Integrated fluid device, and method for manufacturing integrated fluid device
Patent Information
- Application Number
- JP2023129816
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- Filing Date
- 2023-08-09
- Publication Date
- 2025-02-21
Smart Images
Figure 2025025226000001_ABST
Abstract
Description
[Technical field]
[0001] The present invention relates to an integrated fluidic device for amplifying genes and a method for producing the integrated fluidic device. [Background technology]
[0002] In infectious disease testing, rapid pathogen detection is required to hasten the start of appropriate treatment and prevent secondary infection. In particular, bloodstream infections such as sepsis often cause shock symptoms even if only a small number of bacteria are present in the bloodstream, and survival rates drop rapidly if antibiotic treatment is delayed. A wide variety of bacteria cause bloodstream infections, and different antibiotics are effective for each. Therefore, it is important to test for multiple bacteria simultaneously and administer appropriate antibiotics promptly. Genetic testing based on the Polymerase Chain Reaction (PCR) method is faster than conventional tests based on microbial culture, and has been the subject of active research in recent years.
[0003] The blood concentration of bacteria causing bloodstream infections is generally low, and in a few cases, it is said to be about 1 CFU (Colony forming unit) / mL. On the other hand, taking a large amount of blood should be avoided in consideration of the burden on the patient, and the limit is a few mL to several tens of mL. In other words, the number of bacteria that can be used for testing is at most a few to several tens of bacteria. When detecting the 16s ribosomal RNA gene region, since there are six copies of the gene per bacterium, the total number of DNA that can be extracted is several to several tens of copies. Since the concentration is low and detection is difficult in this state, the target bacterial species is detected with high sensitivity by amplifying it by PCR reaction. Therefore, it is necessary to use as much DNA extracted from the bacteria as possible in the PCR reaction without loss.
[0004] In normal PCR, a microtube containing a reaction solution is placed in a heat block, and the temperature of the heat block is changed to repeat the steps of DNA denaturation, annealing, and extension (thermal cycle) to amplify the target DNA. Since the heat capacity of a heat block is generally greater than that of the microtube or reaction solution, the reaction rate is determined by the heating and cooling time of the heat block, not the heating and cooling of the solution, and the reaction time usually takes about an hour.
[0005] In recent years, technology has been developed to perform chemical reactions such as DNA extraction, nucleic acid amplification, and gene detection on integrated fluidic devices. The extracted DNA can be transferred directly to a PCR reaction, which allows amplification while minimizing the loss of small amounts of DNA. In addition, because the reaction is completed in a closed space, the risk of false positives due to contamination (increased foreign DNA other than the DNA of bacteria in the patient sample, resulting in a false positive) is reduced. Another advantage of integrated fluidic devices is that they can be mass-produced in many cases, making them inexpensive to perform.
[0006] Non-Patent Document 1 discloses an integrated fluid device that extracts DNA from bacteria in blood, amplifies the DNA by PCR, and detects the presence or absence of DNA by hybridization. PCR is achieved by changing the temperature of a solution bath installed on a resin chip using a Peltier element separate from the integrated fluid device. In addition, a valve is used that passes an electric current through resistance wiring on a printed circuit board to locally increase the temperature and dissolve paraffin installed in advance in a nearby flow path to conduct the solution. Such integrated fluid devices have a simple configuration consisting of a printed circuit board suitable for mass production and a resin chip, and are characterized by their ability to detect bacterial genes inexpensively. [Prior art documents] [Non-patent literature]
[0007] [Non-Patent Document 1] Robin L., et al., Anal.Chem.2004, 76, 1824-1831 Summary of the Invention [Problem to be solved by the invention]
[0008] In the integrated fluid device disclosed in Non-Patent Document 1, the temperature of the PCR solution bath is controlled by a thermal cycler installed on the device body side. The thermal cycler is usually composed of a metal heat block, a heater for heating the heat block, and a fan or Peltier element for cooling the heat block. It is also possible to use a Peltier element for cooling as a heater. These components have their own heat capacity, and it takes time to heat and cool the heat block, for example. Even if the temperature is controlled by pressing a Peltier element directly against the PCR solution bath without using a heat block, there is a problem that the thermal cycle time is limited by the heat capacity of the Peltier element and the heat capacity of the resin chip itself, since the solution is heated and cooled through the resin chip that forms the PCR solution bath.
[0009] SUMMARY OF THE PRESENT EMBODIMENTS Accordingly, an object of the present invention is to provide an integrated fluid device that is inexpensive and capable of realizing rapid thermal cycling, and a method for manufacturing such an integrated fluid device. [Means for solving the problem]
[0010] The integrated fluid device of the present invention comprises a substrate, a first heater wire and a second heater wire arranged at a predetermined interval on the surface of the substrate, terminals arranged on the substrate for inputting and outputting current to the first heater wire and the second heater wire, a lid arranged on the upper surfaces of the first heater wire and the second heater wire, a solution tank formed by at least the first heater wire, the second heater wire, and the lid, for containing a solution and amplifying genes in the solution by heat generated by the first heater wire and the second heater wire, a pretreatment unit connected upstream of the solution tank, and a gene detection unit connected downstream of the solution tank.
[0011] The method for manufacturing an integrated fluid device of the present invention includes forming, on the back surface of a plate material, a first recess to form a pre-treatment section for pre-treating a solution, a second recess to form a gene amplification section to amplify genes in the solution, and a third recess to form a gene detection section to detect the amplified genes in the solution, forming a first heater wire and a second heater wire at a predetermined interval on the surface of the substrate so as to be accommodated in the second recess, forming terminals on the substrate for inputting and outputting current to the first heater wire and the second heater wire, and joining the back surface of the plate material on which the first recess, second recess, and third recess are formed, to the surface of the substrate on which the first heater wire, second heater wire, and terminals are formed. Effect of the Invention
[0012] According to the present invention, it is possible to realize a thermal cycle that is inexpensive and quick. Problems, configurations and effects other than those described above will become apparent from the following description of the embodiments. [Brief description of the drawings]
[0013] [Figure 1] Configuration diagram of the integrated fluidic device of Example 1 [Diagram 2] Cross-sectional view along line AA' in Figure 1 [Diagram 3] FIG. 1 is a diagram showing the configuration of an integrated fluid device according to a first modified example of the first embodiment. [Figure 4] Cross-sectional view taken along line BB' in Figure 3. [Diagram 5] FIG. 1 is a diagram showing the configuration of an integrated fluid device according to a second modified example of the first embodiment. [Figure 6] Cross-sectional view along line AA' in Figure 5. [Figure 7] FIG. 1 is a diagram showing the configuration of an integrated fluid device according to a third modified example of the first embodiment. [Figure 8] Cross-sectional view taken along line AA' in Figure 7. [Figure 9] FIG. 11 is a cross-sectional view of an integrated fluid device according to a fourth modified example of the first embodiment. [Figure 10] Cross-sectional view taken along line AA' in Figure 9. [Figure 11]FIG. 13 is a diagram showing the configuration of an integrated fluid device according to a fifth modified example of the first embodiment. [Figure 12] FIG. 13 is a diagram showing the configuration of an integrated fluid device according to a sixth modified example of the first embodiment. [Figure 13] Configuration diagram of a gene detection device according to the second embodiment [Figure 14] 1 is a flowchart showing a method for manufacturing an integrated fluidic device according to a third embodiment of the present invention. [Figure 15] A diagram showing details of the process of forming the lid. [Figure 16] A diagram showing the details of the process of forming a substrate. [Figure 17] FIG. 13 is a diagram showing details of a process for bonding a lid and a substrate. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0014] The embodiments of the present invention will be described in detail with reference to the drawings. In the following embodiments, the components (including element steps, etc.) are not necessarily essential unless otherwise specified or considered to be obviously essential in principle.
[0015] In order to facilitate understanding of the invention, the position, size, shape, range, etc. of each component shown in the drawings may not represent the actual position, size, shape, range, etc. Therefore, the present invention is not necessarily limited to the position, size, shape, range, etc. disclosed in the drawings.
[0016] <Example 1> The integrated fluid device (gene detection device) of Example 1 will be described below. The integrated fluid device 116 of Example 1 includes a pretreatment section 111 that performs pretreatment of a biological sample (solution), a gene amplification section 105 that amplifies genes in the biological sample, and a gene detection section 112 that detects genes. The pretreatment section 111 is connected upstream of the gene amplification section 105, and the gene detection section 112 is connected downstream of the gene amplification section 105. The integrated fluid device 116 also includes a solution introduction hole 113 that introduces a biological sample into the pretreatment section 111, and a solution discharge hole 114 that discharges the detected biological sample from the gene detection section 112. The biological sample is introduced into the pretreatment section 111 from the solution introduction hole 113, and the biological sample is pretreated in the pretreatment section 111. The biological sample after the pretreatment is transported to the gene amplification section 105, where a thermal cycle is performed to amplify genes in the biological sample. The biological sample that has been subjected to the thermal cycle is transported to the gene detection section 112, where gene detection is carried out. Then, the biological sample that has already been detected is discharged from the gene detection section 112 to the outside via the solution discharge hole 114.
[0017] Fig. 1 is a plan view of an integrated fluidic device 116 of Example 1, and Fig. 2 is a cross-sectional view taken along line AA' in Fig. 1. The integrated fluidic device 116 includes a lid 100 and a substrate 108 bonded to the lid 100. The integrated fluidic device 116 also includes a heater wire 101 (first heater wire), a heater wire 102 (second heater wire), a terminal 106, and a terminal 107 formed on the surface of the substrate 108.
[0018] The heater wires 101 and 102 are formed on the surface of the substrate 108 at a predetermined distance in a direction (Y direction in the figure) perpendicular to the longitudinal direction of the heater wires 101 and 102 (X direction in the figure).
[0019] The terminals 106 and 107 are provided in an area of the substrate 108 to which the lid 100 is not joined, and are exposed so as to be able to connect to an external power source (for example, a constant current power source). The terminals 106 and 107 may be provided on the front surface of the substrate 108 (the same surface as the heater wires 101 and 102) or on the back surface of the substrate 108. With this configuration, the terminals 106 and 107 can be easily connected to an external power source using an inexpensive connector for printed circuit boards, such as a card edge connector. Furthermore, after gene detection, the used integrated fluidic device can be removed and easily replaced with a new integrated fluidic device.
[0020] Furthermore, integrated fluidic device 116 includes connection wiring 103 and connection wiring 104 provided on an inner layer of substrate 108. Connection wiring 103 electrically connects heater wiring 101, heater wiring 102, and terminal 106. Connection wiring 104 electrically connects heater wiring 101, heater wiring 102, and terminal 107.
[0021] Furthermore, integrated fluidic device 116 includes vias 115 that electrically connect heater wires 101, 102, terminals 106 and 107 formed on the surface of substrate 108 to connection wires 103 and 104 in the inner layer.
[0022] As described above, the integrated fluidic device 116 of Example 1 includes the pretreatment unit 111, the gene amplification unit 105, and the gene detection unit 112. The gene amplification unit 105 is a space formed between the substrate 108, the heater wiring 101 and the heater wiring 102 provided on the surface of the substrate 108, and the lid 100 placed on the upper surfaces of the heater wiring 101 and the heater wiring 102, and is a solution tank capable of containing a biological sample. Hereinafter, the gene amplification unit 105 will be referred to as the solution tank 105 as appropriate.
[0023] A flow channel 109 and a flow channel 110 are formed in the cover 100. The flow channel 109 connects the pretreatment section 111 and the solution tank 105, and the flow channel 110 connects the solution tank 105 and the gene detection section 112.
[0024] The lid 100 is preferably made of a material that is stable to temperature and pressure changes, is not easily affected by the solution used in the chemical reaction, and has good moldability. Suitable materials for this purpose include, for example, cycloolefin polymer (COP), polycarbonate (PC), and acrylic resin (PMMA).
[0025] The substrate 108 is preferably made of a material that is stable to temperature and pressure changes, is not easily corroded by the solutions used in the chemical reactions, and has insulating properties, such as glass epoxy (FR-4) or ceramic.
[0026] The material for the heater wiring 101, the heater wiring 102, the connection wiring 103, the connection wiring 104, the terminal 106, and the terminal 107 is preferably copper, which is conductive and can be wired at low cost, and copper can be formed at low cost using a typical wiring process for printed circuit boards.
[0027] Since the solution tank 105 is formed between the lid 100, the substrate 108, and the heater wiring 101 and heater wiring 102, the depth of the solution tank 105 (the height of the solution tank 105 in a direction perpendicular to the surface of the substrate 108) is determined by the thickness of the heater wiring 101 and heater wiring 102, which are generally copper wiring. The thickness of the copper wiring (heater wiring 101 and heater wiring 102) may be a typical thickness of 18 μm or 35 μm, or may exceed 70 μm to 1000 μm in order to pass a larger current. In other words, the thickness of the heater wiring 101 and heater wiring 102 is 18 μm or more.
[0028] The heater wires 101 and 102 are covered on their surfaces with a chemically stable thin resin film because they are in contact with the solution. Note that the thin resin film is not shown in the figure. It is preferable that the material of the thin resin film is heat resistant and chemically stable, and for example, a material used as a resist for a printed circuit board is suitable.
[0029] The connection wiring 103 and the connection wiring 104 are formed by an inner layer wiring process of the printed circuit board. The connection wiring 103 and the connection wiring 104 may be provided on the lower surface of the printed circuit board. The terminals 106 and 107 are electrically connected to the connection wiring 103 and the connection wiring 104 in the inner layer of the printed circuit board by vias 115, respectively. In addition, the heater wiring 101 and the heater wiring 102 are electrically connected to the connection wiring 103 and the connection wiring 104 in the inner layer of the printed circuit board by vias 115, respectively.
[0030] The specimen targeted by the integrated fluid device 116 of the first embodiment is, for example, blood from a patient with a bloodstream infection. Red blood cells are destroyed in advance with a hemolysis reagent, and then bacteria are concentrated and purified by centrifugation. When a concentrated solution of bacteria is introduced from a solution introduction hole 113, the bacteria are destroyed in the pretreatment section 111, and the DNA of the bacteria is extracted. The DNA of the bacteria is extracted by destroying the cell wall by a chemical means such as a general surfactant or an alkaline reagent, a thermal means by heating, or a physical means by cavitation using a minute blade or ultrasonic waves. The DNA extracted from the bacteria is mixed with reagents required for PCR, specifically, a PCR buffer, a polymerase, a substrate, a primer, and the like. Hereinafter, this mixed solution is referred to as a PCR solution. The general liquid volume of the PCR solution is 50 μL, but amplification is possible with a smaller liquid volume (for example, 1 μL) or a larger liquid volume of 500 μL. To achieve rapid PCR amplification, it is necessary to quickly change the temperature of the PCR solution and reduce the time required for thermal cycling. Therefore, it is desirable that the amount of the PCR solution is small, for example, about 1 μL to 25 μL. The amount of the PCR solution can be appropriately set by appropriately selecting the concentrations of the PCR buffer, polymerase, substrate, and primer to be mixed with the extracted DNA. In addition, it is possible to minimize the increase in the amount of liquid by freeze-drying the salt, polymerase, substrate, or primer, which are components of the buffer, and arranging them on the flow path.
[0031] When the PCR solution is transported to the solution tank 105, a current is applied to the terminal 106 by the gene detection device (see FIG. 13), the heater wires 101 and 102 generate Joule heat, and the PCR solution present between the heater wires 101 and 102 is heated to a predetermined denaturing temperature, for example, 98° C. The solution tank 105 is formed of the heater wires 101 and 102, the lid 100, and the substrate 108, and the PCR solution transported to the solution tank 105 comes into contact with the heater wires 101 and 102 through the resist. With this configuration, the heater wires 101 and 102 come into contact with the PCR solution to be heated through the resist, so that the PCR solution can be heated quickly by the heater wires 101 and 102. As a result, it is possible to administer an appropriate antibacterial drug according to the pathogen more quickly. The gene detection device monitors the temperature of the PCR solution, and stops the current when the temperature reaches a predetermined denaturing temperature. The PCR solution is cooled by thermal conduction to the substrate 108 and the lid 100, and is cooled to a predetermined annealing temperature, for example, 65°C. When the temperature reaches 65°C, a weak current is passed through the heater wires 101 and 102, and the temperature is maintained for a certain period of time so as not to drop any further. This makes it possible to suppress non-specific binding of the primers and to suppress the amplification of DNA other than the target. The solution is heated again to a denaturing temperature, for example, 98°C. The DNA extension reaction is initiated while the solution is held at 65°C, and is carried out during the intermediate stage of heating to 98°C. By carrying out thermal cycling in two temperature zones, 98°C and 65°C, it is possible to amplify genes faster than the PCR reaction in which the temperature is changed in three temperature zones, namely, the denaturing temperature (for example, 98°C), the annealing temperature (for example, 65°C), and the extension temperature (for example, 72°C), which is carried out in classical PCR. However, the integrated fluidic device disclosed in this specification is not specialized for thermal cycling in two temperature zones, and it is also possible to carry out thermal cycling in three temperature zones. By repeating the above process, the PCR solution undergoes a thermal cycle, and the PCR reaction proceeds. The temperature of the PCR solution may be monitored from the outside using infrared rays, or by measuring the resistance or voltage of the heater wiring 101 or the heater wiring 102.When monitoring the temperature of the PCR solution with infrared rays, a very small measurement hole that the PCR solution does not pass through may be opened in the substrate 108 or the lid 100, and the infrared rays emitted from the PCR solution may be measured through the measurement hole. This configuration makes it possible to measure the temperature of the PCR solution more accurately than by observing it from above the lid.
[0032] The number of bacteria contained in the blood of a patient with a bloodstream infection is about 1 bacterium per 1 mL of blood in a few cases. In this case, the minimum number of copies of the bacterial gene extracted from 10 mL of the patient's blood is 10 copies. In order to amplify 10 copies of the gene correctly and quickly by PCR, it is desirable to suppress surface adsorption as much as possible in the flow path from the solution introduction hole 113 to the solution tank 105. This is because DNA is easily lost due to adsorption, and the efficiency of the PCR reaction in the solution tank 105 decreases. Therefore, at least one of the lid 100, the substrate 108, the heater wire 101, and the heater wire 102 is coated with a nucleic acid adsorption prevention coating. One method for preventing surface adsorption is to flow DNA or RNA (blocking DNA or blocking RNA) having a sequence different from the DNA of the bacteria to be amplified into the pretreatment unit 111, flow path 109, solution layer 105, flow path 110, and gene detection unit 112 in advance, and adsorb the blocking DNA to the surface of the substrate 108, lid 100, heater wiring 101, or heater wiring 102, etc., thereby preventing adsorption of the DNA of the target bacteria.
[0033] After the PCR reaction, the PCR solution is transported by a pump of the gene detection device to a gene detection unit 112. The gene detection unit 112 can detect the PCR amplification product by any method, for example, a hybridization reaction by a microarray or nucleic acid chromatography can be detected by a fluorescence or color reaction.
[0034] Furthermore, in the first embodiment, the solution tank 105 for carrying out the thermal cycle can be constructed from the substrate 108, which is a printed circuit board, so that the integrated fluidic device 116 can be obtained at low cost.
[0035] <First Modification of First Embodiment> Fig. 3 is a plan view of an integrated fluid device 116 of a first modified example of the first embodiment. Descriptions of configurations similar to those of the integrated fluid device 116 of the first embodiment in Figs. 1 and 2 will be omitted where appropriate. The first modified example of the first embodiment is characterized in that the heater wiring 101 and the heater wiring 102 have non-uniform wiring widths. The heater wiring 101 and the heater wiring 102 have a wider wiring width in the central portion in the longitudinal direction (X direction in the figure) than in other portions.
[0036] According to this configuration, the heater wiring 101 and the heater wiring 102 have low resistance parts 301 and high resistance parts 302. Since the current (I) flowing through the heater wiring 101 and the heater wiring 102 is constant, the Joule energy P (P=I^2*wiring resistance R) generated in the heater wiring 101 and the heater wiring 102 changes with the position in the longitudinal direction of the heater wiring 101 and the heater wiring 102. That is, the amount of heat generated per unit length is high in the high resistance part 302, and the amount of heat generated per unit length is low in the low resistance part 301. This prevents the central part of the solution bath 105 from being heated intensively, and the solution can be heated more uniformly over the entire area of the solution bath 105, thereby realizing a rapid and efficient PCR reaction. As a result, it is possible to administer an appropriate antibacterial agent according to the pathogen more quickly.
[0037] Although the wiring width of the heater wiring 101 and the heater wiring 102 is increased linearly toward the center in the longitudinal direction (X direction in the figure), the wiring width may be increased in a step-like manner toward the center in the longitudinal direction (X direction in the figure). Also, instead of the wiring width of the heater wiring 101 and the heater wiring 102, the wiring thickness of the heater wiring 101 and the heater wiring 102 may be increased linearly or in a step-like manner toward the center in the longitudinal direction (X direction in the figure). Furthermore, the wiring width or thickness of only one of the heater wiring 101 or the heater wiring 102 may be changed.
[0038] Fig. 4 is a cross-sectional view taken along line BB' in Fig. 3. It is desirable that the thickness of the heater wires 101 and 102, i.e., the depth a of the solution reservoir 105, is greater than the distance between the heater wires 101 and 102, i.e., the width b of the solution reservoir 105. With this configuration, it is possible to bring a larger surface area into contact with the heater wires 101 and 102 relative to the volume of the PCR solution, enabling the PCR solution to be heated more quickly.
[0039] <Second Modification of First Embodiment> 5 is a plan view of an integrated fluid device 116 according to a second modified example of the first embodiment. Descriptions of configurations similar to those of the integrated fluid device 116 according to the first embodiment in FIGS. 1 and 2 will be omitted as appropriate. In the second modified example of the first embodiment, a plurality of rear surface wirings 501 (heat dissipation wirings) are formed on the rear surface of the substrate 108. The plurality of rear surface wirings 501 are electrically connected to the heater wiring 101 and the heater wiring 102 by vias 115.
[0040] According to this configuration, the heater wiring 101 and the heater wiring 102 are thermally coupled by metal to the back surface wiring 501. That is, it is possible to reduce the thermal resistance between the heater wiring 101 and the heater wiring 102 and the back surface wiring 501.
[0041] Fig. 6 is a cross-sectional view taken along line AA' in Fig. 5. When cooling the PCR solution, by cooling the rear surface wiring 501 with a cooling mechanism in the gene detection device, for example, a cooling fan 502 or a Peltier element, it is possible to cool the heater wiring 101 and the heater wiring 102 more effectively than when they are cooled by thermal conduction to the substrate 108. With this configuration, it is possible to cool the PCR solution more quickly, shorten the thermal cycle time, and achieve a more rapid PCR reaction.
[0042] <Third Modification of First Embodiment> FIG. 7 is a plan view of an integrated fluid device 116 of a third modified example of the first embodiment, and FIG. 8 is a cross-sectional view taken along the line AA′ in FIG. 7. Descriptions of the same configuration as the integrated fluid device 116 of the first embodiment in FIG. 1 and FIG. 2 will be omitted as appropriate. In the third modified example of the first embodiment, a groove 701 is formed in a part of the lid 100 to reduce the heat capacity of the lid 100 adjacent to the solution tank 105 and reduce the thermal resistance from the surface of the groove 701 to the solution tank 105. Then, the cooling effect from the upper surface of the solution tank 105 is enhanced by blowing air onto the groove 701 with a cooling fan 702. With this configuration, it is possible to cool the PCR solution more quickly, shorten the thermal cycle time, and realize a more rapid PCR reaction.
[0043] 7 and 8, the rear wiring 501 and the cooling fan 502 are provided as in the second modified example shown in FIGS. 5 and 6, but the rear wiring 501 and the cooling fan 502 do not necessarily have to be provided.
[0044] <Fourth Modification of First Embodiment> FIG. 9 is a plan view of an integrated fluid device 116 of a fourth modified example of the first embodiment, and FIG. 10 is a cross-sectional view taken along line AA′ in FIG. 9. Descriptions of the same configuration as the integrated fluid device 116 of the first embodiment in FIG. 1 and FIG. 2 will be omitted as appropriate. In the fourth modified example of the first embodiment, a slit 901 is provided in a portion between the heater wires 101 and 102 of the substrate 108. Then, the upper surface of the slit 901 is sealed with a thin tape 902. The material of the tape is preferably a material that has low thermal resistance and can withstand temperature changes due to the thermal cycle of PCR, and for example, polyimide, polycarbonate, polyethylene terephthalate, Teflon (registered trademark), silicone rubber, cyclic olefin, etc. can be used.
[0045] In the example of FIG. 10, the gene amplification section 105 is a space formed among the lid 100, the tape 902, the heater wire 101, and the heater wire .
[0046] According to this configuration, by blowing air from cooling fan 502 through slit 901 onto tape 902, it becomes possible to cool the PCR solution contained in solution tank 105 more quickly.
[0047] <Fifth Modification of First Embodiment> FIG. 11 shows an integrated fluid device 116 according to a fifth modification of the first embodiment. The same configuration as that of the integrated fluid device 116 according to the first embodiment shown in FIG. 1 and FIG. 2 will not be described as appropriate. In the fifth modification of the first embodiment, a terminal 1101 (terminal for voltage measurement) and a terminal 1102 (terminal for voltage measurement) for measuring the voltage across the heater wires 101 and 102, a connection wire 1103 for connecting the terminal 1101 and the heater wires 101 and 102, and a connection wire 1104 for connecting the terminal 1102 and the heater wires 101 and 102 are newly provided. An external current source 1105 passes a current from the terminal 107 to the terminal 106 to heat the heater wires 101 and 102. At this time, an external voltmeter 1106 connected to the terminals 1101 and 1102 measures the voltage across the heater wires 101 and 102. The controller 1107 can precisely calculate the resistance values of the heater wires 101 and 102 according to Ohm's law and the principle of the four-terminal method from the current value applied by the current source 1105 during heating and the measurement value of the voltmeter 1106. In general, the resistance value Rt of the heater wire at temperature t can be expressed by the following equation. Rt = R0 * (1 + α0 * t)
[0048] Here, R0 is the resistance value of the heater wire at 0° C., α0 is the temperature coefficient of the heater wire at 0° C., and is a value specific to the metal material (e.g., copper) of the heater wire 101 or the heater wire 102. From the above formula, the temperature t of the heater wire 101 and the heater wire 102 can be calculated by measuring the values of R0 and α0 in advance and measuring the resistance value Rt during PCR.
[0049] According to this configuration, the temperature of the solution can be estimated at low cost without providing an additional temperature measurement means in the integrated fluidic device 116. Note that even during cooling, the temperature can be measured by the same means as above by passing a weak current from the current source 1105.
[0050] <Sixth Modification of First Embodiment> FIG. 12 shows an integrated fluid device 116 according to a sixth modified example of the first embodiment. The same configuration as that of the integrated fluid device 116 according to the first embodiment shown in FIG. 1 and FIG. 2 will not be described. In the sixth modified example of the first embodiment, a temperature measurement wire 1201 (temperature measurement wire), a terminal 1205 (current input terminal) and a terminal 1206 (current input terminal) for applying a current to the temperature measurement wire 1201, and a terminal 1207 (voltage measurement terminal) and a terminal 1208 (voltage measurement terminal) for measuring the voltage at both ends of the temperature measurement wire 1201 are newly provided at the bottom of the solution bath 105. During PCR, a current is applied from an external current source 1202 to the temperature measurement wire 1201, and the voltage at both ends of the temperature measurement wire 1201 is measured by an external voltmeter 1203. The controller 1204 precisely calculates the resistance values of the heater wires 101 and 102 according to Ohm's law and the principle of the four-terminal method from the current value applied by the current source 1202 and the measurement value of the voltmeter 1203. Furthermore, the controller 1204 calculates the temperature of the temperature measurement wire 1201 using the above formula. By measuring in advance the relationship between the temperature of the solution in the solution bath 105 and the resistance value of the temperature measurement wire 1201 at that time, it becomes possible to indirectly estimate the temperature of the PCR solution in the solution bath 105 from the resistance value of the temperature measurement wire 1201.
[0051] According to this configuration, the cost of adding wiring in the manufacturing process of the printed circuit board is at a negligible level, so that the temperature of the solution can be estimated at low cost.
[0052] <Example 2> Fig. 13 is a diagram showing the configuration of a gene detection device 1300 of Example 2. The gene detection device 1300 of Example 2 detects genes using the integrated fluidic device 116 of Example 1 described above. Fig. 13 shows an example in which the gene detection device 1300 uses the integrated fluidic device 116 shown in Fig. 1, but the gene detection device 1300 may use the integrated fluidic devices of the first to sixth modified examples of Example 1 (Figs. 3 to 12).
[0053] The gene detection device 1300 includes a pump 1301, a temperature sensor 1302, a gene detection sensor 1303, a power supply 1304, and a controller 1305. The pump 1301 transports a liquid in the integrated fluid device 116. For example, the pump 1301 transports a PCR solution after a PCR reaction from the solution tank 105 to the gene detection unit 112. The temperature sensor 1302 is, for example, a non-contact infrared sensor, and detects the temperature of the PCR solution. A signal detected by the temperature sensor 1302 is sent to the controller 1305. The gene detection sensor 1303 is a sensor that detects genes in the gene detection unit 112, and acquires information to observe the gene detection reaction in the gene detection unit 112. A signal detected by the gene detection sensor 1303 is sent to the controller 1305. The gene detection sensor 1303 is, for example, a fluorescent detection sensor or an image recognition sensor. The power supply 1304 is a constant current power supply that supplies a current to the integrated fluidic device 116 , and inputs the current to the terminal 106 or the terminal 107 .
[0054] The controller 1305 is a general computer system and includes a processor 1351, a main memory 1352, an auxiliary memory 1353, and an interface 1354. The processor 1351 is a central processing unit (CPU), a graphics processing unit (GPU), a digital signal processor (DSP), an ASIC, or the like. The main memory 1352 is a dynamic random access memory (DRAM) or the like, and is used as a working area for the processor 1351. The auxiliary memory 1353 is a hard disk drive (HDD), a solid state drive (SSD), or a combination thereof, or the like, and stores various programs and various data. The interface 1354 is a device controller or the like that controls the operations of peripheral devices (the pump 1301, the temperature sensor 1302, the gene detection sensor 1303, and the power supply 1304) connected to the controller 1305.
[0055] The controller 1305 controls the output of the power supply 1304 and the pump 1301 in a predetermined procedure based on a signal detected by the temperature sensor 1302. For example, the controller 1305 controls the on / off of the power supply 1304, the amount of current supplied from the power supply 1304, or the duty of the on period to adjust the heating of the heater wiring 101 and the heater wiring 102 and perform a thermal cycle. The controller 1305 also outputs the result of the gene detection sensor 1303 to the outside.
[0056] The gene detection device 1300 may include the voltmeter 1106 in Fig. 11 and the voltmeter 1203 in Fig. 12. The controllers 1107 and 1204 described above may have the same configuration as the controller 1305.
[0057] <Example 3> Fig. 14 is a flowchart showing a method for manufacturing an integrated fluidic device 116 of Example 3. Figs. 15 to 17 are diagrams showing details of each step in the flowchart of Fig. 14. The diagrams on the left side of Figs. 15 to 17 are cross-sectional views taken along line AA' in the same figures. The method for manufacturing an integrated fluidic device 116 mainly includes forming a lid 100 (S1401), forming a substrate 108 (S1402), and bonding the lid 100 and the substrate 108 (S1403).
[0058] (Formation of the lid 100 (S1401)) The formation of the lid 100 in S1401 includes forming a first recess 1501 for forming a pretreatment section 111 for pretreatment of a biological sample (solution), a second recess 1502 for forming a gene detection section 112 for amplifying genes in the solution, and a third recess 1503 for forming a gene detection section 112 for detecting the amplified genes in the solution, on the back surface of a transparent plate material 1500. The formation of the lid 100 in S1401 also includes forming a fourth recess 1504 for forming a flow channel 109 and a fifth recess 1505 for forming a flow channel 110, on the back surface of the transparent plate material 1500. The formation of the lid 100 in S1401 also includes forming a solution inlet hole 113 and a solution outlet hole 114.
[0059] Forming the second recess 1502 on the back surface of the transparent plate material 1500 described above means forming the second recess 1502 with a depth corresponding to the thickness of the heater wires 101 and 102. That is, the depth of the second recess 1502 is determined according to the design values of the thicknesses of the heater wires 101 and 102. The width (W1 in FIG. 15) of the second recess 1502 is greater than the sum of the width (W2 in FIG. 16) of the heater wire 101 and the width (W3 in FIG. 16) of the heater wire 102. W1-(W2+W3) is the width of the gene amplification section 105 (solution tank 105).
[0060] The formation of the lid 100 in S1401 further includes a procedure for forming a reagent spot 1506, a reagent spot 1507, and, when DNA detection is performed using a microarray, a detection spot 1508 for a probe that binds to DNA. The reagent spot 1506 is a freeze-dried reagent required for PCR, specifically a PCR buffer, a polymerase, a substrate, a primer, etc. The reagent spot 1507 is formed as necessary depending on the DNA detection method, and when a microarray is used, a freeze-dried reagent containing salts, surfactants, etc. required for the hybridization reaction conditions to the microarray is used. The detection spot 1508 is a DNA probe that specifically binds to the DNA of the bacteria to be detected, fixed to the lid 100. Although an example of forming two spots is shown in the figure, the number of spots may be increased as appropriate depending on the number of DNA to be detected and the number of positive and negative controls.
[0061] (Formation of Substrate 108 (S1402)) The formation of substrate 108 in S1402 includes forming heater wires 101 and 102 on the surface of substrate 108 at a predetermined interval so as to be accommodated in second recess 1502, and forming terminals 106 and 107 on substrate 108 for inputting and outputting current to and from heater wires 101 and 102. Forming heater wires 101 and 102 may be forming heater wires 101 and 102 having a thickness of 18 μm or more. Forming heater wires 101 and 102 on the surface of substrate 108 may be forming heater wires 101 and 102 such that the thickness of heater wires 101 and 102 is greater than the predetermined interval.
[0062] Forming the heater wiring 101 and the heater wiring 102 on the surface of the substrate 108 may mean forming the heater wiring 101 and the heater wiring 102 such that the wiring width or wiring thickness of the heater wiring 101 or the heater wiring 102 increases toward the center of the heater wiring 101 or the heater wiring 102 in the longitudinal direction (X direction).
[0063] Further, the formation of the substrate 108 in S1402 includes forming the connection wiring 103 and the connection wiring 104 in the inner layer of the substrate 108. The formation of the connection wiring 103 and the connection wiring 104 in the inner layer of the substrate 108 is realized by a general inner layer wiring process of a printed circuit board.
[0064] Furthermore, the formation of the substrate 108 in S1402 includes forming vias 115 that electrically connect the heater wiring 101 and the connection wiring 103, vias 115 that electrically connect the heater wiring 101 and the connection wiring 103, and vias 115 that electrically connect the terminals 106 and the connection wiring 103. The formation of the substrate 108 in S1402 also includes forming vias 115 that electrically connect the heater wiring 101 and the connection wiring 104, vias 115 that electrically connect the heater wiring 101 and the connection wiring 104, and vias 115 that electrically connect the terminals 106 and the connection wiring 104.
[0065] The order in which steps S1401 and S1402 are executed may be reversed.
[0066] (Joining the lid 100 and the substrate 108 (S1403)) The joining of the lid 100 and the substrate 108 in S1403 includes joining the back surface of the lid 100 on which the first recess 1501, the second recess 1502, and the third recess 1503 are formed, to the surface of the substrate 108 on which the heater wiring 101, the heater wiring 102, the terminal 106, and the terminal 107 are formed. This joining forms the gene amplification section 105, which is a space formed between the lid 100 (the second recess 1502), the substrate 108, the heater wiring 101, and the heater wiring 102. This joining also forms the pretreatment section 111, which is a space formed between the lid 100 (the first recess 1501) and the substrate 108, and the gene detection section 112, which is a space formed between the lid 100 (the third recess 1503) and the substrate 108. This bonding also forms flow channel 109, which is a space formed between lid 100 (fourth recess 1504) and substrate 108, and flow channel 110, which is a space formed between lid 100 (fifth recess 1505) and substrate 108. The bonding can be achieved by adhering lid 100 and substrate 108 with an adhesive, double-sided tape cut along the recess of lid 100, or laser welding technology.
[0067] The method for producing integrated fluidic device 116 may further include applying a nucleic acid adsorption prevention coating to the rear surface of lid 100, the front surface of substrate 108, heater wire 101, and heater wire 102.
[0068] 5 and 6, the manufacturing method of the integrated fluidic device 116 may include forming a plurality of rear surface wirings 501 on the rear surface of the substrate 108. The plurality of rear surface wirings 501 are electrically connected to the heater wiring 101 and the heater wiring 102 by vias 115.
[0069] The method for manufacturing the integrated fluidic device 116 may also include forming a groove 701 in a part of the lid 100 as shown in FIGS.
[0070] In addition, the manufacturing method of the integrated fluidic device 116 may include providing a slit 901 in a portion between the heater wires 101 and 102 of the substrate 108, and providing a tape 902 to seal the slit 901, as shown in Figures 9 and 10.
[0071] Furthermore, the method for manufacturing the integrated fluidic device 116 may include providing terminals 1101 and 1102 for measuring the voltage across the heater wires 101 and 102 on the substrate 108, as shown in FIG.
[0072] In addition, the manufacturing method of the integrated fluid device 116 may include forming wiring 1201 for temperature measurement below the solution bath 105 of the substrate 108, terminals 1205 and 1206 for applying a current to the wiring 1201 for temperature measurement, and terminals 1207 and 1208 for measuring the voltage across the wiring 1201 for temperature measurement, as shown in FIG. 12.
[0073] (Modification) The present invention is not limited to the above-mentioned embodiment, but includes various modified examples. The above-mentioned embodiment has been described in detail to clearly explain the present invention, and is not necessarily limited to those having all of the configurations described. It is also possible to replace a part of the configuration of one embodiment with the configuration of another embodiment, and it is also possible to add the configuration of another embodiment to the configuration of one embodiment. It is also possible to add, delete, or replace a part of the configuration of each embodiment with another configuration. [Explanation of symbols]
[0074] 100: Lid 101: Heater wire (first heater wire) 102: Heater wiring (second heater wiring) 103, 104, 1103, 1104: Connection wiring 105: Gene amplification section (solution tank) 106, 107: Terminal 108: Circuit board 109, 110: flow path 111: Pre-processing section 112: Gene detection unit 113: Solution introduction hole 114:Solution drain hole 115: Beer 116: Integrated Fluidic Devices 301: Low resistance part 302: High resistance part 501: Back wiring (heat dissipation wiring) 502, 702: Cooling fan 701: Groove 901: Slit 902: Tape 1101, 1102: Terminals (voltage measurement terminals) 1105, 1202 current source 1106, 1203 Voltmeter 1107, 1204, 1305 Controllers 1201: Wiring (for temperature measurement) 1205, 1206: Terminals (current input terminals) 1207, 1208: Terminals (voltage measurement terminals) 1300: Gene detection device 1301: Pump 1302: Temperature sensor 1303: Gene detection sensor 1304: Power supply 1351: Processor 1352: Main memory 1353: Auxiliary storage 1354: Interface 1500: Board material 1501: Recess (first recess) 1502: Recess (second recess) 1503: Recess (third recess) 1504: Recess (fourth recess) 1505: Recess (fifth recess) 1506, 1507: Reagent spots 1508: Detection spot
Claims
1. A substrate; a first heater wiring and a second heater wiring provided at a predetermined interval on a surface of the substrate; a terminal provided on the substrate for inputting and outputting a current to and from the first heater wiring and the second heater wiring; a lid disposed on an upper surface of the first heater wiring and the second heater wiring; a solution tank formed by at least the first heater wire, the second heater wire, and the lid, which contains a solution and amplifies genes in the solution by heat generated by the first heater wire and the second heater wire; a pre-treatment section connected upstream of the solution bath; A gene detection unit connected downstream of the solution tank. An integrated fluidic device comprising:
2. The first heater wiring and the second heater wiring have a thickness of 18 μm or more. The integrated fluidic device according to claim 1 .
3. The thickness of the first heater wiring and the second heater wiring is greater than the predetermined interval. The integrated fluidic device according to claim 1 .
4. The wiring width or thickness of the first heater wiring or the second heater wiring increases toward the center in the longitudinal direction of the first heater wiring or the second heater wiring. The integrated fluidic device according to claim 1 .
5. The lid, the substrate, the first heater wire, and the second heater wire are coated with a coating to prevent nucleic acid adsorption. The integrated fluidic device according to claim 1 .
6. A measurement hole for measuring the temperature of the solution in the solution tank is formed in the substrate or the lid. The integrated fluidic device according to claim 1 .
7. The heater further includes one or more heat dissipation wires that are connected to the first heater wire and the second heater wire and that are provided below the solution tank. The integrated fluidic device according to claim 1 .
8. A slit is formed in the lower part of the solution bath of the substrate, Further comprising a tape for sealing the slit; The solution tank is formed by the first heater wire, the second heater wire, the lid, and the tape. The integrated fluidic device according to claim 1 .
9. The heater wiring further includes a voltage measurement terminal for measuring a voltage value across both ends of the first heater wiring or the second heater wiring. The integrated fluidic device according to claim 1 .
10. A temperature measurement wiring for measuring a temperature provided at a lower portion of the solution bath; a current input terminal for inputting a current to the temperature measurement wiring; and a voltage measurement terminal for measuring a voltage across both ends of the temperature measurement wiring. The integrated fluidic device according to claim 1 .
11. forming a first recess on the rear surface of a plate material to form a pretreatment section for pretreatment of a solution, a second recess to form a gene amplification section for amplifying genes in the solution, and a third recess to form a gene detection section for detecting the amplified genes in the solution; forming a first heater wire and a second heater wire at a predetermined interval on a surface of a substrate so as to be accommodated in the second recess, and forming terminals on the substrate for inputting and outputting current to the first heater wire and the second heater wire; and joining a rear surface of the plate material on which the first recess, the second recess, and the third recess are formed, to a front surface of the substrate on which the first heater wire, the second heater wire, and the terminal are formed. A method for producing an integrated fluid device comprising the steps of:
12. forming the first heater wiring and the second heater wiring on the surface of the substrate includes forming the first heater wiring and the second heater wiring to a thickness of 18 μm or more, Forming the second recess on the rear surface of the plate material means forming the second recess having a depth corresponding to the thickness of the first heater wire and the second heater wire. The method for producing an integrated fluidic device according to claim 11 .
13. forming the first heater wiring and the second heater wiring on the surface of the substrate includes forming the first heater wiring and the second heater wiring such that thicknesses of the first heater wiring and the second heater wiring are greater than the predetermined interval. The method for producing an integrated fluidic device according to claim 11 .
14. Forming the first heater wiring and the second heater wiring on the surface of the substrate includes forming the first heater wiring and the second heater wiring such that a wiring width or a wiring thickness of the first heater wiring or the second heater wiring increases toward a center in a longitudinal direction of the first heater wiring or the second heater wiring. The method for producing an integrated fluidic device according to claim 11 .
15. and applying a nucleic acid adsorption prevention coating to the back surface of the plate, the front surface of the substrate, the first heater wire, and the second heater wire. The method for producing an integrated fluidic device according to claim 11 .
16. An integrated fluidic device according to any one of claims 1 to 10; A pump for transporting a solution within the integrated fluidic device; a temperature sensor for measuring a temperature of the solution in the solution bath; a gene detection sensor for detecting genes in the gene detection unit; a power source for supplying a current to the integrated fluidic device; a controller that controls the output of the power source and the pump in a predetermined procedure based on information from the temperature sensor and outputs the result of the gene detection sensor to the outside. A gene detection device characterized by: