JP2008128906A - Drive control method for microfluidic chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To obtain a driving method for a microfluidic chip capable of obtaining a precise and reliable analytical result in a short time at low cost, using easy operations that does not require skill. <P>SOLUTION: A drive control method for a micro fluidic chip 100 moves by the application of physical action force, liquid supplied in a liquid passage, to the microfluidic chip 100 in which a liquid processing section is arranged along the liquid passage formed in a substrate, and performs a liquid-process by the liquid processing section. The method detects at least either the top end or the rear end of the liquid, in the liquid passage at a specified position in the liquid passage and decides the control operating conditions of the liquid, according to the timing with which this detection has been made. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a microfluidic chip drive control method for controlling liquid feeding of a liquid supplied into a microfluidic chip.

  Recent advances in molecular biology have shown that by analyzing biological materials 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 this case, a protein such as an antibody or antigen that specifically binds to the substance to be detected, or a single-stranded nucleic acid is used as a probe and immobilized on a solid surface such as a fine particle, a bead, or a glass plate. Perform reaction or nucleic acid hybridization. Then, using a labeled substance having a specific interaction carrying a labeled substance with high detection sensitivity such as an enzyme, such as a labeled antibody, a labeled antigen or a labeled nucleic acid, an antigen antibody compound or a double-stranded Nucleic acids are detected to detect the presence or absence of the test substance or to quantify the test substance.

  As this type of technology, for example, the biological material testing device disclosed in Patent Document 1 is a chip component that is a microreactor for each specimen, and a control / detection component that is a device body, on which elements for reagents and liquid delivery systems are mounted. By configuring the system separately, cross contamination and carry-over contamination are unlikely to occur with respect to trace analysis and amplification reaction.

  Further, the biochemical processing apparatus disclosed in Patent Document 2 includes a stage on which a biochemical reaction cartridge having a chamber and a flow path communicating between the chambers is placed, and a movement for moving a liquid through the flow path. Clogging by providing means, detection means for detecting the presence or absence or amount of liquid in the chamber, and determination means for determining the result of liquid movement based on the information on the liquid in the chamber detected by the detection means, Abnormalities such as leakage are eliminated and the normal flow of liquid can be reliably detected with a simple configuration.

JP 2006-125990 A JP 2006-170654 A JP 2005-160387 A

However, as a conventional method for knowing the base sequence of a specific gene, a DNA sequencer, an NDA microarray, or the like is known. There was a problem of being long. That is, Patent Document 1 discloses a method for branching and feeding liquid reagents. However, when a preset pressure is reached, a liquid feed control unit for allowing passage of liquid, Since a three-dimensionally complicated structure such as a backflow prevention part for preventing the backflow of the liquid is required, the structure of the microfluidic chip is complicated, resulting in a problem that the chip becomes expensive. In addition, since these mechanisms are constituted by movable mechanisms, there is a problem that the reliability of the operation is insufficient due to the influence of variations in manufacturing conditions.
Further, Patent Document 2 discloses a microfluidic chip and a method for detecting a fluid in the chip, but this detection is not for controlling the liquid feeding operation, and the result of liquid feeding is It is for determining whether it is normal or abnormal, and has not been used as a means for obtaining an accurate and reliable analysis result by advanced liquid feeding control. Further, the detection is performed in a chamber, and a method for detecting the position of the liquid in the microchannel is not disclosed. Furthermore, the detection method uses scattering of light by liquid, and it is stable when detecting low-scattering liquids such as water, and when the type and concentration of substances contained in reagents and specimens change. There was a problem that it could not be detected automatically.
On the other hand, Patent Document 3 proposes a nucleic acid amplification method and a nucleic acid amplification primer set in which only a target gene is amplified and analyzed with a relatively simple detection system. When using the pipetting method using the microtubes used in the above, there are problems such as preparation of reagents, pipetting operation, loading and unloading into and from the apparatus, as well as complicated operations. It was. In particular, when analyzing a plurality of target genes, there is a problem in that the complexity of the operation increases the risk of taking a chemical solution and the risk of contamination, resulting in insufficient reliability of the test results.

  The present invention has been made in view of the above circumstances, and provides a microfluidic chip driving method capable of obtaining accurate and reliable analysis results at low cost and in a short time with a simple operation that does not require skill. It is an object.

The above object of the present invention is achieved by the following configurations.
(1) With respect to the microfluidic chip in which the liquid processing unit is disposed along the liquid flow path formed in the substrate, the liquid supplied in the liquid flow path is moved by applying a physical action force. , A microfluidic chip drive control method for performing liquid processing in the liquid processing unit,
Detecting at least one of a front end portion and a rear end portion of the liquid in the liquid flow path at a specific position of the liquid flow path, and determining a control operation condition of the liquid according to the detected timing; A drive control method for a microfluidic chip.

  According to this microfluidic chip drive control method, at least one of the front end and the rear end of the liquid in the liquid flow path is detected, and the liquid control operation condition is determined according to the detection timing of the liquid end. The This eliminates the need for complicated operations such as pipetting and loading / unloading to / from the apparatus, and enables reaction such as extraction or amplification of DNA in the sample in the liquid flow path.

(2) A drive control method for a microfluidic chip according to (1),
The method for driving a microfluidic chip, wherein the liquid control operation condition includes a condition including at least one of a moving direction, a moving speed, and a driving force for movement with respect to the liquid.

  According to this microfluidic chip drive control method, it is possible to control the movement of the liquid feed in the liquid flow path by controlling at least one of the direction of movement with respect to the liquid, the moving speed, and the driving force. And, according to the configuration in which all of the liquid moving direction, moving speed, and driving force can be controlled, these operating conditions can be freely switched to perform pipetting operation, insertion into and removal from the apparatus, etc. Equivalent liquid feeding control is possible.

(3) The microfluidic chip drive control method according to (1) or (2),
The method for driving a microfluidic chip, characterized in that the control operation condition of the liquid treatment includes a heating set temperature for heat-treating the liquid in the liquid treatment unit.

  According to this microfluidic chip drive control method, for example, in a nucleic acid amplification reaction by PCR (Polymerase Chain Reaction) method, etc., by control of liquid feeding in the liquid flow path, template DNA, primer, substrate, heat-resistant polymerase enzyme, etc. The target reaction DNA can be amplified by repeating the temperature adjustment of the mixed reaction solution and sequentially changing the temperature to three predetermined temperatures.

(4) The microfluidic chip drive control method according to (1) or (2),
Irradiating light to the specific position of the liquid flow path to detect reflected light from the liquid flow path, the presence or absence of liquid in the liquid flow path at the specific position is refracted between the air and the liquid of the reflected light A method for driving a microfluidic chip, wherein the determination is based on a change in light quantity based on a change in rate.

  According to this microfluidic chip drive control method, it is possible to make a determination by irradiating light from the outside of the microfluidic chip to the liquid flow path for which the presence or absence of liquid is desired, and by making a change in the refractive index of the reflected light. The sensor or the like is not exposed to the flow path, and the sample liquid is not contaminated.

(5) A method of driving a microfluidic chip according to (4),
A method of driving a microfluidic chip, wherein light for irradiating the specific position of the liquid flow path is supplied through a light-projecting-side optical fiber, and reflected light from the liquid flow path is introduced into a light-receiving-side optical fiber and detected. .

  According to this microfluidic chip drive control method, it is possible to perform irradiation light and reflected light with a small-area fiber front end surface integrated with a light-projecting side optical fiber and a light-receiving side optical fiber. Irradiation and reflected light from the irradiated region can be received, and the presence or absence of liquid at a specific position of the minute liquid flow path can be detected.

(6) A method for driving a microfluidic chip according to (4) or (5),
The light irradiation direction to the specific position of the liquid channel and the detection direction of the reflected light from the specific position are set in directions that are respectively inclined with respect to the normal line of the light irradiation surface of the specific position. A method of driving a microfluidic chip.

  According to this microfluidic chip drive control method, the light irradiation direction and the reflected light detection direction are inclined with respect to the normal line of the light irradiation surface, respectively, and the reflected light is separated from the optical fiber on the light receiving side when air is present, Since the tilt angle at which the reflected light is sometimes incident on the light receiving side optical fiber is used, the liquid can be detected more stably with a simple structure based on the change in the refractive index.

(7) The microfluidic chip drive control method according to any one of (1) to (6),
The microfluidic chip drive control method, wherein the physical acting force is a pneumatic acting force generated by air supply or air suction from connection ports provided at a start point and an end point of the liquid flow path.

  According to this microfluidic chip drive control method, the liquid supplied into the liquid channel is moved to a desired position in the liquid channel by air supply or air suction applied to the start point and end point of the liquid channel. Movement control becomes possible. At this time, the liquid is held in a state of being sandwiched by the gas interposed between the start point and the front end portion and between the rear end portion and the end point, and is not divided in the middle by the action of the pulling force.

(8) The microfluidic chip drive control method according to any one of (1) to (7),
The liquid contains a specimen liquid having ecological cells, a pretreatment reagent, a reaction amplification reagent,
One of the liquid processing units is equipped with a primer that is a fragment of nucleic acid. A predetermined amount of the liquid is dispensed into the liquid processing unit and irradiated with excitation light while being heated. A drive control method for a microfluidic chip, wherein the generated fluorescence is detected.

  According to this microfluidic chip drive control method, a primer, which is a nucleic acid fragment that specifically binds to a substance to be detected, is mounted on one liquid processing unit by drying and solidifying a liquid reagent. Nucleic acid amplification reaction of the substance to be detected is performed by quantitatively dispensing the liquid into the processing unit. At this time, a labeling substance having a specific interaction carrying a luminescent substance that is a labeling substance with high detection sensitivity, such as a labeled antibody, a labeled antigen, or a labeled nucleic acid, is used. Cyber Green emits strong fluorescence by intercalating into double-stranded DNA amplified by the reaction. By measuring the fluorescence intensity, the presence or absence of the target gene sequence can be detected.

  According to the microfluidic chip drive control method of the present invention, the microfluidic chip in which the liquid processing unit is disposed along the liquid flow path formed in the substrate is supplied into the liquid flow path. The liquid is moved by applying a physical action force, liquid processing is performed in the liquid processing section, and at least one of the front end and rear end of the liquid in the liquid flow path is detected and detected at a specific position in the liquid flow path Since the liquid control operation conditions are determined according to the timing of the operation, complicated operations such as pipetting and loading / unloading are not required. High analysis results can be obtained at low cost and in a short time.

DESCRIPTION OF EMBODIMENTS Hereinafter, preferred embodiments of a microfluidic chip drive control method according to the present invention will be described in detail with reference to the drawings.
FIG. 1 is a block diagram showing a microfluidic chip according to the present invention together with a schematic configuration of an inspection apparatus.
A microfluidic chip (hereinafter also simply referred to as “chip”) 100 according to the present invention is set and used in an inspection apparatus 11 and discarded after one use. In the present embodiment, blood (whole blood) as a specimen is injected into the microfluidic chip 100. When the microfluidic chip 100 is set in the testing apparatus 11, a sample liquid is handled by a physical acting force from the outside of the chip, for example, a single nucleotide polymorphism multiple target gene is tested. As shown in 2005-160387, it is possible to realize on the chip 100 a reaction and a detection for specifically amplifying a target sequence nucleic acid isothermally. 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 acting force (pneumatic driving force) generated by air supply or air suction from the port part PT provided at the start point and end point of the liquid flow path. Accordingly, the liquid supplied into the liquid flow path can be controlled to move to a desired position in the liquid flow path by air supply or air suction applied to the start point and end point of the liquid flow path. At this time, the liquid is held in a state of being sandwiched by the gas interposed between the start point and the front end portion and between the rear end portion and the end point, and is not divided in the middle by the action of the pulling force.

  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.

  The inspection apparatus 11 includes a pump PMP that uses air as a working fluid, valves SV1, SV2, SV3, SV4, SV5, a specimen heating unit 13, a temperature adjustment unit 15, a liquid position detection unit 16, and fluorescence detection. A unit 17 and a control unit 19 connected to these for receiving a detection signal or transmitting a control signal are provided as basic components. The valve SV4 is interposed between the pump PMP and the valve SV2, and the valve SV2 is a fourth port PT-C of the chip 100 on the working fluid control side, and the valve SV1 is a second port PT-D of the chip 100 on the working fluid control side. The working fluid control side of the valve SV3 is connected to the first port PT-A, and the working fluid control side of the valve SV2 and the working fluid input side of the valve SV1 are connected to the third port PT-B of the chip 100. The specimen heating unit 13 heats the heated part B of the chip 100, the temperature adjustment unit 15 adjusts the temperature of the reaction unit F of the chip 100, and the fluorescence detection unit 17 can detect the fluorescence of the reaction unit F. Yes. The operations of these components will be described in detail later.

2 is an exploded perspective view of the microfluidic chip shown in FIG. 1, and FIG. 3 is a plan view showing the microfluidic chip in a top view (a) and a bottom view (b).
As shown in FIG. 2, the microfluidic chip 100 includes a flow path substrate 21 and a lid member 23 attached to one surface (lower surface) 22 of the flow path substrate 21. The flow path substrate 21 is manufactured by injection molding of a thermoplastic polymer. The polymer used is not particularly limited, but is preferably optically transparent, highly heat resistant, chemically stable, and easily injection-molded. COP, COC, PMMA, etc. are preferred. . Optically transparent means high transparency at the wavelength of excitation light or fluorescence used for detection, small scattering, and little autofluorescence. The chip 100 has translucency that enables detection of fluorescence. For example, cyber green is used as a detection reagent, and fluorescence emitted by intercalating into double-stranded DNA amplified by reaction is generated. It becomes possible to measure. Thereby, the presence or absence of the target gene sequence can be detected.

  Excavations 29 and 31 are formed in the other surface (upper surface) 28 of the flow path substrate 21, and the excavations 29 and 31 are located corresponding to the heated part B and the reaction part F. Further, as shown in FIG. 3, the lower surface 22 of the flow path substrate 21 communicates with the first port PT-A, the second port PT-D, the third port PT-B, and the fourth port PT-C. Openings 33, 35, 37, and 39 are provided. The flow path substrate 21 has an outer shape of 55 × 91 mm in length and width W2, W1, and a thickness t of about 2 mm, for example.

  The lid member 23 is a member for covering ports, cells, and channels (grooves) formed on the channel surface (lower surface 22) of the channel substrate 21, and the lid member 23 and the channel substrate 21 are adhesives. And bonded with adhesive. As the lid member 23, a sheet-like high molecular polymer that is optically transparent, has high heat resistance, and is chemically stable is used, like the flow path substrate. In this embodiment, a PCR plate seal having a thickness of 100 μm is used (adhesive is applied to a plastic film).

FIG. 4 is an enlarged view of FIG. 3B, FIG. 5 is an exploded perspective view showing the lower surface of the chip before the closing member is attached, and FIG. 6 is an enlarged plan view of the main part showing the vicinity of the port outlet channel.
The flow path substrate 21 includes ports, cells, flow paths, and the like for operations necessary for the liquid (details will be described later). That is, as shown in FIG. 4, the flow path substrate 21 includes a first port PT-A into which a specimen liquid containing biological cells and a pretreatment reagent are introduced, and a second port PT-D into which a reaction amplification reagent is introduced. The third port PT-B for supplying air pressure into the flow path, the fourth port PT-C at the end of the flow path to be depressurized, the sample liquid and the pretreatment reagent introduced from the first port PT-A A first channel (specimen mixing unit) A that mixes to generate a first mixed solution, and a second channel that extracts the DNA from living cells by heating the first mixed solution and decomposes it into single strands ( Heated portion) B, a third flow path (reagent merging portion) C for joining the reaction amplification reagent to the first mixed solution processed in heated portion B, and the second mixing joined at reagent merging portion C Processed by the fourth flow path (enzyme holding part) D, which solidifies and mounts the enzyme whose dissolution progresses as the liquid passes, and the enzyme holding part D A fifth flow path (enzyme mixing section) E that facilitates the mixing of the enzyme into the second mixture, and a primer that is connected to the enzyme mixing section E and solidified and mounted in the flow path, DNA amplification by heating, A plurality of sixth flow paths (reaction units) F that perform DNA amplification detection at the same position, and a second mixed solution that is connected to the flow path of the reaction unit F and processed by the enzyme mixing unit E includes a plurality of reaction units F. A seventh channel (quantitative dispensing channel) G for quantitatively dispensing each reaction detection cell 27 is provided.

  The first port PT-A, the second port PT-D, the third port PT-B, and the fourth port PT-C (port portion PT) are configured by holes penetrating the flow path substrate 21 in the upper and lower surfaces, and the lid By attaching the material 23, a recess connected to the flow path is formed. Each port part PT is slightly thicker than the other part of the flow path substrate 21, and a port pad (not shown) for feeding the inspection apparatus 11 is connected to this part. Each port pad is connected to valves SV1, SV2, SV3, SV4 (valve SV) via piping. The valve SV is connected to the above-described pump PMP for driving liquid feeding. By controlling the operation of the valve SV and the pump PMP by the control unit 19, the air in the port part PT can be decompressed, pressurized, opened to the atmosphere, and sealed, and the droplets in the flow path can be freely conveyed. can do.

  Further, the microfluidic chip 100 seals the chip 100 in a hermetically sealed state by removing the port pad from the port part PT and applying the labels Ra, Rb, Rc, Rd shown in FIG. Can be made. When the amplification reaction is performed in a state where the chip 100 is not sealed, the amplified DNA flows out of the chip, contaminates the environment, and there is a risk of carry-over. To prevent this, the chip 100 is subjected to the amplification reaction before the amplification reaction. Is sealed. As means for sealing the chip 100, in addition to the method of attaching the labels Ra, Rb, Rc, Rd, a method of plugging the port portion PT with a cap (not shown) having a sealing property, A known sealing method can be used, such as solidification by irradiation with UV light after pouring into the part PT.

  The first port PT-A is used as a sample port and is charged with 1 μL of blood and 3 μL of pretreatment reagent. Pretreatment reagents are used to isolate nucleic acid components from white blood cells in the blood. Chemical dissolution treatment is performed using a surfactant or strong alkali. Examples of the surfactant include nonionic surfactants, cationic surfactants, anionic surfactants, and amphoteric surfactants. In order to prevent blood from coagulating, an anticoagulant such as heparin or EDTA may be added.

  The second port PT-D is used as a liquid reagent port, and 56 μL of reaction amplification reagent is charged. Reaction amplification reagents include reagents required for amplification reactions and detection other than enzymes and primers. For example, a catalyst such as magnesium chloride, magnesium acetate, or magnesium sulfate, a substrate such as dNTP mix, or a buffer solution such as Tris-HCl buffer, tricine buffer, sodium phosphate buffer, or potassium phosphate buffer can be used. Furthermore, additives such as dimethyl sulfoxide and betaine (N, N, N-trimethylglycine), acidic substances described in International Publication No. 99/54455 pamphlet, cation complexes and the like may be used.

  Cyber Green can be used as a detection reagent. Cyber Green emits strong fluorescence by intercalating into double-stranded DNA amplified by the reaction. By measuring this fluorescence intensity, the presence or absence of the target gene sequence is detected.

  The third port PT-B and the fourth port PT-C are used as liquid feeding ports, and the liquid droplets in the flow path are switched by depressurization, pressurization, atmospheric release state, and closed state by the pump PMP and the valve SV. Drive.

  As shown in FIG. 6, the sample mixing part A is a flow path in which a plurality of turtle thyroid cells larger than the total amount of the pretreatment reagent and blood introduced into the first port PT-A are connected, and pass through this flow path. As a result, the blood charged into the first port PT-A and the pretreatment reagent are uniformly mixed. That is, the flow path of the specimen mixing unit A is separated from the wide flow channel portion 41 and the wide flow channel portion 41 in which the cross-sectional area in the direction perpendicular to the flow direction of the liquid is larger than the cross-sectional area in the other flow channels. Narrow channel portions 43 having a small area are alternately formed. Therefore, when the blood introduced into the first port PT-A reaches the sample mixing portion A, the wide flow channel portions 4 and the narrow flow channel portions 43 are alternately formed along the direction in which the liquid flows. By passing through the flow path, stirring by the orifice action is performed a plurality of times, and the blood and the pretreatment reagent are uniformly mixed.

  The heated part B is heated to 98 ° C. by the specimen heating part 13 shown in FIG. That is, in the microfluidic chip 100, the liquid processing control operation condition includes a heating set temperature for heating the liquid in the liquid processing unit. For example, in a nucleic acid amplification reaction by PCR (Polymerase Chain Reaction) method, the temperature of the reaction solution mixed with template DNA, primer, substrate, heat-resistant polymerase enzyme, etc. is adjusted by liquid feed control in the liquid flow path. The target DNA can be amplified by repeating the sequential change to the three temperatures. In the present embodiment, the blood and the pretreatment reagent pass through this portion, so that the DNA double strand extracted from the white blood cell by the pretreatment reagent becomes a single strand. In order to heat the heated portion B uniformly, the channel substrate 21 is provided with a dig 29, and the thickness of this portion is as thin as about 1.2 mm.

  The reagent joining part C joins the reaction amplification reagent to the heat-treated blood and the pretreatment reagent. The magnitude of the capillary force of the flow path in the second port PT-D is such that port D outlet flow path 45> main flow path 47> port D flow path (second port PT-D). A connection portion between the flow channel 45 and the main flow channel 47 constitutes a Laplace pressure valve. The reaction amplification reagent charged into the second port PT-D does not flow out to the main channel 47 but stays at the connection surface between the port D outlet channel 45 and the main channel 47. Further, when the mixed solution of blood and pretreatment reagent arrives at the port D outlet channel 45 by an operation described later, the Laplace pressure valve is broken and the two liquids join together.

  As shown in FIG. 4, the enzyme mixing unit E includes a first mixing unit 49 and a second mixing unit 51 that are liquid storage chambers. The enzyme holding unit D is provided between the first mixing unit 49 and the second mixing unit 51 and includes a first holding unit 53 and a second holding unit 55. The first holding unit 53 is a reagent holding cell installed between the first mixing unit 49 and the second mixing unit 51, and after dripping the aqueous solution of polymirase and dextrin, it is dried by lyophilization, The solidified reagent 57 is held.

  The upstream and downstream flow paths of the holding part are narrower than the holding part, and even if there is no adhesion force of the dried and solidified reagent 57 to the flow path, the solidified reagent by vibration such as storage and transportation of the chip 100 57 is prevented from peeling off and flowing out to the front and rear flow paths.

  Any polymerase may be used as long as it has strand displacement activity (strand displacement ability), and any of normal temperature, intermediate temperature, and heat resistance can be suitably used. Further, this polymerase may be either a natural body or a mutant with artificial mutation. Examples of such a polymerase include DNA polymerase. Furthermore, it is preferable that the DNA polymerase has substantially no 5 '→ 3' exonuclease activity. Examples of such a DNA polymerase include thermophilic Bacillus bacteria such as Bacillus stearothermophilus (hereinafter referred to as “B.st”) and Bacillus caldotenax (hereinafter referred to as “B.ca”). Examples include mutants lacking the 5 ′ → 3 ′ exonuclease activity of the derived DNA polymerase, Klenow fragment of E. coli derived DNA polymerase I, and the like.

  Dextrin is used as an enzyme stabilizer. As a result, the enzyme can be stored for a long period of time, and in the amplification reaction, the enzyme in the reaction solution is stabilized, so that the nucleic acid amplification efficiency can be increased. As other enzyme stabilizers, glycerol, bovine serum albumin, saccharides and the like can be used.

  The second holding unit 55 is installed downstream of the first holding unit 53 and has the same structure as the first holding unit 53. The second holding unit 55 holds the reagent 59 that has been dried and solidified by freeze-drying after spotting an aqueous solution of MutS and dextrin. MutS is one of a group of proteins called “mismatch binding proteins” (also called “mismatch recognition proteins”). This is a group of proteins having a function of repairing a base pair that cannot be partially matched (mismatched) in a double strand of DNA. This is a MutS protein (Japanese Patent Publication No. 9-504699). In addition, various mismatch binding proteins such as MutM protein (Japanese Patent Laid-Open No. 2000-300265) are known.

  The enzyme mixing section E reciprocates the blood, the pretreatment reagent, and the reaction amplification reagent between the first mixing section 49 and the second mixing section 51, thereby allowing the reagent 57, which is the first enzyme, The reagent 59 which is an enzyme is dissolved and the liquid is mixed uniformly. In order to stably transport the droplets so as not to entrain the bubbles during reciprocation, it is desirable that the enzyme mixing unit E be water-repellent with respect to the mixed solution. In this embodiment, the material of the flow path substrate 21 is COP. (Contact angle of water about 110 °) was selected.

  In the reaction part F, a target DNA primer and gelatin aqueous solution are cooled, solidified and fixed after spotting. A primer is an oligonucleotide having a length of about 20 bases having a base sequence complementary to a specific portion of a target DNA, and serves as a starting point for DNA synthesis by a polymerase. In the present embodiment, 13 reaction detection cells 27a to 27m are configured, and a primer 61 for amplifying wild in order to perform an amplification reaction specifically on the wild and mutant sequences for the gene to be examined. In addition, a pair of primers 63 for amplifying mutants are fixed to different reaction / detection cells.

  That is, in six reaction detection cells 27a to 27l, six genes D1 to D6 are examined. A primer 65 for amplifying a gene sequence having no polymorphism is fixed to the remaining one reaction detection cell 27m of PD, and this cell is used as a positive control. The sample mixed in the first mixing unit 49 and the second mixing unit 51 is quantitatively dispensed into each of the reaction detection cells 27a to 27m.

FIG. 7 is an explanatory view of a reaction detection cell in which a P2-P2 cross-sectional view of FIG. 4 in which primers are mixed and diffused is shown in FIG.
By heating the reaction detection cells 27a to 27m to 60 ° C., the solidified gelatin is dissolved and dispersed in the reaction detection cells 27a to 27m, and an isothermal amplification reaction is performed. Although only the primer aqueous solution can be spotted on the reaction detection cell 27 and dried and fixed, in this case, when the liquid flows into the cell, the primer is caused to flow in the flow direction. Reaction and detection cannot be performed. For this reason, 0.5% of gelatin that is difficult to dissolve in an aqueous solution at room temperature was contained and spotted and solidified.

  The aqueous solution of the primer and gelatin is spot-fixed to the cell on the flow path substrate 21 side, and is arranged on the upper surface of the flow path as shown in FIG. 7 when the microchip is used. After the liquid has flowed in, the gelatin ge containing the primer 61 dissolved with the temperature rise of the liquid by being heated from the lid 23 side, that is, the lower surface, flows to the lower side in the flow path due to gravity because of its large specific gravity. . Further, the liquid is heated from the lower surface, thereby causing convection 67 in the cell. The primer 61 and the gelatin ge are uniformly mixed and diffused in the reaction detection cell 27 in a short time by the synergistic effect of the flow of the gelatin ge below the flow path due to gravity and the convection 67 due to the heating of the liquid.

FIG. 8 is an enlarged plan view of the reaction detection cell.
A reaction detection cell inlet channel 69 and a reaction detection cell outlet channel 71 are arranged before and after each of the reaction detection cells 27a to 27m, and the inlet / outlet channels 69, 71 are narrow channels. The end face of the liquid after dispensing remains on the connection surface between the inlet channel 69 and the main channel 73 and the connection surface between the outlet channel 71 and the exhaust channel 75. A heating unit 77 is formed in the reaction unit F, and the heating unit 77 has the thickness of the flow path substrate 21 reduced to about 1.2 mm by the digging 31 in order to perform heating uniformly. The heating unit 77 is arranged to heat the entire reaction detection cell 27 and a part of the inlet / outlet channels 69 and 71, and the temperature of the heating unit 77 other than the heating unit 77 is adjusted to room temperature by other temperature adjusting means. . That is, both end surfaces of the liquid in the reaction detection cell 27 are kept at room temperature without being heated. This can prevent moisture from evaporating due to heating. The heating unit 77 and the temperature adjusting means around it constitute the temperature adjusting unit 15 in FIG.

  The inlet / outlet channels 69 and 71, the main channel 73, and the exhaust channel 75 constitute a quantitative dispensing channel G. The quantitative dispensing channel G quantitatively dispenses the second mixed solution processed by the enzyme mixing unit E to each of the plurality of reaction detection cells 27 of the reaction unit F.

  The liquid quantitatively dispensed into the reaction detection cell 27 includes a specimen liquid having ecological cells, a pretreatment reagent, and a reaction amplification reagent. Primers 61, 63,..., Which are nucleic acid fragments, are mounted in the reaction detection cell 27. A liquid is dispensed into the reaction detection cell 27 in a fixed amount, and irradiated with excitation light while being heated. Fluorescence generated in is detected. In the reaction detection cell 27, a diffusion amplification reaction of the substance to be detected is performed. At this time, a labeling substance having a specific interaction carrying a luminescent substance that is a labeling substance with high detection sensitivity, such as a labeled antibody, a labeled antigen, or a labeled nucleic acid, is used. Cyber Green emits strong fluorescence by intercalating into double-stranded DNA amplified by the reaction. By measuring the fluorescence intensity, the presence or absence of the target gene sequence can be detected.

FIG. 9 is a graph of the fluorescence measurement results when (a) shows the case where the target sequence is present and (b) shows the case where there is no target sequence.
The reaction detection cells 27a to 27m are excited by the optical system at a wavelength of about 490 nm, and the amplification of the target DNA is confirmed by measuring the intercalated cyber green fluorescence at about 520 nm. That is, as shown in FIG. 9, when the target nucleic acid sequence is present, an increase in fluorescence intensity I is confirmed, and when it is not present, an increase in fluorescence intensity I is not confirmed.

  In the reaction part F, in order to allow the liquid to smoothly enter during dispensing into the reaction detection cells 27a to 27m and to be stably stopped by the Laplace pressure valve at the outlet part, the reaction detection cell 27 and the narrow inlets before and after the reaction detection cell 27 It is desirable that the outlet channels 69 and 71 are moderately hydrophilic. In the present embodiment, at least the inlet / outlet channels 69 and 71 are hydrophilized by plasma irradiation (water contact angle is about 70 °).

  As a method of partially hydrophilizing or water repelling the flow path substrate 21, a known method (method of applying a hydrophilization / water repellent treatment solution, UV irradiation, vapor deposition or sputtering) may be used in addition to plasma irradiation. / A method of forming a thin film of a water repellent material, a method of molding using resins having different wettability by two-color molding or insert molding, and the like. In the present embodiment, the channel inner wall surface of each channel (at least the inlet / outlet channels 69 and 71) has at least two stages of wettability. As a result, the liquid smoothly enters when dispensing into each of the reaction detection cells 27a to 27m, and can be stably stopped by the Laplace pressure valve at the outlet.

Here, foaming in the reaction detection unit will be described.
FIG. 10 is a plan view showing the foaming state of the reaction detection part in (a) to (c), and FIG. 11 is a cross-sectional view of the principal part showing the foaming prevention measure of the reaction detection part.
When the reaction detection cells 27a to 27m are heated, if bubbles are generated in the reaction detection cell 27, there is a problem that the accuracy of fluorescence detection is lowered. For this reason, it is necessary to prevent bubble generation in the flow path. As shown in FIG. 10, the bubble generation mechanism is as follows. When the mixed solution flows into the reaction detection cell 27, the reaction detection cell 27 is subjected to the flow path cross section R (the chamfer radius of the flow path corner) and the adhesive is applied. If a minute space is formed due to some cause such as unevenness or weld line, the mixed liquid cannot flow into the minute space, and a minute wetting residue, that is, a minute air trap can be formed. Air bubbles are generated by the expansion and increase of the air pool due to heating.

  As a typical example of the minute space, there is a minute space 81 formed by the joint portion of the cross-section R of the flow path shown in part a of FIG. As a means for preventing this, it is effective to make the cross section R of the flow path 83 as small as possible (desirably 100 μm or less, more desirably 10 μm or less), as shown in part b of FIG.

  Further, as another means, as shown in part c of FIG. 11, by optimizing the application condition and the bonding condition of the adhesive 79, the minute structure constituted by the cross section R of the flow path 83 and the lid member 23 is used. There is also a method of filling the space 81 with the adhesive 79.

  As another example of the minute space 81, there are an adhesive 79 for the lid member 23 and uneven application of the adhesive 79, as shown in part d of FIG. If the minute space 81 is formed due to uneven application of the adhesive surface, the minute space 81 is formed in the same manner as R in the cross section of the flow path, which causes foaming. As another example, as shown in part e of FIG. 11, there is a weld line 85 of the flow path substrate 21 manufactured by injection molding. If the weld line 85 forms a similar minute space 81, foaming is performed. Cause. For this reason, in particular, the inner wall surface of the flow path of the reaction section F is preferably formed of a continuous smooth surface that prevents the formation of a minute gap space that is not filled with the liquid when the liquid flows in the flow path. Thereby, bubbles are not generated in the flow path during heating, and a decrease in fluorescence detection accuracy is prevented. Thus, in order to prevent foaming, it is necessary to select a suitable solution from the above-described solution means so that when the liquid flows in, the minute space 81 into which the liquid can flow is not formed. It becomes.

  In the present embodiment, as described above, it is composed of 12 reaction detection cells 27a to 27l for determining six sets of single nucleotide polymorphisms and one reaction detection cell 27m for positive control. In the positive control reaction detection cell 27m, a primer 65 that targets a gene sequence portion where no polymorphism exists is mounted, and an increase in fluorescence intensity is confirmed no matter what sample is examined. By confirming the fluorescence of the positive detection reaction detection cell 27m, it is possible to confirm that a series of liquid feeding operations are normally performed and a normal reaction has been performed, thereby ensuring the reliability of the test results. It becomes possible.

  In addition, as a guarantee method for negative control, water may be used instead of blood to cause a series of reactions to be confirmed that there is no increase in fluorescence intensity, or two sets of circuits are formed on the same substrate. However, inspection and negative control may be performed at the same time.

  When operating a finite liquid with the microfluidic chip 100, in particular, with the microfluidic chip 100 configured by a simple flow path that does not incorporate an active valve or pump, complex handling of liquid is performed from the outside of the chip. It is indispensable to accurately detect the position of the liquid in order to perform the pneumatic driving. For example, if the control is performed with the flow rate of the driving air without detecting the position of the liquid, the temperature change of the volume (dead volume) of the air in the pipe from the pump to the port part PT of the microfluidic chip 100 or the flow path in the chip Handling liquids with good reproducibility due to disturbances such as expansion and contraction due to turbulence, changes in flow resistance due to minute scratches in the flow path and static electricity, or the effect of vapor pressure due to fluid evaporation when the fluid is heated It becomes difficult. For this reason, it is very important to accurately detect the position of the liquid.

  The microfluidic chip 100 detects at least one of a front end portion and a rear end portion of the liquid in the liquid flow path at a specific position of the liquid flow path, and determines a liquid control operation condition according to the detected timing. . This eliminates the need for complicated operations such as pipetting and loading / unloading to / from the apparatus, and enables reaction such as extraction or amplification of DNA in the sample in the liquid flow path. If the liquid control operation condition includes at least one of the direction of movement with respect to the liquid, the movement speed, and the driving force for movement, the movement control of the liquid feeding in the liquid flow path can be performed. In addition, according to the configuration in which all of the liquid moving direction, moving speed, and driving force can be controlled, these operating conditions can be freely switched, so that pipetting operation, insertion / removal to / from the apparatus, and the like are performed. Equivalent liquid feeding control is possible.

  In the present embodiment, the control of the driving speed, driving direction, and driving force of the liquid is switched by the control unit 19 of the inspection apparatus 11 by detecting the position of the liquid. Here, the driving force includes, in addition to suction and pressurization with a constant pressure, the port portion PT opened to the atmosphere, a closed state, and a connected state of the plurality of port portions PT.

FIG. 12 is an explanatory diagram showing a plan view of the liquid position detection unit in (a) and a P1-P1 cross-sectional view in (b). FIG. 13 is a schematic diagram showing incident / reflected light of the liquid position detection unit. Is a graph showing the correlation between the reflectance and the incident angle.
In the microfluidic chip 100, sensing units PH1 to PH5 (see FIGS. 1 and 4) for liquid position detection are arranged. In the present embodiment, the liquid position detection unit 16 is disposed at a position facing the sensing units PH1 to PH5. Although one liquid position detection part 16 is collectively shown in FIG. 1, it arrange | positions facing each of a total of five places of sensing part PH1-5. As a specific example of the liquid position detection unit 16, a reflection type optical fiber sensor 87 shown in FIG. 12 is used. The tip of each fiber sensor 87 is arranged from the lid member 23 side of the chip 100 toward the channel 83 as shown in FIG.

  The reflection type optical fiber sensor 87 detects light reflected from the flow path 83 by irradiating light to a specific position of the flow path 83, and detects the presence or absence of the liquid in the flow path at the specific position between the air of the reflected light and the liquid. Judgment is made from a change in the amount of light based on a change in refractive index. Therefore, since light can be irradiated from the outside of the chip 100 and the determination can be made by the change in the refractive index of the reflected light, the sensor or the like is not exposed to the flow path 83 and the sample liquid is not contaminated.

  Specifically, the reflection type optical fiber sensor 87 supplies light for irradiating a specific position through the light projecting side optical fiber 89 and introduces the reflected light from the flow path 83 into the light receiving side optical fiber 91 for detection. According to the reflection type optical fiber sensor 87, the irradiation light and the reflection light can be performed by a small area fiber front end surface in which the light projecting side optical fiber 89 and the light receiving side optical fiber 91 are integrated. Irradiation and reflected light from the irradiation region can be received, and the presence or absence of liquid at a specific position of the minute flow path 83 can be detected.

The light receiving side optical fiber 91 of the reflection type optical fiber sensor 87 detects the intensity of the reflected light reflected from the chip 100.
The presence / absence of liquid droplets in the channel 83 is mainly detected by the difference in reflectance between the side of the channel of the lid member 23 when air is present in the channel and when there is water. Can do.

  In general, the reflectance for incident light as shown in FIG. 13 is expressed by the following equation.

ここで、Rp：p偏光、Rs：ｓ偏光であり、n=n2/n1とおいた。

Here, Rp: p-polarized light, Rs: s-polarized light, and n = n2 / n1.

When the refractive index of the lid member 23 is n1 = 1.49 and the refractive index of the fluid in the flow path 83 is no liquid droplet, n2 (air) = 1.00
When there are droplets ... n2 (water) = 1.33
In other words, the difference in reflectance as shown in FIG. 14 can be obtained depending on whether the fluid in the flow path 83 is air or water.

  When the light spread angle of the light-projecting side optical fiber 89 used is 60 °, the range of 0 ° to 30 ° in FIG. 14 may be considered. When the fluid in the flow path 83 is air, the reflectance is high. About 4%, in the case of water, it is 0.5% or less. Due to this difference, the amount of light received by the reflective optical fiber sensor 87 changes with and without a droplet, and the arrival and passage of the droplet can be detected.

  Further, as can be seen from FIG. 14, as the incident angle increases, the difference in reflectance between air and water increases, so that the reflection type optical fiber sensor 87 has a fiber on the light emitting side and light receiving side as shown in FIG. 89 and 91 can be separated types and can be arranged with an angle with respect to the chip 100. Here, FIG. 15 is a side view of the liquid position detection unit in which the light projecting side optical fiber and the light receiving side optical fiber are disposed in an inclined manner. In the configuration shown in the figure, the light irradiation direction to the specific position of the flow path 83 and the detection direction of the reflected light from the specific position are set to be inclined with respect to the normal line 93 of the light irradiation surface at the specific position. Has been. According to such a configuration, the reflected light is separated from the light receiving side optical fiber 91 when air is present, and the angle of inclination at which the reflected light is incident on the light receiving side optical fiber 91 when the liquid is present is set. Detection can be performed more stably with a simple structure. The fiber diameter, outgoing light / incident light angle, fiber arrangement, number of fibers used, etc. of the light emitting side optical fiber and the light receiving side optical fiber can be optimized experimentally or by optical simulation according to the shape of the flow path to be detected.

  Thus, the detection by the reflective optical fiber sensor 87 detects the difference in refractive index between air and fluid. Compared with the detection method based on the principle of detecting the light transmittance of fluid or light scattering, Even if there is a kind of dissolved substance in the fluid and its concentration change, it has an advantage that it can be detected stably.

According to the microfluidic chip 100 according to the present embodiment having the above configuration,
(1) a first port PT-A for introducing a sample solution and a reagent,
(2) a second port PT-D into which a reaction amplification reagent is charged;
(3) a third port PT-B for supplying air pressure into the flow path;
(4) Specimen mixing unit A that mixes the sample liquid introduced from the first port PT-A and the pretreatment reagent to generate the first mixed liquid;
(5) A heated portion B that heats the first mixed liquid to extract DNA from living cells and decomposes it into single strands;
(6) Reagent merging portion C for merging the reaction amplification reagent with the first mixed solution treated in heated portion B;
(7) an enzyme holding part D that solidifies and mounts an enzyme that is dissolved by passing through the second mixed liquid joined at the reagent joining part C;
(8) an enzyme mixing unit E for promoting the mixing of the enzyme into the second mixed solution treated in the enzyme holding unit D;
(9) a reaction part F comprising a plurality of reaction detection cells 27 connected to the enzyme mixing part E and dissolved in the primer solidified in the flow path, DNA amplification by heating, and detection of this DNA amplification at the same position; ,
(10) a quantitative dispensing flow path G for quantitatively dispensing the second mixed solution connected to the plurality of reaction detection cells 27 and processed by the enzyme mixing unit E to each of the plurality of reaction detection cells 27;
It does not require a three-dimensionally complicated structure, and it can perform complicated liquid feeding control with a simple structure, and does not require complicated operations such as pipetting and loading / unloading to / from the device. Accurate and reliable analysis results can be obtained at low cost and in a short time with simple operations that are not required.

Next, a liquid feeding flow using the microfluidic chip 100 will be described.
FIG. 16 is a time chart showing the operation state of each element along with the driving control of the microfluidic chip along the time axis, FIG. 17 is an operation explanatory diagram from the liquid setting to the first heating, and FIG. FIG. 19 is an explanatory diagram of the operation until the injection of the reaction part, and FIG.
In the following description, the control operations V1 to V13 in FIG. 16 and the states in steps S1 to S20 in FIGS.
First, the chip 100 is prepared, and the READY switch of the inspection apparatus 11 is pushed (V1, S1). Then, a reaction amplification reagent is introduced into the second port PT-D (S2). The relationship between the capillary forces of the flow paths in the second port PT-D is such that port D outlet flow path 45> main flow path 47> second port PT-D, and port D outlet flow path 45 and main flow path. A connection part 47 is a Laplace pressure valve. For this reason, the reaction amplification reagent does not flow out to the main channel 47 but stays at the connection surface between the port D outlet channel 45 and the main channel 47.

  Next, blood and a pretreatment reagent are introduced into the first port PT-A (S3). The chip 100 is set in the inspection apparatus 11, and the START switch of the inspection apparatus 11 is pressed (V2). Then, the port pad is pressed against the first port PT-A, the second port PT-D, the third port PT-B, and the fourth port PT-C. At this time, the pads corresponding to the first port PT-A, the second port PT-D, the third port PT-B, and the fourth port PT-C are open to the atmosphere, and the chip is pressed by pressing the pad. The liquid put in the tank does not move. When the pad pressing is completed, the third port PT-B is depressurized (V3), and the blood and the pretreatment reagent L are uniformly mixed by passing through the sample mixing part A at a high speed (100 μL / min) ( S4). The second port PT-D is aspirated at the same reduced pressure as the third port PT-B, and the pretreatment reagent in the second port PT-D remains in the second port PT-D even if the liquid feeding resistance between the blood and the pretreatment reagent is large. It does not flow out into the flow path.

The liquid reaches the sensing position PH1, the sensor PH-1 of the liquid position detector is turned on (V4), and the suction speed is switched to a low speed (30 μl / min) (S5).
When the liquid passes through the heated part B at a low speed (30 μl / min) (S6), the mixed liquid L of blood and pretreatment reagent is heated to 98 ° C. for a certain period of time (for example, 15 seconds), and DNA in white blood cells Is extracted and becomes a single strand.
When the liquid reaches the sensing position PH2 and the sensor PH-2 is turned on (V5), the second port PT-D is opened to the atmosphere, and at the same time, the first port PT-A is closed. The amplification reaction reagent flows out only from D (S7), and joins without containing bubbles and the mixed solution L of blood and pretreatment reagent (S8).

When the liquid reaches the sensing position PH3 and the sensor PH-3 is turned on (V6), the suction speed is switched to high speed (100 μl / min), and a constant flow rate (45 μl) is sucked (S9, S10).
Then, the first port PT-A is opened to the atmosphere (V7), and further 15 μL is sucked, and the second port PT-D is emptied and mixed by the first mixing unit 49 (S11).
Furthermore, by sucking 80 μL at a high speed (500 μl / min) (V8), the mixed solution L passes through the first holding unit 53 and the second holding unit 55, the enzyme is dissolved, and mixed in the second mixing unit 51. (S12).

Further, when 80 μL of pressure is applied at a high speed (500 μl / min) (V9), the mixed solution L is conveyed to the first mixing unit 49, the undissolved enzyme is dissolved, and mixed in the first mixing unit 49 ( S13).
Further, by sucking 80 μL at a high speed (500 μl / min) (V10), the enzymes in the first holding unit 53 and the second holding unit 55 are completely dissolved, and the liquid is uniformly mixed in the second mixing unit 51. (S14).
Next, by sucking at 0.2 kPa from the third port PT-B (V11), the mixed liquid L of the second mixing unit 51 is conveyed to the flow path of the reaction unit F (S15).

  When the liquid reaches the sensing position PH5 and the sensor PH-5 is turned on (V12), the third port PT-B is closed and the fourth port PT-C is sucked at 0.3 kPa, and this state is 5 Hold for seconds. The liquid mixture L is conveyed into the reaction detection cell 27 and stops at the narrow reaction detection cell outlet channel 71 downstream of the cell (S16, S17, S18).

At this time, each reaction detection cell 27 is kept at room temperature, and the primer previously immobilized with gelatin is held in the cell without being dissolved.
Next, the 4th port PT-C is made into a closed state (V13), and it mixes in the main flow path 73 which has connected the reaction detection cell 27 by pressurizing from the 3rd port PT-B at a speed | rate of 100 microliters / min. The liquid is pushed back to the second mixing unit 51 (S19), 2.5 μL of the mixed liquid L is weighed and dispensed into each reaction detection cell 27, and these are not connected to each other by the liquid (S20). ).

  Next, the pad device of the inspection apparatus 11 is detached, and labels Ra, Rb, Rc, Rd (see FIG. 5) are attached to the port portions PT-A, B, C, D by a seal device (not shown). In addition, the chip 100 is hermetically sealed, and the amplification product due to the width reaction flows out of the chip, so that there is no fear of polluting the environment.

Next, the reaction part F is rapidly heated to 60 ° C. by a temperature control device (not shown). By heating, the primer immobilized with gelatin is uniformly diffused into the reaction detection cell 27 and the isothermal amplification reaction starts.
At this time, the liquid end surfaces of the thin reaction detection cell inlet flow path 69 and the reaction detection cell outlet flow path 71 at both ends of the reaction detection cell 27 are kept at room temperature without being heated to 60 ° C. The liquid inside does not evaporate.

  The target corresponding to the primer previously mounted in each reaction detection cell 27a-27m by irradiating each reaction detection cell 27a-27m with excitation light by the fluorescence detection part 17 shown in FIG. You can know if a gene sequence exists. When the target gene sequence is present, an increase in fluorescence intensity is confirmed, whereas when the target gene sequence is not present, there is no increase in fluorescence intensity.

  Therefore, according to the driving control method of the microfluidic chip 100 according to the present embodiment, the first port PT-A for feeding the sample liquid and the pretreatment reagent, the second port PT-D for feeding the reaction amplification reagent, In addition to the third port PT-B for supplying air pressure into the passage, a flow path for mixing with various reagents and quantitative dispensing of the mixed liquid is provided as a constituent means. By detecting at least one of the ends and determining the liquid control operating conditions according to the detected timing, a finite liquid, especially without a built-in active valve or pump, is simple The flow path enables complicated handling by pneumatic driving from the outside of the chip 100. That is, liquid feeding control is possible with a simple structure without requiring a three-dimensionally complicated structure. This makes it possible to perform a desired droplet operation and chemical reaction automatically by simply inserting a sample and a liquid reagent, eliminating the need for complicated operations such as pipetting and loading / unloading to / from the device, and providing high analysis results. can get.

  In the microfluidic chip drive control method according to the present invention, at least one of the front end and the rear end of the liquid in the flow path is detected, and the liquid control operation condition is determined according to the detection timing of the liquid end. This eliminates the need for complicated operations such as pipetting and loading / unloading to / from the apparatus, and enables reactions such as extraction or amplification of DNA in the sample in the liquid flow path.

1 is a block diagram illustrating a microfluidic chip according to the present invention together with a schematic configuration of an inspection apparatus. FIG. 2 is an exploded perspective view of the microfluidic chip shown in FIG. 1. It is the top view which represented the top view of the microfluidic chip to (a), and the bottom view was represented to (b). FIG. 4 is an enlarged view of FIG. It is a disassembled perspective view showing the chip | tip lower surface before sticking of a closure member. It is a principal part enlarged plan view showing the vicinity of a port exit flow path. It is explanatory drawing of the reaction detection cell which represented the P2-P2 cross section view of FIG. 4 with which the primer was mixed and diffused, and represented the principal part enlarged view to (b). It is an enlarged plan view of a reaction detection cell. It is a graph of the fluorescence measurement result showing the case where there is a target sequence (a) and the case where there is no target sequence (b). It is the top view which represented the foaming condition of the reaction detection part to (a)-(c). It is principal part sectional drawing showing the foaming prevention measure of the reaction detection part. It is explanatory drawing which represented the planar view of the liquid position detection part to (a), and represented the P1-P1 cross section view to (b). It is a schematic diagram showing the incident / reflected light of the liquid position detection unit. It is a graph showing the correlation between a reflectance and an incident angle. It is a side view of the liquid position detection part by which the light projection side optical fiber and the light reception side optical fiber were inclinedly arranged. It is the time chart which represented the operation state of each element accompanying the drive control of a microfluidic chip along the time axis. It is operation | movement explanatory drawing from a liquid set to the first heating. It is operation | movement explanatory drawing until an enzyme mixing. It is operation | movement explanatory drawing to reaction part injection | pouring. It is operation | movement explanatory drawing from dispensing to completion of an inspection.

Explanation of symbols

21 flow path substrate 61, 63, 65 primer 83 flow path 89 light emitting side optical fiber 91 light receiving side optical fiber 93 normal 100 microfluidic chip PT-A first port (connection port)
PT-B 3rd port (connection port)
PT-C 4th port (connection port)
PT-D 2nd port (connection port)
PH-1 Sensor at sensing position PH1 PH-2 Sensor at sensing position PH2 PH-3 Sensor at sensing position PH3 PH-4 Sensor at sensing position PH4 PH-5 Sensor at sensing position PH5

Claims (8)

The liquid supplied in the liquid channel is moved by applying a physical action force to the microfluidic chip in which the liquid processing unit is disposed along the liquid channel formed in the substrate, and the liquid A microfluidic chip drive control method for performing liquid processing in a processing unit,
Detecting at least one of a front end portion and a rear end portion of the liquid in the liquid flow path at a specific position of the liquid flow path, and determining a control operation condition of the liquid according to the detected timing; A drive control method for a microfluidic chip. A drive control method for a microfluidic chip according to claim 1,
The method for driving a microfluidic chip, wherein the liquid control operation condition includes a condition including at least one of a moving direction, a moving speed, and a driving force for movement with respect to the liquid. A microfluidic chip drive control method according to claim 1 or 2,
The method for driving a microfluidic chip, characterized in that the control operation condition of the liquid treatment includes a heating set temperature for heat-treating the liquid in the liquid treatment unit. A microfluidic chip drive control method according to claim 1 or 2,
Irradiating light to the specific position of the liquid flow path to detect reflected light from the liquid flow path, the presence or absence of liquid in the liquid flow path at the specific position is refracted between the air and the liquid of the reflected light A method for driving a microfluidic chip, wherein the determination is based on a change in light quantity based on a change in rate. A method for driving a microfluidic chip according to claim 4,
A method of driving a microfluidic chip, wherein light for irradiating the specific position of the liquid flow path is supplied through a light-projecting-side optical fiber, and reflected light from the liquid flow path is introduced into a light-receiving-side optical fiber and detected. . A method for driving a microfluidic chip according to claim 4 or 5, comprising:
The light irradiation direction to the specific position of the liquid channel and the detection direction of the reflected light from the specific position are set in directions that are respectively inclined with respect to the normal line of the light irradiation surface of the specific position. A method of driving a microfluidic chip. A microfluidic chip drive control method according to any one of claims 1 to 6,
The microfluidic chip drive control method, wherein the physical acting force is a pneumatic acting force generated by air supply or air suction from connection ports provided at a start point and an end point of the liquid flow path. A microfluidic chip drive control method according to any one of claims 1 to 7,
The liquid contains a specimen liquid having ecological cells, a pretreatment reagent, a reaction amplification reagent,
One of the liquid processing units is equipped with a primer that is a fragment of nucleic acid. A predetermined amount of the liquid is dispensed into the liquid processing unit and irradiated with excitation light while being heated. A drive control method for a microfluidic chip, wherein the generated fluorescence is detected.

Images