JP2004325422A - Method, head, and apparatus for manufacturing microarray - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for manufacturing a microarray capable of uniformly forming the size of each spot when arranging a plurality of spots on a substrate, reliably, quickly performing a cleaning operation prior to the next spot formation after forming the spot, efficiently manufacturing the microarray, and increasing the quality of the microarray and to provide an apparatus for manufacturing the microarray. <P>SOLUTION: In the microarray manufacturing method, the plurality of spots of a solution containing a biological sample are formed on the substrate. In this case, at least one portion of a contact surface with the solution in a spot arranging tool for forming the spots is covered with a surface-active agent. In this state, the solution is supplied for forming the spots. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

表１に示すように、界面活性剤による被覆処理を行うことにより、液溜め部材５２およびニードル５３の表面状態が安定（濡れ性が安定）し、スポットのばらつきが少なくなり、またスポット量が増加した。
実施例２
図１に示すような構成を有する装置において、１６本のスポット配置具（液溜め部材５２およびニードル５３）を有するヘッドを用い、実施例１と同様にして、１００回の試料のスタンピングを行った。なお、試料としては、３ｘＳＳＣバッファー中に蛍光色素（ローダミン）に１０ｐｍｏｌ／μＬ添加したものを用い、各スポットの蛍光強度を測ることにより、各スポットの相対的液量を求めた。
【０１２５】
各スポットのスタンピング操作の後には、スポット配置具は、４０容量％濃度の界面活性剤（Ｔｗｅｅｎ２０）溶液の被覆およびその後の水でのリンスという処理を行われた。なお、比較対照のため、界面活性剤を使用せず同量の水のみにて洗浄操作を行ったものについても同様に測定した。
【０１２６】
その結果、界面活性剤による被覆を行った場合には、各スポットのばらつき（標準偏差）は、０．０７６であり、一方、水のみで洗浄したものにおいては、０．１１７であり、また界面活性剤による被覆を行った場合と水のみで洗浄した場合における蛍光強度比は、水のみで洗浄した場合を１とした場合に、界面活性剤による被覆を行った場合は１．８であった。これらの点から、実施例１の場合と同様に、界面活性剤による被覆処理を行うことにより、液溜め部材５２およびニードル５３の表面状態が安定（濡れ性が安定）し、スポットのばらつきが少なくなり、またスポット量が増加した。
実施例３
界面活性剤被覆による濡れ性の変化を測定した。円筒形の液溜め部材５２内面上での測定は困難であるため、この液溜め部材５２と同じステンレス鋼製のプレートを、同様の面粗度（Ｒａ＝０．１ ＃６００番研磨）としたものを使用し、界面活性剤（Ｔｗｅｅｎ２０）被覆前後の接触角を測定することで、濡れ性の変化を測定した。なお、Ｔｗｅｅｎ２０溶液としては、４０容量％濃度のものを用い、これをプレート上に塗布後、実際のリンス工程と同様となるように３０秒ほどの流水洗浄をおこなって、余剰の界面活性剤を除去した後に、試験を行った。
【０１２７】
なお、測定においては、水滴量は１２μＬとし、顕微鏡による断面観察における任意の３点の平均値として界面活性剤被覆前後による接触角を求めた。
【０１２８】
この結果、界面活性剤被覆前においては、接触角は７４．９５°であったのに対し、被覆後においては６４．４９°となり、その差が１０．４６°と、大きく濡れ性が変化した。
【０１２９】
【発明の効果】
以上説明したように、本発明によれば、基板上に、生体試料を含有する溶液のスポットを、複数形成するマイクロアレイ製作方法において、前記スポットを形成するためのスポット配置具における前記溶液との接触面の少なくとも一部を、界面活性剤にて被覆し、この状態で前記溶液を供給してスポットを形成するものであるので、各スポットの大きさおよび形状等を均一なものとしてマイクロプレートを作製することができ、また、各試料溶液間のコンタミネーションも抑制することができる。
【図面の簡単な説明】
【図１】本発明の第１の実施形態におけるマイクロアレイ作製装置を示す平面図。
【図２】図１におけるＩＩ−ＩＩ線方向から見たマイクロアレイ作製装置の正面図。
【図３】図２におけるＩＩＩ−ＩＩＩ線方向から見たマイクロアレイ作製装置の右側面図。
【図４】図２におけるＩＶ−ＩＶ線方向から見たマイクロアレイ作製装置の左側面図。
【図５】図１におけるＶ−Ｖ線方向から見たマイクロアレイ作製装置の断面図。
【図６】ヘッドの正面図。
【図７】図６におけるＩＸ−ＩＸ線方向から見たヘッドの右側面図。
【図８】図６におけるＸ−Ｘ線方向から見たヘッドの底面図。
【図９】ニードルが液溜め部材から突出している状態を示すヘッドの正面図。
【図１０】液溜め部材及びニードルを示す詳細図。
【図１１】液溜め部材内に溜められた溶液を基板上に配置する方法を示す工程図。
【符号の説明】
３…基板
４…作業台
６…スタンピング領域のＸＹ２軸搬送機構（搬送手段）
２３，９５…Ｚ軸駆動機構（移動手段）
５１…ヘッド（保持手段）
５２…液溜め部材（液溜め部）
５３…ニードル（スポット配置具）
５７…下部プレート（基体部）
５８…洗浄液等供給用空間
７１…超音波洗浄部（洗浄等施工部）
７１ａ…界面活性剤処理部（洗浄等施工部）
７２ｂ…すすぎ洗浄部（洗浄等施工部）
７４…溶液貯留部（洗浄等施工部）
７５…洗浄領域のＸＹ２軸搬送機構（搬送手段）[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a microarray manufacturing method and a microarray manufacturing apparatus for arranging a large number of biological samples such as DNA fragments and oligonucleotides on a substrate.
[0002]
[Prior art]
At present, technology development for efficiently analyzing all gene functions of various organisms is progressing. A DNA microarray (that is, a DNA chip) is one in which a large number of spots containing DNA fragments and the like are arranged on a slide glass or silicon substrate, and is very effective for analyzing gene expression, mutation, diversity, and the like.
[0003]
Typical substrate size is 1 to several tens cm ² In this region, spots of thousands to hundreds of thousands of DNA fragments are aligned. The DNA fragment on the substrate is examined using complementary fluorescently labeled DNA. When hybridization occurs between the DNA fragment on the substrate and the fluorescently labeled DNA, fluorescence is emitted. The spot where this fluorescence occurs is detected with a fluorescence scanner or the like, and the expression, mutation, diversity, etc. of the gene can be analyzed by analyzing the fluorescence image.
[0004]
In order to develop the technology of the DNA microarray, a microarray fabrication technology for precisely arranging spots of densely packed DNA fragments on a substrate is required.
[0005]
For example, Patent Literature 1 discloses a microarray manufacturing apparatus that arranges DNA fragments prepared in advance on a substrate. This apparatus holds a sample in an open capillary channel formed between a pair of elongated members, and places a spot on the substrate by lightly tapping the tip of a head composed of the pair of elongated members onto the substrate. ing. In addition, the head is also known to include a liquid reservoir for holding a solution and a spot arranging device (for example, a pin or a needle) projecting from the liquid reservoir and disposing a spot on the substrate. I have.
[0006]
In such a microarray manufacturing apparatus, it is necessary to control the amount of sample for each spot so as to be equal to each other, but in a conventionally known apparatus, the more spots are formed on a substrate, That is, the smaller the predetermined amount of each spot in the design, the more the variation occurs, and a technique for improving the variation has been desired.
[0007]
In such a microarray manufacturing apparatus, it is necessary to wash the head so that the previous solution is not turbid when the next solution of a different type is held in the head after the spot forming operation. A 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. However, in order to prevent turbidity of the solution, these plurality of spot arranging devices are provided. Everything must be cleaned thoroughly. 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. Then, in order to enhance the cleaning effect, ultrasonic vibration is applied to the cleaning unit in the same manner as in the case of cleaning the dispensing nozzle of a medical analyzer as in Patent Documents 2 and 3, for example. However, 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.
[Patent Document 1]
Japanese Patent Publication No. Hei 10-503841
[Patent Document 2]
JP-A-1-254871
[Patent Document 3]
JP-A-8-21840
[0008]
[Problems to be solved by the invention]
Therefore, an object of the present invention is to provide a microarray manufacturing method and a microarray manufacturing apparatus which can form spots of a uniform size when a plurality of spots are arranged on a substrate. The present invention also provides a microarray production method and a microarray production method that can ensure and promptly perform a washing operation performed prior to the next spot formation after a spot formation operation, thereby improving the efficiency of microarray production and providing a high-quality microarray. It is an object to provide a device.
[0009]
[Means for Solving the Problems]
The present invention for solving the above-mentioned problems is a method for producing a microarray in which a plurality of spots of a solution containing a biological sample are formed on a substrate, and a contact surface with the solution in a spot arrangement tool for forming the spots. Is coated with a surfactant, and the solution is supplied in this state to form spots.
[0010]
As described above, in the present invention, since the contact surface of the spot placement device with the biological sample-containing solution is coated with a surfactant, a member constituting such a spot placement device, for example, a surface of stainless steel or the like. The solution containing the biological sample flows smoothly without forming droplets, etc., even in the narrow flow path of the spot placement device, and as a result, the size of each spot to be formed is equalized. Can be kept.
[0011]
Further, in one embodiment of the method for producing a microarray of the present invention, the coating with the surfactant is carried out such that each time the solution is supplied to form a spot, the surface of the surfactant is applied to the contact surface of the spot arranging tool. And rinsing with water to remove the 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. The solution to be used can be quickly and reliably washed and removed, contamination between samples can be prevented, and the time for preparing a microarray can be shortened.
[0012]
Further, in one embodiment of the method for producing a microarray of the present invention, a method is described in which the concentration of the surfactant used is 5% or more.
[0013]
Also, in one embodiment of the method for producing a microarray of the present invention, it is shown that the rinsing is performed using 100 to 1000 times the volume of the used surfactant.
[0014]
In another embodiment of the method for producing a microarray of the present invention, the coating with the surfactant is performed by applying a surfactant to a contact surface of the spot placement tool and performing an immobilization treatment. It is characterized by. When the surfactant is immobilized in this manner, it is not necessary to perform the treatment using 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.
[0015]
In one embodiment of the method for producing a microarray of the present invention, the contact angle of water on the portion of the spot placement tool coated with the surfactant is determined as an average value of any three points in a cross-sectional observation of the site with a microscope. Is reduced by 10% or more as compared with before the cleaning process. When the contact angle with water is reduced by 10% or more in the portion coated with the surfactant, good circulation of the sample solution is maintained.
[0016]
The present invention for solving the above-mentioned problems also has a spot placement tool that holds a solution containing a biological sample and places a spot of the solution on the substrate by contacting the tip with the substrate. A microarray producing head, wherein at least a part of a contact surface of the spot placement tool with the biological sample is coated with a surfactant.
[0017]
If spots of a biological sample solution are formed using such a microarray manufacturing head, the size of each spot on the microarray can be kept uniform as described above.
[0018]
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 the solution storage section taking in the solution from the solution storage section and holding the substrate. A solution holding means provided with a spot arranging tool for forming a spot of a solution thereon, a cleaning application section for cleaning the holding means, etc., and a direction in which the holding means approaches and separates from the substrate. Moving means for moving the holding means to form a spot by the holding means, and conveying means for transferring the holding means in an area including the solution storage section, the work table, and the cleaning and working section and giving two-dimensional coordinates. Wherein at least a part of a contact surface of the spot placement tool with the biological sample is coated with a surfactant.
[0019]
If spots of a biological sample solution are formed using such a microarray manufacturing apparatus, the size of each spot on the microarray can be kept uniform as described above.
[0020]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail based on embodiments.
[0021]
The microarray manufacturing method of the present invention is a microarray manufacturing method for forming a plurality of spots of a solution containing a biological sample, wherein at least a part of a contact surface with the solution in a spot arrangement tool for forming the spots. And coating with a surfactant, and supplying the solution in this state to form a spot.
[0022]
In the present invention, the surfactant that covers at least a part of the contact surface of the spot placement device with the biological sample-containing solution is not particularly limited, and any known surfactant can be used. However, nonionic surfactants and amphoteric surfactants are desirable from the viewpoint of the effect on biopolymers such as DNA and RNA contained in the biological sample-containing solution.
[0023]
The nonionic surfactant is not particularly limited, for example, fatty acid glycerin ester, sorbitan fatty acid ester, sucrose fatty acid ester, polyglycerin fatty acid ester, higher alcohol ethylene oxide adduct, single long chain polyoxyethylene alkyl ether , Polyoxyethylene alkyl allyl ether, polyoxyethylene lanolin alcohol, polyoxyethylene fatty acid ester, polyoxyethylene glycerin fatty acid, polyoxyethylene propylene glycol fatty acid ester, polyoxyethylene sorbitol fatty acid ester, polyoxyethylene castor oil or hydrogenated castor oil Derivative, polyoxyethylene lanolin derivative, polyoxyethylene fatty acid amide, polyoxyethylene alkylamine, alkyl Pyrrolidone, glucamides, alkylpolyglucosides, mention may be made of mono- or di-alkanolamide, polyoxyethylene alcohols mono- or diamides and alkylamine oxides and the like. Of these, preferred examples are 1 to 10 oxyethylene moieties and C ₁₀ -C ₂₀ And mono-, di- or tri-fatty acid esters of 1 to 20 oxyethylenes, and specifically, for example, polyoxyethylene (8) Octyl phenyl ether (Triton X-114), polyoxyethylene (9) octyl phenyl ether (NP-40), polyoxyethylene (10) octyl phenyl ether (Triton X-100), polyoxyethylene (20) sorbitan monolau Rate (Tween 20), polyoxyethylene (20) sorbitan monopalmitate (Tween 40), polyoxyethylene (20) sorbitan monostearate (Tween 60), polyoxyethylene (20) sorbitan trioleate, and the like. Door can be. In particular, polyoxyethylene sorbitan monofatty acid ester, typically, polyoxyethylene (20) sorbitan monolaurate can be preferably used.
[0024]
Examples of the amphoteric surfactant include, but are not particularly limited to, alkyldimethylaminoacetic acid betaine, alkyldimethylamine oxide, alkylcarboxymethylhydroxyethyl imidazolium betaine, and alkylamidopropyl betaine.
[0025]
As the surfactant, the above-mentioned nonionic surfactant and amphoteric surfactant can be used alone, but if necessary, a plurality of these may be used in combination, or these may be used. Further, it can be used in combination with various anionic surfactants and / or nonionic surfactants.
[0026]
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, but in the first preferred embodiment, The coating with the activator is performed by supplying a surfactant to the contact surface of the spot disposing device and rinsing with water to remove the excess before each supply of the solution to form a spot. Examples of the method include:
[0027]
In the production of a microarray, as described above, in order to form spots of a very large number of kinds of biological sample-containing solutions of several thousand to several hundred thousand kinds, it is general to use a plurality of spot arrangement tools at the same time. However, there is a limit to providing a large number of spot arranging devices so as to correspond to all types of biological sample-containing solutions of spots to be formed. This is also used for spot formation of the sample-containing solution.
[0028]
For this reason, after the formation of the spot of one type of biological sample-containing solution is completed, prior to the formation of the spot of the next type of biological sample-containing solution, the subsequent biological sample-containing solution before adhering and remaining on the spot placement tool is used. Conventionally, a washing operation with water has been performed to prevent contamination of the sample-containing solution. In this embodiment of the present invention, instead of this, by supplying a surfactant to the contact surface of the spot placement tool as described above, by performing an operation of rinsing with water to remove the excess, Biological samples that adhere to the spot placement tool efficiently and in a shorter time than the case where only water is used due to the cleaning action of the surfactant while the contact surface of the spot placement tool is coated with the surfactant. The containing solution can be removed, and the contamination of the biological sample containing solution can also be prevented.
[0029]
As described above, each time a spot is formed by supplying the biological sample-containing solution, a surfactant is supplied to the contact surface of the spot placement tool, and the excess is removed by rinsing with water. In the embodiment in which a surfactant film is formed on the contact surface of the spot arranging device, the concentration of the surfactant used is not particularly limited, and depends to some extent on the type of the surfactant used. For example, it is desirable that the concentration of the surfactant is 5% by volume or more, preferably 20 to 60% by volume, more preferably 30 to 50% by volume. If 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. On the other hand, even when the concentration of the surfactant is extremely high, there is a remarkable difference between the case where the concentration within the above range is used and the action of coating the contact surface and washing and removing the residual solution. This is disadvantageous in terms of economy and the like.
[0030]
When supplying a surfactant to the contact surface of the spot placement device, additional cleaning operations such as ultrasonic vibrations, which are conventionally performed, may be performed as necessary in order to enhance the cleaning effect of the surfactant. May be added.
[0031]
As described above, after the surfactant is supplied to the contact surface of the spot disposition device, rinsing with water is performed to remove the surfactant that has excessively adhered.
[0032]
As the water to be used, water with little impurities such as pure water, deionized water and distilled water is used.
[0033]
Further, the amount of water used for rinsing is not particularly limited, but may be 100 to 1000 times, more preferably 100 to 300 times, the amount of the surfactant used. desirable. If the amount of water used for rinsing is excessively large, the surfactant attached to the contact surface of the spot disposition tool is almost washed away, and there is a possibility that the modifying action of the contact surface by the surfactant cannot be expected. On the other hand, if the amount of water is extremely small, surplus surfactant remains in the spot disposition device, and when a spot of the biological sample-containing solution is subsequently formed, an interface is formed in the spot. A large amount of the active agent flows out, and in some cases, the properties of the obtained microarray may be deteriorated.
[0034]
After rinsing with water, a process such as air drying or heat drying may be performed as necessary.
[0035]
Alternatively, in a second preferred embodiment in which at least a part of the contact surface of the spot placement tool with the biological sample-containing solution is coated with the surfactant as described above, the coating with the surfactant is performed using the spot placement tool. This is performed by applying a surfactant to the contact surface of the substrate and performing a fixing treatment.
[0036]
The method of applying the surfactant to the contact surface of the spot placement tool is not particularly limited, for example, as described above, after contacting the surfactant of a predetermined concentration to the contact surface of the spot placement tool, A method of removing excess surfactant by rinsing with a solvent such as water can be adopted. The method of immobilizing the surfactant on the contact surface of the spot arranging tool is not particularly limited. For example, heat treatment, plasma treatment, physical bonding by treatment such as electron beam irradiation, or use of a linker, Any chemical bonding, such as introduction of a bonding group or a bonding group to an active agent, or a combination thereof may be used, and even if the bonding itself is in a strong form, it may cause a sustained release of the surfactant. A relatively loose connection may be used.
[0037]
When the surfactant is immobilized in this manner, it is not necessary to perform the treatment using 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.
[0038]
Also, in this case, when the type of the biological sample-containing solution that forms the spot is exchanged, it is necessary to wash the spot placement tool after use in the previous sample, but the coating with the surfactant is performed. Therefore, even with washing with water alone, the biological sample-containing solution remaining on the spot placement tool can be removed more easily and more quickly than in the conventional case.
[0039]
In the present invention, at least a part of the contact surface of the spot disposition device with the biological sample-containing solution is coated with the surfactant and made hydrophilic. Although not particularly limited, the contact angle of the coated portion with water is 10% or more as an average value of an arbitrary 3 in a cross-sectional observation of the site with a microscope, as compared with before the cleaning treatment with the surfactant. It is reduced, for example, to such an extent that it falls within the range of 40 to 65 °, more preferably 55 to 65 °.
[0040]
For this reason, using the spot dispenser coated with such a surfactant, the predetermined volume of each spot is 3 to 5 × 10 5 per spot. ^-4 mm ³ When spots of the biological sample-containing solution within the range are formed, the variation rate (variation) of the volume between the spots is typically suppressed to a very low value of 10% or less.
[0041]
In the present invention, the shape of the spot arranging device used for forming the spot of the biological sample-containing solution is not particularly limited, and various types of conventionally known devices can be used. For example, a QUIILL method, a pin & ring method, a spring pin method, a micropipette method using a piezoelectric / electrostrictive element as a micropump, or any other form may be used.
[0042]
Here, the QUIILL method is a method in which a sample is stored in a concave portion formed on a pin tip, and the pin tip is brought into contact with a substrate to transfer the sample in the concave portion onto the substrate to form a minute spot. Is a method in which a sample solution is reserved in a ring, then the solution penetrates the inside of the reserved ring, the sample in the ring is captured with a pin tip, and a spot is formed on a substrate. The method uses a double pin structure with a built-in spring, and transfers the sample attached to the pin tip onto the substrate by pressing the pin tip onto the substrate to form a minute spot. The micropipette system using the element as a micropump is the same system as that generally used in the ink jet recording system.
[0043]
In 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 that becomes a micropore channel.
[0044]
Next, the microarray manufacturing head and the microarray manufacturing apparatus of the present invention will be described with reference to a preferred embodiment, but the microarray manufacturing head and the microarray manufacturing apparatus of the present invention have the There is no particular limitation as long as the contact surface with the biological sample-containing solution has been coated with a surfactant.
[0045]
FIG. 1 is a plan view showing a microarray manufacturing apparatus according to one embodiment of the present invention, FIG. 2 is a front view of the microarray manufacturing apparatus seen from the II-II line direction in FIG. 1, and FIG. FIG. 4 is a left side view of the device as viewed from the line IV-IV in FIG. 2, and FIG. 5 is a cross-sectional view of the device as viewed from the line VV in FIG. is there.
[0046]
This device is mounted on a glass slide or silicon Preparation A number of spots of a solution of a biological sample such as a DNA fragment or an oligonucleotide are arranged. Typical substrate size is 1 to several tens cm ² Then, spots of many DNA fragments are arranged in this region. The spot diameter has a size of, for example, several tens of microns to several hundreds of microns.
[0047]
As shown in FIG. 1, the microarray manufacturing device has two regions. One is a stamping region 1 in which a microarray manufacturing head 51 (hereinafter, simply referred to as a head) holding a solution is hit on a substrate, and a spot of a solution of a biological sample is arranged on the substrate. The other is a cleaning area 2 in which the head 51 after forming the spot is cleaned, and the cleaned head 51 holds the next solution of a different type. The configuration of the head will be described later.
[0048]
First, the stamping area will be described. A large number of substrates 3 are placed on the worktable 4 in a matrix. The substrate 3 is made of a glass slide, silicon, or the like, and the surface of the substrate 3 is subjected to a surface treatment so that a biological sample can be attached thereto.
[0049]
On one part of the work table 4, a test table 5 on which two substrates or dummy substrates for producing a microarray on a trial basis are provided. When the needle of the head 51 holding the solution is suddenly struck against the substrate 3, the needle is struck while the solution is excessively applied. In order to avoid this, a needle is hit on the substrate 3 on the test table 5, and the solution that has excessively adhered to the needle is dropped.
[0050]
An XY two-axis transport mechanism 6 as a second transport unit that transports the head 51 and gives the head 51 two-dimensional coordinates is mounted on the work table 4. The XY two-axis transport mechanism 6 proceeds to receive the head 51 to a transfer position 104 described later, and transports the head 51 after the spot formation is completed to the transfer position 104 again.
[0051]
As shown in FIGS. 1 and 4, the XY two-axis transport mechanism 6 includes an X-axis transport mechanism 6X and a Y-axis transport mechanism 6Y. The X-axis moving mechanism 6X has a longitudinal fixed frame 8 extending in the X-axis direction, rails 9, 9 mounted on the fixed frame so as to extend in the X-axis direction, and movably with respect to the rails 9, 9. It includes a linear guide composed of assembled sliders 10, 10, a table 11 guided by the linear guide, and a linear motor 12 for driving the table 11. On the opposite side of the X-axis transport mechanism 6X across the substrate, a longitudinal fixed frame 13 extending in the X-axis direction, a rail 14 extending on the fixed frame 13 in the X-axis direction, and a rail 14 On the other hand, a linear guide including a slider 15 movably incorporated is provided.
[0052]
As shown in FIGS. 1 and 2, the Y-axis driving mechanism 6Y includes a longitudinal movable frame 17 provided between the table 11 and the slider 15 driven by the X-axis driving mechanism 6X. A linear guide comprising rails 18, 18 mounted to extend in the axial direction, and sliders 19, 19 movably incorporated with respect to the rails 18, 18; a table 20 guided by the linear guide; A linear motor 21 for driving the table 20;
[0053]
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 a Z-axis direction orthogonal to the X-axis and the Y-axis, that is, in a direction approaching / separating from the substrate 3. The Z-axis drive mechanism 23 has a Z1-axis drive mechanism 23Z1 for raising and lowering the entire head 51, and has a Z2-axis drive mechanism 23Z2 for projecting the needle from the liquid reservoir member of the head 51.
[0054]
The Z1-axis drive mechanism 23Z1 is composed of an electric actuator that moves a slider using a feed screw and an electric motor.
[0055]
As shown in FIGS. 1, 2 and 5, a table 41 is attached to the slider of the Z1-axis driving mechanism 23Z1, and a Z2-axis moving mechanism 23Z2 is attached to the table 41. The Z2-axis drive mechanism 23Z2 is composed of an electric actuator having the same configuration as the Z1-axis drive mechanism 23Z1, and is smaller than the Z1-axis drive mechanism 23Z1. An L-shaped arm 42 for moving the needle of the head 51 up and down is attached to the slider of the Z2-axis moving mechanism 23Z2. 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 Z2-axis moving mechanism 23 </ b> Z <b> 2 so that the needle protrudes from the liquid storage member of the head 51.
[0056]
The table 41 of the Z1-axis drive mechanism 23Z1 is provided with a Θ-axis rotation mechanism 43 as posture changing means for changing the posture of the head 51. The Θ-axis rotation mechanism 43 rotates the head 51 in a horizontal plane. The Θ-axis rotating mechanism 43 includes an electric motor 44 attached to the table 41 and a substantially cylindrical chuck portion 45 supported on the table 41 so as to be rotatable around the Z-axis. The chuck part 45 is connected to the electric motor 44 via a joint. The chuck portion 45 detachably holds the head 51.
[0057]
6 and 7 show a head 51 as a holding unit. The head 51 includes 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 a plurality of columns 56 on the upper plate 55. And a lower plate 57 having a substantially rectangular shape as a base portion to be joined through the lower plate 57.
[0058]
A liquid storage member 52 as a liquid storage part for holding a solution to be supplied to the substrate 3 is attached to the lower plate 57 horizontally and vertically. A needle 53 (also referred to as a pin) as a spot placement tool is housed in the liquid reservoir members 52. The needle 53 is guided by a plurality of needle bushes 66 fixed to the lower plate 57 so as to be able to reciprocate in the vertical direction. In this embodiment, a total of 48 liquid reservoir members 52... And needles 53... Of 4 rows and 12 columns are attached. Of course, the number of the liquid reservoir members 52. Can be set in various ways depending on the number of spots forming.
[0059]
The lower plate 57 is provided with a space 58 for supplying a plurality of needles 53... As shown in FIG. 8, the supply space 58 for the cleaning liquid or the like communicates with all of the plurality of liquid storage members 52 arranged vertically and horizontally.
[0060]
In this embodiment, the outer surfaces of the needles 53 and the inner surfaces of the liquid reservoirs 52 are coated with a surfactant at least at the time of the sample spot forming operation. .
[0061]
An intermediate plate 59 is provided between the upper plate 55 and the lower plate 57 so as to be slidable with respect to the columns. A needle support plate 61 is fixed to the lower surface of the intermediate plate 59 via a connecting portion 60, and the plurality of needles 53 are supported by the needle support plate 61. A flange 53d to be placed on the upper surface of the needle support plate 61 is formed above the needle 53, and a coil spring 62 is interposed between the flange 53d and the intermediate plate 59. The coil springs 62 are compressed and deformed when the needle 53 comes into contact with the substrate 3, and adjust the load applied from the needle 53 to the substrate 3. The intermediate plate 59 is provided with a bush 63 for guiding the sliding movement of the intermediate plate 59 with respect to the 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 storage member 52.
[0062]
FIG. 9 shows a state where the needles 53 are lowered. When the intermediate plate 59 is lowered as shown in this figure, the needles 53 are also lowered together with the intermediate plate 59, and the needles 53 project from the lower ends of the liquid reservoir members 52. When the needles 53 contact the substrate 3, the coil springs 62 are compressed and deformed so that no excessive load is applied from the needles 53 to the substrate 3.
[0063]
The operation of the head 51 by each drive mechanism will be described. First, the head 51 is positioned by the XY biaxial transport mechanism 6 in the X and Y directions above the substrate 3. Next, the entire head 51 is lowered by the Z1-axis moving mechanism 23Z1, and the head 51 is positioned at a predetermined distance from the substrate in the Z direction. Next, the L-shaped arm 42 of the Z2-axis moving mechanism 23Z2 is advanced above the intermediate plate 59 in the head 51. When the L-shaped arm 42 is lowered by the Z2-axis moving mechanism 23Z2, the intermediate plate 59 is pushed down by the L-shaped arm 42, whereby the needle 53 protrudes from the liquid storage member 52. On the other hand, when the L-shaped arm 42 is raised by the Z2-axis moving mechanism 23Z2, the intermediate plate 59 is raised by the restoring force of the coil spring 64, whereby the needle 53 is retracted into the liquid storage member 52.
[0064]
FIG. 10 shows a liquid reservoir member 52 and a needle 53 that hold a solution. The liquid storage member 52 is formed in a tapered tapered tube shape, and a needle 53 is stored in the tapered internal space together with the solution. The vertical movement of the needle 53 is also guided at the lower part of the liquid reservoir member 52 having the narrowest width.
[0065]
The distal end portion 53a of the needle 53 also has a tapered outer peripheral surface. Further, the distal end surface 53b of the needle 53 that contacts 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. This surface roughness is such that when the needle 53 projects a predetermined amount from the liquid storage member 52 and the distal end surface 53b of the needle 53 comes into contact with the substrate 3, the solution held on the distal end surface 53b and the solution in the liquid storage member 52 are separated. Set to be connected. Further, this surface roughness is formed by grinding or electric discharge machining. For example, in the case of grindstone processing, by moving the grindstone in the longitudinal direction of the needle 53, the outer peripheral surface of the tip 53a is roughened.
[0066]
FIGS. 11A to 11E are process diagrams illustrating a method of disposing the solution stored in the liquid storage member 52 on the substrate 3. FIGS. 7A to 7E show the state of the solution when the needle 53 moves in the Z-axis direction (that is, the vertical direction) with respect to the liquid storage member 52.
[0067]
First, the liquid storage member 52 is positioned above the substrate 3 as shown in FIG. Next, as shown in (b) and (c) in the figure, the needle 53 is gradually lowered with respect to the liquid storage member 52. Next, the needle 53 is made to protrude from the liquid reservoir member 52 as shown in FIG. At this time, the solution in the liquid storage member 52 is pulled out by the solution adhering to the needle. Then, the distal end face 53 b of the needle 53 is brought into contact with the substrate 3. When the distal end surface 53b of the needle 53 comes into mechanical contact with the substrate 3, the solution moves from the distal end surface 53b of the needle 53 to the substrate 3, and the solution is disposed on the substrate 3. At this time, the solution held at the tip of the needle 53 and the solution in the liquid storage member 52 are connected. Then, as shown in (e) in the figure, when the needle 53 is retracted from the substrate 3, a solution spot is formed on the substrate 3.
[0068]
As described above, the solution held by the liquid storage member 52 and the solution held at the tip of the needle 53 are integrated, and the solution is pulled out from the liquid storage member 52 by utilizing the solution adhering to the needle, and the substrate is removed. By arranging the spots on the substrate 3, the size and shape of the spots arranged on the substrate 3 can be kept constant. In particular, in the present invention, since 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. Further, by making 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.
[0069]
Next, the cleaning region will be described. In this cleaning area, the head 51 after the formation of the spot is ultrasonically cleaned, rinsed and then dried. After the cleaning, the head 51 holds a new solution of the next biological sample.
[0070]
As shown in FIG. 1, an ultrasonic cleaning section 71 as a cleaning section for ultrasonically cleaning the head 51 and a surfactant solution processing for treating the head 51 with a surfactant solution are provided on the cleaning table 96. A section 72a, a rinsing section 72b as a cleaning section for rinsing the head 51, a drying section 73 for drying the head 51, and a solution storage section 74 for storing a solution containing a biological sample are provided.
[0071]
The head 51 is transported between the ultrasonic cleaning unit 71, the surfactant solution processing unit 72a, the rinsing cleaning unit 72b, the drying unit 73, and the solution storage unit 74 on the cleaning table 96. An XY two-axis transport mechanism 75 is provided as first transport means for giving dimensional coordinates.
[0072]
The XY two-axis transport mechanism 75 includes an X-axis transport mechanism 75X and a Y-axis transport mechanism 75Y.
[0073]
As shown in FIGS. 1 and 3, the X-axis moving mechanism 75 </ b> X includes a longitudinal fixed frame 81 extending in the X-axis direction, rails 82, 82 that are attached to the fixed frame 81 so as to extend in the X-axis direction, and A linear guide comprising sliders 83, 83 movably assembled to the rails 82, 82, a table 84 guided by the linear guide, and a feed screw 85 for driving the table 84 are provided.
[0074]
The Y-axis driving mechanism 75Y includes a longitudinal movable frame 87 fixed on a table 84 driven by the X-axis driving mechanism 75X, Y-axis direction A linear guide comprising a rail 88 extended and mounted on the rail and a slider 89 movably mounted on the rail 88, a table 90 guided by the linear guide, and a feed screw 91 for driving the table 90 And
[0075]
As shown in FIGS. 1 and 2, a Z-axis drive mechanism 95 as a moving unit is attached to the XY two-axis transport mechanism 75. The Z-axis drive mechanism 95 moves the head 51 in a Z-axis direction orthogonal to the X-axis and the Y-axis, that is, in a direction orthogonal to the washing table 96. The Z-axis drive mechanism 95 has a Z1-axis drive mechanism 95Z1 and a Z2-axis drive mechanism 95Z2, like the Z-axis drive mechanism 23 in the stamping area. The needle 53 can be projected from the liquid storage member 52 at any position of the ultrasonic cleaning section 71, the surfactant solution processing section 72a, the rinse cleaning section 72b, the drying section 73, and the solution storage section 74. .
[0076]
The Z1-axis drive mechanism 95Z1 is composed of an electric actuator that moves a block using a feed screw and an electric motor, like the Z1-axis drive mechanism 23Z1 in the stamping area.
[0077]
A Z2-axis moving mechanism 95Z2 is attached to the table 97 of the Z1-axis driving mechanism 95Z1. The Z2-axis drive mechanism 95Z2 is composed of an electric actuator having the same configuration as the Z1-axis drive mechanism 95Z1, and is smaller than the Z1-axis drive mechanism 95Z1. An L-shaped arm 99 for raising and lowering the needle 53 of the head 51 is attached to the table 98 of the Z2-axis moving mechanism 95Z2. The L-shaped arm 99 can be inserted into the head 51 by an air cylinder (not shown). The tip of the L-shaped arm 99 is inserted into the head, and the inserted L-shaped arm 99 is lowered by the Z2-axis moving mechanism 95Z2 so that the needle 53 projects from the liquid storage member 52 of the head 51.
[0078]
A turning motor 100 as a turning unit is attached to the table 97 of the Z1-axis drive mechanism, and a disk 101 that turns in a horizontal plane is attached to an output shaft of the turning motor 100. A pair of clamps 102, 102 as gripping portions capable of gripping the head 51 are attached to the lower surface of the disk 101 at intervals of 180 degrees. The clamps 102, 102 are opened and closed by an air cylinder (not shown) or the like, and sandwich a flat portion 103 (see FIGS. 6 and 7) formed on the outer periphery of the head 51.
[0079]
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 stamping area. I do.
[0080]
The turning motor 100 turns by 180 degrees so that the head 51 is transferred from the XY two-axis transport mechanism 6 in the stamping area to the XY two-axis transport mechanism 75 in the cleaning area, and is also transferred from the XY two-axis transport mechanism 75 in the cleaning area to the stamping area. Delivery of the head 51 to the XY two-axis transport mechanism 6 is performed.
[0081]
Specifically, as shown in FIGS. 1 and 2, first, the XY two-axis transport mechanism 6 in the stamping area transports the head 51 after forming the spot to the transfer position 104. On the other hand, the XY two-axis transport mechanism 75 in the cleaning area transports the head 51 holding the new solution to the copy 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. Next, the clamp 102 of the XY two-axis transport mechanism 75 in the cleaning area grips the head 51 after the spot transported to the transfer position 104. Thus, the head is transferred from the XY two-axis transport mechanism 6 in the stamping area to the XY two-axis transport mechanism 75 in the cleaning area. Next, the rotating motor 100 rotates the disk 101 by 180 degrees, and positions the head 51 after spot formation at the copy position 105 and positions the head 51 holding the new solution at the transfer position 104. Next, the chuck portion 45 of the XY biaxial transport mechanism 75 in the stamping area grips the head 51 holding the new solution. Thus, 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.
[0082]
As described above, the heads 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, so that one head 51 forms a spot on the substrate 3. Then, the other head 51 can be cleaned or the like.
[0083]
Next, in the microarray manufacturing apparatus of one embodiment of the present invention as described above, each time a biological sample-containing solution is supplied to form a spot, before this, a surfactant is applied to the contact surface of the spot placement tool. An operation of forming a surfactant film on the contact surface of the spot disposing device by supplying and rinsing with water to remove the excess is described.
[0084]
First, 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 72a has a surfactant solution tank, and moves the head 51 to an upper part of the tank. The tip of the liquid storage member 52 of the spot disposition tool attached to the head 51 is cleaned by the surfactant. It is immersed in the surfactant solution in the solution tank. In this state, if the needle 53 is moved up and down, for example, up and down 1 to 10 times, preferably 3 to 5 times at a speed of about 0.5 to 2 times / second, the movement relatively Even if the surfactant solution has a high concentration, the surfactant solution can be easily carried into the inside of the liquid reservoir member 52, and the inside of the liquid reservoir member 52 and the outer surface of the needle 53 can be coated with the surfactant solution.
[0085]
Next, the head 51 is transported to the ultrasonic cleaning unit 71 by the XY two-axis transport mechanism 75 in the cleaning area. In the ultrasonic cleaning section 71, the liquid storage member 52 is immersed in pure water subjected to ultrasonic vibration to clean the outside thereof. In the ultrasonic cleaning section 71, it is desirable to clean the outside of the needle 53 with the needle 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.
[0086]
Further, the head 51 is transported by the XY two-axis transport mechanism 75 to the rinse cleaning section 72b. The rinsing washing section 72b removes surplus surfactant attached to the inside and outside of the liquid storage member 52 and the outside of the needle 53.
[0087]
By mounting the head 51 in a pure water tank storing ultrapure water as a cleaning liquid and immersing the liquid reservoir member 52 in ultrapure water, the outside of the liquid reservoir member 52 is cleaned. Further, by incorporating the head 51 into the pure water tank, the pure water supply pipe 108 shown in FIG. 6 provided in the pure water tank is connected to the head 51, and the pure water supply pipe communicates with the supply space 58 for cleaning liquid and the like. 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 storage member 52. The cleaning liquid supply space 58 is a wide single space extending over each liquid storage member 52. When pressured pure water 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.
[0088]
By supplying water to the inside of the liquid storage member 52 by applying pressure, it is possible to shorten the time for rinsing excess surfactant or cleaning the sample remaining on the surface. 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 the pressure loss between the liquid storage members 52 is reduced. Becomes substantially equal. Accordingly, 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.
[0089]
As shown in FIG. 1, the head 51 after the rinsing is transported to the drying unit 73 by the XY two-axis transport mechanism 75.
[0090]
In the drying tank of the drying unit 73, a vacuum suction pipe (not shown) having the same configuration as the pure water supply pipe 108 in FIG. 6 is connected to the head 51, and this vacuum suction is connected to the drying tank. The tube communicates with the 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 by vacuum, and the water and the like remaining in the space of the liquid reservoir member 52 are suctioned and removed.
[0091]
If the excess surfactant is not sufficiently removed, the head 51 is again rinsed with the ultrasonic cleaning unit 71 after the vacuum suction of water in the drying unit, if necessary. The cleaning and vacuum suction steps in the ultrasonic cleaning unit 71 and the rinsing cleaning unit 72b can be repeated a plurality of times.
[0092]
Thereafter, in the drying section 73, 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.
[0093]
From the viewpoint of the contamination of the surfactant used, before the head 51 after spot formation is immersed in the surfactant solution tank, first, the ultrasonic cleaning unit 71 or the rinsing cleaning unit 72b is used. A method of once washing with water to remove most of the remaining sample is also conceivable. In this way, once the water is contained in the liquid reservoir member 52, the operation described above is thereafter performed. Therefore, even if an attempt is made to feed the surfactant solution into the inside of the liquid storage member 52, this is hindered by the existing water, and eventually, a sufficient amount or concentration of the surfactant is brought into contact with the inside of the liquid storage member 52 or the like. Since it may be difficult, it is desirable to adopt a method of first supplying a surfactant as described above.
[0094]
Thereafter, as shown in FIGS. 1 and 2, the dried head 51 is transported to the solution storage 74 by the XY biaxial transport mechanism 75. The solution storage unit 74 includes a cassette 122 in which a plurality of titer plates 121 as solution holding plates for storing a solution are stored, a plate transport mechanism 123 that takes out the titer plate 121 from the cassette 122 and transports the titer plate 121 to a load position 132. Is provided. In the solution storage section 74, 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.
[0095]
A plurality of (for example, 384) recesses are arranged in each titer plate 121, and the solution of the biological sample is stored in the recesses. For example, assuming that the head has 48 reservoir members, one titer plate can be loaded eight times. The plurality of recesses may be filled with the same type of solution, or may be filled with different types of solutions.
[0096]
A plurality of (for example, ten) titer plates 121 are stored in the cassette 122 at equal intervals in the Z-axis direction (that is, in the vertical direction). Two cassettes 122 are provided above and below the washing table 96. In this apparatus, a total of 20 titer plates 121 are stored. 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.
[0097]
The cassette 122 is placed on a cassette support 124 slidably mounted on the apparatus. The cassette support base 124 is manually pulled out, the cassette 122 is placed on the cassette support base 124, and the cassette support base 124 is manually returned to the original position, whereby the cassette 122 is incorporated into the apparatus.
[0098]
The plate transport mechanism 123 includes a Z-axis drive mechanism 123Z, a Y-axis drive mechanism 123Y, and an X-axis drive mechanism 123X. The Z-axis drive mechanism 123Z 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 123Z vertically moves a support plate 126 that supports the titer plate 121 between the upper end titer plate 121 and the lower end titer plate 121.
[0099]
The X-axis moving mechanism 123X is attached to the table 127 of the Z-axis driving mechanism 123Z. The X-axis moving mechanism 123X 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 129 that can slide the track rail 128.
[0100]
The Y-axis moving mechanism 123Y is attached to the table 129 of the X-axis moving mechanism 123X. The Y-axis moving mechanism 123Y also includes a so-called rodless cylinder, and moves the table 130 in the Y-axis direction and positions the table 130 at two positions in the Y-axis direction.
[0101]
After the titer plate 121 is transported to the load position 132, the head 51 after washing and drying is also transported to the load position by the two-dimensional transport mechanism 75. At the loading position 132, the solution is aspirated by immersing the liquid reservoir member 52 in the solution of the biological sample.
[0102]
The method for sucking the solution will be described. First, the liquid storage member 52 is inserted into the concave portion in the titer plate 121, and the tip of the liquid storage member 52 is immersed in the solution. Next, when the needle 53 is raised while the position of the liquid reservoir 52 is fixed, the solution is pulled up in accordance with the rise of the needle 53, and the solution is filled in the liquid reservoir 52. When the liquid storage member 52 and the needle 53 are pulled up from this state, the solution filled in the liquid storage member is held as it is.
[0103]
A head storage 135 is provided on the washing table 96. The head 51 is first placed in the head storage 135. The operation of the apparatus starts when the XY two-axis transport mechanism 75 in the cleaning area goes to the head place 135 to pick up the head 51.
[0104]
Next, the overall operation of the microarray manufacturing apparatus of the present embodiment will be described according to the procedure for manufacturing a microarray. In the following steps, the head 51 is moved to a predetermined position by appropriately operating the XY two-axis transport mechanism 6 and the Z-axis drive mechanism 23 in the stamping area and the XY two-axis transport mechanism 75 and the Z-axis drive mechanism 95 in the cleaning area. Are sequentially positioned. Such control is performed by a control device (not shown).
[0105]
First, as a preparation stage, a plurality of substrates 3 are arranged in a stamping area, and a vacuum device is operated to suction and fix the substrates 3. On the test table 5, a substrate or a dummy substrate for forming a microarray on a test basis is fixed. On the other hand, a plurality of titer plates 121 are stored in the cassette 122 of the solution storage section 74 in the cleaning area. In each of the recesses of the titer plates 121, for example, solutions of a plurality of types of DNA fragments are put.
[0106]
Next, the XY two-axis transport mechanism 75 in the cleaning area goes to pick up the head 51 placed in the head place 135. Here, of the pair of clamps 102, only one clamp 102 grips the head 51.
[0107]
Next, the XY two-axis transport mechanism 75 transports the gripped head 51 to the load position 132. Here, 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. After the XY two-axis transport mechanism 75 transports the head 51 to the load position 132, the Z-axis drive mechanism 123Z in the cleaning area raises and lowers the reservoir 52 and the needle 53 so that the reservoir 52 sucks the solution.
[0108]
Next, the XY biaxial transport mechanism 75 in the cleaning area transports the head 51 holding the solution to the transfer position 104.
[0109]
Next, the XY two-axis transport mechanism 6 in the stamping area transports the empty chuck portion 45 to the transfer position 104. Then, the Z1-axis driving mechanism in the stamping area moves down the chuck unit 45, and the chuck unit 45 grips the head holding the solution. Thus, 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.
[0110]
Next, the XY two-axis transport mechanism 6 in the stamping area transports the head 51 to the test table 5. In the test table 5, a test process for adjusting the amount of the solution adhering to the needle 53 is performed.
[0111]
After the test process is completed, a stamping process for forming a spot on the substrate 3 is performed. In this stamping step, first, the XY biaxial transport mechanism 6 in the stamping area moves the head 51 to the spot forming position on the substrate 3. Then, the Z1-axis driving mechanism 23Z1 in the stamping area descends the head 51, and positions the head 51 slightly above the substrate 3. Next, the Z2 axis drive mechanism 23Z2 in the stamping area causes the needle 53 to protrude from the liquid storage member 52, and strikes the needle 53 on the substrate 3.
[0112]
After forming a spot on a predetermined substrate, 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.
[0113]
While the XY two-axis transport mechanism 6 in the stamping area repeats the stamping process, the XY two-axis transport mechanism 75 in the cleaning area grips the remaining head 51 placed on the head storage 135 and transports it to the load position 132. At the loading position 132, a loading step of sucking the solution into the liquid storage member 52 is performed. Then, the XY two-axis transport mechanism 75 in the cleaning area transports the head 51 holding the solution to the copy position 105. At this time, an empty clamp 102 that does not grip the head is located at the transfer position 104.
[0114]
After forming the spots on all the substrates 3 on the worktable 4, the XY two-axis transport mechanism 6 in the stamping area transports the head 51 after the spot formation to the transfer position 104. Then, the heads 51, 51 gripped between the XY two-axis transport mechanism 6 in the stamping area and the XY two-axis transport mechanism 75 in the cleaning area are transferred to each other.
[0115]
The XY two-axis transport mechanism 6 in the stamping area after the transfer step transports the head 51 onto the substrate again. Then, the test step and the stamping step are performed.
[0116]
The XY two-axis transport mechanism 75 in the cleaning area after the delivery step executes the cleaning step at the same time that the XY two-axis transport mechanism 6 in the stamping area executes the test step and the stamping step. In this cleaning step, first, the head 51 after forming the spot is first conveyed to the surfactant solution processing section 72a as described above, and the inner surface and the outer surface of the liquid storage member 52 and the outer surface of the needle 53 are washed with the surfactant. Coating treatment. Next, the head 51 is conveyed to the ultrasonic cleaning section 71, and the outside of the liquid storage member 52 is ultrasonically cleaned. Next, the head 51 is transported to the rinsing section 72, and the inside and outside of the liquid reservoir member 52 and the needle 53 are rinsed. Thereafter, the head 51 is transported to the drying unit 73, and the liquid reservoir 52 and the needle 53 are dried.
[0117]
The XY two-axis transport mechanism 75 in the cleaning area transports the cleaned head 51 to the load position 132 again. At this loading position, the loading process for sucking a new solution into the head 51 after cleaning is performed again.
[0118]
Thereafter, the XY two-axis transport mechanism 6 in the stamping area sequentially executes the head transfer step, the test step, and the stamping step. On the other hand, the XY two-axis transport mechanism 75 in the cleaning area sequentially executes the head transfer step, the cleaning step, and the loading step.
[0119]
In the above embodiment, the holding means (head) provided with the needle and the liquid storage member has been described, but a holding means having only the needle is also applicable.
[0120]
【Example】
Next, the present invention will be specifically described based on examples.
Example 1
In a device having a configuration as shown in FIG. 1, a sample having 100 spots was continuously stamped using a head having 16 spot arranging devices (a liquid reservoir member 52 and a needle 53). In addition, a fluorescent dye (rhodamine) solution was used as a sample, and the tip diameter of the needle was 75 μm.
[0121]
After the stamping operation of each spot, the spot arranging device was subjected to a treatment of coating a surfactant (Tween 20) solution at a concentration of 40% by volume, followed by rinsing with water. In addition, for comparison, the same measurement was performed for the case where the washing operation was performed only with the same amount of water without using a surfactant.
[0122]
In the stamping of 100 spots, the ratio of the largest diameter to the smallest diameter in all the spots, the ratio of the largest amount to the smallest amount in all the spots, the average spot amount in the control and the average spot in the examples The ratio with the amount was determined.
[0123]
The results obtained are shown below.
[0124]
[Table 1]

As shown in Table 1, by performing the coating treatment with the surfactant, the surface states of the liquid reservoir member 52 and the needle 53 are stabilized (the wettability is stable), the dispersion of the spots is reduced, and the spot amount is increased. did.
Example 2
In a device having a configuration as shown in FIG. 1, a sample having 100 spots was stamped 100 times in the same manner as in Example 1 using a head having 16 spot arranging devices (a liquid reservoir member 52 and a needle 53). . As a sample, a fluorescent dye (rhodamine) added at 10 pmol / μL in 3 × SSC buffer was used, and the relative liquid volume of each spot was determined by measuring the fluorescence intensity of each spot.
[0125]
After the stamping operation of each spot, the spot arranging device was subjected to a treatment of coating a surfactant (Tween 20) solution at a concentration of 40% by volume, followed by rinsing with water. In addition, for comparison, the same measurement was performed for the case where the washing operation was performed only with the same amount of water without using a surfactant.
[0126]
As a result, when the coating was carried out with the surfactant, the variation (standard deviation) of each spot was 0.076, while that obtained by washing with water only was 0.117. The fluorescence intensity ratio between the case where the coating with the surfactant was performed and the case where the coating with the surfactant was performed was 1.8 when the case where the coating with the surfactant was performed was 1 when the case where the coating was washed with water alone was 1. . From these points, as in the case of the first embodiment, by performing the coating treatment with the surfactant, the surface states of the liquid storage member 52 and the needle 53 are stable (the wettability is stable), and the dispersion of the spot is small. And the spot volume increased.
Example 3
The change in wettability due to the surfactant coating was measured. Since it is difficult to measure on the inner surface of the cylindrical liquid storage member 52, the same stainless steel plate as this liquid storage member 52 is made to have the same surface roughness (Ra = 0.1 # 600 polishing). The change in wettability was measured by measuring the contact angle before and after coating with a surfactant (Tween 20). As a Tween 20 solution, a solution having a concentration of 40% by volume was used. After applying the solution on a plate, washing with running water was performed for about 30 seconds in the same manner as in the actual rinsing step to remove excess surfactant. After removal, the test was performed.
[0127]
In the measurement, the amount of water droplets was 12 μL, and the contact angle before and after coating with a surfactant was determined as the average value of any three points in cross-sectional observation with a microscope.
[0128]
As a result, before the surfactant was coated, the contact angle was 74.95 °, whereas after the coating, the contact angle was 64.49 °, a difference of 10.46 °, indicating a significant change in wettability. .
[0129]
【The invention's effect】
As described above, according to the present invention, in a microarray manufacturing method for forming a plurality of spots of a solution containing a biological sample on a substrate, contact with the solution in a spot disposing tool for forming the spots Since at least a part of the surface is coated with a surfactant and the solution is supplied in this state to form spots, a microplate is prepared with uniform size and shape of each spot. In addition, contamination between sample solutions can be suppressed.
[Brief description of the drawings]
FIG. 1 is a plan view showing a microarray manufacturing apparatus according to a first embodiment of the present invention.
FIG. 2 is a front view of the microarray manufacturing apparatus as viewed in the direction of the line II-II in FIG.
FIG. 3 is a right side view of the microarray manufacturing apparatus as viewed from the line III-III in FIG. 2;
FIG. 4 is a left side view of the microarray manufacturing apparatus as viewed from the direction of line IV-IV in FIG. 2;
FIG. 5 is a cross-sectional view of the microarray manufacturing apparatus as viewed from the line VV in FIG. 1;
FIG. 6 is a front view of the head.
FIG. 7 is a right side view of the head as seen from the direction of line IX-IX in FIG. 6;
FIG. 8 is a bottom view of the head as seen from the line XX in FIG. 6;
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 a liquid storage member and a needle.
FIG. 11 is a process chart showing a method of arranging a solution stored in a liquid storage member on a substrate.
[Explanation of symbols]
3 ... substrate
4 ... Work table
6. XY biaxial transport mechanism (transport means) in stamping area
23,95 ... Z-axis drive mechanism (moving means)
51 head (holding means)
52: Liquid storage member (liquid storage part)
53 ... Needle (spot arrangement tool)
57 ... Lower plate (base)
58 ... space for supplying cleaning liquid etc.
71 ... Ultrasonic cleaning part (cleaning etc. construction part)
71a: Surfactant processing section (cleaning and other processing section)
72b: Rinse cleaning part (cleaning etc. construction part)
74 ... solution storage part (washing etc. working part)
75: XY two-axis transport mechanism (transport means) for cleaning area

Claims (8)

A microarray manufacturing method for forming a plurality of spots of a solution containing a biological sample on a substrate, wherein at least a part of a contact surface with the solution in a spot arranging device for forming the spots is formed by a surfactant. And forming a spot by supplying the solution in this state. In the coating with the surfactant, each time the solution is supplied to form a spot, a surfactant is supplied to the contact surface of the spot disposing tool prior to this, and the excess is removed by rinsing with water. 2. The method for producing a microarray according to claim 1, wherein the method is performed. The method for producing a microarray according to claim 2, wherein the concentration of the surfactant used is 5% or more. 4. The method according to claim 2, wherein the rinsing is performed using 100 to 1000 times by volume of water with respect to the amount of the surfactant used. 5. The method for producing a microarray according to claim 1, wherein the coating with the surfactant is performed by applying a surfactant to a contact surface of the spot disposition tool and performing a fixing treatment. The contact angle of the spot disposition device with water of the portion covered with the surfactant is reduced by 10% or more as an average value of any three points in a cross-sectional observation of the site with a microscope as compared to before the cleaning treatment with the surfactant. The method for producing a microarray according to claim 1, wherein the method is performed. A microarray manufacturing head comprising one or more spot placement tools for holding a solution containing a biological sample and placing a spot of the solution on the substrate by contacting the tip with the substrate, wherein the spot A microarray producing head, wherein the placement tool covers at least a part of a contact surface with the biological sample with a surfactant. A solution storage portion for storing a solution containing a biological sample, a worktable on which a plurality of substrates can be arranged, and a spot for taking and holding the solution from the solution storage portion and forming a solution spot on the substrate A solution holding unit having an arrangement tool, a cleaning application section for performing washing and the like of the holding unit, and moving the holding unit in a direction of approaching / separating from the substrate; A moving means for forming, and a transport means for transporting the holding means in an area including the solution storage unit, the work table and the cleaning or the like application unit, and providing two-dimensional coordinates,
A microarray manufacturing apparatus, wherein at least a part of a contact surface of the spot placement tool with the biological sample is coated with a surfactant.

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