JP2007322284A - Microchip and filling method of reagent in microchip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a microchip and a filling method of a reagent in the microchip capable of filling quickly and accurately the required trace amount of reagent to each of a plurality of reagent storage parts simultaneously, and moreover, preventing the reagent from leaking out from the reagent storage part into a fine channel communicating therewith, when filling the reagent. <P>SOLUTION: The microchip, having the reagent storage parts for storing an aqueous reagent equipped respectively with a reagent supply part, a liquid sending control part or the like, has characteristics wherein the pressure of the reagent passing through the reagent supply part is higher than the liquid holding pressure, which is the pressure with which the reagent starts to pass the liquid sending control part. The filling method of the reagent has characteristics where the pressure necessary for starting, to pass the reagent supply part is applied to the reagent by a micropump provided in a micropump unit, and after start of inflow of the reagent, a pressure lower than the liquid-holding pressure of the liquid-sending control part is applied to the reagent. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a microchip (analysis chip) used in a micro total analysis system and a method of filling a reagent in the microchip.

In recent years, by making full use of micromachine technology and ultrafine processing technology, devices and means (for example, pumps, valves, flow paths, sensors, etc.) for performing conventional sample preparation, chemical analysis, chemical synthesis, etc. have been miniaturized. A system integrated on a chip has been developed (Patent Document 1). This is also called μ-TAS (Micro total Analysis System), bioreactor, Lab-on-chips, etc., medical examination / diagnosis field, environmental measurement field, agricultural production Its application is expected in the field. In reality, as seen in genetic testing, μ-TAS is automated, accelerated and simplified for complicated processes, skilled procedures, testing that requires operation of equipment, etc. It can be said that it brings great benefits such as enabling analysis not only in the required sample amount and the required time but also in any time and place.

  In various types of analysis and inspection, importance is placed on the quantitativeness of analysis on the above chip (microchip), the accuracy of analysis, and the economy. The present inventors have already proposed a micropump system and a control method thereof for establishing a liquid feeding system having a simple configuration, high accuracy, and high reliability, which is used in combination with a microchip ( Patent Documents 2 to 4).

  The microchip is formed with a series of fine channels including a specimen and reagent storage unit, a reagent mixing unit, a reaction unit, and a channel communicating these. Among these, the reagent storage unit is filled with a predetermined amount of necessary reagents according to the reaction method and detection method used in the analysis in advance, and the microchip is ready for use at the time of analysis. It is desirable that Therefore, there is a demand for a method for quickly and accurately filling a small amount of reagent required in the reagent storage unit.

In addition, if the reagent leaks from the reagent container into the fine flow path communicating with the reagent when filling the reagent, normal liquid feeding cannot be performed at the time of analysis. It is necessary to suppress it. Furthermore, it is necessary to prevent the driving liquid from leaking from the opening used for filling the reagent so as not to hinder normal liquid feeding.
JP 2004-028589 A JP 2001-322099 A JP 2004-108285 A JP 2004-270537 A

  The present invention is capable of quickly and accurately filling a small amount of a required reagent simultaneously into each of a plurality of reagent containers, and when filling a reagent, a fine flow communicating from the reagent container to the reagent container. It is an object of the present invention to provide a microchip capable of suppressing a situation in which a reagent leaks into a path and a method for filling the microchip with the reagent.

The present inventor confirmed that a micropump and a micropump unit, which are also used for feeding a sample and a reagent in a microchannel of a microchip in a micro total analysis system, fill a reagent container having a water repellent valve with a reagent. It was found that it can be suitably used for the above. Furthermore, in the reagent storage unit, by using a microchip in which the pressure at which the reagent passes through the reagent supply unit is higher than the pressure at which the reagent starts to pass through the liquid feeding control unit, filling of the reagent by the micropump unit and The present inventors have found that liquid feeding can be suitably performed and have completed the present invention.

The microchip of the present invention has a reagent container that contains an aqueous reagent, and a site for detecting a reaction product by mixing and reacting the aqueous reagent sent from the reagent container with a sample or another aqueous reagent. A reagent comprising a series of micro flow channels, a liquid feeding control unit provided between the outlet of the reagent storage unit and the inlet of the micro flow channel, and a flow channel communicating the opening and the reagent storage unit A microchip having a supply part,
A pressure at which the reagent passes through the reagent supply unit is higher than a liquid holding pressure that is a pressure at which the reagent starts to pass through the liquid feeding control unit.

The “pressure at which the reagent passes through the reagent supply unit” of such a microchip is preferably 2 to 20 kPa, and the “liquid holding pressure of the water repellent valve” is preferably 1 to 5 kPa.
In the reagent filling method of the present invention, the above-mentioned microchip is filled with a reagent by a micropump unit having a reagent reservoir, a micropump, and an opening connectable to a reagent supply unit of the microchip. It is characterized by.

  In this reagent filling method, (1) the step of connecting the opening of the reagent supply unit in the microchip and the opening of the micropump unit, and (2) the pressure required to pass through the reagent supply unit by the micropump. It is desirable to include a step of applying a reagent, and (3) a step of applying a pressure not higher than the liquid holding pressure of the liquid supply control unit to the reagent by a micropump after the reagent starts to flow.

  For microchips with multiple reagent reservoirs, use a reagent pump, micropump, micropump unit with multiple sets of openings and microchannels to send reagents to multiple reagent reservoirs. It is preferable to liquefy.

  As the micropump used for filling the reagent in the present invention, the first flow path in which the flow path resistance changes according to the differential pressure, and the change rate of the flow path resistance with respect to the change in the differential pressure is greater than that of the first flow path. A small second flow path, a pressurizing chamber positioned between and in communication with the first flow path and the second flow path, an actuator for changing the internal pressure of the pressurizing chamber, and a drive for driving the actuator A piezo pump equipped with a device is preferred.

  According to the present invention, it is possible to quickly and accurately fill a small amount of a required reagent simultaneously into each of a plurality of reagent storage parts of a microchip, and when the reagent is filled, the reagent storage part communicates with this. Since the problem of the reagent leaking into the fine flow path is solved, it becomes possible to efficiently manufacture a microchip filled with the reagent in advance. Furthermore, since the microchip of the present invention can prevent the driving liquid from leaking out from the reagent supply unit used for filling the reagent when the driving liquid is supplied at the time of analysis, Highly reliable analysis is performed.

In the present specification, the “microchip” refers to an analysis chip in a micro total analysis system used for various purposes such as synthesis and testing, for example, for testing for biological substances. “Fine channel” is a minute groove-like channel formed in the microchip of the present invention, and a container, a reaction unit, or a detection unit for reagents and the like communicating with the channel is provided. Even in the case of being formed in a wide liquid reservoir shape having a large capacity, these portions are sometimes referred to as “fine flow paths”. In many cases, the fluid flowing in the fine channel is actually a liquid, and specifically, various reagents, sample liquids, denaturant liquids, cleaning liquids, driving liquids, and the like are applicable.

Micro total analysis system The micro total analysis system integrates the components other than the microchip (equipment necessary for analysis, etc.) into the system unit body, and places the microchip on the chip transport tray as necessary. It is desirable that the system apparatus main body be configured to be attached to and detached from the system apparatus main body.

  A series of analysis steps such as pretreatment, reaction, and detection of a specimen as a measurement sample are mainly performed in a fine channel formed on a microchip. The microchip is filled with a predetermined amount of necessary reagents according to the target reaction method and detection method in advance, and it is not necessary to fill the required amount of reagent each time it is used. It is desirable that In addition, the sample and specimen to be analyzed may be stored in the chip in advance before being mounted on the system apparatus main body, or may be stored in the chip after being mounted on the apparatus main body.

  After mounting the chip on the system apparatus main body, the sample and reagents stored in each microchip storage section by a micropump etc., their merging, predetermined reaction based on mixing, and measurement of reaction products and It is desirable that the measurement data is automatically stored as a series of continuous processes.

  In the conventional analysis system, it is necessary to reconfigure a device corresponding to the changed contents each time a different analysis or the like is performed. On the other hand, the micro integrated analysis system can cope with various types of analysis by exchanging the detachable chip and the system control program.

Microchip The microchip in the present invention is equivalent to what is generally called a test chip, an analysis chip, a microreactor chip, or the like. Usually, the vertical and horizontal sizes of this chip are several tens of mm and the height is about several mm. In microchips, microchannels with channel elements (functional parts) and structures are finely functionally positioned according to applications such as chemical analysis, various inspections, sample processing / separation, and chemical synthesis. It is arranged by processing technology. Desirably, necessary reagents are accommodated in advance in the microchannel of the chip in order to perform the above analysis and the like quickly. Such a chip is connected to a micropump via a pump connection part, and a liquid such as a specimen or a reagent is sent through the microchannel by a driving liquid sent from the micropump.

  The above-mentioned chip preferably has a structure having a basic substrate composed of a groove forming substrate and a covering substrate as a structure. At least on the groove forming substrate, pump connection part, valve base part and liquid reservoir part (respective storage parts such as reagent storage part and specimen storage part, reaction part, detection part, waste liquid storage part etc.), liquid feed control part, backflow prevention A fine channel including a structure part such as a part, a reagent quantitative part, and a mixing part is formed. On the other hand, the coated substrate needs to cover at least the structure portion, the flow path, and the detection portion of the groove forming substrate, and may cover the entire surface of the groove forming substrate. Note that the fine flow path may be formed only on one side of the chip, or the fine flow paths communicating with each other may be formed on both sides.

Microchips are expected to be excellent in processability, non-water absorption, chemical resistance, heat resistance, and low cost. Chip materials are considered in consideration of chip structure, application, detection method, etc. Is required to be selected appropriately. Various known materials can be used as the material, and usually the substrate and the flow path element are formed by appropriately combining one or more materials in accordance with individual material characteristics.

  For example, it is desirable that a chip intended for a large number of measurement specimens, particularly clinical specimens at risk of contamination and infection, be of a disposable type. Therefore, a plastic resin that can be mass-produced, is light in weight, is strong against impact, and can be easily disposed of by incineration. Among them, polystyrene that is excellent in transparency, mechanical properties, and moldability and is easy to be finely processed is preferably used. Polypropylene is preferably used because it has little protein adsorption, is excellent in chemical resistance such as acid and alkali, and is inexpensive.

  When it is necessary to heat the chip to near 100 ° C. in analysis, it is preferable to use a resin having excellent heat resistance (for example, polycarbonate). Resins and glass have low thermal conductivity, and by using these materials in the locally heated region of the microchip, heat conduction in the surface direction is suppressed, and only the heated region can be selectively heated. it can.

  In addition, since the detection unit of the fine channel optically detects a fluorescent substance or a product of a color reaction, it is necessary to use a light transmissive material at least for the substrate at this part. Alkali glass, quartz glass, and transparent plastics can be used as the light transmissive material, but transparent plastics are preferred.

  The microchannel of the microchip is formed on the substrate according to a channel arrangement designed in advance according to the purpose. The flow path through which the fluid flows is, for example, a micro flow path having a width of several micrometers to several hundreds of micrometers, preferably 10 to 500 micrometers, and a depth of about 10 to 1000 micrometers, preferably 10 to 300 micrometers. If the channel width is less than 5 μm, the channel resistance increases, which is inconvenient for fluid delivery and detection. The advantage of the microscale space is diminished in the flow path exceeding 500 μm in width. In addition, the width | variety and depth of the above-mentioned structure part in a fine flow path are good also as a size different from the flow path which connects structure parts as needed, and are not limited to said size.

  A conventional microfabrication technique can be used as a method for forming the microchannel, but a photolithography technique is typically preferable. With this technique, the fine structure is transferred to the photosensitive resin, unnecessary portions are removed, and the fine flow path is formed. As the photosensitive resin used as the material of the groove-formed substrate at this time, a plastic having a good mechanical property and capable of accurately transferring a submicron structure is preferably used. Polystyrene, polydimethylsiloxane, etc. are excellent in shape transferability. If necessary, processing by injection molding, extrusion molding or the like may be used.

Construction 1 near the reagent storage section is a top view showing an embodiment of a microchip having a plurality of reagent storage section. FIGS. 2 (i) and (ii) are cross-sectional views showing the periphery of the reagent container in one embodiment of the microchip. Hereinafter, description will be made with reference to these drawings.

  The reagent storage unit 18 is a part in a fine channel for storing a reagent used for reaction and detection. The volume of the reagent storage unit 18 is appropriately adjusted according to the reagent to be used, and may be formed wider than other fine channels, but is about 3 to 10 μL, for example.

Liquid supply control units 34a and 34b are provided at the upstream end and the downstream end of the reagent storage unit 18, respectively. On the further upstream side of the reagent storage unit 18 and the liquid supply control unit 34a, there is a fine flow path including a pump connection unit 12 connected to a micropump unit for supplying driving liquid and an air vent opening 13 To do. During the analysis, the driving liquid is supplied from the pump connection section, and the liquid feed control section
The reagent passes through 34a and flows into the reagent storage unit 18, and the reagent in the reagent storage unit passes through the liquid supply control unit 34b together with the drive liquid and is pushed out to the downstream microchannel 33 and further supplied.

  On the further downstream side of the reagent storage unit 18 and the liquid feeding control unit 34a, for example, a series of micro flow including a structure unit for detecting reagents by mixing and reacting reagents or a sample and a reagent. There is a road. Such a structure portion may have a predetermined structure in order to perform a desired function. For example, a merging portion 29 as shown in FIG. 1 communicates with a plurality of reagent storage portions and has a slightly wider flow path. It has become. The fine flow path 33 described above may be a flow path itself that constitutes such a structure part, or may be a flow path that interposes the structure part and the liquid feeding control part 34b.

<Reagent supply unit>
The reagent supply unit 30 has a structure including an opening 31 for communicating with the flow path of the micropump unit, and a flow path 32 for communicating the opening 31 and the reagent storage unit 18.

  In the reagent supply unit 30, by applying a pressure higher than a predetermined pressure to the reagent using a micropump described later, the reagent passes through the opening 31 and the flow path 32 of the reagent supply unit 30 and flows into the reagent supply unit 18. In the present invention, the pressure at which the reagent passes through the reagent supply unit 30 is higher than the liquid holding pressure of the liquid feeding control unit described later, preferably 2 to 20 kPa, more preferably 4 to 10 kPa. . In the present invention, “the reagent passes through the reagent supply unit” means that the tip of the supplied reagent enters the reagent storage unit 18 beyond the boundary between the flow path 32 and the reagent storage unit 18, and thereafter It means that the inflow of the reagent can be continued only by applying a pressure smaller than the pressure applied to.

  The pressure at which the reagent passes through the reagent supply unit 30 is set to a desired value by adjusting the cross-sectional area of the opening 31 and the flow path 32 according to the material of the substrate on which the reagent supply unit 30 is formed. Can do. For example, if the reagent supply unit 30 is formed on a plastic substrate, and the opening 31 and the channel 32 have a vertical cross section of 40 μm × 25 μm and a length of about 1 mm, the reagent passes through the reagent supply unit 30. The pressure to be applied is about 16.5 kPa.

The opening 31 and the channel 32 are usually provided in the vicinity of the reagent supply unit such as the top surface of the reagent storage unit 18 as shown in FIGS. 2 (i) and (ii), but the reagent is appropriately supplied to the reagent storage unit. As long as it is possible, it can be provided in a preferable position. In general, since the coated substrate 16 is thinner than the groove forming substrate 17, the reagent supply unit 30 is preferably provided on the upper surface side of the reagent containing unit 18 so as to penetrate the coated substrate 16, but the groove forming You may provide in the lower surface side so that the board | substrate 17 may be penetrated.

  Further, the shape, size, etc. of the opening 31 and the flow path 32, in addition to the pressure at which the reagent starts passing through the reagent supply unit 30 as described above, connectivity with the micropump unit for reagent supply, Considering the form of the fine channel, etc., it can be set as a preferred embodiment. For example, as shown in FIG. 2 (ii), the opening 31 may be made wider than the vertical cross section of the flow path 32 to enhance the connectivity with the micropump unit.

  The opening 31 of the reagent supply unit 30 is connected to an opening provided in the micropump unit when the microchip and a micropump unit described later are aligned and brought into close contact with each other. In the vicinity of the opening 31, it is desirable to provide a means for ensuring a sealing property necessary to prevent leakage of the reagent. For example, it is preferable that the adhesion surface is formed of a resin having flexibility (elasticity, shape following property). Such a close contact surface may be formed by forming a flexible substrate such as polytetrafluoroethylene or silicone resin around at least the periphery of the opening of the microchip constituent substrate (coated substrate 16 and groove forming substrate 17). Alternatively, a separate member made of such a resin may be attached around the opening.

<Liquid feeding control unit>
The liquid feeding control units 34a and 34b are structural units having a function of controlling the timing of liquid feeding such as reagents. The liquid feeding control unit is formed at at least one end of the reagent storage unit, and is generally formed at both the upstream end and the downstream end of the reagent storage unit as shown in FIG.

  The liquid feeding control unit is preferably composed of a “water repellent valve” (also referred to as a “hydrophobic valve”). This water-repellent valve is composed of a “throttle channel” which is a channel having a smaller cross-sectional area than the upstream and downstream channels (reagent storage unit 18 and fine channel 33 in FIG. 2). With such a structure, when the liquid supply pressure does not reach a predetermined pressure (liquid holding pressure), fluids that reach from the upstream side, such as aqueous driving liquid and aqueous reagent, cannot pass through the water repellent valve and stop at the water repellent valve. To do.

  In order to send the fluid from the upstream side to the downstream side of the water repellent valve, a liquid feeding pressure equal to or higher than the liquid holding pressure may be applied by a micropump. As a result, the fluid is pushed out from the throttle channel to the downstream channel against the difference in surface tension with the channel wall. Once the fluid has flowed out into the flow path, the liquid flows into the downstream flow path without maintaining the liquid feeding pressure required to push the tip of the fluid to the downstream flow path.

  Considering the pressure performance generated by the micropump (piezopump) and the sufficient liquid holding capacity at the time of reagent supply or liquid feeding by the driving liquid, the liquid holding pressure of the liquid feeding control unit may be 1 to 5 kPa. preferable.

  Such a liquid holding pressure can be set to a desired value by adjusting the size of the vertical cross section of the water repellent valve according to the material of the flow path wall. As an example, in a microchip formed of a plastic material such as polystyrene or polypropylene, the vertical and horizontal lengths of the upstream and downstream channels are about 150 μm × 300 μm, and the size of the throttle channel is 25. If it is x25 μm, the liquid holding pressure of this water repellent valve is about 2 kPa.

  If the water repellency of the water repellent valve and the flow path communicating with the water repellent valve is inferior, the stopping of the fluid becomes worse and the fluid flows out little by little. For this reason, it is desirable that the wall surface of the fine channel be formed of a highly hydrophobic material such as plastic resin. When the flow path wall is made of a hydrophilic material such as glass, it is necessary to apply a water-repellent coating, for example, a fluorine-based coating, to at least the inner surface of the throttle flow path.

  The formation of such a water-repellent valve is not limited to the reagent container, but is also provided, for example, at the end of the reagent mixing part or the specimen container on the side of the merging part, and the liquid feeding to the flow path beyond that It may be used to control the start timing. By providing such a water repellent valve, it is possible to prevent the fluid from moving to the downstream side by capillary force when the pump is stopped.

Micropump unit structure In the present invention, a micropump unit is used to fill a reagent container with a reagent. This micropump unit is connected to a reagent reservoir, a micropump, and a reagent supply unit in the microchip. It is desirable to be a chip-like micropump unit in which possible openings, reservoirs for these reagents, a micropump and a fine flow path communicating with the openings are formed. When a plurality of reagent storage units provided on the microchip are targeted simultaneously, it is preferable to use a micropump unit having a plurality of sets of reagent reservoirs, micropumps, openings, and microchannels. A similar micropump unit can also be used to supply the driving liquid to the microchip during analysis.

  3 and 4 show an example of such a micropump unit. FIG. 3 is a perspective view showing an embodiment of the micropump unit, and FIG. 4 is a cross-sectional view of the embodiment of the micropump.

The micropump unit 9 includes a silicon substrate 37, a glass substrate 38 thereon,
Furthermore, it is comprised from three board | substrates with the glass-made board | substrate 39 on it. The substrates 37 and 38 are bonded together by anodic bonding or the like, and the substrates 38 and 39 are bonded together by sealing glass, thermal fusion, hydrofluoric acid bonding, or the like.

In producing such a micropump unit 9, it is preferable to use a substrate 37 that is processed by a photolithography technique and that includes a substrate 37 constituting a micropump, a channel groove, and a connection opening. The method for producing the micropump unit is not limited to this. For example, a glass substrate is laminated on a silicon substrate, a photosensitive glass substrate, etc. formed by etching the structure of a micropump, and PDMS is laminated thereon, and further, plastic, glass, silicon, ceramics, etc. The micropump unit can be configured in the same manner by bonding together the substrates on which the flow channel grooves and the connection openings are formed. Further, the micropump provided in the micropump unit may be other than a piezo pump, such as a check valve type micropump.

The present inventors have already proposed a micropump that has been conventionally used for feeding a driving liquid and that can be suitably used for filling a reagent in the present invention. Reference can be made to JP-A-2001-322099, JP-A-2004-108285, JP-A-2004-270537, and the like.

<Mechanism of micro pump>
The internal space between the substrate 37 and the substrate 38 constitutes a micro pump 40 (piezo pump). In the second flow path 47, the width and height of the first flow path 46 and the second flow path 47 are made equal, and the length of the first flow path 46 is made shorter than the length of the second flow path 47. The change rate of the channel resistance with respect to the change in the differential pressure is smaller than that of the first channel 46. That is, in the first flow path 46, when the differential pressure increases, a turbulent flow is generated so as to spiral in the flow path, and the flow path resistance increases, but the second flow path 47 has a long flow path width. Even if the differential pressure increases, laminar flow tends to occur, and the rate of increase in channel resistance is smaller than that in the first channel 46. Note that the difference in flow rate resistance change ratio with respect to the change in differential pressure between the first flow channel 46 and the second flow channel 47 is not necessarily due to the difference in the length of the flow channel, but is different in other shapes. It may be based.

  In addition, a diaphragm (thin film-like diaphragm) is formed at the position of the pressurizing chamber 45 by processing the substrate 37, and a lead zirconate titanate (PZT) ceramic as one embodiment of the actuator is formed on the outer surface thereof. A piezoelectric element 44 made of or the like is attached. The two electrodes on the surface of the piezoelectric element 44 are connected to the drive unit by wiring such as a flexible cable. By applying a voltage having a predetermined waveform to the piezoelectric element 44, it is possible to vibrate the diaphragm and change the volume of the pressurizing chamber 45. By controlling the voltage to the piezoelectric element 44 so that the variation rate of the diaphragm in the direction in which the volume of the pressurization chamber 45 increases and the variation rate of the diaphragm in the direction in which the volume of the pressurization chamber 45 decreases, The pump functions and the reagent is transported.

That is, the volume of the pressurizing chamber 45 is increased while giving a large differential pressure by quickly displacing the diaphragm from the pressurizing chamber 45 toward the outside thereof, and then the diaphragm is slowly moved toward the inside of the pressurizing chamber 45. When the volume of the pressurizing chamber 45 is decreased while giving a small differential pressure by displacing, the reagent is fed in the forward direction (the direction of the arrow) from left to right in the figure. By this reverse operation, it is also possible to send the reagent in the reverse direction from right to left in FIG.

  The two electrodes for driving the piezoelectric element 44 are connected to a flexible wiring. As an example, an ITO film, which is a transparent electrode film, is formed on the surface of the diaphragm, and one surface of the piezoelectric element is bonded to the ITO film with an adhesive on the ITO film. The ITO film and the flexible wiring are connected to each other. The other surface of the piezoelectric element is gold-plated, and the flexible wiring is directly connected to the gold-plated portion.

<Flow path through which reagent is fed>
A flow path 50 is patterned on the substrate 39. As an example, the size and shape of the cross section of the channel 50 is a rectangular shape having a width of about 150 μm and a depth of about 300 μm. As shown in the figure, by providing a flow path and an opening at an appropriate position on the substrate of the micropump unit, the reagent can be fed to a reagent supply unit formed at a desired position on the microchip. In addition, the reagent can be supplied to a plurality of locations efficiently and simply in a short time.

  On the downstream side of the flow channel 50, an opening 51a is provided which is opened from one surface of the substrate 39 to the outside and communicates with the micro flow channel of the microchip. The opening 51a may have a size larger than the width of the flow path 50 if necessary for proper alignment with the opening of the microchip. On the other hand, the upstream side of the flow path 50 communicates with the pressurizing chamber 45 and the first liquid chamber 48 through the second liquid chamber 49 through the through-hole 52 a of the substrate 38. A reagent is stored in the first liquid chamber 48, and the reagent is pushed out by the micropump 40 and fed from the opening 51a of the substrate 39 to the reagent supply part of the microchip.

  In addition, the first liquid chamber 48 is provided in a separate reagent tank (not shown) or the like via an opening 52b of the substrate 38, an opening 51b of the substrate 39, and a packing of polydimethylsiloxane (PDMA) as necessary. It is connected. Each of the reagents in the plurality of first liquid chambers 48 provided in the micropump unit is supplied from a reagent tank in which the corresponding reagent is stored.

<Control of drive voltage>
FIG. 5 shows an example of the waveform of the drive voltage applied to the piezoelectric element. Control of the generated pressure and flow rate by the micropump can be performed by adjusting the voltage applied to the piezoelectric element. In general, the generated pressure and flow rate tend to increase as the voltage increases. The maximum voltage applied to the piezoelectric element is about several volts to several tens of volts, and about 100 volts at the maximum. As an example, the time t1 is about 60 μs, the time t2 is about 20 μs, and the frequency of the drive voltage is about 11 kHz.

  The flow rate of liquid feeding can also be controlled by adjusting the size of the pump chamber with which the piezoelectric element is in contact. For example, by increasing the volume of the pump chamber, the flow rate of the liquid is further increased.

  The drive voltage applied to the piezoelectric element 44 is desirably controlled by a separate device that controls the function of the micropump unit. With this control device, it is possible to control operations such as starting the feeding of the reagent and stopping the feeding after feeding the necessary amount of the reagent.

  When simultaneously filling a plurality of reagent storage parts of a microchip with a plurality of micropumps, the reagent storage part can be adjusted by appropriately adjusting the pressure generated by the micropump and the flow rate of the reagent by controlling the driving voltage. Even when the volumes of the reagent are different, it is possible to complete the filling of the reagent almost simultaneously.

Further, the flow path 50 in the micropump unit is a mode in which a plurality of flow paths communicating with the micropump each having the same pressurizing chamber 45 volume, drive voltage, etc. are joined and communicated with one opening 51a. It may be. The flow rate of the reagent sent from a plurality of micropumps in such a flow path is increased by the quantity ratio of the number of micropumps used compared to the case of a flow path communicating with a single micropump. By adjusting the flow rate using such a method, it is possible to match the timing of completion of reagent filling into a plurality of reagent storage units.

Reagent Filling Method The reagent filling method of the present invention comprises (Step 1) a step of connecting the opening of the reagent supply unit in the microchip and the opening of the micropump unit, and (Step 2) the reagent supply unit using the micropump. A step of applying a pressure necessary to pass through the reagent to the reagent, and (step 3) a step of applying a pressure equal to or lower than the liquid holding pressure of the liquid feeding control unit to the reagent by the micropump. Hereinafter, these steps will be sequentially described.

<Step 1>
FIG. 6 is a diagram showing an aspect of superposition of the microchip and the micropump unit when the reagent is filled.

The opening 31a of the reagent supply part of the microchip and the opening 51a of the micropump unit corresponding to the opening correspond to each other by overlapping and fixing the microchip and the micropump unit in a predetermined positional relationship. Are connected to each other. Note that the microchip 2 in FIG. 6 is superposed on the micropump unit 11 upside down from the microchip 2 shown in FIG. In order to appropriately perform this superposition, for example, a method of providing a positioning guide member on both substrate surfaces to guide the movement, a method of providing a concave portion and a convex portion and fitting them together can be used. In order to prevent liquid leakage around the opening, it is preferable to install a member such as packing 101 around the opening 51a of the micropump unit, and further pressurize from both sides to ensure sealing performance.

By connecting the openings in this manner, the reagent 102 stored in the reagent tank or the like is stored.
A series of flows to be delivered to the reagent container 18 via the openings 51b and 52b of the micropump unit, the micropump 40, the flow path 50 of the micropump unit, the connection part between the microchip and the micropump unit, etc. The road communicates.

<Steps 2 and 3>
Subsequent to Step 1, the reagent starts to flow into the reagent storage unit by applying a pressure higher than the pressure passing through the reagent supply unit (hereinafter referred to as “pressure α”) to the reagent by the micropump. The pressure applied to the reagent in this step 2 is hereinafter referred to as “pressure A”. As in the principle of the water repellent valve, once the reagent starts to pass through the reagent supply unit, the injection can be performed simply by applying a pressure lower than the pressure α (hereinafter referred to as “pressure α ′”). Continue.

  When the inflow starts, the pressure below the liquid holding pressure (hereinafter referred to as “pressure β”) of the liquid feeding control unit provided in the reagent storage unit is maintained until a necessary amount of reagent is filled in the reagent storage unit. Continue to call. The pressure applied to the reagent in this step 3 is hereinafter referred to as “pressure B”. In the case where a plurality of liquid feeding control units having different liquid holding forces are provided in the reagent storage unit, the lowest liquid holding pressure among them is the pressure β.

  If the pressure B is equal to or lower than the pressure β, the filling is completed without the reagent leaking from the liquid feeding control unit when the reagent storage unit is filled with the reagent. However, when the pressure B is higher than the pressure β, the injected reagent passes from the reagent storage section through the liquid supply control section and leaks into the fine flow path, so that the reagent is normally supplied during analysis. It is very inappropriate because it disappears.

  These pressures α, α ′, β, A, and B can vary depending on the mode of the micropump or microchip, etc., but in the reagent filling method of the present invention, the pressure A is equal to or higher than the pressure α, As long as the condition that the pressure B is equal to or higher than the pressure α ′ and equal to or lower than the pressure β is satisfied, the magnitudes of the pressures A and B can be appropriately adjusted.

<Method for controlling the pressure applied to the reagent>
In the filling method of the present invention, the pressure P applied to the reagent at the opening of the micropump unit connected to the opening of the reagent filling portion of the microchip can be adjusted by controlling the driving voltage described above. That is, based on the following principle, the micropump is set so that the pressure P at the time of the step 2 becomes the pressure A and the pressure P at the time of the step 3 becomes the pressure B. The generated pressure P ₀ may be adjusted appropriately.

The pressure P is obtained by subtracting the pressure loss ΔP caused by the fluid flowing through the flow path from the pressure P ₀ generated by the micropump (that is, P = P ₀ −ΔP). This ΔP is
It is obtained by multiplying the internal flow resistance R [N · s / m ⁵ ] of the micropump unit by the flow rate Q which is the volume of the fluid flowing through the flow path per unit time (ie ΔP = R × Q), and R is If laminar flow is dominant in the flow path, it can be calculated by the following formula:
R = ∫ [32 × η / (S × φ ² )] dL
Here, η is the viscosity, S is the channel cross-sectional area, φ is the equivalent diameter (φ = (a × b) / [(a + b) / 2] when the channel cross-section is a rectangle having a width a and a height b) L is the flow path length.

Whether the liquid flow is laminar or not can be estimated from the Reynolds number (= density × speed × representative dimension ÷ viscosity). In general, if the Reynolds number is 2000 or less, the flow is laminar.
Therefore, the pressure P can be set to a desired value by appropriately adjusting each element affecting the pressure P as described above. For example, if the drive voltage is increased to increase the displacement amount of the piezoelectric element and the generated pressure P ₀ is controlled to increase, the pressure P to the reagent increases, and conversely, the generated pressure P ₀ decreases. The pressure P on the reagent decreases.

  The pressure P is derived from the above equation, but can be obtained in advance by experiments or the like. As a method, for example, after filling the flow path of the micro pump unit with a reagent, the micro pump is driven by applying it to the piezoelectric element, and at the same time, compressed air or the like is supplied from the outside of the opening of the micro pump unit. And the pressure of the compressed air when the liquid delivery is stopped despite the driving of the micropump. By performing this measurement for various driving voltages, the characteristics of the driving voltage and the pressure P can be obtained.

<Optional process>
After filling the microchip with the reagent by the above method, it is desirable to immediately seal the opening of the reagent filling part with a sealing member or the like in order to prevent evaporation, leakage, contamination, denaturation or mixing of bubbles of the reagent. .

Furthermore, in order to prevent such a problem more reliably, it is desirable to encapsulate a sealant prior to filling with a reagent.
The above-mentioned sealant is solidified or gelled under the refrigerated condition stored before using the microchip, and prevents the reagent from flowing out. However, it melts at room temperature, becomes a fluid state, and is easily discharged at the time of liquid feeding. It is something that can be done. Examples of such a sealant include fats and oils having a solubility in water of 1% or less and a melting point of 8 ° C. to room temperature (about 25 ° C.). Such a sealant may be filled between the reagent container and the fine channel communicating with the reagent container as long as the upstream and downstream sides of the reagent container are sealed. You may fill the storage part provided for use.

<Reagents to be filled>
The reagent filling method of the present invention is a liquid reagent that can be delivered by a micropump and does not have excessive viscosity in the temperature environment in which the filling step is performed, typically a solution or dispersion using an aqueous solvent. Such aqueous reagents are targeted. In controlling the liquid feeding by the liquid feeding control unit (water repellent valve) or the like in the present invention, the reagent is required to be aqueous.

  Reagents necessary for analyzing a biological substance in a specimen are basically the same as those in the past. For example, various reagents for genetic testing include those used in gene amplification reactions (DNA polymerase, reaction stop solution, denaturing solution, etc.) and those used in detection reactions (hybridization buffer, probe DNA, gold Colloid, probe DNA for internal control, probes such as positive control and negative control, coloring reagent, etc.), and washing solution. In addition, when analyzing an antigen present in a specimen, a reagent containing a labeled antibody (preferably a monoclonal antibody) is used. Furthermore, if necessary, a pretreatment reagent (for example, a lysis agent such as a 1% SDS mixed solution) used for the pretreatment of the specimen is also included.

Prevention of Driving Liquid Leakage FIG. 7 is a diagram showing a state of liquid feeding by the driving liquid around the reagent container of the microchip. At the time of analysis, a micro pump unit for supplying a driving liquid (separately from the one used for filling the reagent as described above) is connected to the above pump connecting portion provided on the microchip 2. As a result, the driving liquid 103 applied with the liquid feeding pressure P ′ by the micropump is fed into the fine flow path. The driving liquid 103 passes through the liquid feeding control unit 34a and flows into the reagent storage unit 18,
The reagent 102 stored in the reagent storage unit is pushed out from the liquid feeding control unit 34b.

  In the microchip of the present invention, the pressure passing through the reagent supply unit (the pressure α) is about 2 to 10 times the liquid holding pressure (the pressure β) of the liquid feeding control unit. In addition, even if a liquid supply pressure P ′ larger than β is applied to the drive liquid and liquid is supplied into the reagent storage unit, almost all of the drive liquid 103 can be supplied in the direction passing through the liquid supply control unit 34b. There is little that can pass through the reagent supply unit 32 and overflow from the opening 31. As described above, when the opening 31 is sealed with a seal member or the like in order to prevent deterioration of the filled reagent, there is a risk that the driving liquid 103 leaks from the opening 31 due to the liquid supply pressure P ′. The sex will be further lowered.

FIG. 1 is a top view showing an embodiment of a microchip provided with a plurality of reagent storage units. 2 (i) and (ii) are cross-sectional views showing one embodiment of the vicinity of the reagent storage unit. FIG. 3 is a perspective view showing an embodiment of the micropump unit. FIG. 4 is a cross-sectional view of an embodiment of a micro pump (piezo pump). FIG. 5 shows an example of the waveform of the drive voltage applied to the piezoelectric element. FIG. 6 is a diagram showing an aspect of superposition of the microchip and the micropump unit when the reagent is filled. FIG. 7 is a diagram showing a state of liquid feeding by the driving liquid around the reagent container of the microchip.

Explanation of symbols

2 Microchip 11 Micro pump unit 12 Pump connection (opening)
Reference Signs List 13 Air venting opening 15 Fine channel 16 Covered substrate 17 Groove forming substrate 18 Reagent storage unit 29 Junction unit 30 Reagent supply unit 31 Opening 32 Channel 33 Mixing reaction channel 34a, 34b Liquid feed control unit (water repellent valve)
37 Substrate 38 Substrate 39 Substrate 44 Piezoelectric element 45 Pressurizing chamber 46 First channel 47 Second channel 48 First fluid chamber 49 Second fluid chamber 50 Channels 51a, 51b Opening 52a, 52b Opening 101 Packing 102 Reagent 103 Driving liquid

Claims (6)

A reagent container for containing an aqueous reagent,
A series of fine flow paths including a part for detecting the reaction product by mixing and reacting the aqueous reagent sent from the reagent container with a sample or other aqueous reagent,
A microchip having a liquid supply control unit provided between an outlet of the reagent storage unit and an inlet of the microchannel, and a reagent supply unit including an opening and a channel communicating the opening and the reagent storage unit There,
The microchip according to claim 1, wherein a pressure at which the reagent passes through the reagent supply unit is higher than a liquid holding pressure, which is a pressure at which the reagent starts to pass through the liquid feeding control unit.   The microchip according to claim 1, wherein a pressure at which the reagent passes through the reagent supply unit is 2 to 20 kPa, and a liquid holding pressure of the liquid feeding control unit is 1 to 5 kPa. In the reagent container of the microchip according to claim 1 or 2,
A reagent filling method comprising filling a reagent through a micropump unit having a reagent reservoir, a micropump, and an opening connectable to the reagent supply part of the microchip according to claim 1 or 2. (1) connecting the opening of the reagent supply unit in the microchip and the opening in the micropump unit;
(2) A step of applying a pressure higher than the pressure at which the reagent passes through the reagent supply unit to the reagent by the micropump, and (3) After the reagent starts to flow in the step (2), the micropump Applying a pressure below the liquid holding pressure of the liquid feeding control unit to the reagent,
The reagent filling method according to claim 3, comprising:   The micropump unit is provided with a plurality of sets of reagent reservoirs, micropumps, openings, and microchannels. The micropump unit is used to send a reagent to a plurality of reagent storage units provided in the microchip. 5. The reagent filling method according to claim 3, wherein the reagent is filled.   The micropump includes a first channel in which channel resistance changes in accordance with a differential pressure, a second channel in which a change rate of channel resistance with respect to a change in differential pressure is smaller than the first channel, and a first flow A piezo pump comprising a pressurizing chamber located between and in communication with the passage and the second flow passage; an actuator for changing the internal pressure of the pressurizing chamber; and a drive device for driving the actuator. The reagent filling method according to any one of claims 3 to 5, wherein the method is filled.

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