WO2004099788A1 - Method for producing microarray, and head and apparatus for producing microarray - Google Patents

Abstract

A method for producing a microarray wherein a plurality of spots of a solution containing plural kinds of biological samples are formed on a substrate using a spot-arranging means is characterized in that cleaning of the spot-arranging means during intervals between formation of respective spots is conducted using a surfactant solution. By this method, a cleaning operation conducted after formation of each spot before forming the next spot is performed surely and quickly, and contamination between spots can be prevented. Further, this method enables to form a high-quality microarray of spots having a uniform size and shape efficiently.

Description

 Itoda »

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. Background art

 At present, technology development for efficiently analyzing the function of all genes in various organisms is progressing. 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. In order to develop this DNA microarray technology, a microarray fabrication technology that precisely arranges the spots of densely packed DNA fragments on a substrate is required.

For example, 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. In addition to the above, there is also known 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.

 In such a microarray manufacturing apparatus, it is necessary to control the amount of the sample for each spot so as to be equal to each other. However, in a conventionally known apparatus, a large number of 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.

In such a microarray manufacturing apparatus, it is necessary to wash the head so that the previous solution does not become cloudy when the next solution of a different type is held in the head after the spot formation operation. is there. 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. However, in order to prevent turbidity of the solution, a plurality of spot arranging tools are required. 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. In order to enhance the washing action, for example, the washing of a dispensing nozzle of a medical analyzer as disclosed in Japanese Patent Application Laid-Open No. Hei 1-25471 and Japanese Patent Application Laid-Open No. Hei 8-21840 As in the case, ultrasonic vibration was applied to the cleaning section. However, even when such ultrasonic vibration is used in combination with the washing operation in order to prevent contamination by the previous solution, water alone cannot perform sufficient washing, and the earlier sample is replaced with the latter sample. Carryover, which was carried over to the inside, occurred, which was a problem. In particular, when the biopolymer contained in the sample has a high molecular weight, for example, a chain length of 100 O mer or more, 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.

Disclosure of the invention

 Therefore, 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.

 It is still another object of the present invention to provide a microarray manufacturing method and a microarray manufacturing apparatus capable of uniformly forming the size of each spot when arranging a plurality of spots on a substrate.

 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. A method for producing a microarray, characterized in that washing of the spot disposition device is performed using a surfactant solution.

As described above, in the present invention, since 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.

 Conventionally, 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.

 Further, by performing the washing using the surfactant in this manner, 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.

 Further, in one embodiment of the method for producing a microarray of the present invention, 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.

 Further, in one embodiment of the microarray manufacturing method of the present invention, 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. Is shown. In the spot disposition tool having such a shape, 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.

In one embodiment of the method for producing a microarray of the present invention, The activator concentration is at least 5%. By applying a surfactant having a relatively high concentration as described above, the cleaning effect can be expected to be greatly improved.

 In another embodiment of the microarray production method of the present invention, 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.

 In one embodiment of the method for producing a microarray of the present invention, the surfactant used in the surfactant solution is a nonionic surfactant or an amphoteric surfactant, and more preferably, polyoxyethylene. A fatty acid ester. By using such a surfactant, the effect of the surfactant on the biological sample to be used can be eliminated.

 Furthermore, in a preferred embodiment of the microarray production method of the present invention, 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. As described above, in the present invention, 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.

 Further, in one embodiment of the method for producing a microarray of the present invention, 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.

 In one embodiment of the method for producing a microarray of the present invention, the rinsing is performed using 100 to 100 times the volume of water with respect to the amount of the surfactant used. Is shown.

 In another embodiment of the method for producing a microarray according to 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 a fixing treatment. It is characterized by. When 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.

In one embodiment of the method for producing a microarray of the present invention, 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.

 If 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.

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. BRIEF DESCRIPTION OF THE FIGURES

 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 reference numerals given in these figures represent the following, respectively. '

 3 substrates,

 4 workbench,

 6 XY biaxial transport mechanism (transportation means) in the stamping area,

 23, 95 Z-axis drive mechanism (moving means),

5 1 head (holding means), 5 2 Liquid reservoir member (liquid reservoir),

 5 3 Needle (spot placement tool)

 5 7 Lower plate (base)

 5 8 Cleaning solution supply space,

 7 1 Ultrasonic cleaning part (cleaning etc. part),

 7 1 a Surfactant treatment section (cleaning section)

 7 2 b Rinse cleaning part (cleaning etc. part),

 7 4 Solution storage section (washing section)

 7 5 XY 2-axis transport mechanism (transportation means) in the cleaning area.

BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in detail based on embodiments. Cleaning with surfactant solution

 As described above, 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.

 In the present invention, 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.

Examples of the nonionic surfactant 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. Of these, preferred examples include 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. Specifically, for example, polyoxyethylene (8) octyl phenyl ether (Triton X-114), polyoxyethylene (9) octyl phenyl ether (NP-40), polyoxyethylene (10) octyl phenyl Monotel (Triton X-100), polyoxyethylene (20) sorbitan monolaurate (Tween20), polyoxyethylene (20) sorbitan monopalmitate (Tween40), polyoxyethylene (20) Sorbitan monostearate (Tween60), polyoxyethylene (20) sorbitan trioleate, etc. . In particular, polyoxyethylene sorbitan monofatty acid ester, typically polyoxyethylene (20) sorbitan monolate, can be preferably used.

Examples of the amphoteric surfactant include, but are not particularly limited to, alkyldimethylaminoacetate betaine, alkyldimethylamine oxide, alkylcarboxymethylhydroxyxethylimidazolymbetaine, Rukylamidopropyl betaine and the like.

 As the surfactant, 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.

 In the production of microarrays, as described above, in order to form spots of a very large number of thousands to hundreds of thousands of biological sample-containing solutions, it is common to use a plurality of spot arranging tools at the same time. However, there is a limit to the number of spot locators that can be provided so as to correspond to all types of biological sample-containing solutions of the spot to be formed. This is also used for spot formation of the biological sample-containing solutions of the above types.

 Therefore, after the formation of the spot of one type of biological sample-containing solution is completed and prior to the formation of the spot of the next type of biological sample-containing solution, the biological sample-containing solution is not attached to the spot placement tool and remains. Conventionally, a washing operation with water has been performed to prevent contamination of a solution containing a biological sample. In the present invention, in place of this, by using 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. Thus, 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. In particular, it is difficult to introduce a relatively high concentration, which is suitable as described later, and there is a possibility that an effective cleaning action may not be obtained. In addition, if a drying treatment is performed to remove water, the solution in the spot placement device dries and adheres to the wall, and is then washed and removed using a surfactant. It becomes difficult. Therefore, as described above, it is desirable to adopt a method of first supplying a surfactant solution.

 In addition, by supplying a surfactant to the contact surface of the spot disposition device and rinsing with water to remove the excess, 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. For example, 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.

When supplying a surfactant to the contact surface of the spot placement tool, conventional methods such as ultrasonic vibrations are used as necessary to enhance the cleaning action of the surfactant. An additional washing operation as described above may be added.

 As described above, after the surfactant solution is supplied to the contact surface of the spot disposition device, washing with water is performed in order to remove the residue released from the contact surface and the used surfactant solution.

 As the 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. On the other hand, if an excessive amount of water is used relative to the amount of the surfactant used, the removal rate of the residue does not increase so much, and the treatment time is prolonged, which is uneconomical. is there. Further, according to the knowledge obtained by the present inventors, as described later, when the surfactant is slightly left on the wall surface of the spot arranging device and becomes in a state of covering the wall surface, such a surfactant becomes However, in the next spot formation, there is no danger of flowing out into the spot, and the surfactant remaining on the wall improves the hydrophilicity of the spot arranging device wall, and the subsequent spot shape The composition is stabilized. Therefore, 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. When the contact surface of the spot placement device with the biological sample-containing solution is hydrophilized by the surfactant in this way, 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 °.

 In this way, after washing with water, treatments such as air drying and heat drying are performed to remove moisture remaining in the spot placement device.

 In the present invention, 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. For example, 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. Here, 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. Is a method in which a sample solution is recovered in a ring, and then the solution penetrates the inside of the reserved ring, captures the sample in the ring with a pin tip, and forms a spot on the substrate. 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.

Regardless of the form of the spot disposition device, it is desirable that 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.

 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. Alternatively, the method can be carried out by forming a surfactant coating on the surface of the spot disposition device as described later.

 In the present invention, 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. Although it is possible, 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 method of cleaning the contact surface of the spot placement device with the biological sample-containing solution using the surfactant in this manner is as described in detail above.

 By supplying the surfactant to the contact surface of the spot placement tool and rinsing it with water to remove the excess, 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. Nation can also be prevented.

Each time a spot containing a biological sample is supplied to form a spot, a surfactant is supplied to the contact surface of the spot locator and rinsed with water to remove the excess. In the embodiment in which a surfactant film is formed on the contact surface of the substrate, 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. 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, 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. In addition, as described above, after supplying the surfactant to the contact surface of the spot disposition device, rinsing with water is performed to remove the excessively attached surfactant. Water used for the rinsing 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.

 Thus, after rinsing with water, if necessary, treatment such as air drying or heat drying may be performed.

 Alternatively, in a second preferred embodiment in which at least a part of the contact surface of the spot placement device 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 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.

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.

 Also, in this case, when changing the type of the biological sample-containing solution that forms the spot, it is necessary to wash the spot placement tool after use with 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 device can be removed more easily and more quickly than in the conventional case. In the present invention, at least a part of the contact surface of the spot placement device with the biological sample-containing solution is thus coated with the surfactant and made hydrophilic. Although not particularly limited, 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 °.

 For this reason, using a spot placement tool coated with such a surfactant, a biological sample containing a spot having a predetermined volume of 3 to 5 × 10-4 mm3 per spot. When spots of the solution are formed, the rate of variation (variation) in the volume between the spots is typically very low, less than 10%.

 In the present invention, 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.

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 serving as a micropore channel. Microarray preparation head and preparation equipment

 Next, 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. As described above, 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, and 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, and 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.

 As shown in FIG. 1, 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.

First, the stamping region will be described. A large number of substrates 3 are arranged on the worktable 4 in a matrix. Substrate 3 is slide glass ゃ silicon etc. The surface of the substrate 3 is surface-treated so that a biological sample can be attached! / Puru.

 On a 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 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.

 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. 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. On the opposite side of the X-axis transport mechanism 6 X across the substrate, 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.

As shown in FIGS. 1 and 2, 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. And 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.

 As shown in Fig. 1, Fig. 2 and Fig. 5, 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. Have been. 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.

Z 1 axis drive mechanism 2 3 The table 41 of Z 1 is provided with a Θ axis rotation mechanism 43 as attitude change means for changing the attitude of the head 51. The Θ-axis rotating mechanism 43 turns the head 51 in a horizontal plane. ΘShaft 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.

 6 and 7 show a head 51 as a holding means. 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.

 To the lower plate 57, 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. In this embodiment, 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.

 Further, 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.

 Thus, in this embodiment, 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.

Slide the support plate between the upper plate 55 and the lower plate 57. 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. At the top of the needle 53, a flange 53d is formed to be placed on the upper surface of the needle support plate 61. Between the flange 53d and the intermediate plate 59, a coil spring 62 is formed. 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. 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 storage members 52. When the needles 53 come into contact with the substrate 3, 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 operation of the head 51 by each drive mechanism will be described. First, the head 51 is positioned by the XY two-axis 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 23 Z1, 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 Z 2-axis moving mechanism 2 3 Z 2 is advanced above the intermediate plate 59 in the head 51. When the L-shaped arm 42 is lowered by the Z two-axis moving mechanism 2 3 Z 2, the intermediate plate 59 is pushed down by the L-shaped arm 42, whereby the needle 53 protrudes from the liquid reservoir member 52. On the other hand, when 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. In addition, 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. When the needle 53 protrudes a predetermined amount from the liquid reservoir member 52 and the distal end surface 53 b of the needle 53 comes into contact with the substrate 3, the surface roughness is determined by the solution held in the distal end surface 53 b and the liquid reservoir. It is set so that the solution in the member 52 is connected. The surface roughness is formed by grinding or discharging. For example, in the case of grindstone processing, the outer peripheral surface of the tip 53 a is roughened by moving the grindstone in the longitudinal direction of the needle 53. 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).

First, as shown in FIG. 11A, the liquid storage member 52 is positioned above the substrate 3. Next, as shown in FIGS. 11A and 11B, the needle 53 is gradually lowered with respect to the liquid storage member 52. Next, as shown in FIG. 11D, the needle 53 is made to protrude from the liquid reservoir member 52. At this time, the solution in the liquid reservoir 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. Needle 5 3 tip When 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. At this time, the solution held at the tip of the udle 53 and the solution in the liquid reservoir 52 are connected. Then, as shown in FIG. 11E, when the needle 53 is retracted from the substrate 3, a solution spot is formed on the substrate 3.

 In this way, 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. By taking out and arranging them 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. In addition, 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.

 Next, the cleaning region will be described. In this cleaning area, 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.

As shown in FIG. 1, on the cleaning table 96, 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. Surfactant solution treatment section 72 a, rinse section 72 b for rinsing head 51, drying section 73 for drying head 51, drying section 73 for drying head 51, and solution containing biological sample A solution storage section 74 for storing is provided. In addition, on the washing table 96, 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}.

 As shown in FIGS. 1 and 3, 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. 82, 82 and a linear guide composed of sliders 83, 83 movably assembled with respect to the rails 82, 82; a table 84 guided by the linear guide; 4 and a feed screw 85 for driving the same.

 The Υ-axis drive mechanism 75 Υ 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 Υ-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.

 As shown in FIG. 1 and FIG. 2, the biaxial transport mechanism 75 is provided with a biaxial drive mechanism 95 as a moving means. The Ζ-axis drive mechanism 95 moves the head 51 in the Ζ-axis direction orthogonal to the X axis and the Υ axis, that is, in the direction orthogonal to the washing table 96. The Ζ-axis drive mechanism 95 has a Ζ1-axis drive mechanism 95 Ζ 1 and a Ζ2-axis drive mechanism 95 Ζ 2, similarly to the Ζ-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.

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.

 Since 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.

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 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. Next, 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. Thus, 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. Next, 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. Next, the chuck portion 45 of the XY two-axis 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.

 In this way, 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.

 Next, in the microarray manufacturing apparatus according to one embodiment of the present invention as described above, every time a biological sample-containing solution is supplied to form a spot, the contact surface of the spot arranging tool is brought into contact with the interface. An operation of cleaning using a surfactant solution, and simultaneously supplying a surfactant to the contact surface of the spot disposition device and rinsing with water to remove an excess, thereby obtaining a contact surface of the spot disposition device. Next, an operation for forming a surfactant film will be described.

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 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. In this state, 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. However, even if 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.

 Then, the head 51 is transferred 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 preferable that 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.

 Further, 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. In addition, by incorporating the head 51 into a pure water tank, 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.

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.

 As shown in FIG. 1, the head 51 after rinsing is transported to the drying unit 73 by the XY two-axis transport mechanism 75.

 In the drying tank of the drying section 73, 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.

 If the surplus surfactant is not sufficiently removed, if necessary, 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.

 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.

From the viewpoint of the contamination of the surfactant to be used, before the head 51 after forming the spot is immersed in the surfactant solution tank, first, the ultrasonic cleaning unit 71 or the rinsing is performed. 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. Since it may be difficult to cause the surfactant to act, it is desirable to adopt a method of first supplying the surfactant as described above. Thereafter, as shown in FIGS. 1 and 2, 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. 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.

 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. For example, if 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.

 After the titer plate 12 1 has been transported to the loading position 13 2, the washed and dried head 51 is also transported to the loading position by the two-dimensional transport mechanism 75. At the loading position 132, the solution is sucked by immersing the reservoir member 52 in the solution of the biological sample.

The method for sucking the solution will be described. First, 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. Next, when 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.

 On the washing table 96, a head storage space 135 is provided. 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.

 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 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).

 First, as 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. On the test stand 5, a substrate for forming a microarray or a dummy substrate is fixed on a test basis. On the other hand, 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.

 Next, 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. Here, of the pair of clamps 102 and 102, only one clamp 102 holds the head 51.

Next, the XY two-axis transfer mechanism 75 transfers the gripped head 51 to the load position 13 2. 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 2-axis transport mechanism 75 transports the head 51 to the load position 1 32, 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.

 Next, the XY two-axis transfer mechanism 75 in the cleaning area transfers the head 51 holding the solution to the transfer position 104.

 Next, the XY two-axis transport mechanism 6 in the stamping area transports the empty check portion 45 to the transfer position 104. Then, 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. 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.

 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.

 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 drive mechanism 23 Z1 in the stamping area descends the head 51 and positions the head 51 slightly above the substrate 3. Next, 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.

 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.

While the XY two-axis transfer mechanism 6 in the stamping area repeats the stamping process, 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.

 After spots are formed on all the substrates 3 on the work table 4, 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 ΥΧ 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. In this cleaning step, first, the head 51 after forming the spot is first treated with the surfactant solution treating section 7 as described above.

2a, and the inner and outer surfaces of the liquid reservoir member 52 and the outer surface of the needle 53 are coated with a surfactant. 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 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.

 XY 2-axis transport mechanism in the washing area 7 5 Loads the head 5 1 after washing 1

3 Transport to 2 again. At this loading position, the loading process for sucking a new solution into the head 51 after cleaning is performed again.

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 above-described head transfer step, the above-described cleaning step, and the opening and closing process.

 In the above embodiment, the holding means (head) provided with a needle and a liquid storage member has been described, but a holding means having only a needle is also applicable. Example

 Next, the present invention will be specifically described based on examples.

Example 1

 In the apparatus having the configuration shown in Fig. 1, a head having sixteen spot arrangement tools (liquid reservoir member 52 and Udle 53) was used, and after the first sample was stamped, the surfactant solution was used. Then, 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 μπι. As a first sample, (1) a fluorescent dye (rhodamine) added at a rate of 10 pmo 1 L in 3 XSSC buffer, and (2) a fluorescently labeled oligo DNA (21 mer) pmo \ / ιχ L, (3) Fluorescently labeled oligo DNA (45mer) added at 割 合 ρ ρ πιο ΐ Ζ μL, and (4) Fluorescently labeled CDNA (40 O mer) was added at a ratio of lpmol / ixL, and only a 3XSSC buffer was used as a second sample. 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).

As the surfactant, 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. For comparison, 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.

 As described above, the carrier-to-bar ratio was significantly reduced when the washing was performed using the surfactant solution.

 Separately, after washing using such a surfactant (biocide), the oligo DNA (21 mer), oligo DNA (45 mer) and cDNA (40 Omer) as described above The solution containing various biological samples was stamped, and the presence or absence of an abnormality in the biological sample contained in each of the obtained spots was examined. No abnormality was found.

Using a head with 16 spot placement tools (liquid reservoir 52 and needle 53), after the first sample was stamped, washing operation was performed using surfactant solutions of various concentrations as shown below. Next, 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 μm. As the first sample, a fluorescent dye (rhodamine) was added in 3 XSSC buffer at a ratio of l Opmol / jL, and the second sample was used. Only 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.

 Table 2

Example 3

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 dollar 53). . A fluorescent dye (rhodamine) solution was used as the sample, and the needle tip diameter was 75 μπι. Was.

 After the stamping operation of each spot, the spot locator was coated with a 40% by volume surfactant (Tween20) solution, followed by rinsing with water. For comparison, 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.

 When stamping 100 spots, 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 The ratio to the average spot amount in the examples was determined.

The results obtained are shown below.

 Table 3

As shown in Table 1, the surface treatment of the liquid storage member 52 and the needle 53 stabilizes (stable wettability), reduces spot variation, and reduces Spot volume increased. 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.

 After the stamping operation of each spot, 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. For comparison, 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.

 As a result, when the coating was performed with the surfactant, the variation (standard deviation) of each spot was 0.076. On the other hand, when the spot was washed with water only, the spot variation was 0.076. The fluorescence intensity ratio between the case where the surface was coated with the surfactant and the case where the surface was washed with water alone was 1. The case was 1.8. From these points, by performing a washing operation using a surfactant, the surface condition of the liquid storage member 52 and the needle 53 becomes stable (stable wettability), and the dispersion of spots is reduced. The spot volume increased. Example 5

The change in wettability due to surfactant washing 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 the liquid storage member 52 is used for the same surface roughness (Ra = 0.1 # 60). 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.

 In the measurement, 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.

 As a result, before the surfactant coating, the contact angle was 74.995 °, but after the coating, it was 64.49 °, and the difference was 10.46 °. The wettability changed greatly. Industrial applicability

 According to the present invention, in forming a plurality of spots of a plurality of types of biological sample-containing solutions on a substrate using a spot arranging device, 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.

 Further, according to the present invention, in 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.

Therefore, according to the present invention, a DNA microarray having more excellent properties and quality can be provided. It can greatly contribute to the efficient analysis of differences and diversity.

Claims

The scope of the claims

1. A method for manufacturing a microarray in which a plurality of spots of a biological sample-containing solution are formed on a substrate using a spot placement tool, wherein the washing of the spot placement tool performed between the formation of the spots is performed using a surfactant. A method for producing a microarray, which is performed using an agent solution.

2. The method for producing a microarray according to claim 1, wherein the washing of the spot disposition device is performed including the steps of supplying a surfactant solution to the spot disposition device, washing with water, and drying.

3. The spot disposition device has a nozzle and a stamping pin disposed inside the nozzle and capable of reciprocating in a nozzle axis direction. 3. The microarray manufacturing method according to claim 2, wherein the supply is performed by moving the stamping pin forward and backward with at least a tip of the nozzle being in contact with a surfactant solution.

4. The method for producing a microarray according to claim 2, wherein the surfactant concentration in the surfactant solution is 5% or more.

5. The water washing step according to any one of claims 2 to 4, wherein a series of operations consisting of ultrasonic cleaning, running water cleaning, and physical moisture removal are sequentially performed once or more than once. The method for producing a microarray according to any one of the first to third aspects.

6. The microarray fabrication according to any one of claims 1 to 5, wherein the surfactant used in the surfactant solution is a nonionic surfactant or an amphoteric surfactant. Method.

7. The method for producing a microarray according to any one of claims 1 to 5, wherein the surfactant used in the surfactant solution is a polyoxyethylene fatty acid ester.

8. The microarray preparation according to any one of claims 1 to 7, wherein a 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. Method.

9. 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 of the spot arranging tool for forming the spot with the solution is formed. A method for producing a microarray, comprising: coating with a surfactant; and supplying the solution in this state to form a spot.

10. In the coating with the surfactant, each time the solution is supplied to form a spot, the surfactant is supplied to the contact surface of the spot placement tool and then rinsed with water. 10. The method for producing a microarray according to claim 9, wherein the method is performed by removing an excess.

11. The method for producing a microarray according to claim 10, wherein the used concentration of the surfactant is 5% or more.

12. The method according to claim 10, wherein the rinsing is performed using 100 to 100 times the volume of water with respect to the amount of the surfactant used. 2. The method for producing a microarray according to 1.

13. The method for producing a microarray according to claim 9, wherein 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.

14 4. The contact angle force S of the spot-covered part with water on the part coated with the surfactant, and the average value of any three points in the cross-sectional observation of the part with a microscope, compared to before the cleaning treatment with the surfactant The method for producing a microarray according to any one of claims 9 to 13, wherein the microarray is reduced by 10% or more.

15 5. A microarray fabrication head that holds one or more spot placement tools that hold a solution containing a biological sample and place the spot of the solution on the substrate when the tip contacts the substrate. A head for producing a microarray, wherein the spot placement tool has at least a part of a contact surface with the biological sample covered with a surfactant.

16. A solution storage section for storing a solution containing a biological sample, a worktable on which a plurality of substrates can be arranged, and taking and holding the solution from the solution storage section to form a spot of the solution on the substrate A solution holding means provided with a spot arranging device for cleaning, a cleaning application section for cleaning the holding means, etc .; and moving the holding means in a direction approaching / separating from the substrate. Moving means for forming a spot by means, and the holding means, Transport means for transporting in a region including the workbench and the cleaning and other construction unit and providing a two-dimensional coordinate,

 A microarray fabrication characterized in that at least a part of a contact surface of the spot placement tool with the biological sample is coated with a surfactant.