strength can be maintained. For this reason, the thickness of the substrate resin film (D) is preferably 1 μm to 200 μm, and more preferably 10 μm to 200 μm.

(Thermal conductivity of substrate resin film (D))
The appropriate value of the thermal conductivity of the substrate resin film (D) varies depending on how the microfluidic device of the present invention is used. When heating only from the substrate resin film (A) side of the microfluidic device of the present invention, the thermal conductivity of the substrate resin film (D) is preferably 2 W / m · K or less, preferably 1 W / m / K or less. It is more preferable that When the thermal conductivity of the substrate resin film (D) is higher than 2 W / m · K, the heat flux in the horizontal direction of the microfluidic device becomes large, and it becomes difficult to control the temperature independently. On the other hand, when the microfluidic device of the present invention is heated from both sides of the substrate resin film (A) and the substrate resin film (D), the substrate resin film (D) has the same thermal conductivity as the substrate resin film (A). The resin film which has can be used preferably. Of course, resin films with improved thermal conductivity, such as those containing a thermally conductive filler in the resin film described above, and those with a thermally conductive substance fixed on the resin film surface, do not inhibit PCR. If it exists, it can be preferably used.

(Lamination of substrate resin film (A), spacer resin film (C), substrate resin film (D))
In order to bond the substrate resin film (A) of the present invention, the spacer resin film (C) from which the flow path equivalent portion is cut out, and the substrate resin film (D) to each other, an adhesive such as heat fusion or ultrasonic fusion is used. It is preferable to adhere by a known method that does not use. Of course, it is also possible to bond using an adhesive, and it is preferable to bond so that the adhesive or the like does not enter the recess. For example, on one side of the resin film (C), preferably on both sides, a portion corresponding to the flow path of the resin film with release paper to which an adhesive has been applied is cut out, the release paper is peeled off, and the substrate resin film (A), What is necessary is just to bond together so that it may pinch | interpose with a board | substrate resin film (D). At this time, the adhesive used preferably has thermosetting property or active energy ray curable property. After bonding, heating or irradiation with active energy rays is performed to cure the adhesive, whereby the adhesive can be prevented from coming into contact with the liquid flowing in the flow path and inhibiting PCR. It goes without saying that any method that does not inhibit PCR can be used other than the methods listed here.

(Method of using the microfluidic device of the second form)
Next, a method for using the microfluidic device according to the second aspect of the present invention will be described. This microfluidic device is used by bringing the substrate resin film (A) side of the microfluidic device into contact with a heat block adjusted to at least two temperatures of annealing and denaturation. As the PCR reaction solution flows through the PCR flow path, nucleic acid amplification is performed through 20 to 50 cycles through three temperature regions suitable for annealing, denaturation, and extension reactions existing between the two temperatures. . Of course, a third heat block adjusted to a temperature suitable for the extension reaction may be used as the heat block. The PCR flow path part of the present invention is disposed close to the thin substrate resin film (A) with a flow path for performing PCR, so that the heat conductivity from the heat block is good, annealing, stretching, The temperature can be accurately controlled to a temperature suitable for denaturation. Further, since the microfluidic device has a thin PCR channel, the heat flux between regions controlled at different temperatures can be suppressed, and independent temperature control can be performed.

In addition, since the microfluidic device of the second aspect of the present invention is disposed close to the substrate resin films (A) and (D) having a thin channel, the substrate resin film (D) is used as an auxiliary material. If a heat block is also prepared on the side, the temperature can be accurately controlled by a temperature suitable for annealing, elongation, and denaturation, which is a preferred method of use.

  In the microfluidic device of the second embodiment of the present invention, the bottom surface, the side surface, etc. are not specified as in the microfluidic device of the first embodiment, and can be used arbitrarily such as being used upright.

Although two embodiments have been described above, the microfluidic device of the present invention may have a flow path for analysis, storage, etc. in addition to the flow path for PCR in any form. Of course, a flow path for joining and separating may be provided. That is, various techniques of known microfluidic devices can be used as long as they do not interfere with nucleic acid amplification.

Hereinafter, the present invention will be described with reference to examples.
[Preparation of active energy ray-curable adhesive]
60 parts by mass of Unidic V-4263 (Dainippon Ink Chemical Co., Ltd. urethane acrylate oligomer), 20 parts by weight of Unidic V-5507 (Dainippon Ink Chemical Co., Ltd. epoxy acrylate oligomer), 2- An active energy ray-curable adhesive is prepared by uniformly mixing 20 parts by weight of hydroxypropyl methacrylate, 20 parts by weight of ethyl acetate, and 3 parts by weight of Irgacure 184 (manufactured by Ciba Specialty Chemicals) as a photopolymerization initiator. did.

[Production of microfluidic devices]
Polycarbonate resin injection-molded, 200 μm wide, 100 μm deep recess, and inlet for introducing a PCR reaction solution into the flow path, or taking out a PCR reaction liquid amplified in the flow path And the resin member shown in FIG. 2 which has a port part as an exit was obtained. The energy ray curable adhesive is applied to the surface of this member so as not to enter the recess, and the PMMA resin film whose surface has been subjected to corona discharge treatment after being irradiated with ultraviolet rays for a short time and semi-curing the adhesive. The target microfluidic device (A-1) shown in FIG. 2 was obtained by bonding and irradiating ultraviolet rays to completely cure the adhesive.

[Preparation of PCR reaction solution]
Genetack (manufactured by Nippon Gene Co., Ltd.) was used as the DNA polymerase, and the PCR buffer solution and dNTP mixed solution attached to the enzyme were used. 10 μl of PCR buffer (concentrated 10 times), 8 μl of dNTP mixture (dATP, dGTP, dCTP, dTTP 2.5 mM each), 5 μl of primer (1) (primer (1)) (10 μM; SEQ ID NO: 1) 5 μl of primer (2) (primer (2) (10 μM; SEQ ID NO: 2), 0.5 μl of 5 units / μl Genetack, 1 μl of ras Mutant Set c-Ki-ras codon 12 Gly (1 ng; Takara) Biotechnology Co., Ltd .; SEQ ID NO: 3), and further sterilized water was added to make 100 μl to prepare a PCR reaction solution.

[Perform PCR]
The microfluidic device (A-1) was fixed at the position shown in FIG. 1 on a heater composed of three heat blocks. The heat block (A) was set at 90 ° C, the heat block (B) at 72 ° C, and the heat block (C) at 55 ° C. 25 μl of the PCR reaction solution described above was injected into the opening (inlet) of the device, and a microsyringe pump (IC3200 manufactured by kd Scientific) was connected to the opening, and the solution was fed at 5 μl / min. Thus, the PCR reaction solution passes over the heat blocks (A), (B), (C), (B), (A), (B), (C). In the process, an enzyme reaction for nucleic acid amplification occurs.

  As a result of the reaction, it was verified by agarose gel electrophoresis according to a conventional method that the nucleic acid was amplified. A band indicating nucleic acid amplification was detected (FIG. 4), thus confirming that PCR using the microfluidic device proceeded normally.

  A microfluidic device (A-2) was obtained in the same manner as in Example 1 except that polymethyl methacrylate resin was used instead of polycarbonate resin and the flow path depth was changed from 100 μm to 200 μm. Using this device (A-2), PCR was performed in the same manner as in Example 1, and it was verified by agarose gel electrophoresis that the nucleic acid was amplified according to a conventional method. A band indicating nucleic acid amplification was detected (FIG. 5), thus confirming that PCR using the microfluidic device proceeded normally.

Laser processing was carried out into a shape in which a channel portion having a width of 200 μm was cut out with a double-sided release paper of a 100 μm-thick PET film coated with an adhesive on both sides. In addition, holes of about 200 μm were provided at two locations on a 100 μm thick PET film. After bonding so that the end of the flow path part of the laser-processed film and the hole of the PET film with holes formed were aligned, it was bonded to another PET film with a thickness of 100 μm to obtain a microfluidic device. A polyvinyl chloride tube having an inner diameter of 3 mm and a height of 5 mm was bonded to these two holes to form a port portion, thereby obtaining a microfluidic device (A-3) shown in FIG. Using this device (A-3), PCR was performed in the same manner as in Example 1, and it was verified by agarose gel electrophoresis according to a conventional method that the nucleic acid was amplified. A band indicating nucleic acid amplification was detected (FIG. 6), and it was thus confirmed that PCR using the microfluidic device proceeded normally.
(Comparative Example 1)

  A comparative microfluidic device was produced by the following operation.

[Preparation of Active Energy Ray Curable Composition (1)]
80 parts by mass of Unidic V-4263 (Dainippon Ink Chemical Co., Ltd. urethane acrylate oligomer), 20 parts by mass of New Frontier HDDA (Daiichi Kogyo Seiyaku Co., Ltd., 1,6-hexanediol diacrylate), light A composition (1) was prepared by uniformly mixing 5 parts by mass of Irgacure 184 (manufactured by Ciba Specialty Chemicals) as a polymerization initiator.

[Preparation of active energy ray-curable composition (2)]
60 parts by mass of Unidic V-4263 (a urethane acrylate oligomer manufactured by Dainippon Ink and Chemicals, Inc.), 40 parts by mass of New Frontier HDDA, 5 parts by mass of Irgacure 184 as a photopolymerization initiator, and 2,4-diphenyl A composition (2) was prepared by uniformly mixing 0.5 parts by mass of -4-methyl-1-pentene (manufactured by Kanto Chemical Co., Inc.).

[Production of microfluidic devices]
The composition (1) was applied to a 1 mm thick substrate made of polyacrylate so as to have a film thickness of 10 μm, and cured by irradiation with ultraviolet rays. On this coating film, the composition (2) was applied so that a film thickness might be 350 micrometers, and the uncured coating film of this composition (2) was formed. Ultraviolet parallel light was irradiated through a photomask in which the flow path forming portion was drawn in black, and portions other than the flow path were cured. Next, the uncured composition (2) in the non-irradiated portion was removed with ethanol to form a coating film having a recess having a width of 200 μm and a depth of 350 μm on the surface. A 300 μm through-hole was provided at both ends of the recess with a drill. Next, the composition (1) was applied onto a 100 μm-thick polypropylene film, and irradiated with ultraviolet rays for 3 seconds to form a semi-cured coating film. Using this as a lid, it was bonded to the coating film having the concave portion, and was completely cured by irradiating with ultraviolet rays to obtain a microfluidic device. A polyvinyl chloride tube having an inner diameter of 3 mm and a height of 5 mm was bonded to each of the two holes to form a port portion, and a comparative microfluidic device (B-1) was obtained. Using this device (B-1), PCR was performed in the same manner as in Example 1, and the presence or absence of nucleic acid amplification was confirmed by agarose gel electrophoresis according to a conventional method. No band showing nucleic acid amplification was detected (FIG. 7), and the nucleic acid amplification reaction did not proceed.
(Comparative Example 2)

  A microfluidic device (B-2) was obtained in the same manner as in Comparative Example 1 except that the flow path depth was changed from 350 μm to 200 μm in Comparative Example 1. Using this device (B-2), PCR was performed in the same manner as in Example 1, and the presence or absence of nucleic acid amplification was confirmed by agarose gel electrophoresis according to a conventional method. No band showing nucleic acid amplification was detected (FIG. 8), and the nucleic acid amplification reaction did not proceed.

The above examples and comparative examples are summarized as shown in the following table, and the usefulness of the present invention is clear.

  From the results of the above examples and comparative examples, the microfluidic device having the PCR flow channel of the present invention is mostly made of resin, so it is lightweight, can be discarded by incineration, and is resistant to impact. It is understood that PCR amplification, which is a problem with conventional resin microfluidic devices, does not occur or hardly occurs, and thus highly accurate nucleic acid amplification is possible.

It is a schematic diagram of the flow-through PCR method using a microfluidic device. 1 is a schematic cross-sectional view of a microfluidic device of Example 1. FIG. 6 is a schematic cross-sectional view of a microfluidic device of Example 3. FIG. It is the photograph which analyzed the result of PCR performed in Example 1 by agarose gel electrophoresis (right column side). The left column is a marker (20bp DNA Ladder from Takara Bio Inc.). It is the photograph which analyzed the result of PCR performed in Example 2 by agarose gel electrophoresis (right column side). The left column is a marker (20bp DNA Ladder from Takara Bio Inc.). It is the photograph which analyzed the result of PCR performed in Example 3 by agarose gel electrophoresis (right column side). The left column is a marker (20bp DNA Ladder from Takara Bio Inc.). It is the photograph which analyzed the result of PCR performed in the comparative example 1 by the agarose gel electrophoresis (right column side). The left column is a marker (20bp DNA Ladder from Takara Bio Inc.). It is the photograph which analyzed the result of PCR performed in the comparative example 2 by the agarose gel electrophoresis (right column side). The left column is a marker (20bp DNA Ladder from Takara Bio Inc.).

Explanation of symbols

1 Heat block set at denaturation temperature (A)
2 Heat block set to elongation reaction temperature (B)
3 Heat block set at annealing temperature (C)
4 Microfluidic device 5 Port (inlet)
6 Port part (exit)
7 Substrate resin film (A)
8 Injection molding member 9 Flow path 10 Resin film (B)
11 Resin film (C)
12 Resin film (D)

Claims (1)

The substrate resin film (A) and the injection molded resin member (B) having a recess on the surface were bonded together.
Or
The substrate resin film (A), the spacer resin film (C), and the other substrate resin film (D) were bonded together in this order.
In a microfluidic device having a polymerase chain reaction channel ,
A microfluidic device characterized by satisfying the following formula 1.
(L + d) / λ ≦ 2000 (Formula 1)
(The symbols in the formula have the following meanings: λ: thermal conductivity (W / m · K) of the substrate resin film (A), l: thickness (μm) of the substrate resin film (A), d: flow Road depth (μm)