WO2006009273A1

WO2006009273A1 - Substrate-fixing device, manufacturing apparatus for test piece, and manufacturing method for test piece

Info

Publication number: WO2006009273A1
Application number: PCT/JP2005/013538
Authority: WO
Inventors: Ryo Tsutsui
Original assignee: Olympus Corporation
Priority date: 2004-07-22
Filing date: 2005-07-19
Publication date: 2006-01-26
Also published as: JP2006038460A

Abstract

A substrate-fixing device 2 fixes a substrate A for detecting an organism-related substance in a state where it is mounted in a predetermined position on a mounting surface 10a. The substrate A is a porous substrate and has at least one probe region T where a known specific binding substance P can be immobilized. The substrate-fixing device 2 includes a mounting stage 10 having a through hole 11 formed with an opening in the mounting surface 10a in a non-probe region T' which is other than the probe region T, and a holding device 15 that holds a lower surface of the substrate A via the through hole 11. A manufacturing apparatus 1 for a test piece is furnished with the substrate-fixing device 2.

Description

DESCRIPTION

SUBSTRATE-FIXING DEVICE, MANUFACTURING APPARATUS FOR TEST PIECE, AND MANUFACTURING METHOD FOR TEST PIECE

TECHNICAL FIELD

The present invention relates to a substrate-fixing device which fixes a substrate used for detecting an organism-related substance, a manufacturing apparatus for a test piece furnished therewith, and a manufacturing method for a test piece.

Priority is claimed on Japanese Patent Application No. 2004-213988, filed July 22, 2004, the content of which is incorporated herein by reference.

BACKGROUND ART Heretofore, there is known a DNA microarray serving as a tool for the comprehensive analysis of information on gene expression level, mutation, and the like, in which minute spots of nucleic acid probe by which a gene is detected are arrayed on a substrate, then the gene being the specimen is made to react with these, and the hybridization reaction is used to detect the hybridization signal. In such a DNA microarray; reaction time shortening, gene highdensifyizing, sensitivity and reproductivity improvement, quality stability, and the like are required. Therefore, various manufacturing apparatus and manufacturing methods for DNA microarrays have been provided to satisfy these requirements.

Generally, in order to manufacture a test piece such as a DNA microarray, a substrate is fixed on a fixing device or the like, then a nucleic acid probe is immobilized into a solid phase on the substrate. However, when manufacturing the test piece, it is necessary to mount a substrate such as a membrane manually one by one on a predetermined position on the fixing device, and to then move the substrate after the probe is immobilized into a solid phase. Therefore this takes time and labor. Here, as one of the fixing devices for solving this, there is known a membrane-fixing device wherein a substrate such as a membrane can be mounted on a predetermined position without touching it by hand (for example, refer to Japanese Patent No. 3305295 which will be referred to as "Patent Document 1" hereinafter). The membrane-fixing device disclosed in Patent Document 1 comprises a lower suction bath having a suction tube, and an upper suction bath that can cover and be fixed to the lower suction bath. The upper suction bath has a backing plate formed with a large number of vent openings, and a porous plate made from a synthetic resin plate or the like and provided with a large number of minute suction holes is mounted on the backing plate. Moreover, the upper suction bath has a magnet sheet at the same height as that of the upper surface of the porous plate, so as to enclose the porous plate. Then, a membrane is mounted on the porous plate and the magnet sheet, so that the membrane is pressed from the above by a magnet sheet for pressing of the same square frame shape as that of the magnet sheet.

In order to fix the membrane by this membrane-fixing device, after the membrane is mounted and pressed by the magnet sheet for pressing, air in the lower suction bath is sucked out by a suction tube. As a result, the membrane is sucked in the whole region (area) of the porous plate via the vent openings in the backing plate, and the minute suction holes in the porous plate. Consequently, the membrane can be reliably fixed. Moreover, after fixing the membrane, a probe is immobilized into a solid phase on the membrane, to thereby manufacture a test piece such as a microarray. Then, after manufacturing the test piece, the upper suction bath and the lower suction bath are separated, and the next upper suction bath is fitted to the lower suction bath, after which the next test piece can be manufactured.

Moreover, generally when manufacturing a test piece, it is necessary, for example, to previously perform a predetermined chemical treatment on a substrate so that a probe can be readily adhered to the substrate (for example, refer to Japanese Unexamined Patent Application, First Publication No. 2003-194811 which will be referred to as "Patent Document 2" hereinafter). Then, after the chemical treatment of the substrate, the substrate is fixed to the fixing device so as to immobilize the probe into a solid phase. However, in the abovementioned conventional fixing device, the following problems remain.

That is, in the membrane-fixing device described in Patent Document 1, when fixing the substrate, the whole substrate is sucked by air and thus fixed, and hence there has been concern that airborne dust is sucked to the substrate and adhered thereto. Therefore, there is a likelihood of the dust becoming noise at the time of analysis, thus affecting the analysis results and disenabling accurate analysis.

Moreover, the membrane-fixing device merely mounts and fixes the substrate, and has not been considered for performing various chemical treatments using solutions for activating the substrate as described in Patent Document 2, in a state where the substrate is mounted. Therefore, the chemical treatment or the like for the substrate must be performed separately using a device other than the fixing device, and it is necessary to frequently remove the substrate from the mounting surface and to move the substrate. Therefore it takes time and labor. Assuming the case where a chemical treatment such as described in Patent

Document 2 is performed in a state where the substrate is mounted on the membrane-fixing device, the solution is adhered to redundant regions other than the probe immobilization region (test site), so that not only is a large amount of solution required, but also there is concern of unevenness of the contact state between the solution and the probe immobilization region.

Furthermore, in the manufacturing method for a test piece using the membrane-fixing device, only one substrate can be fixed to the stage. Therefore, if a plurality of substrates are treated at the same time, it is necessary to prepare the same number of stages as that of the substrates. Consequently, the cost becomes expensive.

DISCLOSURE OF INVENTION

The present invention takes such problems into consideration with an object of providing a substrate-fixing device whereby a plurality of substrates can be fixed while preventing the adherence of dust thereto, and chemical treatment and immobilization of a probe can be performed in a desired region on the substrate, in a state where the substrates are mounted on a mounting surface, and a manufacturing apparatus for a test piece furnished therewith, and a manufacturing method for a test piece.

In order to achieve the above object, the present invention provides the following means.

A first aspect of the invention provides a substrate-fixing device which fixes a substrate for detecting an organism-related substance in a state where it is mounted in a predetermined position on a mounting surface, the substrate being a porous substrate and having at least one probe region where a known specific binding substance can be immobilized, the substrate-fixing device including; a mounting stage having a through hole formed with an opening in the mounting surface in a non-probe region other than the probe region, and a holding device that holds a lower surface of the substrate via the through hole.

In the substrate-fixing device according to the first aspect of the invention, the non-probe region of the substrate is positioned over the opening of the through hole formed in the mounting surface when the substrate is mounted in a predetermined position on the mounting surface of the mounting stage. Then, the holding device holds the lower surface of the substrate, that is, the lower surface of the substrate in the non-probe region, for example, by suction or absorption via the through hole. As a result, the substrate can be reliably fixed onto the mounting surface.

In particular, since the porous probe region is not affected when the substrate is fixed, dust is not sucked to the substrate by sucking the whole substrate as in the conventional case. Accordingly there is no likelihood of dust being adhered to the probe region of the substrate. Consequently, the substrate can be reliably fixed in a state where the influence on detection of the organism-related substance is as little as possible.

A second aspect of the invention provides a substrate-fixing device according to the first aspect, wherein a plurality of the through holes are formed.

In the substrate-fixing device according to the second aspect of the invention, since a plurality of through holes are formed, the substrate can be stably fixed regardless of the number or shape of the probe region.

A third aspect of the invention provides a substrate-fixing device according to either one of the first and second aspects, which includes a contact section which is formed at a height so as not to protrude from an upper surface of the substrate on the mounting surface, and which can contact with a side face of the substrate, and the contact section positions the substrate in the predetermined position.

In the substrate-fixing device according to the third aspect of the invention, when the substrate is mounted on the mounting surface, the positioning can be performed by making the side face of the substrate contact with the contact section, so that the substrate can be mounted in the predetermined position more readily and reliably. Consequently, the non-probe region and the opening of the through hole can be aligned with high accuracy, so that the substrate can be fixed more reliably.

Furthermore, since the contact section is formed at a height so as not to protrude from the upper surface of the substrate, after fixing the substrate, for example, when a specific binding substance or the like is discharged onto the probe region by another device, interference with the contact section can be avoided. In this manner, the positioning of the substrate can be performed reliably without affecting other devices.

A fourth aspect of the invention provides a substrate-fixing device according to any one of the first to third aspects, wherein the mounting stage is made from a material having electroconductivity.

In the substrate-fixing device according to the fourth aspect of the invention, since the mounting stage is formed from a material having electroconductivity, electrostatic charge can be avoided as much as possible. Consequently, after the substrate is mounted on the mounting surface, adherence of dust onto the substrate due to electrostatic charge can be prevented.

A fifth aspect of the invention provides a substrate-fixing device according to any one of the first to fourth aspects, wherein the holding device includes: a base on which the mounting stage is mounted, having a passage that is connectable to the through hole; and a pressure device which is connected to the passage and which sucks air inside the passage to suck and hold a lower surface of the substrate in the non-probe region.

In the substrate-fixing device according to the fifth aspect of the invention, the through hole in the mounting stage mounted on the base and the passage in the base are connected. If the air in the passage is sucked by operating the pressure device, for example, a suction pump, the through hole connected to the passage can be also sucked. That is, the lower surface of the substrate in the non-probe region can be sucked and held. In this manner, the substrate can be reliably fixed using suction force. Moreover, since the non-probe region is sucked, airborne dust is not sucked to the substrate via the porous probe region. Furthermore, since the air is simply sucked, the structure is simple, and there is little affect on the substrate because there is no heat generated.

A sixth aspect of the invention provides a substrate-fixing device according to any one of the first to fifth aspects, which includes a lid member which, in a state where the substrate is mounted on the mounting stage, covers the whole of the mounting surface and is detachably fixed to the mounting stage, and the lid member is fixed to the mounting stage, while pressing the substrate down between itself and the mounting surface.

In the substrate-fixing device according to the sixth aspect of the invention, by fixing the lid member to the mounting stage, the upper surface of the substrate can be covered, while covering the whole mounting surface. As a result, after fixing the substrate, for example, until the specific binding substance is immobilized into a solid phase on the substrate, or after the specific binding substance is immobilized into a solid phase on the substrate, adherence of dust from the air onto the substrate can be more reliably prevented.

In particular, since the lid member is fixed to the mounting stage while pressing the substrate down onto the mounting surface, even if the substrate is released from being held by the holding device, the positional relationship of the mounting surface and the substrate can be maintained. Hence the mounting stage can be solely moved without shaking nor moving the substrate. Moreover, abrasion or scratching of the substrate caused by shaking of the substrate can be prevented, and hence the analysis results are not affected.

A seventh aspect of the invention provides a substrate-fixing device according to any one of the first to fourth aspects, wherein the mounting stage has a solution through hole with an opening formed in the mounting surface in the probe region, and the substrate-fixing device comprises: a jig which, in a state where the substrate is mounted on the mounting stage, covers the whole of the mounting surface and is detachably fixed to the mounting stage while being stuck to the upper surface of the substrate, and has a solution storage hole formed directly above the probe region; a solution supply device which supplies a solution to the solution storage hole, and stores the solution in a space enclosed by an inner peripheral surface of the solution storage hole and an upper surface of the probe region; and a solution removal device which removes the stored solution that has passed through the probe region and the solution through hole. hi the substrate-fixing device according to the seventh aspect of the invention, when the substrate is mounted in a predetermined position on the mounting surface, the probe region is positioned over the opening of the solution through hole formed in the mounting surface. Then, if the jig is fixed to the mounting stage in this condition, since the solution storage hole is formed directly above the probe region, only the probe region can be exposed to the outside air via the solution storage hole. By supplying the solution to the solution storage hole by the supply device, the solution can be stored in a space enclosed by the inner peripheral surface of the solution storage hole and the upper surface of the probe region. At this time, since the jig is fixed to the mounting stage while being stuck to the upper surface of the substrate, the solution can be reliably stored without leaking.

Moreover, the solution removal device removes the solution by letting it pass through the porous probe region and the solution through hole. By so doing, for example, the probe region can be chemically treated and washed by the solution. In this manner, differing from the conventional case where the chemical treatment of the substrate and the mounting and fixing thereof are separately performed, the chemical treatment and the washing can be readily performed on the probe region while the substrate is mounted on the mounting surface, and hence the time and labor can be reduced. Furthermore, the solution can be used for the probe region only, and hence the solution can be used without any waste (efficiently), and costs can be reduced.

An eighth aspect of the invention provides a substrate-fixing device according to the seventh aspect, wherein the solution is a solution for chemically treating the probe region in a predetermined condition, or a cleaning solution for washing the probe region. In the substrate-fixing device according to the eighth aspect of the invention, the probe region can be chemically treated using a predetermined solution, and the probe region can be washed using a predetermined cleaning solution.

A ninth aspect of the invention provides a substrate-fixing device according to the seventh or eighth aspects, wherein the solution removal device comprises: a solution base on which the mounting stage is mounted, and which has a solution passage that is connectable to the solution through hole; a solution suction pump which is connected to the solution passage and which sucks the stored solution; and a solution storage device disposed between the solution suction pump and the solution base, which stores the solution sucked by the solution suction pump. In the substrate-fixing device according to the ninth aspect of the invention, the solution through hole in the mounting stage mounted on the solution base, and the solution passage in the solution base are connected. If the air inside the solution passage is sucked by operating the solution suction pump, the interior of the solution through hole connected to the solution passage can be also sucked. That is, the lower surface of the substrate in the probe region can be sucked, and the stored solution can be sucked via the probe region and the solution through hole using the suction force. Moreover, the sucked solution is discharged and stored in the solution storage device. In this manner, the stored solution can be reliably removed using the suction force.

A tenth aspect of the invention provides a substrate-fixing device according to any one of the seventh to ninth aspects, wherein the mounting stage is made from a material having chemical resistance.

In the substrate-fixing device according to the tenth aspect of the invention, since the mounting stage is chemical resistant, then even if various solutions for chemical treatment or washing of the probe region are used, there is no effect from the solution. Therefore, the reliability over long term use can be improved.

An eleventh aspect of the invention provides a substrate-fixing device according to any one of the first to tenth aspects, wherein the substrate is a membrane substrate.

In the substrate-fixing device according to the eleventh aspect of the invention, the membrane substrate can be reliably fixed in a state where any affect on the detection of the organism-related substance is as little as possible.

A twelfth aspect of the invention provides a manufacturing apparatus for a test piece comprising; a substrate-fixing device according to any one of the first to eleventh aspects, and a probe discharge device which discharges the specific binding substance in droplet form to a predetermined position on the probe region of the substrate, to immobilize the specific binding substance into a solid phase thereon.

In the manufacturing apparatus for a test piece according to the twelfth aspect of the invention, the probe discharge device discharges the specific binding substance in droplet form to the predetermined position on the probe region of the substrate to immobilize the specific binding substance into a solid phase thereon, to thereby enable a test piece such as a microarray to be manufactured. In particular, since the specific binding substance can be immobilized into a sold phase on the substrate which is fixed by the substrate-fixing device, in a state where the adherence of dust thereto is prevented, higher quality test pieces can be manufactured. Consequently, there is no effect of noise, and the organism-related substance can be checked with higher accuracy.

A thirteenth aspect of the invention provides a manufacturing apparatus for a test piece according to the twelfth aspect, which includes a probe checking device which confirms whether or not the specific binding substance discharged to the probe region is discharged to the predetermined position. In the manufacturing apparatus for a test piece according to the thirteenth aspect of the invention, since the probe checking device confirms whether or not the specific binding substance is reliably discharged to a predetermined position by the probe discharge device, higher quality test pieces can be obtained. A fourteenth aspect of the invention provides a manufacturing method for manufacturing a test piece by immobilizing a specific binding substance into a solid phase on a porous substrate for detecting an organism-related substance, which is furnished with at least one probe region where a known specific binding substance can be immobilized, the method comprising: a treatment step for, after mounting the substrate on a mounting surface of a mounting stage, supplying a solution to the probe region and letting it pass through the probe region, to chemically treat the probe region into a predetermined condition; a fixing step for, after the treatment step, sucking a lower surface of the substrate in a non-probe region other than the probe region, to hold and fix the substrate; a spotting step for, after the fixing step, discharging the specific binding substance in droplet form to a predetermined position of the probe region to immobilize the specific binding substance into a solid phase; a checking step for, after the spotting step, checking whether or not the specific binding substance is discharged to the predetermined position; and a washing step for, after the checking step, supplying a cleaning solution to the probe region and letting it pass through the probe region to wash the probe region, so as to rinse out the specific binding substance that is not completely immobilized into a solid phase, and the respective steps are performed in state where the substrate is mounted on the mounting surface.

In the manufacturing method for a test piece according to the fourteenth aspect of the invention, firstly, after mounting the substrate on the mounting surface of the mounting stage, the treatment step for supplying the solution to the probe region and letting the solution pass therethrough to chemically treat the probe region into a predetermined condition, is performed. As a result, the probe region can be pretreated before immobilizing the specific binding substance. After this treatment step, the substrate is fixed onto the mounting surface by the fixing step. That is, the substrate is sucked and held onto the mounting surface, by sucking the lower surface of the substrate in the non-probe region. At this time, since the substrate is fixed only by sucking the non-probe region, the porous probe region is not affected. Therefore airborne dust are not sucked and adhered to the substrate, as with the conventional case. After fixing the substrate, the specific binding substance is discharged in droplet form, for example in an array form, to the predetermined position on the probe region by the spotting step. Then, by the checking step it is checked whether or not the specific binding substance has been reliably discharged to the predetermined position. After checking, the washing step for supplying a cleaning solution to the probe region and letting it pass through the probe region to wash the probe region, so as to rinse out only the specific binding substance that is not completely immobilized into a solid phase, is performed. By the checking step and the washing step, only the specific binding substance that is completely immobilized into a solid phase in the predetermined position in the probe region can be obtained. In particular, at the time of the fixing step, since the substrate is fixed while preventing the adherence of dust, high quality test pieces can be manufactured.

Moreover, since the respective steps can be performed in a state where the substrate is mounted on the mounting surface, test pieces can be manufactured effectively in a sequential flow, and the manufacturing time, the labor, and the cost can be reduced.

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a plan view showing an example of a substrate used for a substrate-fixing device, a manufacturing apparatus for a test piece, and a manufacturing method for a test piece, according to the present invention. FIG. 2 is a cross-sectional view taken along the line C-C of FIG. 1. FIG. 3 shows a state where the substrate is fixed and a probe is discharged in droplet form onto a test site on the substrate, by the substrate-fixing device of the manufacturing apparatus for a test piece according to an embodiment of the present invention. FIG. 4 shows a state where it is confirmed by a microscope of the manufacturing apparatus for a test piece according to the embodiment of the present invention, whether or not the probe discharged onto the test site on the substrate is discharged to a predetermined position. FIG. 5 is a plan view showing an example of a mounting stage of the substrate-fixing device according to the embodiment of the present invention.

FIG. 6 shows a state where a water solution or a cleaning solution stored in a solution storage hole is passed through the test site and removed by a solution removal device of the manufacturing apparatus for a test piece according to the embodiment of the present invention.

FIG. 7 shows a state where the substrate is covered with a lid member of the substrate-fixing device according to the embodiment of the present invention.

FIG. 8 shows a comparison between the time for the manufacturing steps per one substrate in the manufacturing method for a test piece of the present invention, and the time for the manufacturing steps per one substrate in a conventional manufacturing method.

BEST MODE FOR CARRYING OUT THE INVENTION Next is a description of one embodiment of a substrate-fixing device, a manufacturing apparatus for a test piece furnished therewith, and a manufacturing method for a test piece, according to the present invention, with reference to FIG. 1 to FIG. 8.

A membrane substrate (substrate) A used in the present embodiment is formed in a square shape in plan view, as shown in FIG. 1 and FIG. 2, and comprises in the approximate center thereof, one circular test site (probe region) T. The circular test site T has a plurality of holes formed between the upper surface and the lower surface extending in a perpendicular direction to both surfaces, with openings in the upper surface and the lower surface, that is, a porous test site, and can immobilize an amino-labeled probe (known specific binding substance) P into a solid phase. That is to say, the membrane substrate A is formed overall from a porous member (for example, a flow-through type porous filter) B₅ and in a non-probe region T' which is the region other than the circular test site T, the upper surface and the lower surface of the membrane substrate A are coated with a holding member (resin laminate or the like) R for handling. That is, since the porous member B is exposed on the upper surface and the lower surface only in the region of the test site T, the test site T has a plurality of holes with openings in the upper surface and the lower surface. The test site T is not limited to a circular shape, and the number thereof is not limited to one.

As shown in FIG. 3, the manufacturing apparatus for a test piece 1 of the present embodiment comprises: a substrate-fixing device 2; a probe dispensing nozzle (probe discharge device) 3 which discharges a probe P in droplet form to a predetermined position on respective test sites T of a membrane substrate A fixed to the substrate-fixing device 2, so as to immobilize the probe P into a solid phase; and a microscope (for example, BX51 made by Olympus Corp.) (probe checking device) 4 shown in FIG. 4, for confirming whether or not the probe P discharged to the test site T is discharged to the predetermined position.

In the present embodiment, "Organism-oriented substance" includes not only animal cells, plant cells, and microbe cells, but also substances originating from viruses and the like which are unable to proliferate by themselves without parasiting with such cells. The organism-oriented substance includes not only substances extracted or isolated directly from these cells in the natural form, but also includes substances produced using genetic engineering methods, and substances modified chemically. For example, a hormone, a tumor labeling substance, an enzyme, an antibody, an antigen, abzymes, other proteins, a nucleic acid, cDNA, DNA, mRNA, and the like are included.

Moreover, the probe P means a substance bindable specifically with the above organism-oriented substance, for example, including a ligand such as a hormone and the receptor thereof, an enzyme and the substrate thereof, a nucleic acid having a specific sequence and the nucleic acid having the complementary sequence thereof, and any substance having such a relation. The substrate-fixing device 2, as shown in FIG. 3, fixes a membrane substrate A for detecting the organism-related substance, in a state where it is mounted in a predetermined position on a mounting surface 10a. The substrate-fixing device 2 comprises: a mounting stage 10 with an upper surface as the mounting surface 10a, and having through holes 11 formed with openings in the mounting surface 10a in a non-probe region T' other than the test site T; and a holding device 15 that holds a lower surface of the membrane substrate A via the through holes 11.

In FIG. 3 one membrane substrate A out of a plurality of membrane substrates A is illustrated. The mounting stage 10, as shown in FIG. 5, is made from a material having electroconductivity and chemical resistance, for example, a stainless steel such as SUS303, 304, or the like, and is formed in a rectangular shape in plan view. The material of the mounting stage 10 is not limited to a stainless steel. For example, as a solvent to chemically treat the test site T, there is a silane coupling material, poly-L-lysine, alcohol, acetone, buffer solution, and the like, and Teflon® or a stainless steel may be used as a material resistant to these solvents. However, as described above, a stainless steel such as SUS303 or 304 having electroconductivity and chemical resistance, is preferably used. In particular, SUS303 is preferred since it hardly rusts and processing thereof is easy. Moreover, regarding the mounting stage 10, the mounting surface 10a is formed so as to mount a plurality of membrane substrates A. In the present embodiment, as shown in FIG. 5, a total of twenty four membrane substrates A, with four in the vertical direction (up and down direction with respect to the page) and six in the horizontal direction (horizontal direction with respect to the page), can be mounted adjacent to each other. Furthermore, in the mounting stage 10, mounting holes 10b for attaching fastening screws that fix the mounting stage 10, are formed respectively two on each side of the mounting surface 10a. The mounting holes 10b are counterbored so that when the fastening screws 16 shown in FIG. 3 are attached, the fastening screws 16 are buried inside, and the upper parts of the fastening screws 16 do not project from the upper surface of the mounting surface 10a. Moreover, for each membrane substrate A, four (a plurality of) through holes 11 are formed in positions corresponding to four corners of the membrane substrate A. Furthermore, in the mounting stage 10, a solution through hole 12 having an opening formed in the mounting surface 10a, is formed in a position corresponding to the test site T on the membrane substrate A. The opening of this solution through hole 12 is formed to have the same diameter as that of the test site T.

Furthermore, the mounting stage 10 comprises a plurality of pads (contact sections) 13 which are formed at a height so as not to protrude above the upper surface of the membrane substrate A when the membrane substrate A is mounted on the mounting surface 10a, and which can contact with the side face of the membrane substrate A. The pad 13 has a function of positioning the membrane substrate A in a predetermined position on the mounting surface 10a, and is formed so that four of them can contact with one membrane substrate A, to enclose the respective membrane substrates A. As shown in FIG. 3, the holding device 15 comprises: a base 21 on which the mounting stage 10 is mounted, having a passage 20 that is connectable to the respective through holes 11 ; and a vacuum pump (for example, DAl 5D made by Ul vac Techno Ltd.) (pressure device) 22 which is connected to the passage 20 and sucks the air inside the passage 20 to suck and hold a lower surface of the membrane substrate A in the non-probe region T' .

Moreover, in the upper surface of the base 21, threaded holes 21a for screwing the fastening screws 16, are formed in positions opposed to the mounting holes 10b of the mounting stage 10. The base 21 and the mounting stage 10 are joined via the fastening screws 16. When joining the base 21 and the mounting stage 10, they can be joined by interposing a rubber sheet 23 therebetween so that air leakage between the base 21 and the mounting stage 10 can be eliminated as much as possible. In this rubber sheet 23, holes 23 a having the same size as those of the through holes 11 in the mounting stage 10, are formed in positions opposed thereto.

Furthermore, as shown in FIG. 6, the substrate-fixing device 2 of the present embodiment comprises: a jig 30 which, in a state where the membrane substrate A is mounted on the mounting stage 10, covers the whole of the mounting surface 10a and is detachably fixed to the mounting stage 10 while being stuck to the upper surface of the membrane substrate A, and has a solution storage hole 30a formed directly above the test site T; a solution dispensing nozzle (solution supply device) 31 which supplies a solution W to the solution storage hole 30a, and stores the solution W in a space enclosed by an inner peripheral surface of the solution storage hole 30a and an upper surface of the test site T; and a solution removal device 32 which removes the stored solution W that has passed through the test site T and the solution through hole 12.

The solution dispensing nozzle 31 discharges, as the solution W, a solution to chemically treat the test site T into a predetermined condition, for example 10% poly-L-lysine (made by Sigma Corp.), or a cleaning solution to wash the test site T, for example PBS (phosphate-buffered salt solution) according to the condition. The appropriate use thereof is described in detail later.

The solution removal device 32 comprises: a solution base 34 on which the mounting stage 10 is mounted, and which has a solution passage 33 that is connectable to the solution through hole 12; the vacuum pump 22 (solution suction pump) which is connected to the solution passage 33 and sucks the stored solution W; and a discharge tank 35 (solution storage device) disposed between the vacuum pump 22 and the solution base 34, which stores the solution W sucked by the vacuum pump 22. The solution base 34 is set on a spotting apparatus (Bio chip arrayer made by

Perkin Elmer Life Sciences Corp.) (not shown), and has threaded holes 34a for screwing the fastening screws 16, formed in positions opposed to the mounting holes 10b of the mounting stage 10, similarly to the base 21. The solution base 34 and the mounting stage 10 are joined via the fastening screws 16. When joining the solution base 34 and the mounting stage 10, similarly, they can be joined by interposing a rubber sheet 35 therebetween so that air leakage between the solution base 34 and the mounting stage 10 can be eliminated as much as possible. In this rubber sheet 35, holes 35a having the same size as those of the solution through holes 12 in the mounting stage 10, are formed in positions opposed thereto. The structure for the vacuum pump 22 is such that the vacuum pump 22 constituting the holding device 15 is used, however a separate pump may be used.

As shown in FIG. 7, the substrate-fixing device 2 of the present embodiment comprises: a lid member 40 which, in a state where the membrane substrate A is mounted on the mounting stage 10, covers the whole of the mounting surface 10a and is detachably and firmly fixed to the mounting stage 10. The lid member 40 is firmly fixed to the mounting stage 10 while pressing the membrane substrate A down between itself and the mounting surface 10a.

As shown in FIG. 4, the microscope 4 comprises: an X stage 45 for mounting the mounting stage 10, and moving the mounting stage 10 in X direction parallel to the horizontal plane; a Y stage 46 for mounting the X stage 45, and moving the X stage 45 and the mounting stage 10 in Y direction (orthogonal direction to the X direction) parallel to the horizontal plane; and a CCD 47 which irradiates light to the test site T of the membrane substrate A mounted on the mounting stage 10, and captures for example an image of the self-fluorescence of the probe P. Then, using the captured image of the self-fluorescence, and with a setting of WIB cube and 400ms exposure time, it is confirmed whether or not the probe P is immobilized into a solid phase at a predetermined position of the test site T.

Hereunder is a description of a case where a test piece, that is, a microarray, is manufactured by immobilizing an amino-labeled probe P into a solid phase in the test site T of the membrane substrate A, using the substrate-fixing device 2 and the manufacturing apparatus for a test piece 1, constituted in the above manner.

The manufacturing method for a test piece of the present embodiment has: a treatment step for after mounting the membrane substrate A on the mounting surface 10a of the mounting stage 10, supplying the solution W to the test site T and letting it pass through the test site T, to chemically treat the test site T into a predetermined condition; a fixing step for, after the treatment step, sucking the lower surface of the membrane substrate A in the non-probe region T' other than the test site T, to hold and fix the membrane substrate A; a spotting step for, after the fixing step, discharging the probe P in droplet form to a predetermined position of the test site T to immobilize the probe P into a solid phase; a checking step for, after the spotting step, checking whether or not the probe P is discharged to the predetermined position; and a washing step for, after the checking step, supplying a cleaning solution to the test site T and letting it pass through the test site T to wash the test site T, so as to rinse out the probe P that is not completely immobilized into a solid phase. These respective steps are performed in a state where the membrane substrate A is mounted on the mounting surface 10a. Hereunder is a detailed description of the respective steps.

At first, twenty four membrane substrates A are mounted at predetermined positions on the mounting surface 10a of the mounting stage 10, using a pincette (tweezers) or the like. At this time, four pads 13 are formed on the mounting surface 10a per one membrane substrate A so as to enclose the membrane substrate A.

Therefore by making the pads 13 contact with the four side faces of the membrane substrate A, the membrane substrate A can be readily and reliably mounted in a predetermined position.

The treatment step is performed after mounting the membrane substrate A. At first, as shown in FIG. 6, the mounting stage 10 on which the membrane substrate A is mounted, is mounted on top of the solution base 34 with the rubber sheet 35 sandwiched therebetween, and the mounting stage 10 and the solution base 34 are fixed together. That is, the fastening screws 16 are inserted into the mounting holes 10b (counterbored holes) at the four corners of the mounting stage 10, and the fastening screws 16 are screwed into the threaded holes 34a in the solution base 34 to fix the mounting stage 10 to the solution base 34. In particular, since the rubber sheet 35 is interposed between the mounting stage 10 and the solution base 34, the mounting stage 10 and the solution base 34 are fixed in a reliably sealed condition, so that leakage of air from any gaps between the mounting stage 10 and the solution base 34 can be prevented.

Next, the jig 30 is fixed to the mounting stage 10. As a result, the test site T is exposed via the solution storage hole 30a, and the non-probe region T' other than the test site T is covered with the jig 30. Moreover, since the jig 30 is fixed in a sealed condition to the upper surface of the membrane substrate A, a gap does not occur between the solution storage hole 30a and the test site T. Furthermore, since the pads 13 formed on the mounting surface 10a are formed at a height so as not to protrude from the upper surface of the membrane substrate A, there is no influence on the jig 30 which is fixed in a sealed condition to the upper surface of the membrane substrate A.

After fixing the jig 30, a solution (solution W) of 10% poly-L-lysine (made by Sigma Corp.) is sequentially supplied (dispensed) into the respective solution storage holes 30a from the top of the solution storage hole 30a by the solution dispensing nozzle 31, to store the solution in a space enclosed by the inner peripheral surface of the solution storage hole 30a and the upper surface of the test site T.

After storing the solution in all of the solution storage holes 30a, the solution passage 33 in the solution base 34 is sucked by operating the vacuum pump 22, to thereby create a negative pressure. As a result, the stored solution passes through the porous test site T via a plurality of holes while infiltrating the test site T, and is then discharged to the discharge tank 35 through the solution through hole 12 and the solution passage 33. By performing this treatment step, the test site T can be chemically treated into a predetermined condition.

After completing the pretreatment by the chemical treatment, the jig 30 is removed from the mounting stage 10, and the fastening screws 16 are removed. Then the mounting stage 10 is removed from the solution base 34 with the membrane substrate A still mounted. Then, as shown in FIG. 7, the lid member 40 is firmly fixed to the mounting stage 10.

As a result, the mounting surface 10a is sealed off from the outside air, and hence adherence of airborne dust onto the test site T can be prevented. Moreover, since the lid member 40 is fixed while pressing the membrane substrate A down between itself and the mounting surface 10a, the membrane substrate A is not displaced from the predetermined position on the mounting surface 10a, even if the mounting stage 10 is moved.

Then, after attaching the lid member 40, the mounting stage 10 is placed into a vacuum dryer (for example, DP22 made by Yamato Chemical Co.) (not shown), and the mounting stage 10 is dried for 60 minutes. Next, after completing the drying, the mounting stage 10 is taken out from the vacuum dryer, and the lid member 40 is removed, to perform the fixing step.

As shown in FIG. 3, in this fixing step, firstly, the mounting stage 10 is mounted on top of the base 21 that is set on the spotting apparatus, with the rubber sheet 23 sandwiched therebetween, and the mounting stage 10 and the base 21 are fixed together. That is, the fastening screws 16 are inserted into the mounting holes 10b (counterbored holes) at the four corners of the mounting stage 10, and the fastening screws 16 are screwed into the threaded holes 21a in the base 21 to fix the mounting stage 10 to the base 21. At this time, similarly to the abovementioned treatment step, since the rubber sheet 23 is interposed between the mounting stage 10 and the base 21, the mounting stage 10 and the base 21 are fixed in a reliably sealed condition, so that leakage of air from any gaps between the mounting stage 10 and the base 21 can be prevented.

Then, the air in the passage 20 in the base 21 is sucked by operating the vacuum pump 22, to thereby create a negative pressure. As a result, the through holes 11 connected to the passage 20 are also sucked, and hence the lower surface of the membrane substrate A in the non-probe region T' in contact with the openings of the through holes 11 can be sucked and held. By sucking and holding in this way, the respective membrane substrates A can be reliably fixed onto the mounting surface 10a. In particular, since four (a plurality of) through holes 11 are formed at the four corners of one membrane substrate A to enclose the test site T, the whole of the membrane substrate A can be evenly sucked and held, and thus fixed.

Moreover, since the non-probe region T' is sucked, the suction of airborne dust, and its adherence to the membrane substrate A as with the conventional case can be prevented. After fixing the membrane substrate A, the spotting step is performed. That is, by means of the probe dispensing nozzle 3 of the spotting apparatus, the probe P which has been amino-labeled is discharged in droplet form to the predetermined position on the respective test sites T of the membrane substrate A to immobilize it into a solid phase, so as to form a microarray. As this time, as described above, since the pad 13 is formed at a height so as not to protrude from the upper surface of the membrane substrate A, interference with the probe dispensing nozzle 3 can be prevented. That is, the operation of the probe dispensing nozzle 3 is not restricted, and hence the spotting step can be reliably performed.

Then, after immobilization of the probe P, the vacuum pump 22 is stopped to release the fixing of the membrane substrate A. After that, the fastening screws 16 are removed, and the mounting stage 10 is removed from the base 21. Then, the checking step is performed.

As shown in FIG. 4, in the checking step, the removed mounting stage 10 is attached onto the X stage 45 of the microscope 4. Then, by irradiating light to the test site T, and using the self-fluorescence of the probe P with a setting of WIB cube and 400ms exposure time, it is confirmed whether or not the probe P is reliably discharged and immobilized into a solid phase at a predetermined position. At this time, the X stage 45 and the Y stage 46 are operated at appropriate times to move the membrane substrate A in the X and Y horizontal directions, to perform confirmation of the test sites T of all of the membrane substrates A. After performing confirmation of all of the probes P, the washing step is performed.

As shown in FIG. 6, in the washing step, similarly to the abovementioned treatment step, the mounting stage 10 is fixed to the solution base 34 and the jig 30 is fixed onto the mounting stage 10. Then, a cleaning solution (solution W) of 3 ml PBS is sequentially supplied (dispensed) into the respective solution storage holes 30a from the top of the solution storage hole 30a by the solution dispensing nozzle 31, to store the cleaning solution in a space enclosed by the inner peripheral surface of the solution storage hole 30a and the upper surface of the test site T. After storing the cleaning solution in all of the solution storage holes 30a, the solution passage 33 in the solution base 34 is sucked by operating the vacuum pump 22. Due to this suction force, the stored cleaning solution passes through the porous test site T via the plurality of holes while infiltrating the test site T, and is then discharged to the discharge tank 35 through the solution through hole 12 and the solution passage 33. Consequently, the cleaning solution (solution W) of 2 ml sterile water is discharged to the respective solution storage holes 30a by the solution dispensing nozzle 31, to wash the test site T, similarly to the abovementioned case.

By performing the washing step, the probe P that is not completely immobilized into a solid phase in the site T among the immobilized probe P can be rinsed out with the cleaning solution. Consequently, a microarray having the probe P that is completely immobilized into a solid phase in a predetermined position in the site T can be obtained.

After completing the washing step, the mounting stage 10 is removed from the solution base 34, and the lid member 40 is firmly fixed thereto similarly to the abovementioned case. Then, the mounting stage 10 is again placed into the vacuum dryer, and is dried at room temperature for 60 minutes. After completing the drying, the mounting stage 10 is taken out from the vacuum dryer and placed into a refrigerator (not shown) right away, and stored in a temperature at 4⁰C. As a result, the microarray can be stored in the manufactured state.

As described above, according to the substrate-fixing device 2 of the present embodiment, since the non-probe region T' is sucked and held when the membrane substrate A is fixed, suction of airborne dust to the membrane substrate A and adherence thereto can be prevented. Consequently, there is no occurrence of noise caused by dust, and the membrane substrate A can be reliably fixed in a state where the influence on detection of the organism-related substance is as little as possible. In particular, since the membrane substrate A can be fixed simply by sucking the air, the structure can be simplified, and there is little effect on the membrane substrate A because there is no heat generated.

Moreover, since a plurality of (four) through holes 11 are formed when the membrane substrate A is fixed, the membrane substrate A can be fixed more stably. Furthermore, since the pads 13 are formed on the mounting surface 10a, when the membrane substrate A is mounted on the mounting surface 10a, the positioning can be performed by making the side faces of the membrane substrate A contact with the pads 13, so that the membrane substrate A can be mounted in the predetermined position more readily and reliably. Hence, the non-probe region T' and the through holes 11 can be aligned with high accuracy, so that the membrane substrate A can be fixed more reliably. Furthermore, since the pads 13 are formed at a height so as not to protrude from the upper surface of the membrane substrate A, there is no effect when the lid member 40 is fixed to the mounting stage 10, nor effect such as interference with the probe dispensing nozzle 3 which discharges the probe P to the test site T.

Moreover, since there is the lid member 40, the membrane substrate A can be sealed off from contact with outside air. As a result, adherence of dust onto the membrane substrate A can be prevented, for example during the time while the mounting stage 10 is being placed into the vacuum dryer. Furthermore, since the lid member 40 is fixed to the mounting stage 10 while pressing the membrane substrate A down onto the mounting surface 10a, even if the membrane substrate A is released from being sucked and held, the positional relationship of the mounting surface 10a and the membrane substrate A can be maintained. Hence the mounting stage 10 can be solely moved into the vacuum dryer without shaking nor moving the membrane substrate A. Moreover, abrasion or scratching of the membrane substrate A caused by shaking of the membrane substrate A can be prevented, and hence the analysis results are not affected.

Furthermore, since the solution base 34, the jig 30, the probe dispensing nozzle 3, and the solution removal device 32 are provided, the operations such as pretreatment of the test site T by chemical treatment, and washing thereof after discharging the probe, can be readily performed in a state where the membrane substrate A is mounted on the mounting surface 10a of the mounting stage 10, and hence the time and the labor can be reduced. In particular, since the jig 30 having the solution storage hole 30a is used, various solutions W such as water solution, cleaning solution, and the like can be supplied to the test site T only, and hence the solution W can be used without any waste (efficiently), and costs can be reduced.

Moreover, since the solution W can be supplied to the test site T only, the contact state can be uniform and the chemical treatment can be even. As a result, variance in the analysis result can be reduced. In particular, since the solution removal device 32 removes the solution W by simply using the suction force of air similarly to the holding device 15, the structure is simple, and the solution W can be reliably removed.

Moreover, since the mounting stage 10 is formed from a material having electroconductivity and chemical resistance, adherence of dust onto the membrane substrate A due to electrostatic charge can be prevented. Furthermore, even if various solutions for chemical treatment or washing are used in a state with the membrane substrate A mounted, there is no effect from the solution W, and the reliability over long term use can be improved.

According to the manufacturing apparatus for a test piece 1 of the present embodiment, since the probe P can be immobilized into a sold phase on the membrane substrate A which is fixed by the abovementioned substrate-fixing device 2, in a state where adherence of dust thereto is prevented, high quality microarrays can be manufactured. Therefore, there is no effect of noise caused by adhered dust, and the organism-related substance can be tested with higher accuracy. Moreover, since the substrate-fixing device 2 is capable of mounting a plurality of membrane substrates A on the mounting surface 10a, high quality microarrays can be manufactured effectively at once.

Furthermore, since the microscope 4 enables confirmation as to whether or not the probe P is reliably discharged to a predetermined position in the test site T, higher quality microarrays can be obtained.

Moreover, since the membrane substrates A are used, the surface treatment can be performed evenly.

According to the manufacturing method for a test piece of the present embodiment, since the treatment step, the fixing step, the spotting step, the checking step, and the washing step are performed in a state where the membrane substrate A is mounted on the mounting surface 10a of the mounting stage 10, microarrays can be manufactured effectively in a sequential flow, and the manufacturing time, the labor, and the cost can be reduced.

[Comparative Example] Next, a comparison is made between the time for the manufacturing steps per one microarray in the case where the microarray was manufactured by the manufacturing method for a test piece of the present embodiment as described above, and the time for the manufacturing steps per one microarray in the case where the microarray was manufactured by a conventional manufacturing method for a test piece. In the conventional manufacturing method for a test piece, similarly to the manufacturing method for a test piece of the present embodiment, the membrane substrate is chemically treated, the membrane substrate is fixed, the probe is discharged onto the membrane substrate, the position of the discharged probe is checked, the membrane substrate is washed, and the microarray is stored. The spotting described later means both the fixing of the membrane substrate and the discharging of the probe.

First, the membrane substrate was chemically treated. That is, using a pincette or the like, twenty four membrane substrates were arranged in a Petri dish. Then, a prepared silica colloid solution was dispensed onto the whole membrane substrate and air-dried. After that, a water solution of 10% poly-L-lysine (made by Sigma Corp.) was dispensed onto the whole membrane substrate, and the surplus dispensed water solution was removed by a pipette, to thereby perform an amino group treatment. Next, in order to prevent dust from adhering to the membrane substrate, the Petri dish was covered, and was then placed into a vacuum dryer and dried at room temperature for 60 minutes. After drying, the Petri dish was taken out from the vacuum dryer. Next, the spotting was performed.

That is, membrane substrates were arranged one by one from the Petri dish using a pincette, on a stage that was fixed to a spotting apparatus (for example, a Bio chip arrayer made by Perkin Elmer Life Sciences Corp.) Then, using a vacuum pump (for example, DAl 5D made by Ulvac Techno Corp.) connected to the stage, the membrane substrates were sucked and fixed. After fixing them, a probe was discharged from the nozzle of the spotting apparatus onto the test site to form an array, to thereby manufacture the microarray. After completing immobilization of the probe, the sucking wajs stopped, and the membrane substrates were collected one by one by a pincette and arranged in a Petri dish.

After immobilizing the probe into a solid phase in the membrane substrate, it was checked to confirm whether or not the probe was discharged to the correct position. That is, one membrane substrate was taken out by a pincette, and mounted on the stage of the microscope (for example, BX51 made by OLYMPUS Corp.). Then exciting light was irradiated thereon to confirm the discharge position using the self-fluorescence from the probe.

After confirmation, the membrane substrate was collected into the Petri dish. This was repeated for the same number of times as the number of the membrane substrates.

Next, the membrane substrates were washed.

That is, five membrane substrates were taken out from the Petri dish by a pincette and mounted on the stage of a washer. After mounting, the membrane substrates were fixed by placing a lid thereon. Then 3 ml of PBS was dispensed into the solution storage hole, and sucked from the bottom by a vacuum pump (for example, DAl 5D made by Ulvac Techno Corp.) and made to infiltrate the test site, then discharged to a discharge container. Similarly, 2 ml of sterile water was dispensed into the solution storage hole, made to infiltrate the test site, then discharged to a discharge container. After completing the washing, the lid was opened and the membrane substrates were collected into a Petri dish by a pincette, then the lid was covered so as not to allow contamination with dust. After that, the Petri dish was placed into the vacuum dryer to dry the membrane substrates at room temperature for 60 minutes. After drying, the Petri dish was taken out from the vacuum dryer. Finally, the Petri dish was wrapped with aluminum foil so as to cover the whole membrane substrates, and the membrane substrates were placed into a refrigerator and stored in a temperature at 4⁰C.

FIG. 8 shows the results of a comparison of the time for the manufacturing steps per one membrane substrate, for the aforementioned conventional manufacturing method for a test piece, and the manufacturing method for a test piece of the present embodiment.

As shown in FIG. 8, according to the manufacturing method for a test piece of the present embodiment, since the respective steps can be performed in a sequential flow while the membrane substrates are mounted on the mounting surface of the mounting stage, it was confirmed that the time for the manufacturing steps was reduced by about 56% compared to the conventional method.

The scope of the present invention is not limited to the abovementioned embodiment, and various modifications can be made without departing from the scope of the present invention. For example, although a membrane substrate was used as the substrate, the substrate is not limited to the membrane substrate, and can be any substrate as long as it is a porous base material with a probe region and non-probe region.

Moreover, although the holding device fixes the substrate onto the mounting surface using suction force from the suction pump, it is not limited to this, and may be any form as long as the substrate can be fixed by holding the lower surface of the substrate in the non-probe region via the through holes.

Moreover, the mounting stage has a structure in which twenty four membrane substrates can be mounted, however the number is not limited to twenty four. Furthermore, although four through holes are formed per one membrane substrate, the number is not limited to four. Also, although four pads are formed per one membrane substrate, the number is not limited to four.

Moreover although the solution removal device removes the solution using the suction force from the suction pump similarly to the holding device, it is not limited to this and may be any form as long as the solution can be removed. For example, the solution may be removed by centrifugation using an optional centrifuge in an optional cycle, or the solution may be removed by pressurization.

Moreover, although the base and solution base are respectively separate devices, they may be used in common. In this case, a mechanism to selectively shut off the through holes or the solution through holes may be provided. INDUSTRIAL APPLICABILITY

In the substrate-fixing device according to the present invention, since the holding device holds the lower surface of the substrate, that is, the lower surface of the substrate in the non-probe region, by suction or the like via the through hole, the substrate can be reliably fixed onto the mounting surface. In particular, since the porous probe region is not affected when the substrate is fixed, there is no likelihood of dust being sucked to the substrate and adhered to the probe region. Therefore, the substrate can be reliably fixed in a state where the influence on detection of the organism-related substance is as little as possible. Moreover, the chemical treatment and the washing can be readily performed on the probe region while the substrate is mounted on the mounting surface, and hence the time and the labor can be reduced. Furthermore, the solution can be used for the probe region only, and hence the solution can be used without any waste (efficiently), and costs can be reduced. Moreover, in the manufacturing apparatus for a test piece according to the present invention, since the specific binding substance can be immobilized into a sold phase in the substrate which is fixed by the substrate-fixing device, in a state where the adherence of dust thereto is prevented, higher quality test pieces can be manufactured. Consequently, there is no effect of noise, and the organism-related substance can be tested with higher accuracy.

Furthermore, in the manufacturing method for a test piece according to the present invention, since the respective steps can be performed while the substrate is mounted on the mounting surface, test pieces can be manufactured effectively in a sequential flow, and the manufacturing time, the labor, and the cost can be reduced.

Claims

1. A substrate-fixing device which fixes a substrate for detecting an organism-related substance mounted in a predetermined position on a mounting surface, said substrate being a porous substrate and having at least one probe region where a known specific binding substance can be immobilized, said substrate-fixing device comprising: a mounting stage having a through hole formed with an opening in said mounting surface in a non-probe region which is other than said probe region; and a holding device that holds a lower surface of said substrate via said through hole.

2. A substrate-fixing device according to claim 1 , wherein a plurality of said through holes are formed on said mounting stage.

3. A substrate-fixing device according to either one of claim 1 and claim 2, further comprising a contact section which is formed at a height so as not to protrude from an upper surface of said substrate on said mounting surface, and which can contact with a side face of said substrate, wherein said contact section positions said substrate in said predetermined position.

4. A substrate-fixing device according to any one of claim 1 through claim 3, wherein said mounting stage is made from a material having electroconductivity.

5. A substrate-fixing device according to any one of claim 1 through claim 4, wherein said holding device comprises: a base on which said mounting stage is mounted and has a passage that is connectable to said through hole; and a pressure device which is connected to said passage and sucks air inside said passage to suck and hold a lower surface of said substrate in said non-probe region.

6. A substrate-fixing device according to any one of claim 1 through claim 5, further comprising a lid member which covers the whole of said mounting surface in a state where said substrate is mounted on said mounting stage, and is detachably fixed to said mounting stage, wherein said lid member is fixed to said mounting stage while pressing said substrate down between itself and said mounting surface.

7. A substrate-fixing device according to any one of claim 1 through claim 4, further comprising: a solution through hole provided on said mounting stage, said solution through hole having an opening formed in said mounting surface in said probe region; a jig which covers the whole of said mounting surface in a state where said substrate is mounted on said mounting stage, and is detachably fixed to said mounting stage while being stuck to the upper surface of the substrate, and has a solution storage hole formed directly above said probe region; a solution supply device which supplies a solution to said solution storage hole, and stores the solution in a space enclosed by an inner peripheral surface of the solution storage hole and an upper surface of said probe region; and a solution removal device which removes the stored solution that has passed through said probe region and said solution through hole.

8. A substrate-fixing device according to claim 7, wherein said solution is a solution for chemically treating said probe region in a predetermined condition, or a cleaning solution for washing said probe region.

9. A substrate-fixing device according to either one of claim 7 and claim 8, wherein said solution removal device comprises: a solution base on which said mounting stage is mounted, and which has a solution passage that is connectable to said solution through hole; a solution suction pump which is connected to said solution passage to suck the stored solution; and a solution storage device disposed between said solution suction pump and said solution base to store the solution sucked by said solution suction pump.

10. A substrate-fixing device according to any one of claim 7 through claim 9, wherein said mounting stage is made from a material having chemical resistance.

11. A substrate-fixing device according to any one of claim 1 through claim 10, wherein said substrate is a membrane substrate.

12. A manufacturing apparatus for a test piece comprising: the substrate-fixing device according to any one of claim 1 through claim 11 ; and a probe discharge device which discharges said specific binding substance in droplet form to a predetermined position on the probe region of said substrate to immobilize the specific binding substance into a solid phase thereon.

13. A manufacturing apparatus for a test piece according to claim 12, comprising a probe checking device which confirms whether or not said specific binding substance discharged to said probe region is discharged to said predetermined position.

14. A manufacturing method for manufacturing a test piece by immobilizing a specific binding substance into a solid phase in a porous substrate for detecting an organism-related substance, which is furnished with at least one probe region where a known specific binding substance can be immobilized, said method comprising: a treatment step for supplying a solution to said probe region and letting it pass through said probe region to chemically treat said probe region into a predetermined condition after mounting said substrate on a mounting surface of a mounting stage; a fixing step for sucking a lower surface of said substrate in a non-probe region other than said probe region to hold and fix said substrate; a spotting step for discharging said specific binding substance in droplet form to a predetermined position of said probe region to immobilize said specific binding substance into a solid phase; a checking step for checking whether or not said specific binding substance is discharged to said predetermined position; and a washing step for supplying a cleaning solution to said probe region and letting it pass through said probe region to wash said probe region, so as to rinse out said specific binding substance that is not completely immobilized into a solid phase, wherein said respective steps are performed in state where said substrate is mounted on said mounting surface.