JP2006078498A - Manufacturing method of dna chip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To realize the uniformity of a spot diameter formed on a substrate and improve the quality and the reliability of DNA chips, by performing a dosing control in response to the types of sample solutions supplied to the substrate. <P>SOLUTION: When the thickness shape of a spot 80 on the substrate 10 to which the sample solutions are supplied is raised on a fringe portion 130, the so-called "doughnut-shape", the substrate 10 is cooled to 0°C and thereafter treatment for returning to the environment of ambient temperature in which a sufficient volume of gas with a humidity of 30% or higher is present. Moisture is supplied to the spot 80 of the doughnut-shape and the fluidity of the sample solutions in the spot 80 increases to change into hemispherical shape by surface tension force, and then the raise of the fringe portion 130 vanishes. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for producing a DNA chip (DNA microarray) in which thousands to 10,000 or more different types of DNA fragments are aligned and fixed as spots on a substrate such as a microscope slide glass.

  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 a great expectation for the development of a new method with good spot shape controllability and excellent productivity for higher density (see Patent Documents 1 to 3).

JP-A-11-99000 International Publication No. 99/02266 Pamphlet WO 97/44134 pamphlet

  By the way, as shown in FIG. 16, when a spot 202 is formed on the substrate 200 by dropping the sample solution, the spot 202 becomes hemispherical due to the surface tension. In this case, the amount of the substantial sample immobilized on the substrate 200 is only a small portion 204 in contact with the substrate 200, and the amount is a very small part of the whole (hemisphere). Since the remaining portion 206 is not immobilized, the remaining portion 206 is washed away in the subsequent washing step, and there is a problem that the loss of the sample solution is large and the use efficiency of the sample solution is low.

  The cost for manufacturing the DNA chip is substantially controlled by the amount of the sample solution, and in the case of the above example, most of the sample solution is flowed, which is disadvantageous in terms of improving the production efficiency.

  In addition, several thousand to 10,000 or more different types of sample solutions are dropped on a single substrate. However, because different types of sample solutions have different viscosities and surface tensions, they are the same. In order to obtain the spot diameter, it is necessary to change the dropping amount of the sample solution according to the viscosity, the surface tension, and the like.

  However, in the conventional method, since the sample solution adhering to the pins is physically brought into contact with the substrate together with the pins and the sample solution is dropped, the spot is formed on the substrate by one drop. Thus, fine drop control (control of drop amount and drop position) cannot be performed, and there is a problem that the diameter of the spot formed on the substrate varies.

  In addition, in order to more reliably fix the DNA fragment in the sample solution on the substrate, a method of mixing the organic or inorganic polymer in the sample solution and physically holding the DNA fragment during polymer crosslinking However, in this case, the viscosity of the sample solution increases, and it becomes easy to dry, thicken, and solidify, shortening the pot life of the sample at the time of spot formation, and increasing the amount of one drop. There is a problem.

  The present invention has been made in view of such problems, a DNA chip capable of improving the efficiency of use of a high-cost sample solution, and improving the productivity and yield of the DNA chip. It aims at providing the manufacturing method of.

  Another object of the present invention is to control the supply according to the type of sample solution supplied on the substrate, to make the spot diameter formed on the substrate uniform, An object of the present invention is to provide a DNA chip manufacturing method capable of improving quality and reliability.

  The method for producing a DNA chip according to the present invention includes: supplying a sample solution onto a substrate by an inkjet method, and producing a DNA chip in which a large number of spots of the sample solution are arranged on the substrate. When supplying the sample solution onto the substrate, the humidity around the portion where the sample solution is discharged and supplied is selectively made higher than other parts so that the sample solution does not dry, thicken or solidify. It is characterized by that. Thereby, discharge failure can be avoided especially when using a sample solution which is easy to dry, thicken and solidify.

  Further, in the present invention, after the sample solution is supplied onto the substrate to produce a substrate on which a large number of spots are arranged, the substrate is cooled to 0 ° C. or lower, and then has a sufficient volume of 30% or higher. You may employ | adopt the process returned to the room temperature in which gas exists.

  Furthermore, after the sample solution is supplied onto the substrate to produce a substrate on which a large number of spots are arranged, the substrate is exposed to an atmosphere containing a sufficient volume of gas having a humidity of 80% or more, or mist is applied. It may be exposed to the steam contained.

  These inventions use a sample solution in which a polymer or the like is mixed to increase the viscosity, adjust the dropping method of the sample solution, and adjust the shape of the spot on the substrate, for example, the peripheral portion of the spot is raised and the central portion is recessed. However, it is preferably used when presenting a so-called donut shape.

  In the case of such a donut shape, the boundary between the peripheral edge of the spot and the substrate is easily observed, and a spot of a colorless and transparent liquid is placed on a colorless and transparent glass substrate or the like like a sample solution containing a DNA fragment. In the case of forming the film, the spot shape can be easily observed, and it is easy to inspect the quality of the spot shape.

  However, in such a spot having a donut shape, in many cases, even if most of the bulging portion of the peripheral edge is washed away in the subsequent cleaning process at the time of immobilization, the substantial sample to be immobilized is the peripheral edge. Since it becomes large (darker) in the part, when used as a DNA chip, the distribution of the amount of fluorescent light emitted from the spot exhibits a donut shape within the spot, which causes a deterioration in sensitivity and variation. End up.

  Therefore, in order to achieve both the ease of inspection (doughnut shape) and the good spot shape (non-doughnut shape), when supplying the sample solution to the substrate, the sample solution is ejected by the inkjet method or the like. In order to concentrate the sample solution on the peripheral part of the spot by controlling the kinetic energy and the hydrophobicity of the substrate, a donut shape is formed, and then it does not become spherical due to the surface tension of the liquid, While increasing the viscosity of the sample solution in advance, after completion of the inspection, a method of increasing the fluidity of the sample solution forming a spot and changing the donut shape to a non-doughnut shape by surface tension is suitable. The present invention is suitable for realizing this method.

  That is, after cooling the substrate on which the doughnut-shaped spot is formed to 0 ° C. or lower, the substrate is returned to room temperature where a sufficient volume of gas having a humidity of 30% or more exists, or the substrate is sufficiently heated to have a humidity of 80% or more. When exposed to an atmosphere containing a volume of gas or exposed to water vapor containing mist, moisture is taken into the sample solution from the surrounding gas, or the fluidity of the sample solution is affected by contact with the mist. As a result, the spot shape changes to a hemispherical shape that is a non-doughnut shape, thereby improving the sensitivity of the DNA chip and reducing the sensitivity variation. Of course, after the spot shape has become hemispherical, the substrate may be immediately put into a drying process to fix the shape. In addition, when exposed to water vapor | steam, of course, the temperature of water vapor | steam must be below the temperature which does not denature a DNA fragment.

  As described above, according to the method for producing DNA according to the present invention, it is possible to improve the use efficiency of the costly sample solution, and to improve the productivity and yield of the DNA chip. Further, the dropping control according to the type of the sample solution to be dropped can be performed, the spot diameter formed on the substrate can be made uniform, and the quality and reliability of the DNA chip can be improved. .

  Several embodiments of the method for producing a DNA chip according to the present invention will be described below with reference to FIGS.

  As shown in FIG. 1, the method for manufacturing a DNA chip according to the present embodiment includes a pretreatment step S1 for forming a poly-L-lysine layer 12 (see FIG. 10A) on the surface of a substrate 10, and a DNA fragment. A sample preparation step S2 for preparing a sample, a dilution step S3 for diluting the concentration of the obtained sample, and supplying the diluted sample solution onto the substrate 10 (including dripping), as shown in FIG. A supply step S4 for creating a spotted substrate in which a large number of spots 80 are arranged on the substrate 10, a drying step S5 for drying the spot 80 by applying heat to the substrate 10, and DNA fragments in the spots 80 on the substrate 10 The DNA chip 20 shown in FIG. 3 is manufactured.

  Further, as shown in FIG. 2, the sample preparation step S2 includes an amplification step S11 for PCR amplification of a DNA fragment to prepare a PCR product, and a powder generation step S12 for drying the obtained PCR product to obtain a DNA powder. And a mixing step S13 in which the obtained DNA powder is dissolved in a buffer solution.

  Specifically, in the pretreatment step S1, first, the substrate 10 is immersed in an alkaline solution and slowly shaken at room temperature for at least 2 hours. The alkaline solution is, for example, a solution in which NaOH is dissolved in distilled water, ethanol is further added, and the mixture is stirred until it becomes completely transparent.

  Thereafter, the substrate 10 is taken out, moved into distilled water, rinsed, and the alkaline solution is removed. Next, the substrate 10 is immersed in a poly-L-lysine solution prepared by adding poly-L-lysine in distilled water, and left for 1 hour.

  Thereafter, the substrate 10 is taken out and centrifuged in a centrifuge to remove excess poly-L-lysine solution. Next, the substrate 10 is dried at 40 ° C. for about 5 minutes to obtain the substrate 10 on which the poly-L-lysine layer 12 is formed.

  Next, in the sample preparation step S2, first, 3Msodium acetate and isopropanol are added to a PCR product (amplification step S11) amplified by a known PCR machine, and left for several hours. Thereafter, the PCR product solution is centrifuged with a centrifuge to precipitate DNA fragments.

  The precipitated DNA fragments are rinsed with ethanol, centrifuged and then dried to produce DNA powder (powder production step S12). A sample is prepared by adding a buffer solution (for example, TE buffer solution) to the obtained DNA powder and allowing it to stand for several hours to completely dissolve (mixing step S13). The concentration of the sample at this stage is 1 to 10 μg / μl.

  In this embodiment, the concentration of the obtained sample is diluted (dilution step S3). In the dilution step S3, the sample is diluted with an aqueous solution containing water or sodium chloride, an aqueous solution containing a polymer, or the like. The diluted sample solution may be allowed to stand for 1 hour to several hours as necessary, or may be mixed by freezing and thawing to mix the sample and the diluted solution. Thereafter, the diluted sample solution is centrifuged or vacuum defoamed to remove bubbles in the solution, and then supplied onto the substrate 10 to create a spotted substrate (supplying step S4).

  In particular, in this embodiment, a dispensing device 30 as shown in FIGS. 4A to 4C and FIG. 5 is used in the supply step S4.

  In the dispensing device 30, for example, 10 micropipettes 34 are arranged in 5 rows and 2 columns on the upper surface of a rectangular fixed plate 32, and a group of micropipettes 34 aligned in the direction of each column is provided with a fixing jig 36. It has the structure fixed to the fixed plate 32 via.

  As shown in FIGS. 4C and 5, 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, and A cavity 56 formed between the sample injection port 52 and the sample discharge port 54 and an actuator unit 58 that vibrates the base body 50 and changes the volume of the cavity 56 are configured. .

  Therefore, as shown in FIG. 5, 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 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 present embodiment, the first communication hole 62 is formed near the sample injection port 52 in the lower surface of the cavity 56, and the second communication is formed at a position corresponding to the sample discharge port 54 in the lower surface of the cavity 56. A hole 64 is formed.

  Further, in this embodiment, the portion of the base body 50 where the upper surface of the cavity 56 contacts is thin, and is structured to be easily subjected to vibration against external stress, and functions as the vibrating portion 66. Yes. 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. 4C, 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, respectively.

  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 from the sample discharge port 54 communicating with the cavity 56 at a predetermined speed, and the sample solution discharged from the micropipette 34 as shown in FIG. Can be prepared as DNA spots 20 aligned and fixed as spots 80 on a substrate 50 such as a microscope slide glass. Further, as the volume of the cavity 56 increases, a new sample solution is injected and filled into the cavity 56 from the first communication hole 62 to prepare for the next discharge.

  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 under a less disturbed state.

  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. Further, the inner wall of the cavity 56 is preferably smooth so that there are no protrusions that disturb the flow, and the material is preferably made of ceramics having good affinity for 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 third thin plate layer 50E in which the sample discharge port 54 is formed is made of a sheet obtained by processing an organic resin such as a PET film with an excimer laser or a metal such as a stainless film in consideration of matching with the processing method. A sheet punched with a mold or the like is preferable.

  The dimensions of the sample discharge port 54 and the first communication hole 62 are optimally designed depending on the physical properties of the sample solution to be discharged, the discharge amount, the discharge speed, and the like, but are preferably about 10 to 100 μmφ.

  In FIG. 7, two first communication holes 62 communicate with one sample injection port 52 and an introduction hole 60 connected thereto, and each communication hole 62 includes a cavity 56, a second communication hole 64, and Two channels 65 each having 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. 4A, a plurality of pins 38 for positioning and fixing the micropipette 34 are provided on the upper surface of the fixing plate 32. When the micropipette 34 is fixed on the fixing plate 32, the micropipette 34 is inserted into the positioning holes 90 (see FIG. 4C) provided on both sides of the base 50 of the micropipette 34 while the pins 38 of the fixing plate 32 are inserted. Is placed on the fixed plate 32, the plurality of micropipettes 34 are automatically arranged and positioned in a predetermined arrangement.

  Each fixing jig 36 includes a pressing plate 100 that presses the plurality of micropipettes 34 against the fixing plate 32. Insertion holes into which the screws 102 are inserted are formed at both ends of the holding plate 100, and the screws 102 are inserted into the insertion holes and screwed into the fixing plate 32. Can be pressed against the fixed plate 32 at a time. A plurality of micropipettes 34 pressed by one pressing plate 100 constitutes one unit. The example of FIG. 4A shows an example in which one unit is configured by five micropipettes 34 arranged in the row direction.

  In addition, when a plurality of micropipettes 34 are pressed into the holding plate 100, introduction holes 104 (see FIG. 4B) for supplying sample solutions to locations corresponding to the sample injection ports 52 of the micropipettes 34, respectively. The tubes 106 for guiding the sample solution to the introduction holes 104 are held at the upper ends of the introduction holes 104, respectively.

  Note that the width of the pressing plate 100 is determined so that the pads 76 and 78 connected to the electrodes 70 and 74 of the actuator portion 58 when the plurality of micropipettes 34 are pressed against the fixed plate 32 are taken into consideration for the efficiency of wiring work. The dimensions are preferably such that they face upward.

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

  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 30 having such a configuration, as a method of supplying different types of sample solutions corresponding to the respective sample inlets 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. In this method, different types of sample solutions are put in the respective recesses 110 of the cartridge 112, the cartridges 112 are attached so that the respective recesses 110 and the tubes 106 correspond to each other, and the bottom of each recess 110 is opened with a needle or the like. Thus, a method of supplying the sample solution in each recess 110 to each micropipette 34 through the tube 106 can be considered.

  When the tube 106 is not used, the cartridge 112 is attached so that each recess 110 and each introduction hole 104 of the fixing jig 36 correspond to each other, and the bottom of each recess 110 is opened with a needle or the like. In addition to supplying the sample solution in the recess 110 to each micropipette 34 through the introduction hole 104, a needle or the like is previously formed in the vicinity of each introduction hole 104 in the fixing jig 36 to fix the cartridge 112. Each recess 110 may be opened simultaneously with attachment to the tool 36.

  In addition, you may add the mechanism which pumps gas etc. after opening and forcibly pushes out a sample solution. Further, providing a mechanism for cleaning the space from the sample injection port 52 to the sample discharge port 54 formed in the base 50 of each micropipette 34 contaminates thousands to tens of thousands of DNA fragments. In addition, it is desirable to eject the spot 80 with high purity.

  In the example of FIG. 4A, both ends of the pressing plate 100 are fastened to the fixing plate 32 with screws 102. However, the pressing plate 100 is fixed not only mechanically with screws, springs, etc., but also with an adhesive or the like. You may go.

  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. Composite ceramics containing components such as lead, lead antimony stannate, lead manganese tungstate, lead cobalt niobate, barium titanate, etc., or any combination thereof can be used. A material mainly composed of components composed of lead oxide, 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.

  Further, in the present embodiment, the piezoelectric ceramic includes lanthanum, calcium, strontium, molybdenum, tungsten, barium, niobium, zinc, nickel, manganese, cerium, cadmium, chromium, cobalt, antimony, iron, yttrium, tantalum, lithium. Further, ceramics to which oxides such as bismuth and tin, or any combination thereof, or other compounds are 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. A cermet material in which the same material as the layer 72 and the substrate 50 is dispersed may be used.

  Next, in this embodiment, using the dispensing device 30 as described above, the spotted substrate on which the sample solution is supplied onto the substrate 10 is left in a constant temperature bath at 80 ° C. for about 1 hour to dry the spot 80. After (drying step S5), 120 mJ UV irradiation, immersion in NaBr solution for 20 minutes (blocking treatment), boiling for 2 minutes, and ethanol substitution (dehydration) were performed to immobilize DNA fragments on the substrate 10 ( Immobilization step S6), the DNA chip 20 is manufactured.

  Next, several methods for supplying the sample solution onto the substrate 10 using the dispensing device 30 and forming the spots 80 will be described with reference to FIGS.

  First, as shown in FIG. 9, in the first method, different types of sample solutions (diluted) are supplied from the tubes 106 into the cavities 56 of the micropipettes 34 through the introduction holes 104 of the fixing jig 36, respectively. Then, each actuator unit 58 is driven to discharge the sample solution from the sample discharge port 54 of each micropipette 34.

  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 the sample solution is supplied a plurality of times to form one spot 80, the supply position is usually fixed and the supply frequency is repeated. However, the supply position may be shifted for each supply. For example, as shown in FIGS. 10A and 10B, by appropriately changing the supply position of the sample solution, minute spots 80a made of 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), so that one spot 80 is formed as shown in FIGS. 11A and 11B. 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. .

  Furthermore, in this embodiment, when the sample solution is supplied onto the substrate 10, as shown in FIGS. 12A to 13, the portion of the sample solution is dried, thickened, and solidified. This process can be realized, for example, by heating the substrate 10.

  As a method for heating the substrate 10, as shown in FIG. 13, there is a method in which the back surface of the substrate 10 is irradiated with infrared rays emitted from the infrared lamp 122 and heated. As a method for directly heating the sample solution, as shown in FIGS. 12A and 12B, the laser light L and the electromagnetic wave E emitted from the laser light source 120 and the electromagnetic wave generation source 121 are focused on the sample solution and irradiated. There is a method of heating. The sample solution may be heated while being supplied onto the substrate 10, but is discharged from the sample discharge port 54 on the substrate 10 from the viewpoint of stability of the shape during supply and prevention of the spread of the spot 80. It is desirable to irradiate and heat while falling.

  Further, the electromagnetic wave E is more preferable because it can selectively heat a water-containing material such as a sample solution, and therefore can heat only the sample solution forming the spot 80 (the sample solution being supplied). During heating, the sample discharge port 54 that has been discharged may be shielded with a metal shield plate or the like in order to avoid discharge failure due to drying.

  Furthermore, in this embodiment, when the sample solution is supplied onto the substrate 10, the sample solution and the substrate 10 are cooled. In the cooling method, the substrate 10 to which the sample solution is supplied may be cooled in advance to room temperature or lower, or a coolant made of alternative chlorofluorocarbon or liquid nitrogen may be sprayed on the sample solution itself. However, in such a cooling process, water droplets adhere due to dew condensation of moisture from the surrounding gas during cooling, and the spot 80 itself may flow, so the humidity management, cooling, and room temperature of the surrounding gas may occur. It is necessary to manage the speed of return.

  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. Thereby, the DNA fragments contained in the sample solution filled in the cavity are uniformly dispersed, and the amount of the DNA fragments for each dropping does not vary.

  Next, a second method using the dispensing device 30 will be described. In this second method, as shown in FIG. 14, a replacement solution such as purified water or buffer solution is filled into the cavity 56 of each micropipette 34 from each tube 106 through the introduction hole 104 of the fixing jig 36. Then, a pre-diluted sample is injected from the sample inlet 52 into the cavity 56 while being replaced. Then, the actuator unit 58 is driven to discharge and supply the sample solution onto the substrate 10. An intermediate solution (for example, a mixed solution of an aqueous solution containing a buffer solution and a polymer) that does not contain a DNA fragment having a specific gravity substantially the same as that of the sample solution may be interposed between the replacement solution and the sample solution.

  Completion of the laminar flow replacement of the sample in the cavity 56 is preferably grasped by detecting a change in fluid characteristics in the cavity 56.

  The replacement of the replacement liquid and the sample in the cavity 56 is preferably performed in a laminar flow. However, when the type of the sample is changed or the moving speed of the liquid is very high, the first of the cavities 56 is changed. The vicinity of one communication hole 62 may not necessarily be a laminar flow. In this case, the purge amount of the sample solution is increased by mixing the sample solution and the replacement solution. However, the increase in the purge amount can be minimized by determining the completion of the replacement by detecting the change in the fluid characteristics in the cavity 56. .

  Here, the change in the fluid characteristics in the cavity 56 is grasped by applying a voltage that excites vibration to the actuator unit 58 and detecting a change in electrical constant associated with the vibration. Such detection of changes in fluid characteristics is described in, for example, Japanese Patent Application Laid-Open No. 8-201265, and the contents thereof can be referred to.

  Specifically, the electrical connection from the discharge driving power source is disconnected by a relay at a predetermined interval with respect to the actuator unit 58, and at the same time, the means for measuring the resonance frequency is connected by the relay, and at that time Electrically measure impedance or resonant frequency.

  Thereby, it can be grasped whether the viscosity, specific gravity, etc. of a liquid are the target samples (liquid containing a DNA fragment etc.). That is, in each micropipette 34, since the micropipette 34 itself functions as a sensor, the configuration of the micropipette 34 can be simplified.

  After the replacement, the actuator unit 58 is driven under a driving condition corresponding to an appropriate amount of the liquid according to the required spot diameter, and the supply of the sample solution onto the substrate 10 is repeated, whereby the DNA chip 20 Manufacturing. Usually, 1 to several hundred drops are ejected from the micropipette 34 to form one spot 80.

  When the amount of the sample in the sample injection port 52 decreases, an aqueous solution containing a buffer solution, purified water, or sodium chloride is added so that bubbles do not enter the flow path, and the sample solution is continued by discharging. Can be used up without leaving in the micropipette 34. Completion of replacement of the sample with the replacement liquid (end of sample discharge) is similarly performed by detecting the viscosity and specific gravity of the liquid using the actuator unit 58.

  In addition, it is preferable to use a replacement liquid, an intermediate liquid, and a sample solution that are used in advance by removing dissolved gas in the solution through a degassing operation. By using such a solution, when the sample solution is filled in the flow path of the micropipette 34, bubbles are trapped in the middle of the flow path, and even if a filling defect occurs, the bubbles are contained in the sample solution. It is possible to avoid problems by being dissolved in the liquid, and bubbles are not generated in the fluid in the middle of discharge, so that discharge problems do not occur.

  Further, in the second method described above, while discharging the sample solution, a replacement solution such as an aqueous solution containing a buffer solution, purified water, or sodium chloride is injected into the cavity from the sample injection port 52, and similarly, laminar flow replacement is performed. Thus, the sample solution remaining in the cavity 56 can be completely discharged to prepare for the next sample injection.

  Similarly, whether or not the sample solution remains in the cavity 56 (whether or not the sample solution can be discharged) can be detected by detecting a change in fluid characteristics in the cavity 56. In this case, the amount of purge of the sample that is not used by the laminar flow replacement or replacement completion detection mechanism can be extremely reduced, and the use efficiency of the sample solution can be improved.

  Further, when the sample is filled into the cavity 56 from the sample injection port 52, the sample may be laminarly replaced from the sample injection port 52 into the cavity 56 while the actuator unit 58 is driven. In this case, by reliably replacing the interior of the cavity 56 with an inexpensive replacement liquid in advance and then performing laminar flow replacement of an expensive sample, the occurrence of defective discharge can be completely prevented, and an expensive sample solution can be discharged efficiently.

  Next, when the shape of the thickness of the spot 80 on the substrate 10 to which the sample solution has been supplied becomes a so-called donut shape raised at the peripheral portion 130 as shown in FIG. 15, the substrate 10 is brought to 0 ° C. After cooling, a process of returning to room temperature where a sufficient volume of gas with a humidity of 30% or more is present is performed. By doing so, moisture is supplied to the spot 80 having a donut shape, the fluidity of the sample solution in the spot 80 is increased, the hemisphere is changed by the surface tension, and the peripheral portion 130 is not raised.

  In the case of a donut shape, the boundary between the peripheral portion 130 of the spot 80 and the substrate 10 is easily observed, and a colorless and transparent liquid spot 80 is formed on a colorless and transparent glass substrate or the like like a sample solution containing DNA fragments. When formed on the top, it is easy to observe the spot shape, and it is easy to inspect the quality of spot formation.

  However, in many cases, the spot 80 having such a donut shape is fixed even if most of the peripheral portion 130 (swelled portion) is washed away in a cleaning step at the time of subsequent fixing. Since the substantial sample 132 is large (darker) in the peripheral portion 130, the distribution of the amount of fluorescence emitted from the spot 80 when used as the DNA chip 20 exhibits a donut shape in the spot 80. This is a cause of sensitivity deterioration and variation.

  Therefore, in order to achieve both the ease of inspection (doughnut shape) and the good spot shape (non-doughnut shape), when the sample solution is supplied to the substrate 10, the substrate 10 is discharged by an inkjet method or the like. A donut shape is formed by supplying the sample solution to the substrate and concentrating the sample solution on the peripheral portion 130 of the spot 80 by controlling the kinetic energy and hydrophobicity of the substrate 10.

  Thereafter, the viscosity of the sample solution is increased in advance to such an extent that it does not become spherical due to the surface tension of the liquid. On the other hand, after completion of the inspection, the fluidity of the sample solution that forms the spot 80 is increased. A method of changing the shape into a non-doughnut shape is suitable.

  The method according to the present embodiment (the process of cooling the substrate 10 to 0 ° C. and then returning it to room temperature in which a sufficient volume of gas having a humidity of 30% or more exists) can easily realize the above-described method. .

  The water supply to the spot 80 may be made by directly applying a vapor containing mist or the like, but in the step of returning to room temperature after the cooling, it is possible to use the water condensed on the substrate 10; This is preferable in that spots do not flow due to excessive moisture and that fine water droplets are supplied uniformly.

  Then, the spot 80 is dried by baking the substrate 10 at 80 ° C. for 1 hour. After baking at 80 degreeC for 1 hour, you may give the process which returns to cooling-room temperature, In that case, a baking process is needed again.

  Thus, in the DNA chip manufacturing method according to the present embodiment, the concentration of the sample solution is diluted prior to the supply of the sample solution onto the substrate 10. When supplied above, as shown in FIG. 11A, the spot 80 due to the sample solution does not become hemispherical but has a flat shape. In this case, since most of the supplied sample solution is fixed, most of the sample solution is not flowed in the subsequent washing step, and the use efficiency of the sample solution can be improved.

  Further, the diameter of the spot 80 formed on the substrate 10 can be made uniform by changing the degree of dilution according to the type of DNA fragment contained in the sample solution and changing the viscosity and surface tension of the sample solution. Can do.

  Furthermore, the shape of the spot 80 in the thickness direction can be made more uniform by adding a step of supplying moisture to the spot 80 after the sample solution is supplied onto the substrate 10 to form the spot 80. .

  As described above, in this embodiment, it is possible to improve the use efficiency of the costly sample solution, and to improve the productivity and yield of the DNA chip 20. In addition, supply control according to the type of sample solution to be supplied can be performed, the spot diameter formed on the substrate 10 can be made uniform, and the quality and reliability of the DNA chip 20 can be improved. Can do.

  The method for manufacturing a DNA chip according to the present invention is not limited to the above-described embodiment, and it is needless to say that various configurations can be adopted without departing from the gist of the present invention.

It is a process block diagram which shows the manufacturing method of the DNA chip which concerns on this Embodiment. It is a process block diagram which shows the breakdown of a sample preparation process. It is a perspective view which shows the DNA chip manufactured. FIG. 4A is a plan view showing a configuration of a dispensing device used in the method of manufacturing a DNA chip according to the present embodiment, FIG. 4B is a front view thereof, and FIG. 4C is a diagram showing 1 constituting the dispensing device. It is an enlarged plan view showing two micropipettes. It is a longitudinal cross-sectional view which shows the structure of a micropipette. 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 disassembled perspective view of the dispensing apparatus shown with a cartridge. It is explanatory drawing which shows the 1st method in the case of manufacturing a DNA chip using a dispensing apparatus. FIG. 10A 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. 10B is a plan view thereof. FIG. 11A is a cross-sectional view showing a state where a single spot is formed by combining a number of minute spots on a substrate, and FIG. 11B is a plan view thereof. FIG. 12A is an explanatory diagram illustrating an example of a method for heating a sample solution or a substrate, and FIG. 12B is an explanatory diagram illustrating another method. It is explanatory drawing which shows an example of the method of heating a board | substrate. It is explanatory drawing which shows the 2nd method in the case of manufacturing a DNA chip using a dispensing apparatus. It is sectional drawing which shows the example of a donut-shaped spot. It is sectional drawing which shows the shape of the spot formed on the board | substrate by the manufacturing method of the DNA chip concerning a prior art example.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Board | substrate 20 ... DNA chip 30 ... Dispensing apparatus 34 ... Micropipette 50 ... Base | substrate 52 ... Sample injection port 54 ... Sample discharge port 56 ... Cavity 58 ... Actuator part 80 ... Spot 80a ... Minute spot 112 ... Cartridge 120 ... Laser light source 122 ... Infrared lamp S1 ... Pretreatment step S2 ... Sample preparation step S3 ... Dilution step S4 ... Supply step S5 ... Drying step S6 ... Immobilization step S11 ... Amplification step S12 ... Powder production step S13 ... Mixing step

Claims (4)

In a method for manufacturing a DNA chip, a sample solution is supplied onto a substrate by an inkjet method, and a DNA chip in which a number of spots of the sample solution are arranged on the substrate is manufactured.
When supplying the sample solution onto the substrate, the humidity around the portion where the sample solution is discharged and supplied is selectively made higher than other parts so that the sample solution is not dried, thickened or solidified. A method for producing a DNA chip, characterized in that In the manufacturing method of the DNA chip of Claim 1,
After the sample solution is supplied onto the substrate to produce a substrate on which a large number of spots are arranged, the substrate is cooled to 0 ° C. or lower, and then at room temperature where a sufficient volume of gas having a humidity of 30% or more exists. A method for producing a DNA chip, comprising a step of returning. The method for producing a DNA chip according to claim 1 or 2,
And a step of exposing the substrate to an atmosphere containing a sufficient volume of gas having a humidity of 80% or more after the sample solution is supplied onto the substrate to produce a substrate on which a large number of spots are arranged. A method for manufacturing a DNA chip. In the manufacturing method of the DNA chip of any one of Claims 1-3,
A method for producing a DNA chip, comprising the step of exposing the substrate to water vapor containing mist after the sample solution is supplied onto the substrate to produce a substrate on which a large number of spots are arranged.

Images