JP3647369B2 - DNA chip and manufacturing method thereof - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a DNA chip (DNA microarray) in which several thousand to 10,000 or more different types of DNA fragments are aligned and fixed at high density as microspots on a substrate such as a microscope slide glass, and a method for producing the same.
[0002]
[Prior art]
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 such gene structure analysis, a DNA chip (DNA microarray) in which thousands to 10,000 or more different types of DNA fragments are aligned and fixed as microspots on a substrate such as a microscope slide glass is used. It has become to.
[0003]
As a method for forming a microspot in the manufacture of this DNA chip, there are widely used methods such as a QUILL method, a pin & ring method, or a spring pin method for contacting and supplying a sample solution containing a DNA fragment onto a substrate by a so-called pin. Whichever method is used, it is important to keep the distance between the minute spots constant by suppressing the variation in the capacity and shape of each minute spot to a low level.
[0004]
On the other hand, there is a great expectation for the development of a new method that has good controllability of the shape of the microspots and is excellent in productivity for higher density.
[0005]
[Problems to be solved by the invention]
By the way, when a large number of micro spots are formed on a substrate by supplying a sample solution (including dripping), a dispensing device in which a large number of supply nozzles (pins, spring pins, etc.) are arranged in a matrix, for example, is used. I am doing so.
[0006]
Usually, since the arrangement pitch of the supply nozzles is larger than the arrangement pitch of the minute spots formed on the substrate, the sample solution is supplied while shifting the supply position in the dispensing apparatus.
[0007]
At this time, if there is a variation in the shift width of the dispensing device or the arrangement pitch of the supply nozzles, the variation is directly reflected in the arrangement state of the minute spots, and adjacent minute spots join together to form one spot. May be formed.
[0008]
On the other hand, a method of spotting using a so-called ink jet method that has been put into practical use in a printer is also being studied, but when using an ink jet type supply device, the direction of ejected droplets bends, so-called flight bends, satellites and so on. There is a possibility that adjacent minute spots join together to form one spot due to the influence of unnecessary ejection droplets.
[0009]
The present invention has been made in consideration of such a problem, and even when there is variation in the shift width of the dispensing device and the arrangement pitch of the supply nozzles, in the case of using an inkjet type supply device, To provide a DNA chip capable of causing the arrangement state of a large number of micro-spots formed on a substrate to be in a state along a prescribed arrangement pitch even if there is a displacement of the ejected droplets due to flight bends or satellites. Objective.
[0010]
Another object of the present invention is that even if there is a variation in the shift width of the dispensing device or the arrangement pitch of the supply nozzles, and when an inkjet type supply device is used, flight droplets or ejected droplets due to satellites are used. Even if there is a misalignment, the arrangement state of a large number of microspots formed on the substrate can be brought into a state along a prescribed arrangement pitch, and the quality of the DNA chip and the yield can be improved. An object of the present invention is to provide a method for manufacturing a DNA chip capable of
[0011]
[Means for Solving the Problems]
According to the present invention, in a DNA chip in which a large number of microspots are formed on a substrate by supplying a sample solution, the substrate is provided with a misalignment correcting means for automatically correcting misalignment of the microspots.
[0012]
As a result, even when the supply position of the sample solution is shifted from the specified position when the sample solution is supplied onto the substrate, the minute spot due to the supply of the sample solution is moved to the specified position by the position shift correction means. The position shift is corrected.
[0013]
As described above, in the DNA chip according to the present invention, even when the shift width of the dispensing device and the arrangement pitch of the supply nozzles vary, and when the inkjet supply device is used, the flight curve or satellite Even if the position of the ejected droplet is displaced, the arrangement state of a large number of minute spots formed on the substrate can be made to be in a state along a prescribed arrangement pitch.
[0014]
The present invention also provides a DNA chip manufacturing method for manufacturing a DNA chip by supplying a large number of sample solutions onto a substrate, wherein the substrate is a position that automatically corrects a positional deviation of a minute spot due to the supply of the sample solution. A DNA chip is manufactured using a substrate having a deviation correcting means.
[0015]
As a result, even if there are variations in the shift width of the dispensing device and the arrangement pitch of the supply nozzles, and when using an inkjet type supply device, it is assumed that there was a deviation in the position of the ejected droplets due to flight bending or satellites. In addition, the arrangement state of a large number of minute spots formed on the substrate can be brought into a state along a predetermined arrangement pitch, so that the quality of the DNA chip and the yield can be improved.
[0016]
And it is preferable to supply the said sample solution using the supply apparatus of an inkjet system. In this case, the supply device includes an injection port for injecting the sample solution from the outside into at least one or more substrates, a cavity for injecting and filling the sample solution, and an ejection port for discharging the sample solution A piezoelectric / electrostrictive element is provided 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 are arranged. In addition, it is preferably a dispensing device in which different types of sample solutions are discharged from the discharge ports of each micropipette, and more preferably, the movement of the sample solution is a laminar flow.
[0017]
With the inkjet method, minute spots can be formed at high speed, and the speed and liquid volume of the ejected droplets can be freely set. Therefore, the minute spots can be accurately formed in the prescribed liquid amount / shape, and variations between each minute spot can be pinned. There is an advantage that there are few compared with the method performed with a spring pin.
[0018]
In addition, unlike the pin type, the ink jet method forms a minute spot in a non-contact manner, and therefore can be suitably used without physical interference and contact with the position deviation correcting means.
[0019]
Furthermore, with respect to the degree of freedom of design such as the spot amount and discharge speed of the ink jet system, there is an advantage that it is easy to match with the positional deviation correction means. That is, since the amount of positional deviation is large, when large correction is required, for example, by increasing the ejection amount and the ejection speed to increase the spread of the spot on the substrate, it is easy to contact the positional deviation correction means, There is an advantage that the positional deviation correction can be surely performed.
[0020]
As the above-described positional deviation correction means, The surface to be contacted by the minute spot may be a rough surface, and the contact area of the minute spot on the rough surface may be larger than the portion other than the rough surface, The protrusion may be formed at a position where the micro spot is to be formed in the substrate, and the hydrophilic region formed at the position where the micro spot is to be formed in the substrate and the other region. It may be composed of a water-repellent region formed in the part.
[0021]
Further, the above-described misregistration correction means may be a recess formed at a position where the minute spot is to be formed in the substrate, and the minute spot should be formed in the substrate. You may make it make a surface state differ in a part and a part other than that.
[0022]
Further, as the above-mentioned position deviation correcting means, there may be provided an electric field generating means for causing a portion of the substrate where the micro spot is to be formed to be in a charged state opposite to that of the sample solution.
[0023]
For example, when the sample solution is negatively charged (minus charge), the portion where the micro spot is to be formed is in a charged state opposite to the sample solution, that is, a positively charged state (plus charge state). It is preferable to have the electric field generating means for enabling spotting to a specified position with certainty. When the sample solution is in a positively charged state, a portion where a micro spot is to be formed may be in a negatively charged state.
[0024]
Furthermore, when an inkjet method is used as a method for supplying a sample solution, a minute spot can be formed in a non-contact manner, so that the droplet discharge direction is aligned with the electric field direction, and the correction of the dropping position is easy with the electric field. Therefore, it can be used suitably.
[0025]
In addition, when the sample solution is a solution containing a DNA fragment, in order to make the positional displacement correction effect due to an electric field more effective, a functional group for adding a charge is added to the DNA fragment, or the DNA fragment is included in the charged solution. It is also preferable to disperse.
[0026]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, embodiments of the DNA chip and the method for producing the same according to the present invention will be described with reference to FIGS.
[0027]
As shown in FIG. 1, the DNA chip 20 according to the present embodiment is configured by arranging a large number of microspots 80 by supplying (including dropping) a sample solution on a substrate 10. In particular, on the substrate 10, a misalignment correcting means for automatically correcting misalignment of the minute spot 80 is provided.
[0028]
Specifically, as shown in FIG. 2, the substrate 10 has a hydrophilic poly-L-lysine layer 12 formed on the surface thereof, and a position on the substrate 10 where the micro spot 80 is to be formed. A projection 14 is formed at each central portion of the. These protrusions 14 function as misalignment correction means.
[0029]
That is, as shown in FIG. 2, when a micro spot 80 is formed on the substrate 10 by supplying a sample solution, when a part of the micro spot 80 hits the protrusion 14 (see the two-dot chain line), Due to the surface tension of the microspot 80, the microspot 80 moves, and the center position of the microspot 80 and the protrusion 14 are substantially coincident.
[0030]
As described above, in the DNA chip 20 according to the present embodiment, even if the minute spot 80 is dropped from the prescribed position, the spot is moved to the prescribed position by the protrusion 14 formed on the substrate 10, The positional deviation is corrected.
[0031]
By the way, in order to manufacture the DNA chip 20 by forming the micro spot 80 by supplying the sample solution on the substrate 10, for example, the manufacturing process shown in FIG. 3 is performed.
[0032]
That is, a pretreatment step S1 for forming a poly-L-lysine layer 12 (see FIG. 2) on the surface of the substrate 10, a sample preparation step S2 for preparing a sample solution containing DNA fragments, and the obtained sample solution as a substrate The DNA chip 20 is manufactured through the supply process S3 to be supplied onto the substrate 10.
[0033]
As shown in FIG. 4, the sample preparation step S2 includes an amplification step S11 for PCR amplification of a DNA fragment to prepare a PCR product, 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.
[0034]
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 is completely transparent.
[0035]
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.
[0036]
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.
[0037]
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.
[0038]
The precipitated DNA fragment is rinsed with ethanol, centrifuged, and dried to generate DNA powder (powder generation step S12). A sample solution is prepared by adding × 1 TE buffer to the obtained DNA powder and allowing it to stand for several hours to completely dissolve (mixing step S13). The concentration of the sample solution at this stage is 1 to 10 μg / μl. After this, the immobilization liquid may be supplied from the sample injection port 52 into the cavity 56.
[0039]
And in this Embodiment, the obtained sample solution is supplied on the board | substrate 10, and the DNA chip 20 is manufactured (supply process S3). The immobilization solution may be mixed with the sample solution obtained through the sample preparation step S2, or the sample solution may be diluted. In this case, an aqueous solution containing water or NaCl or an aqueous solution containing a polymer can be used as a diluent.
[0040]
Furthermore, when manufacturing the DNA chip 20 in this embodiment, for example, the dispensing device 30 shown in FIGS. 5A to 7 is effectively used.
[0041]
As shown in FIGS. 5A and 5B, the dispensing device 30 has, for example, ten micropipettes 34 arranged in five rows and two columns on the upper surface of a rectangular fixed plate 32 and arranged in the direction of each column. The pipette 34 group is configured to be fixed to the fixing plate 32 via the fixing jig 36.
[0042]
As shown in FIGS. 5C and 6, 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. .
[0043]
Accordingly, as shown in FIG. 6, 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 20 fixed below, for example, the fixed plate 32 through the through hole 40.
[0044]
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.
[0045]
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.
[0046]
Further, in this embodiment, the portion of the base body 50 that is in contact with the upper surface of the cavity 56 is thin, and has a structure that is susceptible 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.
[0047]
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.
[0048]
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.
[0049]
In addition to the vibration part 66, the actuator part 58 includes a lower electrode 70 directly formed on the vibration part 66, and a piezoelectric / electrostrictive layer, an antiferroelectric layer, or the like formed on the lower electrode 70. The layer 72 includes an upper electrode 74 formed on the upper surface of the piezoelectric layer 72.
[0050]
As shown in FIG. 5C, 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.
[0051]
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 cavity (pressurizing chamber) 56 in contact is reduced.
[0052]
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. The DNA chip 20 can be fabricated in which the microspots 80 are aligned and fixed on the substrate 50 such as a microscope slide glass.
[0053]
In this case, since the arrangement pitch of the sample discharge ports 54 in the dispensing apparatus 30 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 30. It will be.
[0054]
At this time, even if the shift width of the dispensing device 30 and the arrangement pitch of the sample discharge ports 54 vary, the minute spot 80 due to the supply of the sample solution is defined by the protrusion 14 formed on the substrate 10. And the positional deviation is corrected. Therefore, in this embodiment, the arrangement state of a large number of minute spots 80 formed on the substrate 10 can be brought into a state along a prescribed arrangement pitch.
[0055]
Furthermore, after the position correction, it is also preferable to add the moisture to the substrate 10 to facilitate the movement of the minute spot 80 and to move the minute spot 80 to the protrusion 14. Moreover, the effect that the cross-sectional shape of the minute spots 80 concentrated on the protrusions 14 can be corrected to a hemispherical shape can be expected, which is preferable.
[0056]
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).
[0057]
The cavity (pressurizing chamber) 56 is preferably formed with a flow path dimension so that the sample solution containing DNA fragments and the like moves in a laminar flow.
[0058]
That is, the size of the cavity 56 differs 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 is buffered (TE buffer) at a concentration of 1 μg / μl. When the sample dispersed in () is dropped at a pitch of several hundred μm at a pitch of several hundred μm, the cavity length (L) is 1 to 5 mm and the cavity width (W) is as shown in FIG. 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.
[0059]
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.
[0060]
Incidentally, as shown in FIG. 5A, 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. 5C) 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.
[0061]
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. 5A shows an example in which one unit is configured by five micropipettes 34 arranged in the row direction.
[0062]
In addition, when a plurality of micropipettes 34 are pressed into the holding plate 100, the introduction holes 104 (see FIG. 5B) for supplying the sample solution 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.
[0063]
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.
[0064]
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.
[0065]
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.
[0066]
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.
[0067]
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.
[0068]
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 fine spot 80 with high purity.
[0069]
In the example of FIG. 5A, both ends of the pressing plate 100 are fastened to the fixing plate 20 with screws 102, but the fixing method of the pressing plate 100 is not only mechanically performed with screws, springs, but also with an adhesive or the like. You may go.
[0070]
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.
[0071]
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.
[0072]
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.
[0073]
In addition, the piezoelectric layer 72 constituting the actuator unit 58 is made of, 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, etc., or a combination of any of these can be used. A material mainly composed of components composed of lead oxide, lead titanate and lead magnesium niobate is preferably used.
[0074]
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.
[0075]
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.
[0076]
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.
[0077]
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.
[0078]
Next, the first and second methods for manufacturing the DNA chip 20 using the dispensing device 30 will be described with reference to FIGS.
[0079]
First, as shown in FIG. 9, different types of sample solutions are filled into the cavities 56 of the micropipettes 34 from the tubes 106 through the introduction holes 104 of the fixing jig 36, respectively, as shown in FIG. Next, each actuator unit 58 is driven to discharge the sample solution from the sample discharge port 54 of each micropipette 34. As a method of filling the solution into the cavity 56, the solution may be injected by the capillary force of the solution introduced from the sample injection port 52, but a method of filling the solution by vacuum suction from the sample discharge port 54 is reliable.
[0080]
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 increasing the amplitude of the pulse, the deformation of the vibrating section 66 increases, and the amount of sample solution discharged increases accordingly. In addition, when a plurality of pulses are applied during a certain period, a large amount of a small amount of sample solution can be discharged by shortening the pulse period and reducing the amplitude of each pulse.
[0081]
At this time, by appropriately changing the supply position, the supplied sample solutions are combined on the substrate 10 (combined) to become a sample solution having one spot diameter, but depending on the type of the sample solution to be supplied, The spot diameter formed on the substrate 10 can be made uniform by controlling the number of supply, the supply position, and the amount of supply at one time.
[0082]
Next, a second method using the dispensing device 30 will be described. In this second method, as shown in FIG. 10, an aqueous solution or a polymer containing a buffer solution or NaCl is introduced from each tube 106 into the cavity 56 of each micropipette 34 through the introduction hole 104 of the fixing jig 36. A replacement liquid such as an aqueous solution is filled, and then the sample is injected from the sample injection port 52 into the cavity 56 while performing laminar flow replacement, and then the completion of the replacement is awaited. Thereafter, the actuator unit 58 is driven to discharge and supply the sample solution onto the substrate 10.
[0083]
The 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.
[0084]
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 increases due to the mixing of the sample and the replacement solution, but the increase in the purge amount can be suppressed to a minimum by detecting the change in the fluid characteristics in the cavity 56 and determining the completion of the replacement. it can.
[0085]
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 disclosed in, for example, Japanese Patent Application Laid-Open No. 8-201265.
[0086]
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 Impedance or resonance characteristics such as resonance frequency and attenuation rate are electrically measured.
[0087]
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.
[0088]
Then, the DNA chip 20 is manufactured by driving the actuator unit 58 under a driving condition corresponding to the amount of liquid droplets corresponding to the required spot diameter and repeating the supply of the sample solution. Usually, one microspot 80 is ejected from the micropipette 34 to form one minute spot 80.
[0089]
When the amount of the sample in the sample injection port 52 decreases, a buffer solution is added so that bubbles do not enter the flow path, and the discharge is continued, so that the sample solution is put into the micropipette 34. Can be used up without leaving. 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.
[0090]
Moreover, as a substitution liquid and a sample to be used, it is preferable to use a solution in which dissolved gas in the solution is removed in advance through a degassing operation. By using such a solution, when filling the solution into the flow path of the micropipette 34, even if bubbles get caught in the middle of the flow path and the filling is inadequate, the bubbles are dissolved in the solution. In addition to being avoidable, bubbles are not generated in the fluid in the middle of discharge, and no discharge failure occurs.
[0091]
In the second method described above, while discharging the sample solution, a replacement solution such as an aqueous solution containing a buffer solution or NaCl, or an aqueous solution containing a polymer is injected into the cavity 56 from the sample injection port 52, and the cavity 56 is discharged. The sample solution remaining inside can be completely discharged to prepare for the next sample injection.
[0092]
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 purge amount of the sample that is not used by the replacement completion detection mechanism can be extremely reduced, and the use efficiency of the sample solution can be improved.
[0093]
Further, when the sample is filled into the cavity 56 from the sample injection port 52, the sample may be replaced from the sample injection port 52 into the cavity 56 while the actuator unit 58 is driven. In this case, the inside of the cavity 56 can be surely replaced with an inexpensive replacement liquid in advance, and as a result, the occurrence of defective discharge can be completely prevented, and an expensive sample can be discharged efficiently.
[0094]
Further, the cavity 56 is filled with a replacement solution such as an aqueous solution containing a buffer solution or NaCl, or an aqueous solution containing a polymer, and the amount of the replacement solution in the cavity 56 and the sample inlet 52 is adjusted to a predetermined amount. Next, after injecting the sample solution by a predetermined amount from the sample injection port 52, the actuator unit 58 is driven by a predetermined number of pulses, and the amount of the replacement liquid in the cavity 56 and the sample injection port 52 is increased. It may be discharged.
[0095]
By doing so, the amount of the replacement liquid in the cavity 56 and the sample injection port 52 is accurately discharged, and the filling of the sample solution can be completed without waste.
[0096]
As described above, in the DNA chip 20 according to the present embodiment, the substrate 10 is provided with the protrusions 14 as the positional deviation correction means for automatically correcting the positional deviation of the minute spot 80. When the sample solution is supplied to the microspot 80, even if the supply position is deviated from the specified position, the microspot 80 due to the supply of the sample solution is moved to the specified position by the projection 14, and the misalignment is corrected. Will be.
[0097]
As described above, in the DNA chip 20 and the manufacturing method thereof according to the present embodiment, even if there are variations in the shifting width of the dispensing device 30 and the arrangement pitch of the sample discharge ports 54, they are formed on the substrate 10. The arrangement state of a large number of microspots 80 can be brought into a state along a predetermined arrangement pitch, and the quality of the DNA chip 20 and the yield can be improved.
[0098]
Next, a modified example of the positional deviation correction means provided on the substrate 10 will be described with reference to FIGS.
[0099]
In the first modification, as shown in FIG. 11, a hydrophilic region Z1 is formed at a position where a minute spot 80 is to be formed in the substrate 10, and a water-repellent region Z2 is formed at other portions. It is different. Specifically, this is achieved by forming the water repellent film 16 in a portion other than the position where the micro spot 80 is to be formed. As the water repellent film 16, for example, Si coat, fluororesin, or the like can be used.
[0100]
According to this, as shown in FIG. 11, when the micro spot 80 is formed on the substrate 10 by supplying the sample solution, a part of the micro spot 80 is applied to the water-repellent film 16 (2 The fine spot 80 is moved by the surface tension of the fine spot 80 and the water repellent action of the film 16, and the center of the fine spot 80 can be positioned at a predetermined position. In this case, even when a part of the minute spot 80 is applied to the water-repellent film 16, the film 16 is water-repellent, so there is no trace after the sample solution has moved, and the spot shape after immobilization is hydrophilic. Only the portion has a shape, and variations in shape are reduced.
[0101]
In the second modification, as shown in FIG. 12, in the water repellent film 16, a tapered surface 16a inclined downward toward the hydrophilic region Z1 is provided only in the peripheral portion of the hydrophilic region Z1. An example is shown. In this case, as indicated by the alternate long and short dash line, even when the minute spot 80 is applied to the water-repellent film 16, the water-repellent action of the water-repellent film 16, the surface tension of the minute spot 80, and the gravity due to the inclination of the tapered surface. Thus, the micro spot 80 moves to a position where the micro spot 80 is to be formed, and the center of the micro spot 80 can be positioned at a predetermined position.
[0102]
As shown in FIG. 13, the third modification is different in that a recess 18 is formed at a position on the substrate 10 where the minute spot 80 is to be formed.
[0103]
According to this, as shown in FIG. 13, when the micro spot 80 is formed on the substrate 80 by supplying the sample solution, a part of the micro spot 80 is applied to the shoulder portion of the recess 18 (2 The fine spot 80 is moved by the surface tension of the fine spot 80, and the fine spot 80 can be positioned in the recess 18. In this case, when the sample solution enters the recess 18, the sample film thickness is made uniform, and the variation in spot thickness can be reduced. As a result, there is an advantage that it is possible to suppress sensitivity variations and sensitivity degradation when inspecting fluorescence emitted by spots.
[0104]
As shown in FIG. 14, the fourth modification is an example in which a plurality of convex portions 150 are formed in the peripheral portion of the portion where the micro spot 80 is formed, or an annular convex portion 152 is formed in the peripheral portion. Show. In the case of the annular convex portion 152, the portion where the minute spot 80 is formed is formed in a crater shape. In the case of the fourth modified example, in addition to the effect of the third modified example described above, there is an effect that the minute spots 80 are easily collected at a predetermined position (position where the minute spots 80 are to be formed). In particular, if the skirt portions of the convex portions 150 and 152 are formed on the gentle inclined surface 154, the above effect becomes even more remarkable.
[0105]
The convex portions 150 and 152 as described above are formed on the substrate 12 by spotting by appropriately adjusting the jetting pressure, the distance between the jet nozzle and the substrate 12, etc., by an ink jet system that has been put to practical use in a printer. can do.
[0106]
As shown in FIG. 15, the fifth modification is different in that the surface state of the substrate 10 is different between the portion where the minute spot 80 is to be formed and the other portion.
[0107]
According to this, as shown in FIG. 15, when the micro spot 80 is formed on the substrate 10 by supplying the sample solution, a part of the micro spot 80 is applied to a portion other than the rough surface 22 ( The fine spot 80 is moved by the surface tension of the fine spot 80, and the center of the fine spot 80 can be positioned at a predetermined position. In this case, as in the first modified example described above, even when a part of the minute spot 80 is applied to a portion other than the rough surface 22, the portion other than the rough surface 22 has no trace after the sample has moved, The spot shape after immobilization becomes the shape of only the rough surface 22, and variation in shape is reduced. Further, since the contact surface of the micro spot 80 is a rough surface, the contact area is large, and the micro spot 80 is firmly fixed to the substrate 10, so that the sample solution can be prevented from flowing out at the time of fixation after spotting.
[0108]
The surface state changes such as the formation of the tapered surface 16a and the formation of the concave portion 18 and the rough surface 22 in the second, third, and fifth modified examples described above are processes such as blasting, laser processing, and cutting. Can process a large number of substrates at a time, which is preferable because it can be implemented at low cost.
[0109]
In the sixth modified example, misalignment correction is performed by applying an electric field. Specifically, for example, as shown in FIG. 16, a large number of electrode patterns 130 made of a metal film are formed in portions corresponding to locations where the micro spots 80 are to be formed in the lower layer of the poly-L-lysine layer 12 in the substrate 10. Further, a common electrode pattern 132 made of a metal film common to the electrode patterns 130 is formed in the lower layer.
[0110]
Then, a power source 134 is connected between the common electrode pattern 132 and, for example, ground, and each electrode pattern 130 is charged, for example, to the + side. In this state, when the micro spot 80 is formed on the substrate 10 by supplying the sample solution, the sample solution constituting the micro spot 80 is charged to the negative side, so that a part of the micro spot 80 is an electrode pattern. When placed on 130 (see the two-dot chain line), the fine spot 80 moves onto the electrode pattern 130 by the attractive force of the electric field, and the center of the fine spot 80 is positioned at a specified position.
[0111]
Further, if the electric field strength is increased, the droplet 140 ejected from the sample ejection port 54 of each micropipette 34 is corrected so that its flight direction is directed to the corresponding electrode pattern 130 in the air. The spot 80 will accurately fall on the corresponding electrode pattern 130. In this case, since the droplet 140 does not move on the surface of the substrate 10, there is no trace due to the droplet movement, and the spot shape can be stabilized (uniformized).
[0112]
As shown in FIG. 17, the seventh modified example has substantially the same principle as the sixth modified example (see FIG. 16) described above, but a predetermined electrode pattern 130 is placed on the stage 142 on which the substrate 10 is placed and fixed. And 132 are provided. In this case, since it is not necessary to provide the electrode patterns 130 and 132 on the substrate 10, there is an advantage that it can be implemented at low cost.
[0113]
In the eighth modification, as shown in FIG. 18, the substrate 10 is made of glass, for example, and predetermined electrode patterns 130 and 132 are formed in the substrate 10. In particular, the electrode pattern 130 is formed on the substrate 10. It is an example exposed on the surface. The voltage supply to the electrode patterns 130 and 132 is performed through the electrode 144 formed on the stage 142.
[0114]
In the eighth modified example, the glass surface of the substrate 10 exhibits water repellency, and the electrode pattern 130 exposed on the glass surface exhibits hydrophilicity. Therefore, for example, as shown in FIG. As shown, when the microspot 80 is formed on the substrate 10 by supplying the sample solution, when a part of the microspot 80 is applied to the water-repellent glass surface (see the two-dot chain line), Due to the surface tension of 80 and the water repellent action of the glass surface, the minute spot 80 moves and the center of the minute spot 80 can be positioned at a predetermined position. In this case, even when a part of the minute spot 80 is applied to the glass surface, the glass surface is water-repellent, so there is no trace after the sample solution has moved, and the spot shape after immobilization is only a hydrophilic part. It becomes a shape and variation in shape is reduced.
[0115]
Further, when the electric field strength is increased, as shown in FIG. 19B, as in the above-described sixth modification, the droplet 140 that is in the process of dropping should have its flight direction directed toward the corresponding electrode pattern 130 in the air. As a result of the correction, the minute spot 80 by the droplet 140 accurately falls on the corresponding electrode pattern 130. In this case, since the droplet 140 does not move on the surface of the substrate 10, there is no trace due to the droplet movement, and the spot shape can be stabilized (uniformized).
[0116]
As described above, the eighth modification example is more preferable because it combines the effects of the first modification example and the sixth modification example described above. The material of the substrate 10 is not limited to glass but may be an insulator such as ceramics or plastic.
[0117]
As described above, even when the positional deviation correction means according to the first to eighth modifications are used, the shift width of the dispensing device 30 and the arrangement pitch of the sample discharge ports 54 vary as in the above-described embodiment. Even if there is, the arrangement state of a large number of microspots 80 formed on the substrate 10 can be brought into a state along a prescribed arrangement pitch, and the quality of the DNA chip 20 and the yield can be improved. Can do.
[0118]
In addition, the DNA chip and the manufacturing method thereof according to the present invention are not limited to the above-described embodiments, and various configurations can be adopted without departing from the gist of the present invention.
[0119]
【The invention's effect】
As described above, according to the DNA of the present invention, even if there are variations in the shifting width of the dispensing device and the arrangement pitch of the dropping nozzles, the arrangement state of a large number of minute spots formed on the substrate is defined. It can be made to be in a state along the arrangement pitch.
[0120]
In addition, according to the DNA production method of the present invention, even if there are variations in the shifting width of the dispensing device and the arrangement pitch of the dropping nozzles, the arrangement state of a large number of minute spots formed on the substrate is defined. It is possible to achieve a state along the arrangement pitch, and it is possible to improve the quality of the DNA chip and the yield.
[Brief description of the drawings]
FIG. 1 is a perspective view showing a DNA chip according to the present embodiment (a DNA chip manufactured by the manufacturing method according to the present embodiment).
FIG. 2 is an enlarged cross-sectional view showing a configuration of a DNA chip according to the present embodiment.
FIG. 3 is a process block diagram showing a DNA chip manufacturing method according to the present embodiment.
FIG. 4 is a process block diagram showing a breakdown of a sample preparation process.
FIG. 5A is a plan view showing a configuration of a dispensing apparatus used in the method for producing a DNA chip according to the first embodiment, FIG. 5B is a front view thereof, and FIG. It is an enlarged plan view which shows one micropipette which comprises an injection apparatus.
FIG. 6 is a longitudinal sectional view showing a configuration of a micropipette.
FIG. 7 is a perspective view showing the shape of a flow path including a cavity formed in a base of a micropipette.
FIG. 8 is an exploded perspective view of the dispensing device shown together with the cartridge.
FIG. 9 is an explanatory diagram showing a first method when a DNA chip is manufactured using a dispensing apparatus.
FIG. 10 is an explanatory view showing a second method when a DNA chip is manufactured using a dispensing apparatus.
FIG. 11 is a cross-sectional view showing a misregistration correction unit according to a first modification.
FIG. 12 is a cross-sectional view showing a misregistration correction unit according to a second modification.
FIG. 13 is a cross-sectional view showing a misregistration correction unit according to a third modification.
FIG. 14 is a cross-sectional view showing a misregistration correction unit according to a fourth modification.
FIG. 15 is a cross-sectional view showing a misregistration correction unit according to a fifth modification.
FIG. 16 is a cross-sectional view showing a misregistration correction unit according to a sixth modification.
FIG. 17 is a cross-sectional view showing a misregistration correction unit according to a seventh modification.
FIG. 18 is a cross-sectional view showing a misregistration correction unit according to an eighth modification.
FIG. 19A is a diagram showing that the misalignment correction unit according to the eighth modification has the same effect as the first modification, and FIG. 19B is a misalignment correction according to the eighth modification. It is a figure which shows having the effect similar to the 6th modification in a means.
[Explanation of symbols]
10 ... Substrate 12 ... poly-L-lysine layer
14 ... Protrusions 16 ... Water-repellent film
18 ... recess 20 ... DNA chip
22 ... Rough surface 30 ... Dispensing device
34 ... Micro pipette 54 ... Sample outlet
58 ... Actuator part 80 ... Minute spot
130 ... Electrode pattern 132 ... Common electrode pattern
134 ... Power supply 140 ... Droplet
142 ... stage S1 ... pretreatment process
S2 ... Sample preparation process S3 ... Supply process
S11 ... Amplification process S12 ... Powder production process
S13 ... mixing process

Claims (7)

In a DNA chip in which a large number of micro spots are formed on a substrate by supplying a sample solution,
The substrate may have a positional deviation correcting means for automatically correcting the positional deviation of the minute spot, the positional deviation correcting means includes a surface to be contacted of the minute spot is rough, the in crude surface A DNA chip characterized in that a contact area of a minute spot is larger than a portion other than the rough surface . In a DNA chip in which a large number of micro spots are formed on a substrate by supplying a sample solution,
The substrate includes a position deviation correction unit that automatically corrects a position deviation of the minute spot, and the position deviation correction unit is a protrusion formed at a position on the substrate where the minute spot is to be formed. A DNA chip characterized by In a method for producing a DNA chip in which a large number of sample solutions are supplied onto a substrate to produce a DNA chip,
As the substrate, it has a positional deviation correction means for automatically correcting the positional deviation of the micro spot due to the supply of the sample solution, the positional deviation correction means, the surface to be contacted by the micro spot is a rough surface, A method for producing a DNA chip, wherein a contact area of the minute spot on the rough surface is larger than that of a portion other than the rough surface. In a method for producing a DNA chip in which a large number of sample solutions are supplied onto a substrate to produce a DNA chip,
The substrate includes a position deviation correction unit that automatically corrects a position deviation of the minute spot, and the position deviation correction unit is a protrusion formed at a position on the substrate where the minute spot is to be formed. A method for producing a DNA chip, characterized in that The method for producing a DNA chip according to claim 3 or 4 ,
A method for producing a DNA chip, wherein the sample solution is supplied by an ink jet method using a supply device. In the manufacturing method of the DNA chip of Claim 5 ,
The supply device includes:
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. A piezoelectric / electrostrictive element is provided on at least one wall surface of the substrate to be formed, and a plurality of micropipettes configured to move the sample solution are arranged in the cavity, and each micropipette has a discharge port. A method for producing a DNA chip, characterized by being a dispensing device that discharges different types of sample solutions. In the manufacturing method of the DNA chip of Claim 6 ,
The method of manufacturing a DNA chip, wherein the dispensing device is configured by arranging a plurality of micropipettes configured so that the sample solution moves in a laminar flow in the cavity.

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