JP2007292594A - Liquid imparting device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To shorten a DNA chip production time to enhance productivity by promoting a reaction of a substrate with a probe in a probe solution, and to reduce a required concentration of the probe in the probe solution to reduce a cost of a DNA chip by promoting the reaction of the substrate with the probe in the probe solution, in a liquid imparting device used in DNA chip production. <P>SOLUTION: This liquid imparting device includes an oscillator in a substrate mounting part, and a mechanism for controlling a frequency/amplitude/time of the oscillator inside or outside the liquid imparting device, and the reaction of the substrate with the probe in the probe solution is promoted by imparting vibration to the substrate. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a liquid application apparatus for applying a probe solution on a substrate and a method for manufacturing a probe carrier on which a probe is fixed.

  Probes for detecting target nucleic acids are placed on a solid-phase carrier, such as a DNA chip, as one of the technologies that enables rapid and accurate determination of nucleic acid base sequences, detection of target nucleic acids in samples, and identification of various bacteria. Then, there is a method of reacting with a sample to detect or analyze nucleic acids in the sample. A probe is a substance that specifically binds to a target nucleic acid having a specific base sequence, and a probe array carrier is formed by arranging a plurality of types thereof in an array on a solid phase. By using this carrier, it is possible to simultaneously evaluate the specific binding ability of a nucleic acid in a sample to a plurality of types of probes. A probe carrier, also called a probe array, is a glass substrate, plastic substrate, membrane, etc., which is a probe that consists of several tens to thousands of different types of DNA fragments, etc., that are fixed at high density as spots. is there.

  In recent years, research on detection and quantification of target substances using such probe arrays has been energetically performed. For example, Patent Document 1 discloses a method for preparing a probe array by sequential DNA extension reaction on a solid support using photolithography. Patent Document 2 discloses a probe array manufacturing method for supplying DNA onto a membrane using a capillary. Patent Document 3 discloses a method for preparing a probe array for solid-phase synthesis of multiple types of DNA using a piezo jet nozzle, and Patent Document 4 discloses that a liquid containing a probe is applied to a solid phase as a droplet by an inkjet head. A method of making a probe array to be attached is described. Furthermore, Patent Document 5 describes a method of immobilizing a gene by immersing a carrier containing a functional group in a solution containing the gene and accelerating the reaction between the functional group and the gene while irradiating the solution with ultrasonic waves. Has been.

  A conventional discharge device (bubble jet method) will be described below as an example of producing a DNA chip. FIG. 1 is a perspective view of the discharge device. On the surface plate 80, a Y-axis stage 81 and a guide rail 82 are fixed in parallel. An X-axis stage 83 is attached to the movable parts of the Y-axis stage 81 and the guide rail 82, and the X-axis stage 83 is movable in the Y-axis direction. A chuck 84 is fixed to the X-axis stage 81. The chuck 84 is connected to a pump (not shown) by a tube, and the substrate 85 is adsorbed to the chuck 84 when the pump sucks air.

  Posts 86 and 87 are fixed on the surface plate 80, and bridges 88 and 89 are fixed to the columns 86 and 87, respectively. The bridges 88 and 89 are fixed by a stay 92, and the strength of the structures of the columns 86 and 87 and the bridges 88 and 89 is maintained. A head mounting base 90 is fixed between the bridges 88 and 89, and a head 91 is fixed to the head mounting base 90.

  The probe solution is injected into the head 91, and the head 91 is mounted on the discharge device. The probe solution is discharged to a predetermined position on the substrate 85 by operating the Y-axis stage 81 and the X-axis stage 83 and discharging the probe solution from the head 91.

Since the mechanism for mounting the substrate 85 is not shown in detail in FIG. 1, this mechanism will be described in detail with reference to FIG. FIG. 2 is a sectional view of the periphery of the substrate. A substrate 85 is disposed on the chuck 84. The head 91 is provided with a plurality of nozzles 93. A probe solution supply port 94 communicates with each nozzle 93. A heater unit (not shown) is provided in the vicinity of the nozzle 93. By filling the probe solution supply port 94 with the probe solution 95, the probe solution 95 is filled into the nozzle 93. The probe solution 95 is discharged onto the substrate 85 from the nozzle 93 by causing film boiling in the probe solution 95 by a heater (not shown). The discharged probe solution 95 is disposed on the substrate 85 as a spot 96. FIG. 3 is a layout view of the substrate 85 on the chuck 84. As shown in FIG. 3, the substrate 85 is disposed on the chuck 84, and the spot 96 is disposed on the substrate 85.
US Pat. No. 5,424,1865 WO95 / 35505 pamphlet European Patent No. 0703825 JP 11-187900 A JP 04-045800

  As described above, in the ejection device, the probe solution is ejected onto the substrate, and the probe is fixed on the substrate by reacting the substrate with the probe in the probe solution at room temperature. In the spotting apparatus, the probe solution is spotted on the substrate with a pin, and the probe is fixed on the substrate by bringing the substrate into contact with the probe in the probe solution at room temperature. The probe may be fixed to the substrate from within the probe solution depending on the type of substrate or sample, the concentration, the fixing method, and the like, but may require 12 hours at room temperature. Therefore, in order to improve the productivity of the probe array in the liquid application device including the discharge device and the spotting device, it is necessary to shorten the time required for the processing for fixing the probe from the probe solution to the substrate. .

  This processing time depends on the time required for these reactions in the case where the probe is immobilized on the substrate surface using a chemical reaction. In general, immobilization of a probe on a substrate by a chemical reaction cannot be performed unless the probe can contact the substrate surface. Unreacted probes are finally washed away from the substrate. If the reaction rate is low, an expensive sample such as DNA cannot be used effectively. Therefore, it is possible to increase the amount of probe immobilized on the substrate by increasing the chance of the probe contacting the substrate surface.

  Patent Document 5 describes a method of immobilizing a probe by immersing a substrate containing a functional group in a solution containing the probe and promoting the reaction between the functional group and the probe while irradiating the solution with ultrasonic waves. Yes. However, in this method, since the substrate is immersed in a solution containing the probe, a large amount of probe is required. Further, when different types of probes are immobilized on a substrate, it is necessary to change and immerse the solution many times, which is not suitable for improving the productivity of the probe array.

  An object of the present invention is to provide a liquid application apparatus and a probe carrier manufacturing method capable of reducing the time required for fixing a probe to a substrate at the time of manufacturing the probe carrier.

The liquid application apparatus of the present invention capable of achieving the above object is a liquid application apparatus for applying a probe solution containing a probe capable of specifically binding to a target substance on a substrate.
A substrate mounting portion that can removably fix the substrate;
A liquid application unit for applying a probe solution to the substrate fixed to the substrate mounting unit;
A vibrator for applying vibration to a substrate fixed to the substrate mounting portion;
It is a liquid application apparatus characterized by having.

One aspect of the method for producing a probe carrier of the present invention is a method for producing a probe carrier in which a probe capable of specifically binding to a target substance is immobilized on a substrate,
A) providing a probe solution containing a probe that can specifically bind to a target substance on a substrate;
B) The probe solution imparts vibration to the applied substrate;
It is a manufacturing method of the probe carrier characterized by having.

Another aspect of the method for producing a probe carrier of the present invention is a method for producing a probe carrier in which a probe capable of specifically binding to a target substance is immobilized on a substrate,
It is a method for producing a probe carrier, comprising a step of applying a probe solution containing a probe capable of specifically binding to a target substance on a substrate in a state where vibration is applied.

  By applying vibration to the substrate during or after application of the solution containing the probe on the substrate, the chance of contact between the probe in the solution applied to the substrate surface and the substrate surface increases. The amount of probe immobilized increases. In addition, since the reaction time between the substrate surface and the probe is shorter than before, the productivity of the probe array can be improved. Further, since the reaction efficiency between the substrate and the probe in the probe solution is improved by promoting the reaction between the substrate and the probe in the probe solution, the probe concentration in the probe solution can be reduced. As a result, the amount of the probe in the probe solution can be reduced and the cost can be reduced. Further, the increase in the amount of immobilization and the improvement in productivity described above can be carried out with many types of probes, which is advantageous for the production of probe arrays.

  The liquid application apparatus according to the present invention is fixed to the substrate mounting unit, the substrate mounting unit capable of detachably fixing the substrate, the liquid application unit for applying the probe solution to the substrate fixed to the substrate mounting unit, and the substrate mounting unit. And at least a vibrator for applying vibration to the substrate. With the probe solution spot placed on the substrate, the substrate is vibrated to increase the chance of contact between the probe in the probe solution and the substrate, thereby shortening the processing time for immobilizing the probe. be able to. This application of vibration to the substrate is particularly effective when the probe is fixed to the substrate using a chemical reaction, for example, when using a covalent bond. The application of vibration to the substrate may be from the start to the completion of the application operation of the probe solution to the substrate from the liquid application unit or after the spot of the probe solution is formed on the substrate. That is, the vibration may be applied in a state where the probe solution spot is on the substrate. In this way, by applying vibration to the substrate during or after applying the probe-containing solution on the substrate, the chance of contact between the substrate surface and the probe increases, and the amount of probe immobilized on the substrate increases. did. Moreover, the productivity of the probe array can be improved by shortening the reaction time compared to the conventional case.

  The substrate mounting portion in which the substrate can be detachably fixed in the liquid application apparatus of the present invention preferably has a configuration in which the substrate and the substrate mounting portion can be detachably attached. For example, after mounting the substrate on the substrate mounting part, the form in which the substrate is brought into close contact using the suction force from the substrate mounting part side, or the form in which the substrate is pressed from the substrate surface side to the substrate mounting part with a jig or the like is mentioned. It is done.

  Examples of the vibrator include those having various configurations such as a piezoelectric vibrator, an attractive force, and an electromagnetic force, and there is no particular limitation as long as it can vibrate the substrate. If the vibrator is provided on the substrate mounting portion, an object smaller than the size of the substrate is preferable. In the substrate mounting portion and the vibrator of the present invention, one vibrator may be provided in one substrate mounting portion, or a plurality of vibrators may be provided in one substrate mounting portion. Furthermore, one vibrator may be provided for a plurality of substrate mounting portions, or a vibrator may be provided for each of the plurality of substrate mounting portions. Although a plurality of these substrate mounting portions and vibrators may be provided, it is preferable that the substrate is provided with the most efficient vibration. Alternatively, the substrate may be vibrated by bringing a vibrator into contact with the substrate mounting portion or the substrate fixed to the substrate mounting portion when applying vibration.

  There are several timings for applying vibration to the substrate of the present invention. When a vibrator is provided on the substrate mounting portion, vibration is started after the substrate is mounted on the substrate mounting portion, and vibration is continuously applied while the probe solution is applied onto the substrate, and the substrate is recovered after application. There is a way to stop the vibration. Alternatively, there is a method in which vibration is not applied while the probe solution is applied to the substrate, and vibration is applied for a certain time after application. Further, without providing the vibrator on the substrate mounting portion, the substrate may be moved to another place where the vibration is applied after the probe solution is applied to the substrate to apply the vibration. For the movement of the substrate, a method of moving the whole substrate mounting portion may be adopted, or a method of moving the substrate to another substrate mounting portion may be adopted. For applying vibration at another location, it is possible to use a method in which a vibrator is provided in the substrate mounting portion for applying liquid, and after applying the liquid, the substrate mounting portion is moved to another location to apply vibration. it can. Alternatively, a substrate mounting portion for applying vibration is prepared, and a vibration is applied by providing a vibrator on the substrate mounting portion, or indirectly through the substrate mounting portion by a separately provided vibrator, Alternatively, a method of directly applying vibration to the substrate may be used.

  A method of collecting the substrate immediately after application by applying vibration during application of the probe solution is preferable because the production rate of the probe carrier is the shortest.

The position where the vibrator is provided on the board mounting portion is not particularly limited as long as it is a position where vibration can be applied to the board, but the displacement of the natural mode of the board mounted on the board mounting portion is substantially the maximum position. Is more preferable. For example, in a synthetic quartz substrate having the following size and physical properties, when a secondary bending mode is desired to be generated, the position of the arrow in FIG. 4 is the maximum position of the displacement of the substrate eigenmode. Is preferably arranged.
Synthetic quartz substrate:
Size: 1 x 3 inches (25.4 mm wide x 76.2 mm long x 1.0 mm thick)
Physical properties: longitudinal elastic modulus (7.0E10Pa), density (2201kg / m ³ ), Poisson's ratio (0.16)
The number of vibrators may be one, and more vibrations can be generated by arranging a plurality of vibrators.

  In addition, when the vertical length of the substrate is L and the probe solution is applied at an Xmm pitch as shown in FIG. 6, the vibrator is arranged at a position where a vibration mode of Lmm / Xmm = Y order or higher is generated. It is preferable because the probe solution applied on the substrate can be vibrated uniformly. As the vibration mode, a different mode can be generated depending on the position of the vibrator, and either the torsion mode as shown in FIG. 5 or the bending mode as shown in FIG. 4 may be used.

  The mechanism for controlling the vibrator of the present invention may be provided in the liquid applying apparatus, or may be provided so as to control only separately. The control item targeted by the control mechanism is characterized by controlling at least one of the frequency (frequency), amplitude, and time of the vibrator. Two or more items may be controlled simultaneously.

  The frequency of the vibrator that can achieve the effect of the present invention is a frequency that substantially matches the natural frequency of the substrate. When a secondary bending mode as shown in FIG. 4 is desired to be generated in the synthetic quartz substrate, vibration is preferably applied to the substrate at a frequency of 2.75 kHz. In addition, when the primary torsional mode as shown in FIG. 5 is desired to be generated in the synthetic quartz substrate, vibration is preferably applied to the substrate at a frequency of 1.92 kHz. When it is desired to generate a higher-order vibration mode as shown in FIG. 6 on the synthetic quartz substrate, it is preferable to apply vibration to the substrate at a frequency in the ultrasonic region of 20 kHz or higher. Furthermore, it is more preferable to apply vibration to the substrate at a frequency of 20 kHz to 80 kHz. When the vibration is applied to the substrate at a high frequency, the amplitude of the substrate is reduced, and when the vibration is applied to the substrate at a low frequency, the amplitude of the substrate is increased. When it is desired to uniformly apply vibration to the probe solution applied to the substrate, it is more preferable to apply vibration to the substrate at a high frequency.

  When applying vibration using a plurality of vibrators, it is necessary to consider the phase difference between the amplitudes of vibrations given by the vibrators.

  The time for vibrating the vibrator is a time necessary for effectively increasing the chance of contact between the probe in the probe solution applied on the substrate and the substrate surface. By increasing the chance of contact between the probe and the substrate surface due to vibration, more probe molecules can be brought into contact with the substrate surface in a shorter time. As a result, the probe can be fixed to the substrate surface by a chemical reaction in a shorter time.

  In the immobilization by chemical reaction, the binding reaction between the functional group on the substrate surface and the functional group of the probe is promoted, and the amount of probe immobilization increases. Further, the activation energy required for the reaction is exceeded by the vibration energy, and the reaction rate is accelerated. Thus, by promoting the binding reaction, the reaction time between the substrate surface and the probe is shortened, and the productivity of the probe array can be improved.

  The time required for the binding reaction in a state where no vibration is applied varies depending on the combination of the substrate and the probe. For example, as disclosed in JP-A-11-187900, when an oligonucleotide having a maleimide group introduced on a quartz substrate and a mercapto group introduced at the end via a linker is used, no vibration is applied. Then, it reacts in about 30 minutes and is fixed.

  In addition, as disclosed in JP-A-2003-161731, when an oligonucleotide having a formyl group introduced into a plastic substrate and an amino group introduced into the terminal via a linker is used, the reaction is performed for 30 minutes to 60 minutes. I am letting. In addition, although the example of the DNA chip which fixed several DNA to the fixed board | substrate was given here, as a board | substrate described here, a probe is fixed and a target substance is detected or isolate | separated using the obtained probe fixed board | substrate. There is no particular limitation as long as there is no problem. For example, taking a microarray as an example, in consideration of detection of target substances and versatility, a glass substrate and a plastic substrate are preferable, and an alkali-free glass substrate and a quartz substrate that do not contain an alkali component are particularly preferable. Probes are not limited to DNA, but are specific to target substances including biopolymers such as proteins, peptides, antigens, antibodies, PNA, RNA, and sugar chains (including complex sugar chains). A substance that can be bound is selected according to the type of target substance and used as a probe.

  Thus, the reaction time in a state where vibration is not applied varies depending on the combination of the substrate and the sample. In addition, physical properties such as sample concentration and viscosity of the probe solution are also affected. For this reason, since the vibration time applied to the substrate also differs depending on the combination of the substrate and the sample, it is preferable to set the time for applying the vibration according to the combination of the substrate and the probe.

  In addition, since it is possible to set multiple types of vibration application time for each substrate depending on the combination of the substrate mounting part and the vibrator installation, the time for promoting the reaction between the substrate and the probe in the probe solution is set for each substrate. It is possible to simultaneously manufacture different combinations of probe arrays.

  As a device for applying liquid, as described above, a device that discharges a solution onto a substrate from a nozzle provided in a discharge portion (a discharge device), a device that spots a solution on a substrate by a pin method or the like (spotting device) Etc. are known. The method for applying the probe solution onto the substrate is not limited. A discharge device based on a so-called inkjet method, in which energy for discharge is applied to the liquid in the nozzle from a piezoelectric body or a heating element to discharge from the nozzle opening (discharge port), is a substrate and a discharge unit (liquid application unit). The liquid can be applied as a non-contact state. Among these, the liquid application method by the discharge unit including the electrothermal converter for generating thermal energy for liquid discharge uses the film boiling caused by the thermal energy applied by the electrothermal converter to the liquid application unit. In this method, liquid is ejected from a nozzle provided. According to the ejection method using this thermal energy, the liquid application part and the substrate can be applied in a non-contact state, and the amount of liquid discharged from the nozzle can be precisely controlled. It is particularly suitable. In addition, since the liquid can be applied in a non-contact state with the substrate as described above, it is possible to eliminate the influence on the liquid application portion due to the vibration of the substrate when the liquid is applied to the substrate in the vibration state.

  The liquid applicator according to the present invention may be provided with a plurality of liquid applicators.

  The liquid applicator of the present invention can further be provided with means for preventing the liquid from evaporating. As described above, if vibration is applied to promote the reaction, energy is transmitted to the droplets on the substrate, heat is generated, and components such as water in the droplets are likely to evaporate. In the case of immobilization by a chemical reaction, when a component such as water evaporates from a droplet, the reaction between the substrate and the probe may not easily proceed. In order to cope with such a case, it is preferable that the liquid application apparatus has a humidifying function or the substrate mounting portion of the liquid application apparatus is a closed system in order to prevent evaporation of water or the like. Alternatively, there is a method of providing a cooling means for cooling the substrate or the substrate mounting portion for the purpose of preventing evaporation.

  Embodiments of the present invention will be described below, but the technical scope of the present invention is not limited thereto.

  First, a method for manufacturing a probe carrier using a liquid application apparatus having a configuration in which a vibrator is provided on a substrate mounting portion will be described below with reference to FIGS.

  FIG. 7 is a perspective view of a liquid application device (bubble jet system) that uses thermal energy generated by the heating element as liquid ejection energy. On the surface plate 80, a Y-axis stage 81 and a guide rail 82 are fixed in parallel. An X-axis stage 83 is attached to the movable parts of the Y-axis stage 81 and the guide rail 82, and the X-axis stage 83 is movable in the Y-axis direction. A chuck 84 is fixed to the movable part of the X-axis stage 81, and a substrate fixing part (not shown) is disposed on the chuck 84. The substrate fixing portion on the chuck 84 is connected to a pump (not shown) by a tube (not shown), and the substrate 85 is adsorbed to the substrate fixing portion on the chuck 84 by the pump sucking air. The substrate fixing portion on the chuck 84 is provided with a vibrator (not shown) and a mechanism for controlling the vibrator. The mechanism for controlling the vibrator may be provided on the chuck 84, the substrate fixing portion, or on a discharge device other than the chuck 84. Further, the vibrator and the mechanism for controlling the vibrator may be provided at another place in the discharge device.

  Posts 86 and 87 are fixed on the surface plate 80, and bridges 88 and 89 are fixed to the columns 86 and 87, respectively. The bridges 88 and 89 are fixed by a stay 92, and the strength of the structures of the columns 86 and 87 and the bridges 88 and 89 is maintained. A head mounting base 90 is fixed between the bridges 88 and 89, and a head 91 is fixed to the head mounting base 90.

  The probe solution is injected into the head 91, and the head 91 is mounted on the discharge device. The probe solution is discharged to a predetermined position on the substrate 85 by operating the Y-axis stage 81 and the X-axis stage 83 and discharging the probe solution from the head 91. In order to prevent the probe solution on the substrate 85 from drying, a cover may be disposed on the substrate 85 after the probe solution is discharged onto the substrate 85. Further, the vicinity of the probe solution on the substrate 85 may be humidified by a humidifier (not shown).

  FIG. 8 is a sectional view of the periphery of the substrate. A substrate 85 is disposed on the chuck 84. The head 91 is provided with a plurality of nozzles 93. A probe solution supply port 94 communicates with each nozzle 93. In the vicinity of the nozzle 93, a heater unit (not shown) for generating thermal energy for liquid discharge is provided. By filling the probe solution supply port 94 with the probe solution 95, the probe solution 95 is filled into the nozzle 93. The probe solution 95 is discharged onto the substrate 85 from the nozzle 93 by causing film boiling in the probe solution 95 by a heater (not shown). The discharged probe solution 95 is disposed on the substrate 85 as a spot 96.

  Below the substrate 85, the vibrator 1 is provided near the position where the displacement of the eigenmode of the substrate is substantially maximum. A substrate mounting portion 2 is provided around the vibrator 1, and a substrate fixing portion (suction hole) 12 is provided inside the substrate mounting portion 2. The vibrator 1 and the substrate fixing unit 12 are electrically connected to a feedback control unit (not shown). The vibrator 1, the substrate fixing unit 12, and the feedback control unit (not shown) may be provided for each substrate or may be provided for each of a plurality of substrates.

  FIG. 9 is a layout view of the substrate 85 on the chuck 84. As shown in FIG. 9, the substrate 85 is disposed on the chuck 84, and the spot 96 is disposed on the substrate 85. The vibrator 1 and the substrate mounting portion 2 are provided on the chuck 84. In order to show the positional relationship among the vibrator 1, the substrate mounting portion 2, the substrate 85, and the spot 96, a part of the substrate 85 and the spot 96 are removed. A substrate fixing portion (suction hole) 12 is disposed inside the substrate mounting portion 2. When the substrate 85 is installed on the substrate mounting portion 2, the substrate is fixed on the chuck 84 by suction at the substrate fixing portion 12. As shown in FIG. 9, the vibrators 1 may be arranged one by one at both ends in the longitudinal direction of the substrate 85 or at four corners of the substrate 85. Further, the vibrator 1 may be a size that is in contact with a part of the substrate, or may be disposed so as to be in contact with the substrate 85 as a whole like the vibrator 1-a. The vibrator 1-a shows a state in which two piezoelectric elements that expand and contract in the longitudinal direction when a voltage is applied are combined.

  Although the apparatus having the configuration shown in FIGS. 7 to 9 has been described as having a configuration in which a transducer is arranged in the device, the transducer is arranged in such a device for manufacturing the probe carrier of the present invention. An apparatus having a configuration is preferred. However, a vibration applying device for applying vibration to the substrate may be prepared separately, and the probe solution may be applied to the substrate and then the vibration may be applied to the substrate in the vibration applying device.

  FIG. 10 is a flowchart of an example of the control program for the liquid application operation and the vibration application operation according to the present invention. First, in step 1, the substrate 85 is installed on the substrate mounting unit 2 and fixed by the substrate fixing unit 12. In step 2, it is determined whether vibration is applied during ejection of the probe solution. If vibration is to be applied while the probe solution is being discharged, the frequency and vibration time of the vibrator are set in step 3. The frequency and vibration time can be set to different values for each substrate. In step 4, vibration is started. When the vibration is started, the probe solution 95 is discharged from the nozzle 93 onto the substrate 85 in step 5. In step 6, it is determined whether or not the set vibration time has ended (in this case, if the probe solution discharge has ended), the vibration ends. If the set time has expired, the process proceeds to steps 7 and 8 and is completed. At this time, when the vibration time is set for each substrate 85 in the apparatus in which the vibrator 1 is provided for each substrate 85, the process proceeds to steps 7, 8, and 9 in order from the substrate 85 from which the probe solution has been discharged.

  In Step 2, when vibration is not given during ejection of the probe solution and vibration is given after ejection, the process proceeds to Step 10. In step 10, the probe solution 95 is discharged from the nozzle 93 onto the substrate 85. When the discharge to all the substrates 85 is completed, the process proceeds to step 12. In step 12, it is determined whether or not vibration is applied by the vibrator 1 on the discharge device. If vibration is to be applied by the vibrator on the discharge device, the process proceeds to step 13 where the frequency and vibration time are set. The frequency and vibration time can be set to different values for each substrate. In step 14, vibration is started, and it is determined whether or not the time set in step 15 has ended. If it has ended, the process proceeds to steps 7, 8, and 9. If not completed in step 15, the process returns to step 14, and step 15 and subsequent steps are repeated. At this time, when the vibration time is set for each substrate 85 in the apparatus in which the vibrator 1 is provided for each substrate 85, the process proceeds to steps 7, 8, and 9 in order from the substrate 85 that has completed the set time.

  If it is determined in step 12 that vibration is applied to the substrate 85 without using the vibrator 1 on the ejection device, the process proceeds to step 16. In steps 16 and 17, the substrate fixing is released, and the substrate 85 is moved to a device that can be subjected to another vibration. In step 18, the substrate 85 is installed and fixed, and the process proceeds to step 13. The following goes through the same steps.

  The above-described configuration in which the vibrator is provided on the substrate mounting portion can also be used for applying vibration at another place in Step 18.

  Note that the flowchart shown in FIG. 10 can be variously changed. Further, the execution of each step based on this flowchart can be automatically performed by an instruction from a computer based on a preset program. Based on an instruction from the computer, a vibrator control mechanism having means for applying a signal for vibration such as an electric signal to the vibrator can be operated.

Hereinafter, the present invention will be described in more detail with reference to examples.
Example 1
The probe carrier prepared by executing the above steps 1, 2, 10-15, and 7-9 will be described.
(1) Preparation of probe Using a DNA automatic synthesizer (Thiol-Modifier (manufactured by Glen Research)), single-stranded DNA with SEQ ID NO: 1 having a mercapto (SH) group introduced at the end was prepared. Ordinary deprotection was performed, and DNA was collected and purified by high performance liquid chromatography to obtain a probe.
5'HS- (CH2) 6-O-PO2-O-ACTGGCCGTCGTTTTACA3 '(SEQ ID NO: 1)
(2) Preparation of target substance (target) Using an automatic DNA synthesizer, a single-stranded DNA having a base sequence complementary to the single-stranded DNA of SEQ ID NO: 1 is synthesized, and Cy3 is bound to the 5 'end. The target was labeled with a fluorescent substance. The target labeled with this fluorescent substance was dissolved in a 1M NaCl / 50 mM phosphate buffer solution (pH 7.0) to a final concentration of 5 nM to prepare a sample solution.
(3) Fixation of probe to carrier (3-1) Washing of carrier As a carrier, a synthetic quartz glass substrate of 1 inch × 3 inch square is used, and pure water brush washing, pure water rinsing, alkalinity is applied to this substrate. Detergent ultrasonic cleaning, pure water rinse, pure water ultrasonic cleaning, pure water rinse, and nitrogen blow drying were sequentially performed to prepare a quartz glass substrate having a clean surface.
(3-2) Surface treatment Aminosilane coupling agent (trade name: KBM-603: manufactured by Shin-Etsu Chemical Co., Ltd.) is dissolved in water so as to be 1 wt%, and stirred for 30 minutes to hydrolyze the methoxy group. The quartz glass substrate subjected to the above cleaning was immersed in the aqueous solution for 30 minutes. Thereafter, the substrate was washed with pure water and heated in an oven at 120 ° C. for 1 hour to obtain a quartz glass substrate into which amino groups had been introduced. Next, 2.7 mg of N-maleimidocaproyloxysuccinimide (Dojin) (hereinafter abbreviated as EMCS) is weighed and dissolved in a solvent to a final concentration of 0.3 mg / ml to prepare an EMCS solution. did. As a solvent, a 1: 1 solution (volume basis) of dimethyl sulfoxide (DMSO) / ethanol was used. The quartz glass substrate with amino groups introduced into the EMCS solution is immersed for 2 hours at room temperature, then washed sequentially with a DMSO / ethanol mixed solution and ethanol, dried in a nitrogen atmosphere, and maleimide is added to the quartz glass substrate with amino groups introduced. A group was introduced to obtain a surface-treated quartz glass substrate.
(3-3) Probe fixation The probe solution was prepared by dissolving the single-stranded DNA (SEQ ID NO: 1) prepared in (1) above in a solvent to a concentration of 4.38 mol / L. As a solvent, an aqueous solution containing glycerin 7.5 wt%, urea 7.5 wt%, thiodiglycol 7.5 wt%, acetylene alcohol (trade name: acetylenol E100: manufactured by Kawaken Fine Chemicals Co., Ltd.) 1.0 wt% Using. The probe solution was filled into the head 91 shown in FIG. A surface-treated quartz glass substrate was placed on the substrate fixing unit 2, and the substrate was fixed by suction from the bottom of the substrate. Next, the probe solution was spotted on the substrate from the head 91. Here, the distance between the liquid ejection surface of the head 91 and the liquid adhesion surface of the substrate was 1.2 to 1.5 mm. After the spotting, a 1M-NaCl / 50 mM phosphate buffer solution (pH 7) was prepared by applying a vibration to the substrate for 30 seconds, a thing left at room temperature for 30 seconds, and a thing left for 20 minutes at room temperature. 0), lightly washed with pure water, and then blown with nitrogen to obtain a probe carrier. For applying vibration to the substrate, the substrate was placed on a separately provided ultrasonic vibration device, and vibration was applied to the substrate for 30 seconds from a piezoelectric element that vibrates at a frequency of 38 kHz.

(4) Hybridization and measurement of fluorescence intensity Hybridization was performed by immersing the probe carrier in the sample solution of the target substance prepared in (1) above at 45 ° C. for 2 hours. Thereafter, the DNA of the sample that was not hybridized with the probe carrier using 1M-NaCl / 50 mM phosphate buffer solution (pH 7.0) was removed, the salt content was removed with pure water, and dried by nitrogen blowing. I let you. The fluorescence intensity of the probe carrier spot at a wavelength of 532 nm was measured using a fluorescence scanner (trade name: GenePix4000B: manufactured by Axon Instruments, Inc.). The result is shown in the graph of FIG. The average value of the fluorescence intensity of the 52 spots on the substrate subjected to vibration for 30 seconds was 5569, and the average value of the fluorescence intensity of the 52 spots on the substrate left at room temperature for 30 seconds without vibration was 4331. The average value of the fluorescence intensities of the 52 spots on the substrate left at room temperature for 20 minutes without vibration was 5524.

  From the above, since the luminance value increases by about 30% by applying vibration for 30 seconds with the same concentration of probe solution for the same reaction time, more probes can be fixed within the same reaction time. I understood. In addition, it has been found that the reaction time which has conventionally required 20 minutes to obtain the same luminance value can be shortened to 30 seconds by applying vibration, so that the productivity of the probe carrier can be improved. It was.

(Example 2)
The probe carrier prepared by executing the above steps 1 to 9 will be described.
(1) Fixing of probe to carrier A substrate manufactured by the same method as in Example 1 was placed on the substrate mounting portion 2 on which the vibrator 1 was installed. The vibrator 1 is a piezoelectric element that vibrates at 38 kHz, and the discharge device is the same discharge device as in the first embodiment. The probe solution of Example 1 (however, the probe concentration was 4.38 mol / L) was spotted while applying vibration from the vibrator to the substrate. The vibration was stopped after spotting.

  When these substrates are observed with a magnifying glass of 15 times, satellite spots (spots derived from droplets when the liquid landed on the solid phase surface) are not observed, and the circular shape is maintained. I found that there was no effect.

The first spotted substrate was vibrated for about 5 minutes, and the last spotted substrate was vibrated for about 30 seconds. Subsequently, these substrates were cleaned in the same manner as in Example 1.
(2) Hybridization and fluorescence intensity measurement The substrate on which the probe was immobilized in (1) was hybridized under the same conditions as in Example 1 to measure fluorescence. The result is shown in FIG. The average value of the fluorescence intensity of 52 spots on the first substrate (vibration time: about 5 minutes) was 5600. The average value of the fluorescence intensity of 52 spots on the last spotted substrate (vibration time of about 30 seconds) was 5556.

  From the above results, it was found that spotting can be performed while vibrating the substrate, and the difference in time for applying vibration due to the difference in time required for spotting has little effect.

(Example 3)
In the probe carrier prepared by executing the above steps 1 to 9, the effect when the probe concentration is reduced will be described.
(1) Fixing of probe to carrier A substrate prepared by the same method as in Example 1 was set in a discharge device equivalent to that in Example 2. In the probe solution of Example 1, spotting was performed using two probe concentrations of 35.04 mol / L and 4.38 mol / L this time. After the spotting, 38 kHz vibration was applied to the substrate for 30 seconds from the vibrator on the discharge device.
(2) Hybridization and fluorescence intensity measurement The substrate on which the probe was immobilized in (1) was hybridized under the same conditions as in Example 1 to measure fluorescence. The result is shown in FIG. The average value of the fluorescence intensity of 52 spots with a probe concentration of 35.04 mol / L was 5589. The average value of the fluorescence intensity of 52 spots with a probe concentration of 4.38 mol / L was 5569.

  From the above results, it was found that almost the same fluorescence intensity can be obtained by applying vibration even when the concentration becomes 1/8. From this, it was found that the probe concentration can be lowered, the probe cost can be reduced, and the production cost of the probe array can be reduced.

  As described above, it has been clarified that the immobilization reaction can be speeded up by applying a vibration to the substrate when the probe solution is applied onto the substrate and the immobilization reaction is performed. Furthermore, not only the speed was increased, but also the fixing efficiency was improved, and it became possible to fix the same probe even if the concentration was lowered. In addition, if vibration is applied for a certain period of time, the difference in vibration application time will not affect the probe fixing amount, and spotting will not be affected even if spotting is performed while applying vibration. It is possible not only to produce by the given method, but also to produce by spotting while applying vibration, and this method has better production efficiency than the former method. According to the present invention, in the production of a probe carrier, it is possible to increase the amount of immobilization, reduce the probe cost, and shorten the probe immobilization reaction.

It is a perspective view of the discharge apparatus of a prior art example. FIG. 2 is a sectional view of the periphery of the substrate in FIG. 1. FIG. 6 is a layout view of substrates on a chuck. It is a figure which shows the mode of a secondary bending mode. It is a figure which shows the mode of a primary torsion mode. It is a figure for demonstrating a high-order bending mode. It is a perspective view of the liquid application apparatus concerning the example of one embodiment of the present invention. It is a board | substrate periphery sectional drawing in the apparatus of FIG. FIG. 8 is a layout view of substrates on a chuck in the apparatus of FIG. 7. 3 is a flowchart of an example embodiment of the present invention. 2 is a graph of fluorescence intensity obtained in the experiment of Example 1. 4 is a graph of fluorescence intensity obtained in the experiment of Example 2. 6 is a graph of fluorescence intensity obtained in the experiment of Example 3.

Explanation of symbols

1: Vibrator 1-a: Vibrator (piezoelectric element)
2: Substrate placement unit 3: Pitch of probe solution spotted on the substrate (Xmm)
4: Length in the longitudinal direction of the substrate (Lmm)
5: Fluorescence intensity of the substrate left at room temperature for 30 seconds 6: Fluorescence intensity of the substrate subjected to vibration for 30 seconds 7: Fluorescence intensity of the substrate left at room temperature for 20 minutes 8: Fluorescence intensity of the substrate subjected to vibration for 5 minutes 9: Fluorescence intensity of substrate subjected to vibration for 30 seconds 10: Probe concentration 35.04 μM, fluorescence intensity of substrate subjected to vibration for 30 seconds 11: Probe concentration 4.38 μM, fluorescence intensity of substrate subjected to vibration for 30 seconds 12: Fixed substrate Section (suction hole)
80: Surface plate 81: Y axis stage 82: Guide rail 83: X axis stage 84: Chuck 85: Substrate 86/87: Post 88/89: Bridge 90: Head mounting base 91: Head 92: Stay 93: Nozzle 94: Probe solution supply port 95: Probe solution 96: Spot

Claims (14)

In a liquid application apparatus for applying a probe solution containing a probe that can specifically bind to a target substance on a substrate,
A substrate mounting portion that can removably fix the substrate;
A liquid application unit for applying the probe solution to the substrate fixed to the substrate mounting unit;
A vibrator for applying vibration to the substrate;
A liquid applicator characterized by comprising:   The liquid applying apparatus according to claim 1, wherein a vibrator is provided on the substrate mounting portion.   The liquid application apparatus for applying a probe solution to a plurality of substrates, wherein the substrate mounting portion and the vibrator are provided for each substrate.   The liquid applying apparatus according to claim 1, further comprising a vibrator control mechanism that controls driving of the vibrator.   5. The liquid application according to claim 1, wherein the vibrator control mechanism is capable of executing a program for applying vibration to the substrate while applying a probe solution onto the substrate fixed to the substrate mounting portion. apparatus.   The liquid according to claim 1, wherein the vibrator control mechanism can execute a program for applying vibration to the substrate after applying the probe solution onto the substrate fixed to the substrate mounting portion. Granting device.   The liquid applying apparatus according to claim 1, wherein the position where the vibrator is provided is a position where the displacement of the natural mode of the substrate is the maximum.   The liquid applying apparatus according to claim 4, wherein at least one of a frequency, an amplitude, and a vibration time of the vibrator is controlled by the vibrator control mechanism.   The liquid applicator according to claim 1, wherein a frequency of the vibrator applied to the substrate is 20 kHz to 80 kHz.   The liquid applying apparatus according to claim 1, wherein the probe is a nucleic acid.   The liquid application apparatus according to claim 1, wherein the liquid application unit is configured to spot a probe solution on a substrate fixed to the substrate mounting unit by an inkjet method or a pin method.   The liquid applying apparatus according to claim 1, further comprising means for preventing evaporation of the liquid from the probe solution. A method for producing a probe carrier in which a probe capable of specifically binding to a target substance is immobilized on a substrate,
A) providing a probe solution containing a probe that can specifically bind to a target substance on a substrate;
B) The probe solution imparts vibration to the applied substrate;
A method for producing a probe carrier, comprising: A method for producing a probe carrier in which a probe capable of specifically binding to a target substance is immobilized on a substrate,
A method for producing a probe carrier, comprising a step of applying a probe solution containing a probe capable of specifically binding to a target substance on a substrate in a state where vibration is applied.

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