JP2006234536A - Micro fluid mixer - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a shape of a passage confluence part for easily realizing uniform mixture without precise liquid feed control for a plurality of samples as to chemical analysis or chemical reaction by means of a micro fluid chip. <P>SOLUTION: The micro fluid chip comprises a plurality of sample introduction ports and passages extending therefrom, and is equipped with the passage confluence part where the respective passages together flow. A certain amount of sample is introduced thereinto from each of the introduction ports and then sent. When a chemical reaction is about to be induced through the mixture of the samples at the confluence part, uniform mixture can not be made because of air bubbles formed therein, etc. during the mixture of the samples unless the liquid front ends of the fed samples are trued up, and in its turn, the chemical reaction can end up in failure. This provided shape of the confluence part allows uniform mixture without forming air bubbles in a mixed solution by giving triangular form to the confluence part even without precisely truing up the front ends of the fed samples. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention belongs to the field of microfluidic chips, and relates to a microfluidic chip suitable for biochemical tests and diagnoses such as genetic tests and immunological tests, or chemical analysis and chemical reactions.

  In recent years, μ-TAS (μ-Total Analysis Systems), which performs fine analysis of chemical analysis, chemical reaction, genetic testing, immunological testing, etc., using microfabrication technology represented by nanotechnology, has attracted attention. ing. In μ-TAS, these reactions are integrated on a microchip to reduce the amount of reagents required, thereby reducing industrial waste, ease of operation, and stirring efficiency and extraction efficiency due to increased specific surface area. Improvements in performance are expected to significantly reduce analysis time.

  In addition, space saving is achieved by downsizing the apparatus, and further, point of care testing (in-situ analysis) is expected to be realized by making it portable. For example, in the diagnosis and treatment of infectious diseases, it is necessary to identify the bacteria and virus species that are infected in order to perform appropriate treatment, and immunological tests and genetic tests are performed. In the latter case, application of a PCR (polymerase chain reaction) method is being studied in order to amplify nucleic acids derived from bacteria or viruses. However, conventional immunological tests and genetic tests require specimen pretreatment, storage and preparation of necessary reagents, and a large-scale detection device. It has been mainly used in medical institutions.

  On the other hand, if it is possible to integrate the above-mentioned inspection items into a microfluidic chip, not only the introduction of various inspection methods to small hospitals, but also in-patients can analyze by themselves at the bedside, and eventually at home. It is no longer impossible to grasp the health condition, and it is expected to contribute to a prosperous life by improving QOL (Quality Of Life).

  In fact, several microfluidic chips that perform genetic analysis and chemical analysis have been reported so far, but no examples have been used as practical clinical diagnostic tools.

  One of the reasons why it is difficult to realize a practical microfluidic chip is that it is difficult to control the fluid on the microfluidic chip with the conventional technology. In particular, a plurality of samples are mixed quantitatively and uniformly at the flow path junction. It is not easy. Therefore, a method for mixing a plurality of solutions on a chip has been proposed. A method of laminating a plurality of substrates having vertical and horizontal channels behind the mixing unit (Patent Documents 1 and 2), and a method of attaching a piezoelectric element to the reaction channel unit and mixing them by deformation of the channel (Patent Document 3) In addition, a method of forming a mixer having a fine uneven surface behind the merging portion (Patent Document 4) has been proposed. Many of the proposals are methods for promoting mixing by dividing and merging fluid solutions, and the realization of such proposals still has problems such as difficulty in chip production and cost increase. Furthermore, the above means is based on a continuous fluid that continuously delivers the sample solution, and it is difficult to apply to a discontinuous fluid in which the amount of sample that can be handled is very small as in the clinical diagnosis field and analysis field. There is a desire for a mixing mechanism that can achieve the above objectives in structure.

JP 2004-113967 A JP 2004-113968 A JP 2004-184315 A JP 2004-016870 A JP 2000-014400 A Ishiguro T. et al. , Anal. Biochem. , 314 (2003) 77-86 Romano J. et al. W. et al. , Clin. Lab. Med. (1996) Mar; 16 (1); 89-103.

  In microfluidic chips that require a small amount of sample and need to mix discontinuous fluids uniformly, in systems where the reaction starts when the introduced sample is mixed on the microfluidic chip, especially in gene amplification reactions When mixing specimens, various enzymes, and substrate solutions on a microfluidic chip, each sample should be mixed quantitatively, as shown in FIG. 3, the arrival time of each sample to the flow path junction If there is a deviation, it was difficult to make uniform mixing, for example, by sandwiching air while the sample was mixed, causing the reaction not to proceed. In addition, in order to match the arrival time of a plurality of samples that flow through the flow path to the flow path confluence, it is necessary to align the liquid tips of the samples that flow through the flow path. Requires a plurality of pumps for controlling each flow path separately, and the apparatus becomes large, which makes it impossible to cope with in-situ analysis.

  The present invention provides a simple and low-fluidity microfluidic mixer having a shape of a flow path merging section that can uniformly mix a sample introduced into a microfluidic chip without requiring strict liquid feeding control. A microfluidic chip that can be manufactured at low cost is provided.

  As a result of intensive studies to solve the above problems, the present inventor conducted a reaction system in which a chemical reaction is started by mixing a plurality of samples on a microfluidic chip. Because the shape of the section is generally triangular with the downstream end of the flow path merge section as the apex, the sample introduced quantitatively into the flow path can be mixed uniformly without requiring complicated liquid feeding control I found out that

  Hereinafter, the present invention will be described in detail.

  The present invention relates to the shape of a flow path merging section for uniformly mixing a plurality of samples in a microfluidic chip, and the shape of the flow path merging section where a plurality of flow paths merge is the downstream end of the flow path merging section It is characterized by a generally triangular shape with the apex as the apex.

  In the microfluidic chip 1 shown in FIG. 1, the sample is quantitatively introduced into the sample introduction ports 2 and 3, the sample is sent to the introduction channel 4 extending from the introduction port, and the sample is mixed at the channel junction 5. When performing a chemical reaction that starts the reaction, the sample is fed by pressurizing or depressurizing with a pump connected to the microfluidic chip. If it does not reach the flow path merging section 5, air bubbles will be caught in the sample mixture as shown in FIG. In this state, a mixed liquid different from the intended reagent composition is generated, and this causes the uniform mixing or chemical reaction not to proceed. In order to prevent this, it is necessary to strictly control the liquid tips of a plurality of samples that are fed through the flow path. However, for that purpose, the same number of control devices as the pressure pumps as the number of samples is required, which hinders miniaturization and simplification. Therefore, this problem can be solved by making the flow path confluence portion into a characteristic shape. That is, as shown in FIG. 4, even if one sample reaches the flow path merging portion without the sample liquid tips being aligned, the flow passage merging portion is generally triangular with the downstream end of the flow channel merging portion as the apex. By adopting the shape, the sample that has reached first stays without flowing downstream from the flow path merging section, and mixes with the sample that arrives late, increasing the volume to mix inside the flow path merging section. The liquid was filled and sent to the discharge path, and it was possible to detect it with a downstream detection unit or the like. Thereby, the liquid mixture of the intended reagent composition produces | generates and the reaction efficiency of the target chemical reaction improves.

  As shown in FIG. 2B, the shape of the flow path merging portion is an isosceles triangle having a shape in which the flow paths to be merged are two sides and the downstream end of the flow path merging portion is a vertex. More preferably, as shown in FIG. 2C, the base of the isosceles triangle is preferably convex in the liquid feeding direction. Specifically, the angle θ formed by the base is preferably 150 ° or more and 180 ° or less. Further, θ is preferably larger than an angle θ ′ formed by the apex of the downstream end of the substantially triangular channel merging portion of the channel merging portion, and θ ′ is preferably 150 ° or less. These (B) and (C) in FIG. 2 (A), (D), and (E), (A) and (E) have no bottom at the flow path confluence, and (D) has a bottom that is convex toward the sample liquid feeding direction. Therefore, it is not included in each triangle. Moreover, even if it is a shape different from (B) and (C) in FIG. 2, the flow path merging portion only needs to be approximately triangular, for example, is not an isosceles triangle as shown in FIG. Samples such as (G) where the two sides forming the apex are not straight have an effect of mixing the samples uniformly, and are generally included in a triangular shape. In the case of (H), since the base is a curve but is convex in the liquid feeding direction, there is an effect of uniformly mixing the sample, and it is assumed that it is generally included in a triangular shape. In the present invention, the generally triangular shape means a generally triangular shape as viewed from above the microfluidic chip as shown in FIG.

  The volume of the flow path merging portion in the present invention is not particularly limited, but is preferably the same as the total amount of the sample sent to the flow path merging portion, and particularly preferably 50% or more and 150% or less. .

  In the present invention, it is preferable that two introduction paths and one discharge path connected to the flow path merging section are connected to each vertex of the triangular shape of the flow path merging section which is generally triangular.

  The material of the microfluidic chip is not particularly limited, and any material can be used as long as it can produce a desired shape and does not react with the liquid fed into the flow path. Absent. Examples thereof include synthetic resins such as acrylic resin, vinyl chloride resin and polyacetal, porcelain, metal, and glass. Further, the width and depth of the flow path of the microfluidic chip are not particularly limited.

  In the present invention, the kind of sample to be mixed is not particularly limited. For example, a combination of an enzyme and a substrate that initiates a reaction after mixing, an antigen and an antibody, or a combination of substances that do not initiate a reaction is mixed. Just be fine.

  In the present invention, two introduction paths are connected to one flow path merging section and fed to one discharge path. When mixing three or more samples, first, the two samples are first mixed. The discharge path through which the mixed liquid is fed at the flow path merging portion again becomes the introduction path of the next flow path merging portion, and can be mixed with the third sample. By sequentially mixing the samples in this way, in the present invention, it is also possible to uniformly mix three or more samples on one microfluidic chip.

  According to the present invention, a microfluid having a plurality of introduction paths capable of feeding a plurality of samples, and a system in which the introduction paths merge and the reaction proceeds by mixing the plurality of samples. In the chip, it is possible to easily mix a plurality of samples uniformly by making the shape of the flow path merging portion approximately triangular with the downstream end of the flow path merging portion as a vertex. In particular, when it is necessary to mix a certain amount of samples introduced into the flow path of the microfluidic chip at a certain ratio, it has been necessary to align the liquid tip of the sample to be fed conventionally. According to this, even if the liquid tip of the sample to be fed is not strictly aligned, the sample solution that has reached first stays at the flow path merging portion, mixes with the sample that has arrived late, and then flows to the downstream flow path. Thereby, mixing according to the intended reaction reagent composition becomes possible, and it can be expected that the reaction efficiency of various chemical reactions performed on the microfluidic chip is improved. Uniform mixing can be achieved with such a simple mechanism, which eliminates the need for complicated liquid feeding control, leading to simplification, miniaturization, and cost reduction of the device, and application of microfluidic chips is expected. The possibility of dealing with the existing POC field has improved. In addition, the microfluidic mixer according to the present invention enables uniform mixing of discontinuous fluids with a small amount of sample, and can be applied to clinical diagnostic fields and analytical fields.

  Hereinafter, although it demonstrates still in detail using an Example, this invention is not a thing limited to these.

Nucleic acid amplification reactions in microfluidic chips with various shapes of flow path confluence.
(1) It has a channel and a channel merging portion in the shape of (A), (B), (D) or (E) of FIG. 2, and an introduction port through which a sample can be introduced into each introduction channel, and , An acrylic resin microfluidic chip having a discharge path through which the sample is fed from the flow path junction, and reagents used in the TRC reaction (Patent Document 5 and Non-Patent Document 1) (substrate reagent, enzyme reagent, primer reagent, Target nucleic acid solution) was prepared. A substrate reagent and an enzyme reagent, and a primer reagent and a target nucleic acid solution were mixed in advance to prepare A solution and B solution, respectively. The flow path of the microfluidic chip is 300 μm in both width and depth, and the internal volume of all the mixers is 12 μl.

Solution A 120 mM Tris-HCl buffer (pH 8.6)
34 mM magnesium chloride 2 mM DTT
0.4 U / μl RNase Inhibitor
0.5 mM each of dATP, dCTP, dGTP, dTTP
7.2 mM ITP
6 mM ATP, CTP, GTP, UTP each
4% sorbitol 0.24 mg / ml bovine serum albumin 11.36 U / μl T7 RNA polymerase 0.53 U / μl distilled water for adjusting AMV reverse transcriptase volume B solution 220 mM potassium chloride 2.0 μM first oligonucleotide (T7 RNA polymerase Sense primer containing promoter sequence)
2.0 μM second oligonucleotide (antisense primer)
0.32 μM third oligonucleotide (3 ′ end modified with amino group)
20.8% DMSO
1000 copies / 15 μl standard RNA
Volumetric distilled water (2) The standard RNA used was mecA (RNA of 2183 bases containing the methicillin-resistant Staphylococcus aureus cell wall synthase PBP2 ′ gene sequence (see Non-Patent Document 2)). This standard RNA was prepared in an RNA dilution solution (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 1 mM DTT, 0.5 U / μl RNase Inhibitor) at 1000 copies / 5 μl to prepare an RNA sample.
(3) Nucleotide base sequence First oligonucleotide (sense primer containing T7 RNA polymerase promoter sequence)
5'-AAT TCT AAT ACG ACT CAC TAT AGG GAG ACT AAC TAT TGA TGC TAA AGT TCA AA-3 '
Second oligonucleotide (antisense primer)
5'-TTC TTT TTT ATC TTC GGT-3 '
Third oligonucleotide (3 'end is modified with an amino group)
5'-GTT AGT TGA ATA TCT TTG CCA TCT TTT TTC TTT TTC TCT ATT AAT GTA T-3'NH2
(4) 5 μl each of the liquid A and the liquid B prepared previously were introduced into different sample introduction ports, and syringe pumps were connected to the respective introduction ports in order to send the sample by air pressure.
(5) After the introduction of the reagent, the microfluidic chip was placed on a hot plate set at 43 ° C., and the sample and the microfluidic chip that were introduced after being allowed to stand for 5 minutes were waited for warming.
(6) The syringe pump set to a liquid feed speed of 10 μl per minute was operated, and the reagent introduced into the inlet was fed by air pressure. The liquid A and the liquid B were mixed at the flow path merging section, and further fed to the downstream discharge path. After the entire amount of the mixed liquid passed through the flow path merging section, the liquid feeding by the syringe pump was temporarily stopped. However, in the microfluidic chip having the shape of the flow path merging portion of (E), the liquid feeding was stopped after the total amount of the mixed liquid passed through the mixing portion installed downstream of the flow path merging portion. This state was left for 20 minutes to allow the TRC reaction to proceed.
(7) After 20 minutes, the syringe pump set at a liquid feed rate of 50 μl per minute is operated, the liquid mixture of liquid A and liquid B is collected from the sample outlet, electrophoresed on an agarose gel, and SYBR Green After staining with II, the band concentration of the RNA amplification product was quantified with a densitometer.

  As shown in FIG. 5, the amplification product amount in the microfluidic chip in which the flow path merging portion is in the shape of (B) as a result of quantifying the amplification product amount by agarose gel electrophoresis is approximately triangular channel mixing. It was found that the amount of the amplified product was significantly larger than that of the microfluidic chips of (A), (D), and (E) having no part, and the reproducibility of the amount of the amplified product was high. In addition, as shown in (D) and (E), those equipped with a round channel merging section or a round-shaped mixing section are micros without a channel merging section that is characteristic in both amplification product amount and reproducibility. It was almost the same as the fluid chip. In addition, when the liquid A and the liquid B were merged, it was observed that the mixing of bubbles was remarkably suppressed by the flow path merge portion having the shape of (B) and was uniformly mixed. From these facts, by making the flow path confluence part as shown in (B), it becomes easy to mix the liquid A and the liquid B introduced quantitatively and improve the success rate of the TRC reaction. I understood. Thus, not only the TRC reaction but also a chemical reaction in which the reaction starts by mixing the reagent introduced into the flow channel on the microfluidic chip, the introduction sample can be uniformly mixed according to the present invention. It is expected to improve the reaction efficiency of the target reaction.

A plurality of sample introduction ports (2), (3) and an introduction channel (4) extending therefrom, a flow channel joining portion (5) where the flow channels merge, and a discharge channel (6) and a discharge port (7) further downstream ) Is a schematic view of a microfluidic chip having). It is an enlarged view of a flow path confluence | merging part, and has shown the following characteristics, respectively. (A) Y-shaped flow path having a Y-shaped flow path merging section where two flow paths merge (B) A flow path where the flow path merging section has a triangular shape with the downstream end of the flow path merging section as a vertex ( C) The flow path merging portion is generally triangular, but has an angle at the bottom, and the angle θ is θ ′ <θ and 150 ° ≦ θ <180 ° (D) The flow path merging The round channel (E) and the channel merge part are Y-shaped like (A), but the channel (F) channel merge has a round mixing part downstream of the channel merge part. The channel (G) flow channel merged portion having a triangular shape is generally triangular, but the channel (H) flow channel merged portion where the two sides forming the apex are not straight is substantially triangular, A channel whose bottom is a curve that is convex in the liquid feeding direction It is a schematic diagram of the state of the merging of the two liquids at the flow path merging portion in the Y-shaped flow path ((A) in FIG. 2). Thus, it is shown that air is sandwiched in the mixed solution (the flow of time is from Phase 1 to Phase 4). It is a schematic diagram of the mode of a two-liquid confluence | merging in a triangular shape ((B) in FIG. 2) which a flow path confluence part makes a vertex the flow path confluence part downstream end. In this way, even if the tips of the two liquids are not aligned, the sample that has reached the first stays in the flow path merging section, and at the same time the other sample arrives, the two liquids merge to form a uniform mixed liquid that is sent downstream. (The flow of time is in the direction from phase 1 to phase 4). It should be noted that the same result is obtained in the flow path mixing section having the shapes (C), (F), (G), and (H) in FIG. RNA amplification efficiency when performing a TRC reaction on a microfluidic chip, indicating that the amount of amplification product is improved and the reproducibility is improved because the flow path confluence is triangular. .

Claims (5)

A plurality of sample introduction ports capable of introducing a sample, a plurality of introduction passages for feeding the introduced sample, a discharge passage connected after the introduction passages join, and a discharge port connected to the discharge passage to take out a sample. In a microfluidic chip having a flow path pattern,
A microfluidic mixer in which the shape of the flow path merging portion is generally triangular with the downstream end of the flow path merging portion as a vertex. 2. The microfluidic fluid according to claim 1, wherein the internal volume of the flow path merging portion is 50 to 150% of the total volume of the introduced sample, that is, 50 to 150% with respect to the volume of the sample solution after mixing. Mixer. 3. The microfluidic mixer according to claim 1, wherein two introduction paths and one discharge path are connected to each vertex of the flow path junction. In Claim 1 thru | or 3, the base of the substantially triangular flow path confluence | merging part is more than the angle | corner which the straight line or the angle | corner of the substantially triangular apex of a flow path confluence part makes, and the magnitude | size of 150 degrees or more and less than 180 degrees. A microfluidic mixer comprising two sides that are convex toward the liquid feeding direction. 4. The microfluidic mixer according to claim 1, wherein the bottom of the substantially triangular flow path converging portion is configured by a curve that is convex toward the liquid feeding direction.

Images