WO2004099788A1 - Procede de production de micro-reseau et tete et appareil de production du micro-reseau - Google Patents

Procede de production de micro-reseau et tete et appareil de production du micro-reseau Download PDF

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Publication number
WO2004099788A1
WO2004099788A1 PCT/JP2004/006205 JP2004006205W WO2004099788A1 WO 2004099788 A1 WO2004099788 A1 WO 2004099788A1 JP 2004006205 W JP2004006205 W JP 2004006205W WO 2004099788 A1 WO2004099788 A1 WO 2004099788A1
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WO
WIPO (PCT)
Prior art keywords
spot
surfactant
solution
microarray
head
Prior art date
Application number
PCT/JP2004/006205
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English (en)
Japanese (ja)
Inventor
Hideo Tashiro
Tokuji Kitsunai
Chikara Koike
Akihiro Iimura
Motoki Abe
Yoshihiro Kimura
Original Assignee
Riken
Thk Co., Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from JP2003124654A external-priority patent/JP2004325421A/ja
Priority claimed from JP2003124655A external-priority patent/JP2004325422A/ja
Application filed by Riken, Thk Co., Ltd. filed Critical Riken
Publication of WO2004099788A1 publication Critical patent/WO2004099788A1/fr

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Classifications

    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/02—Burettes; Pipettes
    • B01L3/0241—Drop counters; Drop formers
    • B01L3/0244—Drop counters; Drop formers using pins
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J19/0046—Sequential or parallel reactions, e.g. for the synthesis of polypeptides or polynucleotides; Apparatus and devices for combinatorial chemistry or for making molecular arrays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L13/00—Cleaning or rinsing apparatus
    • B01L13/02—Cleaning or rinsing apparatus for receptacle or instruments
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00351—Means for dispensing and evacuation of reagents
    • B01J2219/00387—Applications using probes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00277—Apparatus
    • B01J2219/00497—Features relating to the solid phase supports
    • B01J2219/00527—Sheets
    • B01J2219/00533—Sheets essentially rectangular
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00585—Parallel processes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J2219/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus
    • B01J2219/00274—Sequential or parallel reactions; Apparatus and devices for combinatorial chemistry or for making arrays; Chemical library technology
    • B01J2219/00583—Features relative to the processes being carried out
    • B01J2219/00603—Making arrays on substantially continuous surfaces
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0809—Geometry, shape and general structure rectangular shaped
    • B01L2300/0819—Microarrays; Biochips
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00—Moving or stopping fluids
    • B01L2400/02—Drop detachment mechanisms of single droplets from nozzles or pins
    • B01L2400/028—Pin is moved through a ring which is filled with a fluid
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B60/00—Apparatus specially adapted for use in combinatorial chemistry or with libraries
    • C40B60/14—Apparatus specially adapted for use in combinatorial chemistry or with libraries for creating libraries

Definitions

  • Microarray fabrication method Microarray fabrication head and device
  • the present invention relates to a microarray production method and a microarray production apparatus for arranging a large number of biological samples such as DNA fragments and oligonucleotides on a substrate.
  • a DNA microarray (that is, a DNA chip) is an array of numerous spots containing DNA fragments and the like on a slide glass-silicon substrate, which is extremely effective in analyzing gene expression, mutation, diversity, etc. .
  • the size of a typical substrate is one to several tens of cm 2 , and spots of thousands to hundreds of thousands of DNA fragments are arranged in this area.
  • DNA fragments on the substrate are examined using a fluorescently labeled DNA having complementarity. Fluorescence occurs when hybridization occurs between the DNA fragment on the substrate and the fluorescently labeled DNA.
  • the spot where this fluorescence occurs is detected by a fluorescence scanner or the like, and the gene expression, mutation, diversity, etc. can be analyzed by analyzing the fluorescence image.
  • a microarray fabrication technology that precisely arranges the spots of densely packed DNA fragments on a substrate is required.
  • WO 95/35505 Japanese Unexamined Patent Publication No. 10-503841 discloses a microarray production apparatus that arranges DNA fragments prepared in advance on a substrate. This device is formed between a pair of elongated members A spot is placed on the substrate by holding the sample in the open capillary channel and lightly tapping the tip of a head composed of a pair of elongated members onto the substrate.
  • a head that includes a liquid reservoir for holding a solution, and a spot arranging device (for example, a pin or a needle) protruding from the liquid reservoir and disposing a spot on the substrate. ing.
  • the head is often provided with a plurality of spot arranging devices (for example, pins or needles) for simultaneously forming spots on a plurality of substrates.
  • spot arranging devices for example, pins or needles
  • All spot locators must be thoroughly cleaned. Conventionally, washing of the spot arrangement tool of such a microarray manufacturing apparatus has been performed using only water in consideration of the influence on a biological sample.
  • the carrier there was a tendency for the par to be noticeable, and improvements were required. Also, the washing operation to prevent contamination by the previous solution requires a considerable amount of time with water alone even when such ultrasonic vibration is used in combination. There was a place where it was limited.
  • the present invention provides a method for preparing a microarray in which a plurality of spots are arranged on a substrate, whereby contamination of a later spot of a biological sample-containing solution by a previously used biological sample-containing solution can be effectively prevented. It is an object to provide a method and a microarray manufacturing apparatus.
  • the present invention also provides a microarray production method and a microarray which can ensure the washing operation performed prior to the formation of the next spot after the spot formation operation reliably and promptly, increase the efficiency of microarray production, and provide a high quality microarray. It is an object to provide a manufacturing device.
  • the present invention for solving the above problems is a method for producing a plurality of microarrays using a spot disposition tool, wherein a plurality of spots of a biological sample-containing solution are formed on a substrate, which is performed between the formation of each spot.
  • the washing operation of the spot locating device which is performed during the formation of each spot, is performed using a surfactant, the cleaning operation is performed better and faster than when only water is used. Can prevent contamination between samples and reduce the time required for microarray production. It becomes.
  • washing was performed using only water in consideration of the effect on biological samples.However, if an appropriate surfactant is selected, the surfactant is used as in the present invention. The present inventors have found that even with the use of an agent, a reliable washing operation can be performed without adversely affecting a biological sample.
  • the hydrophilicity of the surface of the member constituting such a spot disposition device is improved, and the biological sample-containing solution can be conveyed even in the narrow channel of the spot disposition device. It has also been found that the fluid flows smoothly without forming droplets and the like, and as a result, the size of each spot to be formed can be kept uniform.
  • the cleaning of the spot placement device is performed in the steps of supplying a surfactant solution to the spot placement device, washing with water, and drying. Is shown. As described above, in the cleaning, a more effective cleaning effect can be obtained by first supplying the surfactant to the spot placement device.
  • the spot disposition tool has a nozzle and a stamping pin disposed inside the nozzle and capable of reciprocating in a nozzle axis direction.
  • the supply of the surfactant solution to the spot disposition device is performed by reciprocating the stamping pin while at least the tip of the nozzle is in contact with the surfactant solution.
  • the reciprocating motion of the stamping pin allows the relatively easy concentration of the surfactant in the narrow flow path of the spot disposition tool easily and reliably even when the surfactant has a relatively high concentration.
  • An activator can be provided.
  • the activator concentration is at least 5%.
  • the water washing step is performed by repeating a series of operations consisting of ultrasonic cleaning, running water cleaning, and physical water removal one or more times. It is what is done. Through such a water washing step, the surfactant used for the washing can be quickly and reliably removed.
  • the surfactant used in the surfactant solution is a nonionic surfactant or an amphoteric surfactant, and more preferably, polyoxyethylene. A fatty acid ester.
  • the carry-over rate of the solution containing the biological sample used in the formation of the spot in the subsequent spot is 1% or less.
  • the present invention for solving the above-mentioned problems further provides a microarray manufacturing method for forming a plurality of spots of a solution containing a biological sample on a substrate, wherein the solution in the spot arrangement tool for forming the spots is provided. At least a part of the contact surface of the microarray is coated with a surfactant, and the solution is supplied in this state to form a spot.
  • the surface of the spot placement tool that contacts the biological sample-containing solution is coated with the surfactant, and thus the members constituting such a spot placement tool, for example, stainless steel, etc.
  • the surface is more hydrophilic than that of the surface of the specimen, and even the flow path of the stenosis in the spot placement device contains the biological sample.
  • the solution has a smooth flow without forming droplets and the like, and as a result, the size of each spot to be formed can be kept uniform.
  • the coating with the surfactant is performed by: And what is done by supplying excess water and rinsing with water to remove excess. As described above, if the coating with the surfactant is performed prior to each spot formation, the coating with the surfactant can be performed more reliably and stably. This enables quick and reliable washing and removal of the attached solution from the previous use, prevents contamination between samples, and shortens the microarray preparation time. Further, in one embodiment of the method for producing a microarray of the present invention, a method is described in which the use concentration of the surfactant is 5% or more.
  • the rinsing is performed using 100 to 100 times the volume of water with respect to the amount of the surfactant used. Is shown.
  • the coating with the surfactant is performed by applying a surfactant to a contact surface of the spot placement tool and performing a fixing treatment. It is characterized by.
  • the surfactant is immobilized in this manner, it is not necessary to perform the treatment with the surfactant every time after spot formation, as in the case of the above-described embodiment, and the amount of the surfactant used Can be reduced.
  • the contact angle of the portion of the spot dispenser coated with the surfactant with respect to water is defined as an average value of three arbitrary points in a cross-sectional observation of the site by a microscope. This is a reduction of 10% or more compared to before the cleaning treatment with the activator. Surface activity If the angle of contact with water is reduced by 10% or more in the area covered with the agent, good flow of the sample solution is maintained.
  • the present invention for solving the above-mentioned problems further comprises one or a plurality of spot arranging devices for holding a solution containing a biological sample and arranging a spot of the solution on the substrate by contacting the tip with the substrate.
  • a microarray manufacturing head comprising: a spot arranging device, wherein at least a part of a contact surface with the biological sample is coated with a surfactant. Is.
  • spots of a biological sample solution are formed using such a microarray production head, the size of each spot on the microarray can be kept uniform as described above.
  • the present invention for solving the above-mentioned problems further includes a solution storage section for storing a solution containing a biological sample, a worktable on which a plurality of substrates can be arranged, and holding and taking in the solution from the solution storage section.
  • a solution holding means provided with a spot arranging device for forming a spot of a solution on the substrate; a cleaning application section for cleaning the holding means, etc .;
  • a moving means for moving in the direction of separation, forming a spot by using the holding means, and transporting the holding means in an area including the solution storage section, the work table and the cleaning and application section, and two-dimensional coordinates.
  • Transport means for providing a microarray producing device, wherein at least a part of the contact surface of the spot placement tool with the biological sample is coated with a surfactant. Der Ru.
  • FIG. 1 is a plan view showing a microarray manufacturing device according to the first embodiment of the present invention.
  • FIG. 2 is a front view of the microarray manufacturing apparatus as viewed from the direction of line II-II in FIG.
  • FIG. 3 is a right side view of the microarray manufacturing apparatus as viewed from the direction of line III-III in FIG.
  • FIG. 4 is a left side view of the microarray manufacturing apparatus viewed from the direction of the line IV-IV in FIG.
  • FIG. 5 is a cross-sectional view of the microarray manufacturing apparatus viewed from the line VV in FIG.
  • FIG. 6 is a front view of the head.
  • FIG. 7 is a right side view of the head as viewed from the line IX-IX in FIG.
  • FIG. 8 is a bottom view of the head as viewed in the X-X line direction in FIG.
  • FIG. 9 is a front view of the head showing a state where the needle protrudes from the liquid storage member.
  • FIG. 10 is a detailed view showing the liquid storage member and the edle.
  • FIG. 11 to FIG. 11E are process diagrams each showing a method of arranging the solution stored in the liquid storage member on the substrate.
  • the first microarray manufacturing method of the present invention is a microarray manufacturing method in which a plurality of spots of a plurality of types of biological sample-containing solutions are formed on a substrate by using a spot arrangement tool.
  • the spot arranging device is washed during the formation using a surfactant solution.
  • the surfactant used for washing the spot disposition device is not particularly limited, and any known surfactant can be used.
  • Nonionic surfactants and amphoteric surfactants are desirable in view of their effect on biopolymers such as DNA and RNA contained in them.
  • nonionic surfactant examples include, but are not limited to, fatty acid glycerin ester, sorbitan fatty acid ester, sucrose fatty acid ester, polyglycerin fatty acid ester, and higher alcohol ethylene glycol. 6205
  • Oxide adducts single long-chain polyoxyethylene alkyl ether, polyoxyethylene alkylaryl ether, polyoxyethylene lanolin alcohol, polyoxyethylene fatty acid ester, polyoxyethylene glycerin fatty acid, polyoxyethylene propylene dalycol fatty acid ester, poly Xyethylene sorbitol fatty acid ester, polyoxyethylene castor oil or hydrogenated castor oil derivative, polyoxyethylene lanolin derivative, polyoxyethylene fatty acid amide, polyoxyethylene alkylamine, alkylpyrrolidone, glucamide, alkyl polydarcoside, Mono- or dialkanol amides, polyoxyethylene alcohol mono- or di-amides and alkylamine oxides can be mentioned. That.
  • polyoxyethylene ethers containing 1 to 10 oxyethylene moieties and a C10-C20 linear or branched alkyl chain and 1-20 oxyethylene mono-, di- or tri-fatty acid esters.
  • polyoxyethylene (8) octyl phenyl ether Triton X-114
  • polyoxyethylene 9
  • octyl phenyl ether NP-40
  • polyoxyethylene 10
  • octyl phenyl Monotel Triton X-100
  • polyoxyethylene sorbitan monofatty acid ester typically polyoxyethylene (20) sorbitan monolate, can be preferably used.
  • amphoteric surfactant examples include, but are not particularly limited to, alkyldimethylaminoacetate betaine, alkyldimethylamine oxide, alkylcarboxymethylhydroxyxethylimidazolymbetaine, Rukylamidopropyl betaine and the like.
  • the above-mentioned nonionic surfactants and amphoteric surfactants can be used alone, but if necessary, a plurality of these may be used in combination. Further, it can be used in combination with various Ayuon surfactants or non-ionic surfactants.
  • the biological sample-containing solution is not attached to the spot placement tool and remains.
  • a washing operation with water has been performed to prevent contamination of a solution containing a biological sample.
  • a surfactant solution for cleaning the contact surface of the spot placement tool as described above, it is more efficient in a shorter time than when only water is used.
  • the biological sample-containing solution remaining on the spot placement device can be removed, and contamination of the biological sample-containing solution can be effectively prevented.
  • the method of cleaning the contact surface of the spot placement device with the biological sample-containing solution using such a surfactant is not particularly limited, but preferably, first, the contact of the spot placement device is performed. It is desirable that the surface be supplied with a surfactant, followed by washing with water and drying. Considering the reuse of the surfactant, when washing the spot placement tool after forming the spots, first wash with water before applying the surfactant solution to the spot placement tool with the remaining sample remaining. It would be more advantageous to do so and remove most of the remaining sample. However, in this way, once water has entered the interior of the spot placement device, any subsequent attempts to feed the surfactant solution into the interior of the spot placement device are impeded by the water present.
  • the concentration of the surfactant solution applied to the contact surface of the spot disposition device is particularly high.
  • the force is not limited and depends to some extent on the type of surfactant used.
  • the concentration of the surfactant is 5% or more, preferably 20 to 60% by volume, more preferably. It is desirable that the concentration be relatively high, such as 30 to 50% by volume. If the concentration of the surfactant is extremely low, it is easy to supply the surfactant solution to the contact surface of the spot placement tool, but the cleaning effect of removing the remaining biological sample-containing solution well. On the other hand, even when the concentration of the surfactant is extremely high, a marked difference in the effect of washing and removing the residual solution is obtained when the concentration within the above range is used. This is disadvantageous in terms of economy and other factors.
  • washing with water is performed in order to remove the residue released from the contact surface and the used surfactant solution.
  • water to be used pure water, deionized water, distilled water or the like having a small amount of impurities is used.
  • the amount of water used for the washing is not particularly limited as long as the amount of the residue can be sufficiently removed. Although it depends on whether or not the operation is applied, it is desirable to use water at least 100 times the volume of the surfactant used. If the amount of water is extremely small, the residue cannot be washed out sufficiently, or surplus surfactant remains in the spot disposition device, so that a spot of a biological sample-containing solution is subsequently formed. In this case, a large amount of surfactant flows out into the spot, and in some cases, the characteristics of the obtained microarray may be deteriorated.
  • the amount of water used for washing is preferably 100 to 100 times, more preferably 100 to 300 times the amount of the surfactant used. It is desirable that the surface active agent remains slightly on the wall surface of the spot disposition device.
  • the contact angle of the coated portion with water is not particularly limited, and As an average value of any 3 in cross-sectional observation of the site with a microscope, the angle is reduced by 10% or more compared to that before the cleaning treatment with the surfactant, for example, 40 to 65 °, more preferably Is reformed to an extent within the range of 55 to 65 °.
  • the shape of the spot arranging device used to form the spot of the biological sample-containing solution is not particularly limited, and various types of conventionally known devices can be used.
  • a QUILL system a pin & ring system, a spring pin system, a micropit system using a piezoelectric / electrostrictive element as a micropump, or any other form may be used.
  • the QUILL method is a method in which a sample is stored in a recess formed in a pin tip, and the sample in the recess is transferred to the substrate by contacting the pin tip with the substrate to form a minute spot.
  • the spring pin method is a double pin structure with a built-in spring, in which a sample attached to the pin tip is transferred onto the substrate by pressing the pin tip onto the substrate to form minute spots.
  • a micropit method using a piezoelectric electrostrictive element as a micropump is a method similar to a technique widely used in a general ink jet recording method.
  • the contact surface to the solution containing the biological sample, particularly the inner surface portion which becomes the micropore be cleaned using a surfactant solution. Coating with surfactant
  • a second microarray production method of the present invention is a microarray production method for forming a plurality of spots of a solution containing a biological sample, wherein the spot arrangement tool for forming the spot has a contact surface with the solution. At least a portion is coated with a surfactant, and the solution is supplied in this state to form a spot.
  • the method in the second method for producing a microarray of the present invention, in the first method for producing a microarray, when the spot disposing device is washed with a surfactant solution during the formation of each spot, the method is used for washing.
  • the surfactant can be implemented by supplying the biological sample-containing solution and performing spot formation while maintaining the state in which the surfactant in the solution adheres and remains on the surface of the spot disposition device.
  • the method can be carried out by forming a surfactant coating on the surface of the spot disposition device as described later.
  • the surfactant that covers at least a part of the contact surface of the spot placement device with the solution containing a biological sample is not particularly limited, and any known surfactant may be used.
  • nonionic surfactants and amphoteric surfactants are desirable from the viewpoint of their effect on biological macromolecules such as DNA and RNA contained in the biological sample-containing solution.
  • Specific examples of the nonionic surfactant and the amphoteric surfactant are the same as those described above, and a description thereof will not be repeated.
  • the method for coating at least a part of the contact surface of the spot placement device with the biological sample-containing solution with such a surfactant is not particularly limited. Each time the solution is supplied to form a spot, a surfactant is supplied to the contact surface of the spot placement tool, and the surfactant is rinsed with water to remove the excess. Can be mentioned.
  • the contact surface of the spot placement tool is coated with the surfactant at the same time. Due to the cleaning action of the surfactant, the biological sample-containing solution remaining on the spot placement tool can be removed more efficiently and in a shorter time than when only water is used. National can also be prevented.
  • the concentration of the surfactant to be used is not particularly limited, and is somewhat influenced by the type of the surfactant to be used. although, for example, the concentration of the surfactant 5 volume% or more, preferable properly is 2 0-6 0 volume 0/0, and particularly preferably in the range of 3 0-5 0 volume 0/0.
  • the concentration of the surfactant is extremely low, the contact surface of the spot disposition device cannot be covered with the surfactant satisfactorily, and a cleaning effect of satisfactorily removing the remaining biological sample-containing solution can be expected.
  • the concentration in the above range is more remarkable in the case of using the concentration in the above range and in the effect of coating the contact surface and cleaning and removing the residual solution. This is because there is no significant difference, which is disadvantageous in terms of economics.
  • rinsing with water is performed to remove the excessively attached surfactant.
  • the amount of the surfactant is not particularly limited, but is preferably 100 to 100 times the volume of the surfactant used, more preferably. More preferably, it is 100 to 300 times the capacity. If the amount of water used for rinsing is extremely large, the surfactant adhering to the contact surface of the spot placement device will be almost washed off, and the modification of the contact surface by the surfactant may not be expected. On the other hand, if the amount of water is extremely small, excess surfactant will remain in the spot placement device, and when a spot of a biological sample-containing solution is subsequently formed, this A large amount of surfactant flows out into the spot, and in some cases, the properties of the obtained microarray may be degraded.
  • treatment such as air drying or heat drying may be performed.
  • the coating with the surfactant is performed using the spot placement device. This is performed by applying a surfactant to the contact surface of the substrate and performing immobilization treatment.
  • the method for applying the surfactant to the contact surface of the spot disposition device is not particularly limited, but, for example, a surfactant of a predetermined concentration is brought into contact with the contact surface of the spot disposition device as described above. Thereafter, a method can be adopted in which excess surfactant is removed by rinsing with a solvent such as water.
  • the method of immobilizing the surfactant on the contact surface of the spot dispenser is not particularly limited. For example, physical bonding by heat treatment, plasma treatment, electron beam irradiation, or the like, Any chemical bonding, such as use, introduction of a bonding group or a bonding group to a surfactant, or a combination thereof may be used. It may be one with a relatively loose bond that provides sustained release.
  • the surfactant When the surfactant is immobilized in this way, it is not necessary to use a surfactant every time after spot formation as in the above-described embodiment. In addition, the amount of surfactant used can be reduced.
  • the contact surface of the spot placement device with the biological sample-containing solution is thus coated with the surfactant and made hydrophilic.
  • the contact angle of the coated portion with water is 10% as an average of an arbitrary value of 3 in the cross-sectional observation of the site by microscopic observation as compared with that before the cleaning treatment with the surfactant. It is reduced as described above, and is reformed, for example, to a degree within the range of 40 to 65 °, more preferably 55 to 65 °.
  • a biological sample containing a spot having a predetermined volume of 3 to 5 ⁇ 10-4 mm3 per spot having a predetermined volume of 3 to 5 ⁇ 10-4 mm3 per spot.
  • the rate of variation (variation) in the volume between the spots is typically very low, less than 10%.
  • the shape of the spot arranging device used to form the spot of the biological sample-containing solution is not particularly limited, and various types of conventionally known devices can be used. Specific examples thereof are the same as those described above, and a description thereof will be omitted.
  • any of the spot arranging devices it is effective to perform a coating treatment with a surfactant on a contact surface with the biological sample-containing solution, particularly on an inner surface portion serving as a micropore channel.
  • Microarray preparation head and preparation equipment are effective to perform a coating treatment with a surfactant on a contact surface with the biological sample-containing solution, particularly on an inner surface portion serving as a micropore channel.
  • the head microarray manufacturing apparatus for manufacturing a microarray of the present invention will be described with reference to a preferred embodiment together with an operation example in the above-described microarray manufacturing method of the present invention.
  • the head for microarray production and the microarray production equipment are limited as long as the surface of the spot placement tool that contacts the biological sample-containing solution is coated with a surfactant. It is not done.
  • FIG. 1 is a plan view showing a microarray manufacturing apparatus according to an embodiment of the present invention
  • FIG. 2 is a front view of the apparatus viewed from the II-II line direction in FIG. 1
  • FIG. 3 is a III-III line direction in FIG.
  • Fig. 4 is a left side view of the device seen from the IV-IV line in Fig. 2
  • Fig. 5 is a cross-sectional view of the device seen from the V-V line in Fig. 1. It is.
  • This device arranges a large number of spots of a solution of a biological sample such as a DNA fragment or an oligonucleotide prepared in advance on a substrate made of a glass slide or silicon.
  • a typical substrate size is 1 to several tens cm2, and a large number of DNA fragment spots are arranged in this area.
  • the spot diameter has a size of, for example, several tens to hundreds of microliters.
  • the microarray fabrication device has two regions.
  • One is a stamping area 1 in which a microarray manufacturing head 51 (hereinafter, simply referred to as a head) for holding a solution is hit on a substrate, and a spot of a biological sample solution is arranged on the substrate.
  • the other is a washing area 2 in which the head 51 after forming the spot is washed, and the washed head 51 holds the next solution of a different type.
  • the configuration of the head will be described later.
  • a large number of substrates 3 are arranged on the worktable 4 in a matrix.
  • Substrate 3 is slide glass ⁇ silicon etc.
  • the surface of the substrate 3 is surface-treated so that a biological sample can be attached! / Puru.
  • a test table 5 on which two substrates or dummy substrates for producing a microarray on a trial basis are provided.
  • the needle of the head 51 holding the solution is suddenly struck against the substrate 3, the needle is struck while the solution is still attached. In order to avoid this, a needle is hit on the substrate 3 on the test stand 5, and the solution that has excessively adhered to the needle is dropped.
  • An XY two-axis transport mechanism 6 as a second transport means for transporting the head 51 and giving two-dimensional coordinates to the head 51 is mounted on the work table 4.
  • the XY two-axis transport mechanism 6 proceeds to receive the head 51 up to a transfer position 104 described later, and transports the head 51 after the spot formation is completed to the transfer position 104.
  • the XY two-axis transport mechanism 6 includes an X-axis transport mechanism 6X and a Y-axis transport mechanism 6Y.
  • X-axis moving mechanism 6 X has a longitudinal fixed frame 8 extending in the X-axis direction, and rails 9, 9 mounted on the fixed frame so as to extend in the X-axis direction, and are movable with respect to the rails 9, 9.
  • a linear guide composed of sliders 10 and 10 assembled in a linear guide; a table 11 guided by the linear guide; and a reuer motor 12 for driving the table 11.
  • a longitudinal fixed frame 13 extending in the X-axis direction, rails 14 extending in the X-axis direction and mounted on the fixed frame 13 and A re-air guide including a slider 15 incorporated movably with respect to the rail 14 is provided.
  • the Y-axis driving mechanism 6 Y includes a longitudinal movable frame 17 erected between a table 11 driven by the X-axis driving mechanism 6 and a slider 15, It is attached to this movable frame 17 extending in the Y-axis direction.
  • a linear guide comprising rails 18 and 18 and sliders 19 and 19 movably mounted on the rails 18 and 18; and a table 20 guided by the linear guide.
  • a return motor 21 for driving the table 20.
  • the XY two-axis transport mechanism 6 supports a Z-axis drive mechanism 23 as moving means.
  • the Z-axis drive mechanism 23 moves the head 51 in the Z-axis direction orthogonal to the X-axis and the Y-axis, that is, in the direction approaching / separating from the substrate 3.
  • the Z-axis drive mechanism 23 has a Z1-axis drive mechanism 23 for raising and lowering the entire head 51, and a Z-axis drive mechanism for projecting the needle from the liquid storage member of the head 51. It has a mechanism 2 3 Z 2.
  • the Z1-axis drive mechanism 23 Z1 is composed of an electric actuator that moves a slider using a feed screw and an electric motor.
  • a table 41 is mounted on the slider of the Z1-axis driving mechanism 23 Z1.
  • a Z2-axis moving mechanism 23 3 Z2 is mounted on this table 41.
  • the Z two-axis drive mechanism 23 Z 2 comprises an electric actuator having a configuration similar to that of the Z 1 axis drive mechanism 23 Z 1, and is smaller than the Z 1 axis drive mechanism 23 Z 1.
  • An L-shaped arm 42 for moving the needle of the head 51 up and down is attached to the slider of the Z two-axis moving mechanism 2 3 Z 2.
  • the L-shaped arm 42 can be inserted into the head 51 by an air cylinder (not shown) (see FIG. 7).
  • the tip of the L-shaped arm 42 is inserted into the head 51, and the inserted L-shaped arm 42 is lowered by the Z-axis moving mechanism 23 Z2 to remove the head from the liquid storage member of the head 51.
  • the needle protrudes.
  • the table 41 of Z 1 is provided with a ⁇ axis rotation mechanism 43 as attitude change means for changing the attitude of the head 51.
  • the ⁇ -axis rotating mechanism 43 turns the head 51 in a horizontal plane.
  • ⁇ Shaft rotation mechanism 4 3 The table includes an electric motor 44 attached to a table 41, and a substantially cylindrical chuck portion 45 rotatably supported on the table 41 around the Z axis.
  • the chuck portion 45 is connected to the electric motor 44 via a joint.
  • the chuck portion 45 grips the head 51 detachably.
  • the head 51 has a cylindrical chucked portion 54 attached to the chuck portion 45, a substantially rectangular upper plate 55 fixed to the lower surface of the chucked portion 54, and an upper plate 5.
  • 5 is provided with a substantially rectangular lower plate 57 as a base portion which is connected via a plurality of columns 56.
  • liquid storage members 52 as liquid storage portions for holding a solution to be supplied to the substrates 3 are attached vertically and horizontally in parallel with each other.
  • the needles 5 3... ′ (Also called pins) are housed in the liquid reservoir members 52.
  • the needle 53 is guided by a plurality of needle bushes 66 fixed to a lower plate 57 so as to be able to reciprocate upward and downward.
  • a total of four (4) rows and a horizontal (12) rows and a total of four (8) liquid reservoir members (52) and needles (53) are mounted. Needless to say, the number of liquid reservoir members (52) and needles (53) is one.
  • Various settings can be made according to the number of spots that simultaneously form spots on the substrate 3.
  • the lower plate 57 is provided with a plurality of supply spaces 58 for supplying the cleaning liquid etc. over the entirety of the dollar 53.
  • the cleaning liquid supply space 58 communicates with all of the plurality of liquid storage members 52 arranged vertically and horizontally as shown in FIG.
  • the outer surface of the dollar 53 and the inner surface of the liquid reservoir 52 are coated with a surfactant at least at the time of the sample spot forming operation. It has been done.
  • An intermediate plate 59 is provided so as to be movable.
  • a needle support plate 61 is fixed to the lower surface of the intermediate plate 59 via a connecting portion 60, and a plurality of need nozzles 53 are supported by the needle support plate 61.
  • a flange 53d is formed to be placed on the upper surface of the needle support plate 61.
  • a coil spring 62 is formed between the flange 53d and the intermediate plate 59. Intervened. This coil spring 62 compresses and deforms when the needle 53 comes into contact with the substrate 3, and adjusts the load applied to the substrate 3 from the needle 53.
  • the intermediate plate 59 is provided with a bush 63 for guiding the sliding movement of the intermediate plate 59 with respect to the support column 56.
  • a coil spring 64 is provided between the intermediate plate 59 and the lower plate 57 for raising the needle 53 and retracting the needle 53 in the liquid reservoir member 52.
  • FIG. 9 shows a state where the needles 53 are lowered.
  • the needles 53 are also lowered together with the intermediate plate 59, and the needles 53 project from the lower ends of the liquid storage members 52.
  • the coil springs 62 are compressed and deformed so that no excessive load is applied from the udle 5 3 to the substrate 3.
  • the head 51 is positioned by the XY two-axis transport mechanism 6 in the X and Y directions above the substrate 3.
  • the entire head 51 is lowered by the Z1-axis moving mechanism 23 Z1, and the head 51 is positioned at a predetermined distance from the substrate in the Z direction.
  • the L-shaped arm 42 of the Z 2-axis moving mechanism 2 3 Z 2 is advanced above the intermediate plate 59 in the head 51.
  • the intermediate plate 59 is pushed down by the L-shaped arm 42, whereby the needle 53 protrudes from the liquid reservoir member 52.
  • the L-shaped arm 42 is raised by the Z
  • the intermediate plate 59 is raised by the restoring force of the ring 64, whereby the needle 53 is retracted into the liquid storage member 52.
  • FIG. 10 shows a liquid reservoir member 52 and a dollar 53 holding a solution.
  • the liquid storage member 52 is formed in a tapered tapered tube shape, and the needle 53 is stored in the tapered internal space together with the solution.
  • the lower part of the liquid reservoir member 52 which is the narrowest, also guides the vertical movement of the needle 53.
  • the distal end portion 53 a of the needle 53 also has a tapered outer peripheral surface.
  • the tip surface 53b of the dollar 53 that comes into contact with the substrate 3 is formed as a circular or polygonal flat surface.
  • the outer peripheral surface of the distal end portion 53a is rougher than the outer peripheral surface of the straight portion 53c and the distal end surface 53b of the needle 53 so as to hold the solution.
  • FIG. 11A to FIG. 11E are process diagrams showing a method of arranging the solution stored in the liquid storage member 52 on the substrate 3.
  • FIGS. 11 to 11E show the state of the solution when the dollar 53 moves with respect to the liquid storage member 52 in the Z-axis direction (that is, the vertical direction).
  • the liquid storage member 52 is positioned above the substrate 3.
  • the needle 53 is gradually lowered with respect to the liquid storage member 52.
  • the needle 53 is made to protrude from the liquid reservoir member 52.
  • the solution in the liquid reservoir member 52 is pulled out by the solution adhering to the needle.
  • the distal end face 53 b of the needle 53 is brought into contact with the substrate 3. Needle 5 3 tip
  • the surface 53 b mechanically contacts the substrate 3
  • the solution moves to the substrate 3 from the tip surface 53 b of the needle 53, and the solution is placed on the substrate 3.
  • the solution held at the tip of the udle 53 and the solution in the liquid reservoir 52 are connected.
  • FIG. 11E when the needle 53 is retracted from the substrate 3, a solution spot is formed on the substrate 3.
  • the solution held in the liquid storage member 52 and the solution held in the tip of the needle 53 are integrated, and the solution is pulled from the liquid storage member 52 by utilizing the fact that the solution adheres to the needle.
  • the size and shape of the spots arranged on the substrate 3 can be kept constant.
  • the contact surfaces of these members with the solution are coated with a surfactant, the size and shape of the spot can be kept constant.
  • the outer peripheral surface of the needle 53 rough, the amount of the solution held on the outer peripheral surface of the needle 53 is stabilized. For this reason, the size and shape of the spot arranged on the substrate 3 can be kept more constant.
  • the cleaning region will be described.
  • the head 51 after forming the spot is ultrasonically cleaned, rinsed, and then dried.
  • the washed head 51 holds a new solution of the next biological sample.
  • an ultrasonic cleaning section 71 as a cleaning section for ultrasonically cleaning the head 51, and the head 51 are treated with a surfactant solution.
  • a solution storage section 74 for storing is provided.
  • the ultrasonic cleaning section 71, the surfactant solution processing section 72a, the rinsing section 72b, the drying section 73, and the solution storage section 74 are provided.
  • An XY two-axis transfer mechanism 75 is provided as first transfer means for transferring the head 51 and giving the head 51 two-dimensional coordinates.
  • the XY two-axis transport mechanism 75 is composed of an X-axis transport mechanism 75 X and a ⁇ axis transport mechanism 75 ⁇ .
  • the X-axis moving mechanism 75 X is a longitudinal fixed frame 81 extending in the X-axis direction, and a rail extending and attached to the fixed frame 81 in the X-axis direction.
  • the ⁇ -axis drive mechanism 75 ⁇ is mounted on a movable movable frame 87 fixed on a table 84 driven by the X-axis drive mechanism 75 X, and is mounted on the movable frame 87 by extending in the ⁇ -axis direction.
  • Linear guide consisting of a rail 88 and a slider 89 movably incorporated with respect to the rail 88, a table 90 guided by the linear guide, and a feed for driving the table 90.
  • Screw 9 1 is provided.
  • the biaxial transport mechanism 75 is provided with a biaxial drive mechanism 95 as a moving means.
  • the ⁇ -axis drive mechanism 95 moves the head 51 in the ⁇ -axis direction orthogonal to the X axis and the ⁇ axis, that is, in the direction orthogonal to the washing table 96.
  • the ⁇ -axis drive mechanism 95 has a ⁇ 1-axis drive mechanism 95 ⁇ 1 and a ⁇ 2-axis drive mechanism 95 ⁇ 2, similarly to the ⁇ -axis drive mechanism 23 in the stamping area.
  • the ultrasonic cleaning section 71, the surfactant solution processing section 72a, the rinsing section 72b, the drying section 73, and the solution storage section 74 at any position with respect to the liquid storage member 52.
  • the needle 53 can be protruded.
  • the Z1-axis drive mechanism 95 Z1 is composed of an electric actuator that moves the block using a feed screw and an electric motor, like the Z1-axis drive mechanism 23Z1 in the stamping area.
  • the Z 1 axis drive mechanism 9 5 Z 1 table 9 7 has Z 2 axis moving mechanism 9 5 Z 2 Is attached.
  • the Z two-axis drive mechanism 95 Z 2 is composed of an electric actuator having the same configuration as the 21-axis drive mechanism 95 ⁇ 1, and is smaller than the Z 1-axis drive mechanism 95 Z 1.
  • An L-shaped arm 990 for raising and lowering the needle 53 of the head 51 is attached to the table 98 of the Z two-axis moving mechanism 95 Z2.
  • the L-shaped arm 99 can be inserted into the head 51 by an air cylinder (not shown). Insert the end of the L-shaped arm 99 into the head, and lower the inserted L-shaped arm 99 by the Z-axis moving mechanism 95 Z2 to remove the liquid from the liquid storage member 52 of the head 51. Needle 53 protrudes.
  • a turning motor 100 as a turning unit is attached to the table 97 of the Z1-axis drive mechanism, and a disc 101 that turns in a horizontal plane is provided on the output shaft of the turning motor 100. It is attached.
  • a pair of clamps 102, 102 as a holding portion capable of holding the head 51 at 180 ° intervals is attached to the lower surface of the disk 101.
  • the clamps 102 and 102 are opened and closed by an air cylinder or the like (not shown), and sandwich a flat portion 103 (see FIGS. 6 and 7) formed on the outer periphery of the head 51.
  • the head 51 transported by the XY two-axis transport mechanism 75 in the cleaning area has the same configuration as the head 51 transported by the two-axis transport mechanism 6 in the above-mentioned stamming area, The same reference numerals are given and the description is omitted.
  • the turning motor 100 turns 180 degrees at a time, thereby transferring the head 51 from the XY two-axis transfer mechanism 6 in the stamping area to the XY two-axis transfer mechanism 75 in the washing area, and cleaning. Transfer of the head 51 from the XY two-axis transport mechanism 75 in the area to the XY two-axis transport mechanism 6 in the stamping area is performed.
  • the XY two-axis transport mechanism 6 in the stamping area transports the head 51 after forming the spot to the transfer position 104.
  • the XY 2-axis transport mechanism 75 in the cleaning area The held head 51 is transferred from the transfer position 104 to the reserve position 105 shifted 180 degrees from the transfer position 104. At this time, an empty clamp that does not grip the head is located at the transfer position 104.
  • the clamp 51 of the XY two-axis transport mechanism 75 in the cleaning area grips the head 51 after the spot is transported to the transfer position 104.
  • the head is transferred from the XY two-axis transfer mechanism 6 in the stamping area to the XY two-axis transfer mechanism 75 in the cleaning area.
  • the rotation motor 100 rotates the disk 101 by 180 degrees, and the head 51 after the spot formation is located at the standby position 105 and a head holding a new solution. 5 Position 1 at the transfer position 104.
  • the chuck portion 45 of the XY two-axis transport mechanism 75 in the stamping area grips the head 51 holding the new solution.
  • the head is transferred from the XY two-axis transport mechanism 75 in the cleaning area to the XY two-axis transport mechanism 6 in the stamping area.
  • the head 51 can be transferred between the XY two-axis transport mechanism 6 in the stamping area and the XY two-axis transport mechanism 75 in the cleaning area. While the spot is formed on the substrate 3, the other head 51 can be cleaned or the like.
  • the contact surface of the spot arranging tool is brought into contact with the interface.
  • an operation for forming a surfactant film will be described.
  • the head 51 after spot formation is transported to the surfactant solution processing section 72a by the XY two-axis transport mechanism 75.
  • the surfactant solution processing section 7 2 a has a surfactant solution tank, and the head 51 is moved to an upper part of the tank, and the liquid storage member 5 2 of the spot disposing tool attached to the head 51 is provided.
  • the tip of It is immersed in the surfactant solution in the surfactant solution tank.
  • the needle 53 is moved up and down, for example, at a speed of about 0.5 to 2 times / second, 1 to 10 times, preferably about 3 to 5 times.
  • the surfactant solution has a relatively high concentration, the surfactant solution is easily transported to the inside of the liquid reservoir member 52, and the surfactant solution is applied to the inside of the liquid reservoir member 52 and the outer surface of the needle 53. It can be applied or coated.
  • the head 51 is transferred to the ultrasonic cleaning unit 71 by the XY two-axis transport mechanism 75 in the cleaning area.
  • the liquid storage member 52 is immersed in pure water subjected to ultrasonic vibration to clean the outside thereof.
  • the outside of the needle 53 is also cleaned with the dollar 53 protruding from the liquid storage member 52. By this operation, surplus surfactant mainly adhered to the outside of the liquid storage member 52 is removed.
  • the head 51 is transferred to the rinsing section 72 b by the XY two-axis transfer mechanism 75.
  • the rinsing section 72b removes excess surfactant adhered to the inside and outside of the liquid storage member 52 and the outside of the needle 53.
  • the outside of the liquid storage member 52 is cleaned by incorporating the head 51 into a pure water tank storing ultrapure water as a cleaning liquid and immersing the liquid storage member 52 in ultrapure water.
  • the pure water supply pipe 108 in FIG. 6 provided in the pure water tank is connected to the head 51, and the pure water supply pipe and the supply space 5 Communicates with
  • the lower plate 57 of the head 51 is provided with a space 58 for supplying a cleaning liquid or the like corresponding to the rear end of the liquid reservoir member 52.
  • the cleaning liquid supply space 58 is a wide single space extending over each liquid storage member 52. When pure water with pressure is supplied from the pure water supply pipe 108, the pure water spreads in this single space. The pure water filling the single space is supplied to each liquid storage member 52.
  • the excess interface By applying pressure to the inside of the liquid storage member 52 and supplying water, the excess interface The rinse time of the activator or the washing time of the residual sample can be shortened. Further, by forming a single space extending over the plurality of liquid storage members 52, the pressure loss of pure water supplied into the plurality of liquid storage members 52 is reduced, and each liquid storage member 52 is formed. The pressure loss becomes substantially equal between them. Therefore, the inside of the plurality of liquid reservoir members 52 and the outside of the plurality of needles 53 can be washed by applying substantially equal pressure.
  • the head 51 after rinsing is transported to the drying unit 73 by the XY two-axis transport mechanism 75.
  • a vacuum suction pipe (not shown) having the same arrangement as the pure water supply pipe 108 in FIG. 6 is connected to the head 51, and is connected to the drying tank.
  • This vacuum suction tube communicates with a space 58 for supplying a cleaning liquid or the like. In this state, the space 58 for supplying the cleaning liquid or the like is suctioned under vacuum, and the water remaining in the space of the liquid storage member 52 is sucked and removed.
  • the head 51 is returned to the ultrasonic cleaning section 71 again after vacuum suction of moisture in the drying section. Then, it is sent to the rinsing section 72b and the drying section 73, and the steps of cleaning and vacuum suction in the ultrasonic cleaning section 71 and the rinsing section 72b can be repeated a plurality of times.
  • the inside and outside of the liquid storage member 52 and the outside of the needle 53 are sufficiently dried using, for example, dry compressed air, vacuum suction, or the like.
  • the ultrasonic cleaning unit 71 or the rinsing is performed before the head 51 after forming the spot is immersed in the surfactant solution tank.
  • a method of once washing with water in the washing section 72b to remove most of the remaining sample is also conceivable.However, once water enters the inside of the liquid storage member 52 in this way, it is thereafter The operation as described above Attempts to feed the surfactant solution into the inside of the liquid storage member 52 will be hindered by the existing water, and eventually a sufficient amount or concentration of the surfactant will come into contact with the inside of the liquid storage member 52, etc.
  • the dried head 51 is transferred to the solution storage section 74 by the XY two-axis transfer mechanism 75.
  • the solution storage unit 74 takes out a cassette 122 containing a plurality of titer plates 1 2 1... as a solution holding plate for storing a solution, and takes out the titer plate 121 from the cassette 122 and loads it. And a plate transport mechanism 1 2 3 for transporting to a position 1 32.
  • the head 51 after washing is filled with a new biological sample solution. The operation of immersing the head 51 in the solution and sucking the solution is called loading.
  • a plurality of (for example, 384) recesses are arranged in each titer plate 121, and the solution of the biological sample is stored in these recesses.
  • the head has 48 reservoirs, it can be loaded eight times with one titer plate.
  • the plurality of recesses may be filled with the same type of solution, or may be filled with different types of solutions.
  • a plurality (for example, 10) of titer plates 1 2 1... are stored in the cassette 1 2 at equal intervals in the Z-axis direction (ie, in the vertical direction).
  • Two sets of cassettes 122 are provided above and below the washing table 96, and this device stores a total of 20 titer plates 121.
  • An opening for taking in and out the titer plate 121 is formed on the side of the transport mechanism 123 of the cassette 122. Further, a handle 125 for grasping by hand is provided on the upper surface of the cassette 122.
  • the cassettes 122 are mounted on a cassette support 124 that is slidably mounted on the apparatus. Pull out the cassette support 1 2 4 by hand and press The cassette 1 2 2 is mounted on the set support 1 2 4, and the cassette 1 2 4 is manually returned to the original position, whereby the cassette 1 2 2 is incorporated into the apparatus.
  • the plate transport mechanism 123 includes a Z-axis drive mechanism 123, a Y-axis drive mechanism 123, and an X-axis drive mechanism 123X.
  • the Z-axis drive mechanism 123 has the same configuration as the above-described electric actuator that moves the slider using a feed screw and an electric motor.
  • the Z-axis drive mechanism 1 2 3 Z moves up and down the support plate 1 26 supporting the titer plate 121 between the upper end titer plate 121 and the lower end titer plate 121.
  • the Z-axis drive mechanism 1 2 3 X is attached to the Z-axis drive mechanism 1 2 3 Z table 1 2 7.
  • This X-axis moving mechanism 123 is composed of a so-called rodless cylinder.
  • the rodless cylinder includes a track rail 128 extending in the X-axis direction, and a table 127 capable of sliding the track rail 128.
  • X-axis moving mechanism 1 2 3 Y-axis moving mechanism 1 2 3 Y is attached to X table 1 2 9.
  • the Y-axis moving mechanism 123 Y also comprises a so-called rodless cylinder, which moves the table 130 in the Y-axis direction and positions the table 130 at two positions in the Y-axis direction.
  • the washed and dried head 51 is also transported to the loading position by the two-dimensional transport mechanism 75.
  • the solution is sucked by immersing the reservoir member 52 in the solution of the biological sample.
  • the liquid reservoir member 52 is inserted into the concave portion in the titer plate 121, and the tip of the liquid reservoir member 52 is immersed in the solution.
  • the needle 53 is raised with the position of the liquid reservoir member 52 fixed, the solution is pulled up in accordance with the rise of the dollar 53, and the liquid is filled in the liquid reservoir member 52. From this state, pull up the liquid storage member 52 and needle 53 Then, the solution filled in the liquid storage member is held as it is.
  • a head storage space 135 is provided on the washing table 96. Head 51 is first placed in this headroom 1 35. The operation of the apparatus starts when the XY two-axis transport mechanism 75 in the cleaning area is placed in the head storage space 135 to pick up the head 51.
  • the overall operation of the microarray manufacturing apparatus of the present embodiment will be described according to the procedure for manufacturing a microarray.
  • the XY two-axis transport mechanism 6 and Z-axis drive mechanism 23 in the stamping area, and the XY two-axis transport mechanism 75 and Z-axis drive mechanism 95 in the cleaning area are appropriately operated.
  • the head 51 is sequentially positioned at a predetermined position. Such control is performed by a control device (not shown).
  • a preparation stage a plurality of substrates 3 are arranged in the stamping area, and the vacuum device is operated to suction and fix the substrates 3.
  • a substrate for forming a microarray or a dummy substrate is fixed on a test basis.
  • a plurality of titer plates 1 2 1... are stored in the cassette 1 2 2 of the solution storage section 74 in the washing area. For example, solutions of plural kinds of DNA fragments are put into each recess of the titer plate 1 2 1.
  • the XY two-axis transport mechanism 75 in the cleaning area goes to pick up the head 51 placed in the head storage space 135.
  • the pair of clamps 102 and 102 only one clamp 102 holds the head 51.
  • the XY two-axis transfer mechanism 75 transfers the gripped head 51 to the load position 13 2.
  • a loading step of sucking the solution into the liquid storage member 52 is performed.
  • the plate transport mechanism 123 transports the titer plate 121 containing the required solution to the load position 132 before the head 51 is transported to the load position 132.
  • the Z-axis drive mechanism 1 2 3 Z in the cleaning area The liquid storage member 52 and the needle 53 are raised and lowered so that the material 52 sucks the solution.
  • the XY two-axis transfer mechanism 75 in the cleaning area transfers the head 51 holding the solution to the transfer position 104.
  • the XY two-axis transport mechanism 6 in the stamping area transports the empty check portion 45 to the transfer position 104.
  • the Z1-axis drive mechanism in the stamping area moves down the chuck section 45, and the chuck section 45 holds the head holding the solution.
  • the head is transferred from the XY two-axis transport mechanism 75 in the cleaning area to the XY two-axis transport mechanism 6 in the stamping area.
  • the XY two-axis transport mechanism 6 in the stamping area transports the head 51 to the test table 5.
  • the test table 5 a test process for adjusting the amount of the solution adhering to the needle 53 is performed.
  • a stamping process for forming a spot on the substrate 3 is performed.
  • the XY biaxial transport mechanism 6 in the stamping area moves the head 51 to the spot forming position on the substrate 3.
  • the Z1-axis drive mechanism 23 Z1 in the stamping area descends the head 51 and positions the head 51 slightly above the substrate 3.
  • the Z 2 axis drive mechanism 2 3 Z 2 in the stamping area protrudes the dollar 53 from the liquid reservoir member 52, and strikes the needle 53 on the substrate 3.
  • the XY biaxial transport mechanism in the stamping area moves the head to the next substrate. Then, the above-described stamping step is repeated again.
  • the XY two-axis transfer mechanism 75 in the cleaning area grips the remaining head 51 placed in the head storage area 135, It is transported to load position 1 32. At this loading position 1 32, a loading process for sucking the solution into the liquid storage member 52 is performed. You. Then, the XY two-axis transport mechanism 75 in the cleaning area transports the head 51 holding the solution to the standby position 105. At this time, an empty clamp 102 that does not grip the head is located at the transfer position 104.
  • the biaxial transport mechanism 6 in the stamping area transfers the heads 51 after forming the spots and transports them to the transfer position 104. Then, the heads 51 and 51 gripped between the X X two-axis transfer mechanism 6 in the stamping area and the ⁇ two-axis transfer mechanism 75 in the cleaning area are transferred to each other.
  • the biaxial transport mechanism 6 in the stamping area after the transfer process transports the head 51 again onto the substrate. Then, the test step and the stamping step are performed.
  • the biaxial transport mechanism 75 in the cleaning area after the transfer step executes the cleaning step at the same time that the X-biaxial transport mechanism 6 in the stamping area executes the test step and the stamping step.
  • this cleaning step first, the head 51 after forming the spot is first treated with the surfactant solution treating section 7 as described above.
  • the head 51 is conveyed to the ultrasonic cleaning section 71, and the outside of the liquid storage member 52 is ultrasonically cleaned.
  • the head 51 is conveyed to the rinsing section 72, and the inside, outside, and the needle 53 of the liquid storage member 52 are rinsed. Thereafter, the head 51 is transported to the drying section 73, and the liquid storage member 52 and the dollar 53 are dried.
  • the XY two-axis transport mechanism 6 in the stamping area sequentially executes the head transfer step, the test step, and the stamping step.
  • the XY two-axis transport mechanism 75 in the cleaning area sequentially executes the above-described head transfer step, the above-described cleaning step, and the opening and closing process.
  • a head having sixteen spot arrangement tools liquid reservoir member 52 and Udle 53
  • the surfactant solution was used.
  • the second sample was stamped, and the carry-over rate of the first sample at the stamping spot of the second sample was measured.
  • the tip diameter of the needle used was 75 ⁇ .
  • a fluorescent dye rhodamine
  • a fluorescently labeled oligo DNA 21 mer
  • Fluorescently labeled oligo DNA 45mer
  • Fluorescently labeled CDNA 40 O mer
  • the carry-over rate was calculated by measuring the fluorescence intensity of each spot of the first sample and the fluorescence intensity of each spot of the second sample (there was no fluorescence if there was no carry-over).
  • a surfactant (Tween 20) having a concentration of 40% by volume was used, and after using the surfactant, washing was performed with ultrapure water for about 30 seconds.
  • the carry-over rate was similarly measured for a sample that had been washed only with ultrapure water for about 30 seconds without using a surfactant. The results obtained are shown below.
  • the carrier-to-bar ratio was significantly reduced when the washing was performed using the surfactant solution.
  • washing operation was performed using surfactant solutions of various concentrations as shown below.
  • stamping of the second sample was performed, and the carry-over rate of the first sample at the stamping spot of the second sample was measured, and the difference in the cleaning effect due to the difference in the surfactant concentration was measured. I investigated the difference.
  • the washing operation with the surfactant solution in Example 2 was performed under the condition that the treatment time and the amount of liquid used were increased compared to those in Example 1.
  • the tip diameter of the needle used was 75 ⁇ m.
  • a fluorescent dye rhodamine
  • 3 XSSC buffer was used as a sample.
  • the carryover rate was calculated by measuring the fluorescence intensity of each spot of the first sample and the fluorescence intensity of each spot of the second sample (no fluorescence if there was no carryover), as in Example 1. did.
  • Tween 20 was used as the surfactant, and its concentration was set to 20, 30, 40, 50, and% by volume, respectively. Then, after using the surfactant, cleaning was performed for about 30 seconds with ultrapure water. The results obtained are shown below.
  • a sample having 100 spots was continuously stamped using a head having 16 spot arranging devices (a liquid reservoir member 52 and a dollar 53).
  • a fluorescent dye (rhodamine) solution was used as the sample, and the needle tip diameter was 75 ⁇ . Was.
  • the spot locator was coated with a 40% by volume surfactant (Tween20) solution, followed by rinsing with water.
  • Tween20 40% by volume surfactant
  • the ratio of the largest diameter to the smallest diameter in all spots, the ratio of the largest to the smallest in all spots, and the average spot volume for comparison was determined.
  • Example 4 In an apparatus having a configuration as shown in FIG. 1, stamping of the sample was performed 100 times using a head having 16 spot arranging devices (a liquid reservoir member 52 and a needle 53). . The sample was prepared by adding 10 pmo1 / VL to a fluorescent dye (rhodamine) in 3 XSSC buffer, and measuring the fluorescence intensity of each spot. The amount was determined.
  • a fluorescent dye rhodamine
  • the spot placement device was subjected to a treatment of a surfactant (Tween20) solution at a concentration of 40% by volume and a subsequent washing with ultrapure water.
  • a surfactant Teween20
  • the same measurement was carried out for the case where the washing operation was performed using only the same amount of water without using a surfactant.
  • the change in wettability was measured by measuring the contact angle before and after coating with a surfactant (Tween 20), using the sample that had been subjected to No. 0 polishing. A Tween 20 solution with a concentration of 40% by volume was used. After applying this solution on a plate, the actual rinse was performed. The test was performed after about 30 seconds of washing with running water in the same manner as in the process.
  • the amount of water droplets was set to 12 L, and the contact angle before and after the surfactant was coated was determined as the average value of any three points in cross-sectional observation using a microscope.
  • the washing of the spot arranging device performed between the formation of the spots is performed using a surfactant. Since it is carried out using a solution, contamination between sample solutions can be effectively suppressed, and after a spot forming operation, a washing operation performed prior to the next spot formation can be performed reliably and promptly. It is possible to improve the efficiency of microarray production and provide a high-quality microarray.
  • the microarray manufacturing method for forming a plurality of spots of a solution containing a biological sample on a substrate at least one of contact surfaces of the spot arranging tool for forming the spots with the solution is provided.
  • the surface is coated with a surfactant, and the solution is supplied in this state to form spots. Therefore, the size and shape of each spot are made uniform to produce a microarray. In addition, contamination between sample solutions can be suppressed.
  • a DNA microarray having more excellent properties and quality can be provided. It can greatly contribute to the efficient analysis of differences and diversity.

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  • Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Health & Medical Sciences (AREA)
  • Clinical Laboratory Science (AREA)
  • Organic Chemistry (AREA)
  • Apparatus Associated With Microorganisms And Enzymes (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

L'invention concerne un procédé de production d'un micro-réseau, au cours duquel une pluralité de points d'une solution contenant plusieurs types d'échantillons biologiques sont formés sur un substrat à l'aide de moyens de disposition de points. Le procédé est caractérisé en ce que le nettoyage des moyens précités pendant les intervalles entre la formation de points respectifs est mené à l'aide d'une solution de tensioactif. Grâce à ce procédé, l'opération de nettoyage exécutée après la formation de chaque point et avant la formation du point suivant est effectuée de manière sûre et rapide et la contamination entre les points peut être évitée. Par ailleurs, ce procédé permet de former un micro-réseau de points de qualité ayant une taille et une forme uniformes.
PCT/JP2004/006205 2003-04-30 2004-04-28 Procede de production de micro-reseau et tete et appareil de production du micro-reseau WO2004099788A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
JP2003124654A JP2004325421A (ja) 2003-04-30 2003-04-30 マイクロアレイ作製方法
JP2003-124655 2003-04-30
JP2003-124654 2003-04-30
JP2003124655A JP2004325422A (ja) 2003-04-30 2003-04-30 マイクロアレイ作製方法、マイクロアレイ作製用ヘッドおよび装置

Publications (1)

Publication Number Publication Date
WO2004099788A1 true WO2004099788A1 (fr) 2004-11-18

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PCT/JP2004/006205 WO2004099788A1 (fr) 2003-04-30 2004-04-28 Procede de production de micro-reseau et tete et appareil de production du micro-reseau

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WO (1) WO2004099788A1 (fr)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0721157B2 (ja) * 1987-08-07 1995-03-08 三愛石油株式会社 恒温槽用洗浄剤
JP2001242184A (ja) * 2000-02-29 2001-09-07 Shinya Watanabe 試料チップ作製用分注針の洗浄方法
JP2002323507A (ja) * 2001-04-26 2002-11-08 Thk Co Ltd マイクロアレイ作製装置及び方法
JP2003505711A (ja) * 1999-08-02 2003-02-12 モレキュラー・ダイナミックス・インコーポレイテッド 低容量の化学反応および生化学反応システム

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0721157B2 (ja) * 1987-08-07 1995-03-08 三愛石油株式会社 恒温槽用洗浄剤
JP2003505711A (ja) * 1999-08-02 2003-02-12 モレキュラー・ダイナミックス・インコーポレイテッド 低容量の化学反応および生化学反応システム
JP2001242184A (ja) * 2000-02-29 2001-09-07 Shinya Watanabe 試料チップ作製用分注針の洗浄方法
JP2002323507A (ja) * 2001-04-26 2002-11-08 Thk Co Ltd マイクロアレイ作製装置及び方法

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