JP2005121672A - Dispensing device and method for manufacturing dna chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To make smooth a process from the supply of a solution to its supply onto a substrate by making rapid, efficient, and certain, the supply of the solution to each micro-pipet. <P>SOLUTION: In this dispensing device, a specimen injection port 52 for injecting a specimen solution thereinto from the exterior, a cavity 56 filled with the specimen solution injected thereinto, and a specimen ejection port 54 for ejecting the specimen solution therefrom, are formed in each of at least one substrate 50; an actuator part 58 is provided on at least one wall surface of the substrate 50 forming the cavity 56; a plurality of micro-pipets 34 are arranged which are structured so that the specimen solution moves in the cavity 56; and the specimen solution is ejected from the ejection port 54 of each micro-pipet 34. The injection port 52 of each micro-pipet 34 is provided with an upward projecting pin 100. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention is used for manufacturing a DNA chip (DNA microarray) in which several thousand to 10,000 or more different types of DNA fragments are aligned and fixed as microspots at high density on a substrate such as a microscope slide glass. The present invention relates to a pouring device and a method for producing a DNA chip using the dispensing device.

  In recent years, there has been a remarkable progress in gene structure analysis methods, and a large number of gene structures including human genes have been revealed. For analysis of such a gene structure, a DNA chip (DNA microarray) in which thousands to 10,000 or more different kinds of DNA fragments are aligned and fixed as spots on a substrate such as a microscope slide glass has come to be used. It is coming.

  As a method for forming spots in the manufacture of this DNA chip, there is a method of supplying (driving) a sample solution containing a DNA fragment onto a substrate by a so-called pin, such as a QUILL method, a pin & ring method, or a spring pin method. Regardless of which method is used, it is important to keep the distance between each spot constant by keeping the variation in the capacity and shape of each spot low.

  On the other hand, there is great expectation for the development of a new method with good spot shape controllability and excellent productivity for higher density.

  Here, the QUILL method is a method in which a sample is stored in a recess formed in a pin tip and the sample in the recess is moved onto the substrate by bringing the pin tip into contact with the substrate to form a micro spot. However, there is a problem of durability such as deformation or damage due to contact with the substrate, and a problem that cross contamination is likely to occur due to incomplete cleaning of the sample stored in the recess.

  In the pin and ring method, after the sample solution in the microplate is reserved by the ring, the sample in the ring is caught by the pin tip so that the solution penetrates the inside of the reserved ring, and a spot is formed on the substrate. However, since the number of samples that can be reserved at one time depends on the number of rings, and the number of samples is conventionally several, in order to form microspots of samples of thousands to tens of thousands of samples. In this case, cleaning and drying steps of several hundred to several thousand times are also required, and therefore, productivity is not necessarily high.

  In addition, the spring pin method is a method in which the sample attached to the pin tip is moved onto the substrate by pressing the pin tip against the substrate to form a minute spot. The pin and substrate have a double pin structure with a built-in spring. However, it is basically inferior in productivity because it can only be spotted once per reserve.

  Furthermore, these conventional methods for forming microspots all carry the sample solution on the substrate while being exposed to the atmosphere. This causes problems such as drying of the sample during transportation and the inability to spot, resulting in very expensive samples. There is a problem that the use efficiency of the solution is poor.

  On the other hand, a method of spotting using a so-called ink jet method that has been put to practical use in printers is also being studied, but forming several thousand to tens of thousands of samples in individual flow paths is a problem in terms of size and cost. In addition, in the inkjet method, it is necessary to fill the sample in advance so that there are no bubbles in the pump before spotting. Therefore, a large amount of purge sample is required, and the use efficiency of the sample is extremely inferior. It was. In general, it is preferable for the liquid to move at high speed in the flow path including the pump chamber, so that the sample is stirred in the flow path. For example, a delicate DNA solution is used as the sample. DNA was sometimes damaged.

Japanese Patent Laid-Open No. 6-40030

  The present invention has been made in consideration of such problems, and is configured by arranging a large number of micropipettes that enable the formation of microspots with high accuracy and at high speed, and supplying a solution to each micropipette. It is an object of the present invention to provide a dispensing apparatus that can quickly and reliably perform the steps from the solution supply to the substrate supply.

  Another object of the present invention is to produce a DNA chip that can smoothly perform the steps from the supply of the solution to the supply onto the substrate, and can improve the quality and yield of the DNA chip. It is to provide a method.

  In the present invention, an injection port for injecting a sample solution from the outside, a cavity into which the sample solution is injected and filled, and a discharge port for discharging the sample solution are formed in at least one substrate. A plurality of micropipettes each including a piezoelectric / electrostrictive element provided on at least one wall surface of the substrate forming the cavity and configured to move the sample solution in the cavity, and each micropipette In the dispensing apparatus in which the sample solution is discharged from the discharge port, a pin protruding upward is provided at the injection port of each micropipette.

  In the present invention, an injection port for injecting a sample solution from the outside, a cavity into which the sample solution is injected and filled, and a discharge port for discharging the sample solution are formed in at least one substrate. A dispensing device comprising a piezoelectric / electrostrictive element on at least one wall surface of the substrate forming the cavity, and a plurality of micropipettes configured to move the sample solution in the cavity In the method of manufacturing a DNA chip in which the sample solution is discharged onto the substrate from the discharge port of each micropipette to manufacture a DNA chip, the dispensing device projects upward into the injection port of each micropipette It is characterized by using a pin provided.

  As a result, a hole is formed in the solution reservoir portion of the cartridge positioned above the injection port by the pin, and the solution stored in the solution reservoir portion can be introduced into the injection port.

  That is, a cartridge in which a large number of solution reservoirs are arranged is positioned above the dispensing device, and the cartridge is moved to the dispensing device side. At this time, a hole is made in each solution reservoir by the pin, so that the solution stored in the solution reservoir is introduced into the inlet through the pin. In this way, when the sample solution is injected from the solution reservoir of the cartridge into the dispensing device, it is not necessary to go through a special device, so that the sample solution remains in the special device and the usage efficiency of the sample solution is increased. Will not drop.

  In this case, when the solution stored in the solution reservoir is introduced into the inlet, gas may be pumped from above each solution reservoir. Thereby, the injection time can be shortened.

  Further, in the present invention, a hole is formed in the film material coated so as to close the solution reservoir portion of the cartridge positioned above the inlet, and the solution stored in the solution reservoir portion is added to the solution reservoir portion. It can be introduced at the entrance.

  That is, a film material is coated on a cartridge in which a large number of solution reservoirs are arranged so as to close the solution reservoir, and the cartridge faces the dispensing device above the dispensing device. The cartridge is moved to the dispensing device side. At this time, a hole is made in the portion of the film material corresponding to each solution reservoir portion by the pin, so that the solution stored in the solution reservoir portion passes through the pin and passes through the injection port. Will be introduced.

  Opening a hole in the film material can be carried out relatively easily by opening a hole in the solution reservoir of the cartridge, thereby simplifying the introduction of the sample solution.

  Thus, in the dispensing apparatus according to the present invention, the solution can be supplied to each micropipette quickly and reliably, and the process from the solution supply to the supply onto the substrate can be smoothly performed. Can be done.

  In addition, the said pin refers to the protrusion-shaped part which has the part protruded from the plane, and it is preferable that the front-end | tip is sharp. The arrangement position of each injection port of the micropipette constituting the dispensing device is equal to the arrangement position of the solution reservoir portion of the cartridge, or an integer multiple of the arrangement pitch of the solution reservoir portion, or an arrangement pitch of 1 / integer It is preferable that

  And the said pin may be provided in the position included in the said inlet on a plane, and may be provided in the periphery of the said inlet. By providing the pin at a position included in the injection port on a plane, the hole into which the sample solution is introduced can be positioned directly above the injection port, and the sample solution can be more reliably introduced. . In particular, if the base of the pin is positioned in the injection port, the sample solution can be reliably guided into the injection port. Further, by providing the pins around the injection port, the pins can be easily formed, and the manufacturing cost of the dispensing device can be reduced.

  Further, according to the present invention, a holding portion for holding a pipette for injecting a solution from the injection port or a tube for receiving the pipette is provided at the periphery of the injection port of each micropipette constituting the dispensing device. May be.

  Thereby, when injecting the solution into each micropipette of the dispensing device using the pipette, the pipette or the tube for receiving the pipette is held by the holding portion, so that the solution is surely contained in the micropipette. The solution can be effectively prevented from leaking.

  In particular, by hydrophilizing at least the inner wall of the pipe for receiving the pipette, the solution discharged from the pipette can be reliably guided to the inlet of the micropipette without introducing bubbles or the like.

  Further, in the present invention, a graduation for measuring the amount of liquid injected into the pipe is formed in a part of the pipe for receiving the pipette, or a part of the inner wall of the pipe for receiving the pipette, The portion provided with the protrusion and the portion not provided may be formed at the same distance from the injection port.

  By forming the scale, it is possible to measure and confirm the amount of the injected sample solution and the discharged sample solution on the spot, which is useful for quality control of product manufacturing management, and at the time of injecting and filling the sample solution into the micropipette. This is effective for managing the volume of the replacement liquid and intermediate liquid when using the method of filling the replacement liquid and intermediate liquid in advance, and as a result, the replacement of the replacement liquid and the intermediate liquid into the sample solution is ensured. The concentration variation of the supplied sample solution can be reduced, and the quality of the product is improved.

  In addition, a part of the inner wall of the pipe for receiving the pipette is formed with a part provided with a protrusion and a part not provided at the same distance from the injection port, whereby the tip of the pipette for introducing the sample solution into the protrusion is provided. It is possible to perform the introduction work so as to be brought into contact with each other, the pipette injection position can be always constant, and variations in the introduction work are reduced.

  Further, since there are a portion provided with a protrusion and a portion not provided, a passage for gas during injection is ensured, and the introduction operation can be performed without entraining bubbles or the like. Such an effect is not exhibited only when introducing (injecting) a sample solution, a replacement solution, or the like, but pipetting to remove an excessive amount of the sample solution, the replacement solution, or the intermediate solution. It is also effective when performing.

  In the present invention, an opening having an opening area equal to or smaller than the opening area of the discharge port is provided between the tube for receiving the pipette and the injection port for the purpose of removing foreign substances in the sample solution to be injected. It is preferable that a large number of formed filters are attached. By doing so, it is possible to prevent the foreign matter from entering the micropipette and clogging the discharge port or the like, thereby making it impossible to supply the sample solution.

  Further, the present invention may have a pitch variable mechanism for changing the arrangement pitch of each micropipette constituting the dispensing device.

  Thereby, when supplying the solution to the dispensing device, the arrangement pitch of the micropipettes in the dispensing device is set to the pipette of the solution supply means for supplying the solution to the dispensing device or the inlet of each pipette. When the sample solution is supplied from the dispensing device onto the substrate, the arrangement pitch of each micropipette in the dispensing device is different from the arrangement pitch of each pipette in the solution supply means. The pitch can be set and the process from the supply of the solution to the supply onto the substrate can be performed smoothly.

  That is, in general, the supply (injection or introduction) of the sample solution to the micropipette and the dispensing apparatus is often limited in terms of the dimensions of the solution supply means or the cartridge having the solution reservoir. Alternatively, the arrangement pitch of the inlets must be relatively large. On the other hand, when supplying the sample solution onto the substrate, it is better to reduce the supply pitch in terms of spot density and the number of spots that can be supplied at one time. In many cases, the dispensing device according to the present invention is preferably employed.

  Further, the present invention uses a solution supply means having a pitch variable mechanism for arranging a large number of pipettes for supplying a solution to the dispensing device and making the arrangement pitch of each pipette variable. When the solution is supplied to the pipette, the arrangement pitch of each pipette is adjusted to the arrangement pitch of the solution reservoir portion of the cartridge in which a large number of solution reservoir portions are arranged, and the solution is supplied from the solution supply means to the dispensing device. In this case, the arrangement pitch of each pipette may be adjusted in accordance with the arrangement pitch of the micropipette in the dispensing device.

  In this case, the process of supplying the solution stored in each solution reservoir of the cartridge to the dispensing device can be performed smoothly, and the manufacturing time can be shortened effectively.

  Further, in the present invention, when the dispensing device is used, a cartridge in which a large number of solution reservoirs are arranged is positioned above the dispensing device without providing a pin in the dispensing device, and each solution A hole may be formed in the reservoir portion with a pin from the outside, and the solution stored in the solution reservoir portion may be introduced into the injection port. In this case, each micropipette of the dispensing device can have a simple configuration.

  Since the sample solution supplied through the injection port can be smoothly guided to the cavity side by hydrophilic treatment of the injection port in the above-described dispensing apparatus, the supply time of the sample solution can be shortened.

  As described above, according to the dispensing apparatus and the method for producing a DNA chip according to the present invention, the solution can be supplied to each micropipette quickly, efficiently, and reliably. The process from supply to supply onto the substrate can be performed smoothly.

  Embodiments of a dispensing apparatus and a DNA chip manufacturing method according to the present invention will be described below with reference to FIGS.

  First, the dispensing apparatus 30A according to the first embodiment is configured by arranging a plurality of micropipettes 34 in a matrix on the upper surface of a rectangular fixed plate 32, as shown in FIG. In the example of FIG. 1, ten micropipettes 34 are arranged in 5 rows and 2 columns.

  As shown in FIGS. 2 and 3, the micropipette 34 includes a sample injection port 52 formed on the upper surface of the substrate 50 having a substantially rectangular parallelepiped shape, a sample discharge port 54 formed on the lower surface of the substrate 50, A cavity 56 formed between the sample injection port 52 and the sample discharge port 54 and an actuator unit that vibrates the base body 50 (more precisely, a vibration unit 66 described later) or changes the volume of the cavity 56. 58.

  Therefore, as shown in FIG. 3, the fixing plate 32 is provided with through holes 40 at locations corresponding to the sample discharge ports 54 of the micropipette 34. As a result, the sample solution discharged from the sample discharge port 54 of the micropipette 34 is supplied (including dripping) to the substrate 10 fixed below, for example, the fixed plate 32 through the through hole 40.

  The micropipette 34 has a substantially L-shaped introduction hole 60 having a large opening width from the sample injection port 52 to the inside of the substrate 50. A first communication hole 62 having a small diameter is formed between the introduction hole 60 and the cavity 56, and the sample solution injected from the sample injection port 52 passes through the introduction hole 60 and the first communication hole 62 to form the cavity 56. To be introduced.

  A second communication hole 64 communicating with the sample discharge port 54 and having a diameter larger than that of the first communication hole 62 is formed at a position different from the first communication hole 62 in the cavity 56. . In the first embodiment, the first communication hole 62 is formed near the sample injection port 52 in the lower surface of the cavity 56, and the first communication hole 62 is similarly formed on the lower surface of the cavity 56 at a position corresponding to the sample discharge port 54. Two communication holes 64 are formed.

  Furthermore, in the first embodiment, the portion of the base body 50 that is in contact with the upper surface of the cavity 56 is thin, and is structured to be susceptible to vibration against external stress, so that it functions as the vibrating portion 66. It has become. The actuator portion 58 is formed on the upper surface of the vibration portion 66.

  The substrate 50 is formed by laminating a plurality of zirconia ceramic green sheets (first thin plate layer 50A, first spacer layer 50B, second thin plate layer 50C, second spacer layer 50D, and third thin plate layer 50E). And it is configured to be integrally fired.

  That is, the base 50 is formed with a window portion that constitutes the sample injection port 52, and a thin first thin plate layer 50 </ b> A that constitutes the vibrating portion 66, a part of the introduction hole 60, and the cavity 56. A plurality of window portions constituting the thick first spacer layer 50B formed with a plurality of window portions, a part of the introduction hole 60, a part of the first communication hole 62 and a part of the second communication hole 64, respectively. And a second thin plate layer 50C formed with a plurality of window portions constituting a part of the introduction hole 60 and a part of the second communication hole 64, respectively. The layer 50D and the thin third thin plate layer 50E in which the window constituting the sample discharge port 54 is formed are laminated and integrally fired.

  In addition to the vibration part 66, the actuator part 58 includes a lower electrode 70 formed directly on the vibration part 66, and a piezoelectric / electrostrictive element or antiferroelectric material formed on the lower electrode 70. The layer 72 includes an upper electrode 74 formed on the upper surface of the piezoelectric layer 72.

  As shown in FIG. 2, the lower electrode 70 and the upper electrode 74 are electrically connected to a drive circuit (not shown) through a plurality of pads 76 and 78 formed on the upper surface of the substrate 50.

  According to the micropipette 34 having the above-described configuration, when an electric field is generated between the upper electrode 74 and the lower electrode 70, the piezoelectric layer 72 is deformed, and accordingly, the vibrating portion 66 is deformed, and the vibrating portion 66 is deformed. The volume of the contacting cavity (pressurizing chamber) 56 is reduced or increased.

  As the volume of the cavity 56 is reduced, the sample solution filled in the cavity 56 is discharged at a predetermined speed from the sample discharge port 54 communicating with the cavity 56, and the sample solution discharged from the micropipette 34 as shown in FIG. The DNA chip 20 can be fabricated in which the microspots 80 are aligned and fixed on the substrate 10 such as a microscope slide glass. In addition, as the volume of the cavity 56 is increased, a new sample solution is injected into the cavity 56 from the communication hole 62, and is prepared for the next discharge.

  In this case, since the arrangement pitch of the sample discharge ports 54 in the dispensing apparatus 30A is larger than the arrangement pitch of the micro spots 80 formed on the substrate 10, the sample solution is supplied while shifting the supply position in the dispensing apparatus 30A. It will be.

  In addition, as a structure in which the volume of the cavity 56 is reduced by driving the actuator portion 58, a so-called ink jet type apparatus structure can be adopted (see Japanese Patent Laid-Open No. 6-40030).

  The cavity (pressurizing chamber) 56 is formed with a flow path dimension so that the sample solution containing the DNA fragment and the like moves with less disturbance.

  In other words, the size of the cavity 56 varies depending on the type of sample, the size of the droplet to be created, and the formation density. For example, a DNA fragment of about 1 to 10,000 base pairs at a concentration of 100 μg / μl or less × 1 TE buffer solution When a sample dissolved in (buffer solution) and mixed with an aqueous solution containing an equal amount of polymer is dropped at a pitch of 30 to 500 μmφ at a pitch of 50 to 600 μm, as shown in FIG. The length (L) is preferably 1 to 5 mm, the cavity width (W) is preferably 0.1 to 1 mm, and the cavity depth (D) is preferably 0.1 to 0.5 mm. Moreover, it is preferable that the inner wall of the cavity 56 is smooth so that there is no protrusion that disturbs the flow, and the material is more preferably made of ceramics having good affinity with the sample solution.

  With this shape, the cavity 56 is used as a part of the flow path from the sample injection port 52 to the sample discharge port 54, and enters the cavity 56 from the sample injection port 52 through the introduction hole 60 and the first communication hole 62. It is possible to guide the sample solution to the sample discharge port 54 without disturbing the flow of the sample solution.

  As described above, the substrate 50 is an integrally laminated and fired body of zirconia ceramics, or may be an adhesive body of a zirconia ceramic sintered body on which the actuator portion 58 is formed and a metal, a resin film, or the like. . In particular, the thin plate layer 50E in which the sample discharge port 54 is formed is a sheet obtained by processing an organic resin such as a PET film with an excimer laser or the like, or a metal such as a stainless steel film, in consideration of matching with the processing method. It is preferable that the sheet is punched in

  Further, the dimensions of the sample discharge port 54 and the first communication hole 62 are optimally designed according to the physical properties, discharge amount, discharge speed, etc. of the sample solution to be discharged, but it is preferable to be about 10 to 100 μmφ.

  In FIG. 5, two first communication holes 62 communicate with one sample injection port 52 and the introduction hole 60 connected thereto, and each of the first communication holes 62 includes a cavity 56 and a second communication hole. Two channels 65 each having a hole 64 and a sample discharge port 54 formed continuously are formed independently. Actuators 58 (not shown) for wiring and driving independently are formed on the upper surface of each cavity 56. According to the micropipette 34 having such a configuration, the same sample solution can be supplied onto the substrate 10 simultaneously or at different timings.

  By the way, as shown in FIG. 1, a plurality of pins 38 for positioning and fixing the micropipette 34 are provided on the upper surface of the fixing plate 32. When fixing the micropipette 34 on the fixing plate 32, the micropipette 34 is inserted into the positioning holes 90 (see FIG. 2) provided on both sides of the base 50 of the micropipette 34 while inserting the pins 38 of the fixing plate 32. Is placed on the fixed plate 32, the plurality of micropipettes 34 are automatically positioned in a predetermined arrangement.

  And in this 1st Embodiment, as shown in FIG.2 and FIG.3, the pin 100 which protrudes upwards from the sample injection port 52 is provided, and is comprised. In the example of FIGS. 2 and 3, among the layers 50 </ b> A to 50 </ b> E constituting the base body 50, the four layers 50 </ b> A to 50 </ b> D excluding the lowermost third thin plate layer 50 </ b> E are directed to, for example, the central portion of the sample inlet 52. The overhanging portions 50Aa, 50Ba, 50Ca, 50Da are integrally provided, and the pin 100 is fixed to the upper surface of the overhanging portion 50Aa of the upper layer (first thin plate layer 50A) with an adhesive, for example.

  As other configurations, for example, as shown in FIGS. 7 and 8, a configuration in which the pin 100 is bonded to the bottom of the introduction hole 60 communicating with the sample injection port 52 (first modification), or as shown in FIG. In addition, there is a configuration in which the peripheral portion 52a of the sample injection port 52 is chamfered and the pin 100 is bonded to a part of the peripheral portion 52a (second modified example). The pin 100 may be formed by, for example, integral firing of zirconia ceramics in addition to bonding with an adhesive.

  The above-described dispensing device 30A is configured by standing a plurality of micropipettes 34 each having a sample injection port 52 and a sample discharge port 54 with the sample discharge port 54 facing downward.

  That is, each micropipette 34 has the respective sample injection ports 52 on the upper side, the sample discharge ports 54 on the lower side, and the sample discharge ports 54 arranged vertically and horizontally. Different sample solutions are discharged.

  In the dispensing apparatus 30A having such a configuration, as a method of supplying different types of sample solutions corresponding to the respective sample injection ports 52, for example, as shown in FIG. There is a method of using a cartridge 112 in which concave portions (reservoir portions) 110 are arranged.

  Specifically, several methods for injecting a sample solution into each micropipette 34 of the dispensing device 30A using the cartridge 112 will be described with reference to FIGS. 1, 3, 8, 9, and 10 to 12. explain.

  In the first method, first, different types of sample solutions are put in the respective reservoirs 110 of the cartridge 112. Thereafter, as shown in FIG. 1, the cartridge 112 is positioned above the dispensing device 30A with the tip (top) of the reservoir 110 facing downward.

  Thereafter, the cartridge 112 is moved to the dispensing device 30A side. As shown in FIGS. 3, 8, and 9, when the distance between the cartridge 112 and the dispensing device 30 </ b> A reaches a predetermined distance, the top of the reservoir 110 is provided with the pin 100 provided on each micropipette 34. When the cartridge 112 further contacts and moves downward, the pin 100 pierces the top of the reservoir 110, and as a result, a hole is formed in each reservoir 110.

  When the hole is opened in the reservoir 110, the sample solution leaks from the gap between the hole and the pin 100 by moving the cartridge 112 slightly upward. The leaked sample solution is introduced into the sample injection port 52 through the pin 100 and guided to the cavity 56 through the introduction hole 60 and the first communication hole 62. In particular, in the example of FIG. 8, since the base portion of the pin 100 exists in the introduction hole 60, the sample solution in each reservoir 110 of the cartridge 112 can be guided to the introduction hole 60 without leakage.

  In the first method, it is preferable that gas is pumped toward the cartridge 112 from above the cartridge 112 at least after the hole is formed at the top of the reservoir 110. Thereby, the injection time can be shortened.

  Next, in the second method, first, different types of sample solutions are put in the respective reservoirs 110 of the cartridge 112. Thereafter, as shown in FIG. 10, a thin film material 130 is attached so as to close each reservoir 110 of the cartridge 112. Thereafter, as shown in FIG. 11, the cartridge 112 is positioned above the dispensing device 30A with the tip (top) of the reservoir 110 facing upward. That is, the film material 130 and the dispensing device 30A are opposed to each other.

  Thereafter, the cartridge 112 is moved to the dispensing device 30A side. As shown in FIG. 12, when the distance between the cartridge 112 and the dispensing device 30A reaches a predetermined distance, the film material 130 comes into contact with the pins 100 provided in each micropipette 34, and the cartridge 112 further moves downward. As a result, the pins 100 are pierced into the film material 130, and as a result, holes are formed in portions of the film material 130 corresponding to the reservoirs 110.

  When the hole is formed in the film material 130, the sample solution leaks from the gap between the hole and the pin 100 by moving the cartridge 112 slightly upward. The leaked sample solution is introduced into the sample injection port 52 through the pin 100 and guided to the cavity 56 through the introduction hole 60 and the first communication hole 62.

  In the second method, it is preferable that the cartridge 112 is heated at the stage where a hole is formed at least at the top of the reservoir 110. As a result, the sample solution and gas in each reservoir 110 expand, so that the sample solution is rapidly introduced into the sample injection port 52 from the hole opened in the film material 130. As a result, the sample solution is injected. Time can be shortened.

  Thus, in the dispensing apparatus 30A according to the first embodiment and the first and second methods described above, the supply of the sample solution to each micropipette 34 can be performed quickly and efficiently, and Thus, the steps from the supply of the sample solution to the supply onto the substrate 10 can be performed smoothly, and the quality of the DNA chip 20 and the yield can be improved.

  In the first and second methods described above, as shown in FIGS. 3 and 12, a dispensing apparatus having a micropipette 34 in which a pin 100 is fixed on an overhang 50Aa provided in the sample injection port 52. Although the example applied to 30A was shown, in addition, as shown in FIG. 8, the dispensing device 30A having the micropipette 34 provided with the pin 100 at the bottom of the introduction hole 60 and the peripheral edge 52a of the sample injection port 52 are pinned. The same can be applied to the dispensing apparatus 30A having the micropipette 34 provided with 100.

  In the first and second methods, a mechanism for cleaning the space from the sample inlet 52 to the sample outlet 54 formed in the base 50 of each micropipette 34 may be provided. In this case, it is preferable that thousands of tens of thousands of DNA fragments are discharged as fine spots 80 without contamination and with high purity.

  Moreover, the base | substrate 50 which comprises the micropipette 34 is formed with ceramics as mentioned above, for example, stabilized zirconia, partially stabilized zirconia, alumina, magnesia, silicon nitride etc. can be used.

  Of these, stabilized / partially stabilized zirconia is most preferably employed because of its high mechanical strength, high toughness, and low reactivity with the piezoelectric layer 72 and the electrode material even in a thin plate.

  When stabilized / partially stabilized zirconia is used as the material for the substrate 50 or the like, an additive such as alumina or titania is contained at least in the portion where the actuator portion 58 is formed (vibrating portion 66). It is preferable.

  In addition, the piezoelectric layer 72 constituting the actuator unit 58 is, for example, lead zirconate, lead titanate, lead magnesium niobate, lead magnesium tantalate, lead nickel niobate, lead zinc niobate, manganese niobate as piezoelectric ceramics. A composite ceramic containing lead, lead antimony stannate, lead manganese tungstate, lead cobalt niobate, barium titanate, or any combination thereof can be used. In the first embodiment, A material mainly composed of components composed of lead zirconate, lead titanate and lead magnesium niobate is preferably used.

  This is because such a material has a high electromechanical coupling coefficient and a piezoelectric constant, and has low reactivity with the base material during sintering of the piezoelectric layer 72, so that a material having a predetermined composition can be stably formed. Because it is based on what can be done.

  Furthermore, in the first embodiment, the piezoelectric ceramic includes lanthanum, calcium, strontium, molybdenum, tungsten, barium, niobium, zinc, nickel, manganese, cerium, cadmium, chromium, cobalt, antimony, iron, yttrium, Ceramics to which an oxide such as tantalum, lithium, bismuth, tin, or any combination thereof, or another compound is appropriately added may be used.

  For example, it is also preferable to use a ceramic containing lead zirconate, lead titanate and lead magnesium niobate as main components and containing lanthanum or strontium.

  On the other hand, the upper electrode 74 and the lower electrode 70 in the actuator unit 58 are preferably made of a solid and conductive metal at room temperature, for example, aluminum, titanium, chromium, iron, cobalt, nickel, copper Zinc, Niobium, Molybdenum, Ruthenium, Palladium, Rhodium, Silver, Tin, Tantalum, Tungsten, Iridium, Platinum, Gold, Lead, etc. are used alone or in combination with any of these metals. A cermet material in which the same material as the layer 72 and the substrate 50 is dispersed may be used.

  Then, after each micropipette 34 is filled with different types of sample solutions by the first method or the second method described above, each actuator unit 58 is driven, and the sample discharge port of each micropipette 34 is driven. The sample solution is discharged from 54.

  Here, of the voltage waveforms applied to the electrodes 70 and 74 of the actuator unit 58, when the actuator unit 58 is turned on to reduce the volume of the cavity 56, a pulsed voltage is applied to the electrodes 70 and 74. Will be applied. In this case, by changing the amplitude (voltage) of the pulse, the amount of change per unit time (rising angle of the voltage waveform), the pulse width, etc., the deformation amount, the deformation speed, etc. of the vibration part 66 change, The discharge amount of the sample solution can be controlled. Further, the number of drops of the sample solution per unit time can be changed by changing the number of pulses generated in a certain period.

  When a single spot 80 is formed by supplying a plurality of sample solutions, the supply position is usually fixed and the number of times of supply is repeated, but the supply position may be shifted for each supply. For example, as shown in FIGS. 13A and 13B, by appropriately changing the supply position of the sample solution, minute spots 80a formed by a plurality of sample solutions are formed in one spot 80 (indicated by a two-dot chain line) to be formed. These fine spots 80a are combined on the substrate 10 (merging), whereby one spot 80 is formed as shown in FIGS. 14A and 14B. In this case, the diameter of each spot 80 formed on the substrate 10 can be made uniform by controlling the number of times of supply, the supply position, and the amount of supply at one time according to the type of sample solution to be supplied. .

  In addition, it is preferable to apply a voltage that excites vibration to the actuator unit 58 after the cavity 56 is filled with the sample solution. As a result, the DNA fragments contained in the sample solution filled in the cavity are uniformly dispersed, and the amount of DNA fragments for each supply does not vary.

  By the way, in the above-mentioned example, the pin 100 is provided in each micropipette 34 of the dispensing device 30A, but in addition, as shown in FIG. A hole may be provided in each reservoir 110 of the cartridge 112 with the pin 100 (third method).

  That is, as shown in FIG. 15, as the dispensing device 30A, one in which the pin 100 is not provided at the sample injection port 52 of each micropipette 34 is used. As indicated by a two-dot chain line, for example, when each reservoir 110 of the cartridge 112 is in contact with or close to the sample inlet 52 of the micropipette 34, the pin 100 is inserted into the reservoir 110 from above each reservoir 110. Pierce and make a hole in the reservoir 110.

  When the hole is opened in the reservoir 110, the pin 100 is pulled out, whereby the sample solution is discharged from the hole and introduced into the sample injection port 52, and is introduced into the cavity 56 through the introduction hole 60 and the first communication hole 62. Will be.

  In the dispensing apparatus 30A according to the first embodiment described above, the arrangement pitch of the micropipettes 34 is substantially the same as the arrangement pitch of the reservoirs 110 of the cartridge 112. In addition, FIG. As in the dispensing device 30B according to the second embodiment in FIG. 16B, the arrangement pitch of the micropipettes 34 may be variable.

  That is, the dispensing device 30B according to the second embodiment is configured by being provided with a pitch variable mechanism 140 that makes the arrangement pitch of each micropipette 34 variable as shown in FIGS. 16A and 16B. . As the pitch variable mechanism 140, a mechanism mainly including screws, a mechanism mainly including springs, or a combination mechanism thereof can be employed.

  In particular, when using the dispensing device 30B according to the second embodiment, as shown in FIG. 16A, a solution supply device 144 in which a large number of pipettes 142 are arranged can be used.

  As the variable setting of the arrangement pitch of each micropipette 34, when the arrangement pitch of each micropipette 34 is minimized by the pitch variable mechanism 140, as shown in FIG. When the arrangement pitch is maximized, for example, as shown in FIG. 16A, it is preferable that the arrangement pitch of the pipettes 142 of the solution supply apparatus 144 is substantially the same. This is because variations in pitch between the micropipettes 34 can be suppressed.

  When this dispensing device 30B is used, first, as shown in FIG. 16A, the arrangement pitch of each micropipette 34 is maximized by the pitch variable mechanism 140, and the arrangement pitch of each pipette 142 in the solution supply device 144 is set. In this state, the sample solution is supplied from the solution supply device 144 to each micropipette 34 of the dispensing device 30B.

  When the supply of the sample solution to the dispensing apparatus 30B is completed, the arrangement pitch of the micropipettes 34 is minimized by the pitch variable mechanism 140 as shown in FIG. 16B. Next, the dispensing device 30 </ b> B is transported onto the substrate 10, and then the actuator unit 58 of each micropipette 34 is driven to discharge and supply the sample solution onto the substrate 10, thereby forming a micro spot 80 on the substrate 10. .

  In the dispensing device 30B and the manufacturing method using the dispensing device 30B according to the second embodiment, the supply of the sample solution from the solution supply device 144 to the dispensing device 30B, and the substrate from the dispensing device 30B. The supply onto the substrate 10 can be performed quickly, efficiently and reliably, the process from the supply of the sample solution to the supply onto the substrate 10 can be performed smoothly, and the DNA chip 20 Quality and yield can be improved.

  In the second embodiment described above, the variable pitch mechanism 140 is provided in the dispensing device 30B. In addition, as shown in FIGS. 17A and 17B, the variable pitch mechanism 150 is provided in the solution supply device 144. You may do it. The pitch variable mechanism 150 has a mechanism for changing the arrangement pitch of the pipettes 142 constituting the solution supply apparatus 144. As the pitch variable mechanism 150, a mechanism mainly including a screw, a mechanism mainly including a spring, or a combination mechanism thereof may be employed.

  In this case, as the dispensing device 30, as shown in FIG. 17B, an arrangement pitch of each micropipette fixed to an optimum pitch for supplying the sample solution onto the substrate 10 can be used. .

  As the variable setting of the arrangement pitch of each pipette 142 in the solution supply device 144, when the arrangement pitch of each pipette 142 is minimized by the pitch variable mechanism 150, for example, as shown in FIG. When the arrangement pitch of the pipettes 142 is maximized, for example, as shown in FIG. 17A, the arrangement pitch of the reservoirs 110 of the cartridge 112 is substantially the same. It is preferable. This is because variations in pitch between the pipettes 142 in the solution supply apparatus 144 can be suppressed.

  When this solution supply device 144 is used, first, as shown in FIG. 17A, the arrangement pitch of the pipettes 142 is maximized by the pitch variable mechanism 150 so as to be substantially equal to the arrangement pitch of the reservoirs 110 in the cartridge 112. In this state, the sample solution stored in the reservoir 110 of the cartridge 112 is sucked into the solution supply device 144 via each pipette 142 in this state.

  When the supply of the sample solution to the solution supply device 144 is completed, as shown in FIG. 17B, the arrangement pitch of the pipettes 142 is minimized by the pitch variable mechanism 150, and the arrangement of the micropipettes 34 in the dispensing device 30 is achieved. In this state, the sample solution is supplied from the solution supply device 144 to each micropipette 34 of the dispensing device 30.

  When the supply of the sample solution to the dispensing device 30 is completed, as shown in FIG. 17C, the dispensing device 30 is transported onto the substrate 10, and then the actuator unit 58 of each micropipette 34 is driven, The sample solution is discharged and supplied onto the substrate 10 to form a micro spot 80 on the substrate 10.

  Thus, in the manufacturing method using the solution supply device 144 having the pitch variable mechanism 150, the sample solution is supplied from the cartridge 112 to the solution supply device 144, and the sample solution is supplied from the solution supply device 144 to the dispensing device 30. The supply and the supply from the dispensing device 30 onto the substrate 10 can be performed quickly, efficiently and reliably, and the process from the supply of the sample solution to the supply onto the substrate 10 is smoothly performed. The quality of the DNA chip 20 and the yield can be improved.

  Next, a dispensing device 30C according to a third embodiment will be described with reference to FIGS.

  As shown in FIG. 18, the dispensing device 30C according to the third embodiment is applied particularly to the dispensing device 30B according to the second embodiment and the dispensing device 30 of FIG. 17B. Thus, a holding portion 160 for holding each pipette 142 of the solution supply device 144 is provided at the peripheral edge portion of the sample injection port 52 of each micropipette 34. The holding unit 160 includes, for example, an O-ring 162 provided at the peripheral edge of the sample injection port 52 and a fixing unit 164 that fixes the O-ring 162.

  For example, as shown in FIG. 19, the fixing portion 164 is integrally formed with a side wall 166 formed in a ring shape and an upper wall 170 formed with a circular hole 168 for preventing the O-ring 162 from being detached upward. Has been configured. The fixing portion 164 may be formed integrally with the base body 50, or may be formed separately from the base body 50 and fixed onto the base body 50 with an adhesive or the like. In FIG. 18, the example fixed with the adhesive agent is shown.

  As another example of the fixing portion 164, for example, as shown in FIG. 20, a plurality of L-shaped holding pieces 172 whose upper portions are bent toward the axis m of the sample inlet 52 (four in the example of FIG. 20). ) And may be configured.

  When the sample solution is used for each micropipette 34 of the dispensing device 30C according to the third embodiment using the solution supply device 144, as shown in FIG. This is done by inserting the tip of each pipette 142 into the O-ring 162 in the holding section 160 of the corresponding micropipette 34.

  As another example of the supply of the sample solution, as shown in FIG. 21, for example, the pipe 180 into which each pipette 142 of the solution supply apparatus 144 can be inserted is held by the holding unit 160, and the sample solution is supplied to the micropipette 34. You may make it do. As this pipe | tube 180, what was set so that a diameter may become large gradually toward upper direction, and the diameter of a lower end part was set to be substantially the same as the internal diameter of the O-ring 162 can be used.

  When this pipe 180 is used, it is preferable that the tip of the pipette 142 is made close to the inner wall of the pipe 180, because there is no inconvenience such as the sample solution discharged from the pipette 142 hitting the inner wall of the pipe 180 and scattering.

  In the dispensing device 30C according to the third embodiment, when the sample solution is injected into each micropipette 34 of the dispensing device 30C using the pipette 142, the pipette 142 or the tube 180 is attached to the holding unit 160. Therefore, the sample solution can be reliably injected into the micropipette 34, and solution leakage and the like can be effectively prevented.

  In particular, it is preferable to hydrophilize at least the inner wall of the tube 180 because the sample solution discharged from the pipette 142 can be reliably guided to the sample inlet 52 of the micropipette 34.

  As still another example of the supply of the sample solution, for example, as shown in FIG. 22, the amount of liquid injected into the pipe 180 is inserted into a part of the pipe 180 for receiving each pipette 142 of the solution supply apparatus 144. A scale 182 to be measured is formed, and further, a portion provided with a protrusion 184 for contacting a part of each pipette 142 on a part of the inner wall of the tube 180 and a part not provided are located at the same distance from the sample inlet 52. You may make it form in.

  In FIG. 22, a large number of openings having an opening area equal to or smaller than the opening area of the sample discharge port 54 are formed between the tube 180 and the sample injection port 52 in order to remove foreign substances in the sample solution to be injected. The filtered filter 186 is attached with its periphery held by the holding portion 160, the base body 50, and the adhesive 188.

  Here, the holding part 160 is entirely composed of an elastic body such as rubber, and the pipe 180 is airtightly held only by the holding part. The scale 182 allows the amount of the sample solution to be injected to be confirmed on the spot, and by injecting each pipette 142 in contact with the projection 184, the injection position is always constant, and variations in the injection operation are reduced. In addition, the portion where the projection 184 is not formed constitutes a path for releasing the gas at the time of injection, so that the sample solution can be reliably injected without entraining bubbles.

  Further, the filter 186 can block the entry of foreign matter into the micropipette 34, thereby avoiding ejection failure due to the clogging of foreign matter. The size (diameter) of the opening portion of the filter 186 is preferably equal to or smaller than the size (diameter) of the discharge port. However, if the opening is too small, it is difficult to inject the sample solution. Therefore, the opening is more preferably about 70% of the opening diameter of the discharge port.

  Thus, by manufacturing the DNA chip 20 using the dispensing devices 30A to 30C according to the first to third embodiments and the solution supply device 144 shown in FIG. The process up to the supply onto the substrate 10 can be performed smoothly, so that the quality of the DNA chip 20 and the yield can be improved.

  In particular, in each of the dispensing devices 30A to 30C according to the first to third embodiments and the dispensing device 30 shown in FIG. 17B, each sample inlet 52 is subjected to a hydrophilic treatment so that the sample inlet 52 passes through the sample inlet 52. Since the supplied sample solution can be smoothly guided to the cavity 56 side, the supply time of the sample solution can be shortened.

  In addition, the dispensing device and the method for manufacturing the DNA chip according to the present invention are not limited to the above-described embodiments, and it is needless to say that various configurations can be adopted without departing from the gist of the present invention.

FIG. 2 is a perspective view for explaining a first method for producing a DNA chip using a dispensing device, showing the configuration of the dispensing device according to the first embodiment together with a cartridge. It is a top view which shows the structure of the micropipette which comprises the dispensing apparatus which concerns on 1st Embodiment. It is sectional drawing on the III-III line in FIG. It is a perspective view which shows the shape of the flow path containing the cavity formed in the base | substrate of a micropipette. It is a perspective view which shows the other shape of the flow path containing the cavity formed in the base | substrate of a micropipette. It is a perspective view which shows the DNA chip manufactured. It is a top view which shows the structure of the micropipette which concerns on a 1st modification. It is sectional drawing on the VIII-VIII line in FIG. It is sectional drawing which shows the structure of the micropipette which concerns on a 2nd modification. It is for demonstrating the 2nd method of manufacturing a DNA chip using a dispensing apparatus, Comprising: It is explanatory drawing which shows the state which affixes a film material so that each reservoir part of a cartridge may be obstruct | occluded. It is explanatory drawing which shows the state which conveyed the cartridge which stuck the film material on the dispensing apparatus. It is sectional drawing which shows the state which opens a hole in a film material with a micropipette. FIG. 13A is a cross-sectional view showing a process in which a sample solution is supplied onto a substrate and a large number of minute spots are formed in one spot to be formed, and FIG. 13B is a plan view thereof. FIG. 14A is a cross-sectional view showing a state in which a large number of minute spots are combined to form one spot on the substrate, and FIG. 14B is a plan view thereof. It is explanatory drawing which shows the 3rd method of manufacturing a DNA chip using a dispensing apparatus. FIG. 16A is an explanatory diagram illustrating a state in which a sample solution is supplied from the solution supply apparatus to the dispensing apparatus according to the second embodiment, and FIG. 16B is a diagram illustrating a sample solution from the dispensing apparatus according to the second embodiment. It is explanatory drawing which shows the state supplied on a board | substrate. FIG. 17A is an explanatory view showing a state in which the sample solution is supplied from each reservoir of the cartridge to the solution supply device, and FIG. 17B is an explanatory view showing a state in which the sample solution is supplied from the solution supply device to the dispensing device. FIG. 17C is an explanatory view showing a state in which the sample solution is supplied from the dispensing device onto the substrate. It is sectional drawing which shows the structure of the micropipette in the dispensing apparatus which concerns on 3rd Embodiment. It is a perspective view which shows an example of a holding | maintenance part. It is a perspective view which shows the other example of a holding | maintenance part. It is sectional drawing which shows the structure of the other example of the micropipette in the dispensing apparatus which concerns on 3rd Embodiment. It is sectional drawing which shows the structure of the further another example of the micropipette in the dispensing apparatus which concerns on 3rd Embodiment.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Board | substrate 20 ... DNA chip 30, 30A-30C ... Dispensing apparatus 34 ... Micropipette 50 ... Base | substrate 52 ... Sample injection port 52a ... Peripheral part 54 ... Sample discharge port 56 ... Cavity 58 ... Actuator part 80 ... Minute spot 100 ... Pin 110 ... Recessed part (reservoir) 112 ... Cartridge 130 ... Film material 140 ... Pitch variable mechanism 144 ... Solution supply device 150 ... Pitch variable mechanism 160 ... Holding part 180 ... Pipe 182 ... Scale 184 ... Projection 186 ... Filter

Claims (9)

An injection port for injecting a sample solution from the outside into at least one substrate, a cavity into which the sample solution is injected and filled, and a discharge port for discharging the sample solution are formed to form the cavity. A piezoelectric / electrostrictive element is provided on at least one wall surface of the substrate, and a plurality of micropipettes configured to move the sample solution in the cavity are arranged, and from the discharge port of each micropipette In a dispensing apparatus for discharging the sample solution,
A dispensing device, characterized in that a holding portion for holding a pipette for injecting a solution from the injection port or a tube for receiving the pipette is provided at the periphery of the injection port of each micropipette. The dispensing device according to claim 1,
A dispensing apparatus, wherein at least an inner wall of a pipe for receiving the pipette is subjected to a hydrophilic treatment. The dispensing device according to claim 1 or 2,
A scale for measuring the amount of liquid injected into the pipe is formed in a part of the pipe for receiving the pipette. In the dispensing apparatus of any one of Claims 1-3,
2. A dispensing apparatus according to claim 1, wherein a portion provided with a protrusion and a portion not provided are formed on a part of an inner wall of a pipe for receiving the pipette at a position at the same distance from an injection port. In the dispensing apparatus of any one of Claims 1-4,
A dispensing device in which a filter having a large number of openings having an opening area equal to or smaller than the opening area of the discharge port is attached between the pipe for receiving the pipette and the injection port. An inlet for injecting the sample solution from the outside, a cavity for injecting and filling the sample solution, and an outlet for discharging the sample solution are formed in at least one substrate. Using a dispensing device comprising a piezoelectric / electrostrictive element on at least one wall surface of the substrate to be formed, and a plurality of micropipettes configured to move the sample solution in the cavity. In a DNA chip manufacturing method of manufacturing a DNA chip by discharging the sample solution onto a substrate from a discharge port of a micropipette,
The dispensing device is provided with a holding portion for holding a pipette for injecting a solution from the injection port or a tube for receiving the pipette at the periphery of the injection port of each micropipette,
A method for producing a DNA chip, wherein the solution is supplied to the dispensing device while the pipette or the tube is held by the holding unit. In the manufacturing method of the DNA chip of Claim 6,
A method for producing a DNA chip, wherein a tube for receiving the pipette is partially formed with a graduation for measuring the amount of liquid injected into the tube. The method for producing a DNA chip according to claim 6 or 7,
A method for producing a DNA chip, characterized in that a pipe for receiving the pipette uses a part of an inner wall in which a part provided with a protrusion and a part not provided are formed at the same distance from the injection port . In the manufacturing method of the DNA chip of any one of Claims 6-8,
A method for producing a DNA chip, wherein a filter having a large number of openings having an opening area equal to or smaller than the opening area of the discharge port is attached between a pipe for receiving the pipette and the injection port.

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