JP2008134188A - Probe solidifying reaction array and manufacturing method of array - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bio-chip capable of entirely reducing the effort, cost, time, and resource required for chip manufacturing. <P>SOLUTION: This probe solidifying reaction array comprises a probe solidified linearly on the base of the array and a linear channel formed in the array, and the linearly solidified probe and the linear channel are arranged so that they intersect. The present invention provides a method for manufacturing the probe solidifying reaction array, and the method including the steps of linearly solidifying the probe on the base of the array and forming the channel in the array so that it crosses the linear solidified probe. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a probe-immobilized reaction array. More specifically, the present invention relates to a probe-immobilized reaction array for use in analysis of nucleic acids and proteins.

  In recent years, a technique for analyzing a large amount of specific biomolecules and cells called biochips in parallel has attracted attention. A biochip is a biomolecule of DNA, protein, sugar chain sugar, or a cell immobilized on a support, an immobilized biomolecule (referred to as a probe), a biomolecule or other compound (target) Among the biochemical methods that detect the specific interactions that have occurred, especially in large quantities and in parallel, enabling high-throughput detection / analysis. Is. Specifically, a DNA chip (also called a DNA microarray) that detects the presence of complementary sequences by hybridization by immobilizing nucleic acids on a substrate that has already been widely used in the field of genetic analysis. There is a protein chip (protein chip) that immobilizes proteins expected to be used in the future and detects interactions.

  The types of biochips are divided into flat plate, beads, capillaries / fibers, etc. depending on the shape of the support to which the probe is fixed, but these elements are combined (the flat plate is divided into compartments, and the flat plate is cut into grooves. The beads are fixed in place on the flat plate, the beads are fixed inside or at the tip of the capillary, the capillaries are bundled, or further sliced in a cross section and processed into a flat plate shape Etc.) is also possible. It can also be combined with microfluidic devices using microfabrication technology. In addition, some electronic devices are composed of arrayed electrodes and further incorporate functions such as memory elements / transmitters. A system in which a probe is fixed to a porous substrate and interacts three-dimensionally has also been studied. Typical types of probes are nucleic acids, (poly) peptides, sugars and the like.

  In recent years, attempts have been made to use such biochips in clinical, clinical trials, drug discovery, screening, and the like. On December 23, 2004, Roche Molecular Systems received FDA approval for the first clinical test using a DNA chip. This biochip “AmpliChip P450” is manufactured by Affymetrix and has more than 8000 types of probes ranging from 18mer to 22mer. It is possible to examine polymorphisms of CYP2D6 (34 types) and CYP2C19 (2 types) in cytochrome P450 using this biochip.

  In this way, biochips that were used in the research stage are now being used in clinical, clinical trials, drug discovery, screening, etc. in a more complete form. It is getting demanded.

  The focus array is a reduced number array, which is different from the array for wide-area screening that has been used so far. However, the size of the array is mostly the same as that of the conventional one due to the convenience of the detection system, etc., and there are many useless parts and the price is not lowered so much.

  In order to deal with such a problem, an attempt has been made to divide one array into sections and inject different samples into each array for detection. Such a method is performed by both the Affymetrix method and the Stanford method. As a result, resource saving and low price can be realized at the same time.

  Here, there are roughly two types of biochip manufacturing methods, which are sometimes referred to as the Affymetrix method and the Stanford method, respectively. The Affymetrix method is a technology that sequentially synthesizes probes on a support, diverts photolithographic techniques used in semiconductor manufacturing represented by Affymetrix, and uses an illumination matrix such as a micromirror array or liquid crystal as an exposure technology. and so on. This technique can be used not only for DNA but also for the production of high-density peptide chips. However, a probe that is too long may increase the number of synthesis steps and reduce the synthesis yield, and is large and expensive in terms of apparatus. In the case of the Affymetrix method, the synthesis procedure is repeated for the length of the probe. For example, with a 25mer probe used in Affymetrix, synthesis by photolithography is repeated 25 times. However, this process requires large-scale equipment and specific facilities, and furthermore, it is difficult to maintain the performance of the product, so that it must be very expensive. In addition, since the detection method and the hybridization method also use their own methods, it is not possible to use a widely used Stanford array processing device or detection device. Therefore, it is necessary to make a separate investment for equipment purchase, and the number of people who can use this method is very limited. Furthermore, since there is a limit to the probe length that can be synthesized, it is not suitable for complex detection targets.

  In contrast, the Stanford method is a technology that places a separately prepared probe at a predetermined position using a robot-type arrayer that can drive a spotting pin freely in the XY-axis direction. Things are prevalent in public research institutions such as universities. Some use means such as ink jet or stamp instead of pins. For the above reasons, the most widely used Stanford method is desirable for producing a biochip at a low cost. However, in the Stanford method, it is necessary to directly arrange the probe solution at a specific position, and it is extremely difficult to stably arrange a spot having a diameter of 50 to 100 μm or less because of the characteristics of the probe solution. In addition, spotting must be repeated at least 55,000 times to place 55,000 types of probes on a single substrate. Further, it is a very labor-intensive operation to clean the pins or the inkjet head each time the probe is changed.

  However, as described above, in order to place a partition that divides one array into sections, more accurate spotting and jigs that accurately place sections are required, and the manufacturing process tends to be complicated, as expected. The reality is that it is not possible to reduce costs.

In either case, the effort, cost, time, and resources involved in chip fabrication are enormous, and there are still many issues left for future labor saving, cost saving, time saving, and resource saving.
JP 2000-135081 A

  In view of the above situation, an object of the present invention is to provide a biochip that can reduce the labor, cost, time, and resources involved in chip production as a whole.

  Specifically, the probes are not spotted one by one, but are spotted in a “line” shape, and the partition is separated so as to intersect the linear spot so that a large number of specimens can be viewed simultaneously on one array. The means for solving the above problems and achieving the purpose was developed rather than arranging and detecting using the probe at the intersection. More specifically, when using a line, a thread, a plate, or a rod with a probe attached to it when spotting in a line, and using a groove in which a linear groove is dug as a partition delimiter, the two linear structures are in close contact with each other. In this way, a biochip characterized by being produced by the process has been completed.

That is, in one embodiment of the present invention, a probe-immobilized reaction array in which probes are immobilized on a substrate,
Probes linearly immobilized on the base of the array;
A linear flow path formed in the array;
A probe-immobilized reaction array is provided, wherein the linearly immobilized probe and the linear flow path are arranged so as to intersect with each other.

  The probe-immobilized reaction array, wherein the linearly-immobilized probe has a plurality of different linear probes arranged in parallel with each other. A reaction array is provided.

  Furthermore, the probe solid phase reaction array, wherein the linear flow path formed in the array is formed by a partition-separated base material in which linear grooves are formed. A reaction reaction array is provided.

  Furthermore, the probe-immobilized reaction array is provided, wherein the probe is directly immobilized on a substrate.

  Furthermore, the probe-immobilized reaction array is provided, wherein the probe is indirectly immobilized on a substrate.

  Furthermore, the probe-immobilized reaction array is provided, wherein the linearly immobilized probe and the linear flow path are arranged orthogonally. To do.

  The probe-immobilized reaction array, wherein the probe is one or more probes selected from a probe group consisting of a nucleic acid, protein, antibody or antigen. provide.

  Furthermore, the probe-immobilized reaction array is provided, wherein the probe is a nucleic acid probe.

A method for producing a probe-immobilized reaction array,
Linearly immobilizing probes on the base of the array;
Forming a flow path in the array so as to intersect with the solid-phased linear probe;
A method comprising:

Further, the above method,
The probe provides a probe-immobilized reaction array in which a plurality of different linear probes are arranged in parallel with each other.

  Furthermore, in the above method, the linear flow path formed in the array is formed by a partition-separated base material in which a linear groove is formed.

  Further, the present invention provides a method characterized in that the probe is directly immobilized on a base.

  Further, the present invention provides a method characterized in that the probe is indirectly immobilized on a base.

  Furthermore, the above method is provided, wherein the linearly immobilized probe and the linear flow path are formed in an orthogonal arrangement.

  Furthermore, it is the above method, wherein the probe is one or more probes selected from a probe group consisting of a nucleic acid, protein, antibody or antigen.

  Furthermore, in the above method, the probe is a nucleic acid probe.

  The probe-immobilized reaction array of the present invention comprises a probe linearly immobilized on the base of the array and a linear flow path formed in the array, The linear flow paths are arranged so as to intersect with each other.

  Hereinafter, an embodiment of the probe-immobilized reaction array according to one embodiment of the present invention will be described with reference to an array in which the reaction part is a capillary shape as an example (FIG. 1).

  FIG. 1 (right) is a plan view of a probe-immobilized reaction array according to one embodiment of the present invention as viewed from above. The probe-immobilized reaction array 100 is an example of a probe-immobilized reaction array manufactured using a transparent glass substrate 102 and a partition-separated base material 103.

  A plurality of probes are linearly immobilized on the substrate 102 of the probe-immobilized reaction array 100.

  Here, the “probe” used in the present specification can use not only nucleic acids such as DNA and RNA as probes but also proteins and other substances as probes depending on the item to be analyzed. For example, peptides, antigens or antibodies, or combinations thereof can be used. In that case, a substance that specifically binds to the target substance to be detected is used as a probe. For example, when an antigen is used as a probe, an antibody that specifically binds to the antigen can be detected. What is necessary is just to change the probe used for every capillary according to the item to analyze, and to fix a desired probe to a base | substrate by desired arrangement | positioning. Those skilled in the art will be able to easily select the probe to be used according to the analysis item. A preferred probe is a nucleic acid probe.

  As used herein, the term “nucleic acid” refers to various naturally occurring DNAs and RNAs, as well as artificially synthesized nucleic acids such as peptide nucleic acids, morpholino nucleic acids, methyl phosphonate nucleic acids and S-oligo nucleic acids. Refers to analogs.

  The term “target substance” refers to a substance to be detected by the probe. For example, nucleic acids, proteins and the like are included, and more specifically, peptides, antigens and antibodies are also included. In general, a nucleic acid probe is designed to have a base sequence complementary to a target nucleic acid. If the target substance is an antigen, an antibody that can specifically bind to the antigen is used as a probe. When the test nucleic acid contained in the test sample has the base sequence of the target sequence, hybridization occurs between the nucleic acid probe and the target sequence. Therefore, the nucleic acid contained in the test sample can be analyzed by detecting this hybridization. Hybridization may be detected by means known per se.

  In addition, the term “test sample” refers to a sample prepared from a biological sample such as cells, tissues, organs, blood, serum, lymph, tissue, hair and earwax collected from an individual, as desired, or artificially. Refers to a sample to be subjected to a test containing a synthesized or manufactured substance. The “test sample” may be a sample obtained by subjecting a biological sample to any necessary pretreatment such as homogenate and extraction as necessary. Such pretreatment could be selected by one skilled in the art depending on the biological sample of interest.

  The term “individual” refers to any mammal, including humans, dogs, cats, cows, goats, pigs, sheep, and monkeys, and organisms other than mammals such as plants and insects. The analysis item may be any analysis as long as the analysis is performed by detecting hybridization or binding between the probe and the target substance. For example, gene expression frequency analysis, gene polymorphism analysis, protein expression frequency analysis, or a combination of these may be used.

  Although six types of probes are arranged in parallel on the substrate 102, the probes can be arranged in any shape (for example, length, orientation, and shape) as long as they are arranged in a line. It may be. Therefore, as shown in FIG. 1 (left), it is not necessarily arranged on a straight line.

  In addition, the linear probe can be set in various sizes according to the application, but practically, the width is 10 μm to several mm, the depth is 1 μm to 500 μm, the length is several mm to 100 mm, and the capillary interval is 10 μm. About several mm is sufficient.

  A flow path is formed in the partition-separated base material 103 of the probe-immobilized reaction array 100. As long as the flow paths formed in the array 100 are arranged so as to intersect with the linear probes solid-phased on the probe substrate 102, in any shape (for example, length, orientation, and shape) It may be formed. Further, although the flow path is linear, it is not necessarily arranged on a straight line as shown in FIG. 1 (middle). Further, the channel can be formed in a capillary shape. The size of the flow path can be set to various sizes according to the application, but practically, the width is 10 μm to several mm, the depth is 1 μm to 500 μm, the length is several mm to 100 mm, and the capillary interval is 10 μm to A few mm is sufficient. However, considering the efficiency of the reaction, the diffusion rate of mRNA to be measured, or nucleic acids such as cDNA synthesized or reverse transcribed from mRNA, is as slow as several μm per second. By adopting a flat structure that makes the depth shallow even if it is widened, shortening of the reaction time, miniaturization of the sample, increase in the observation field of view, etc. can be expected.

  In addition, the term “intersect” between the flow path and the linearly arranged probe means that the flow path and the probe are linear, so that the flow path and the probe are arranged to intersect each other and have at least one intersection. It means arranging. For example, as shown in FIG. 1, the flow path and the probe can be linear and arranged so as to be orthogonal to each other. Moreover, although the flow path is provided with an opening, it is usually preferable that the opening be provided at the end of the flow path.

  FIG. 2 is a cross-sectional view of the probe-immobilized reaction array of the present invention shown in FIG. 1 (right) when cut along the probe 107.

  Hereinafter, the probe-immobilized reaction array of the present invention will be described in more detail together with its production method.

  The probe-immobilized reaction array of the present invention can be produced, for example, as shown in FIG. 1 (center) by forming a groove in the partition-separated substrate 103 using the partition-separated substrate. . That is, a partition-separated base material 103 having the same size as the substrate 102 is prepared, and grooves are formed in the partition-separated base material 103 by etching. Then, the flow path can be formed in the array by joining the substrate 102 and the partition-separating base material 103 (FIGS. 1 and 2).

  A method for linearly immobilizing the probe on the substrate 102 will be described below.

Linear spotting
In a Stanford biochip, as a general method when using oligo DNA, there is a method of immobilizing an oligo DNA modified with an amino group on a substrate. Many substrates on which an oligo DNA modified with an amino group can be bound are commercially available (for example, TaKaRa-Hubble Slide Glass, TaKaRa). For spotting, the amino group-modified oligo DNA diluted with a linear spotting solution need only be attached to the substrate. Spotting is performed at room temperature. After the spotting, the oligo DNA that has not been immobilized on the substrate is removed, and blocking is performed so that the target does not adhere to any portion other than the spotted portion.

  In addition to spotting by physical contact such as stamping using threads, plates, rods, etc., spotting may be performed linearly by spotting a small amount of spotting solution. A method is conceivable in which masking is performed linearly, a spotting solution is attached from above, and then the mask is removed. At this time, the spotting liquid does not need to be a liquid, and it may be more suitable for spotting a gel containing a probe to be less affected by drying or the like. These methods are all the Stanford biochip manufacturing methods, but may be manufactured by the Affymetrix method using masking.

  In addition, as shown in FIG. 1, the work efficiency is better and the automation is easier when the linear spot (probe) 101 is orthogonal to the substrate 102. When there are many samples to be detected, the number of partition spaces (grooves) 104 is increased, and the number of detection spots (probes) 105 that are intersections with the linear spots (probes) 101 may be increased (FIGS. 4 and 5). reference).

Next, a case where the probe 101 is spotted linearly on the substrate 102 as shown in FIG. 1 (left) using a linear spotting apparatus will be described as an example. FIG. 3 shows a conceptual diagram of a linear spotting device using yarn. The linear spotting device has telescopic arms 202 and 207, and a spot string 203 is coupled to the ends thereof. This spot string 203 can be easily attached to and detached from the ends of the telescopic arms 202 and 207. With the spot string 203 coupled to the end of the telescopic arm (contracted) 202, the telescopic arm (contracted) 202 extends to a length necessary for spotting on the substrate 201. At that time, the missing spot string 203 is replenished from the spot string stock 205, and when it is replenished, it passes through the probe stock I206, and the probe is attached to the spot string 203 (FIG. 4). In the probe stock I206, probe molecules diluted to an appropriate concentration in a spotting buffer are stored in a liquid state, a gel state, or held in some form of a moisture absorber. The spot string 203 uses a natural fiber, a synthetic fiber, a polymer, or the like depending on the shape and thickness of the spot. In order to prevent drying of the spot string 203 to which the probe is attached, it is necessary to keep the humidity of the spot area of the linear spotting device or the room where the linear spotting device is installed appropriately. The humidity is set according to the shape and thickness of the spot and the material of the spot string 203. A linear spot is formed by pressing a spot string (probe attachment) 208 to which a probe is attached to the substrate 201. At that time, the spot head 204 is operated or the substrate 201 is operated to bring it into close contact (FIG. 5). In FIG. 5, the Z · X stage (Z = 0) is lifted to bring the Z · X stage (Z> 0) into contact with the spot string (probe attachment) 208 and the substrate 201 to form a linear spot. Is formed. Adjustment of the pressure to be brought into contact requires adjustment according to the shape and thickness of the spot, the material of the spot string 203, and the like. At this time, the spot head 204 is operated or the substrate 201 is operated (FIG. 2-3). Adjustment of the pressure to be brought into contact requires adjustment according to the shape and thickness of the spot, the material of the spot string 203, and the like. At this time, the time for keeping the contact is important together with the pressure for making contact. If it is too tightly adhered, the amount of solid phase in the vicinity of the center line of the spot string (probe attachment) 208 may be lowered. However, if it is too loose, the spot shape may be irregularly disturbed. Also important is the time after the probe is deposited from the probe stock I 206 to the spot string 203. Although it depends on the spot environment, particularly humidity and temperature, and the state of the probe (solution, gel, etc.), it is desirable to perform spotting as quickly as possible in order to suppress spot shape variations due to drying or the like. Based on these factors, it is important to perform a spot test to determine the appropriate conditions before the actual spotting. The spot string (probe attachment) 208 and the substrate 201 are separated by operating the spot head 204 at the stage where the spot is finished (FIG. 2-4) or by lowering the Z · X stage (Z> 0) 211. The retractable arm (extension) 207 is contracted while being wound around the waste string stock 209 so that the spot string (probe attachment) 208 that is no longer needed is slackened, and returned to the initial state of FIG. If the same probe is continuously spotted, it is possible to rewind and reuse the spot string stock 205 without winding it with the waste string stock 209. However, in actual use, it is desirable to discard them one by one in consideration of contamination and spot uniformity.

  The means for fixing the probe on the line is not limited to the above method, and any method known to those skilled in the art can be used. For example, the substrate can be subjected to an appropriate surface treatment for immobilizing the probe, such as poly-L-lysine treatment, aminosilane treatment, and oxide film treatment.

  Furthermore, the probe to be immobilized may be indirectly immobilized, for example, using a probe previously immobilized on beads. In this case, the beads on which the probes are immobilized are immobilized on the substrate. As the probe supply means for immobilizing a desired nucleic acid probe on such beads, any means known per se can be used depending on the material of the substrate. It is possible to improve the known means so that it can be applied to the curved surface of a three-dimensional or granular substrate, in particular a spherical surface. For example, in order to place a nucleic acid probe on a substrate having a curved body processed, a combination of two methods, a photosolid phase method and a spotting method, can be used quantitatively with respect to a curved partial region. The nucleic acid probe can be solid-phased. The means for fixing the beads on the wire is not limited to the above method, and any method known to those skilled in the art can be used. For example, the above linear spotting apparatus can be used to immobilize beads using probes immobilized thereon instead of probes.

  On the other hand, a method for forming the flow path of the partition-separating base material 103 will be described below.

Fractionation by division
Several breaks have been proposed as breaks on the chip. Several Affymetrix biochips (also applicable to Stanford biochips) have been proposed (Zarrinkar, P. et al., Arrays or Arrays for High-Throughput Gene Expression Profiling. Genome) Research 1256 (2001)), and many breaks processed into grooves have been proposed (Japanese Patent Laid-Open No. 11-75812).

  For example, the latter groove-shaped partition is made of PDMS (polydimethylsiloxan) material, and the PDMS material is soft and self-adsorbing and does not require a special joining process. (Figure 7). The flow path made of such a material can be easily used because it only needs to be attached to a normal glass slide or a biochip spotted on a silicon substrate, and no special bonding is required.

  In addition to the PDMS material, a channel made of silicon, metal, glass, plastic, or the like may be bonded to the substrate using an adhesive or the like to form the channel. It is also possible to form a flow path by sticking a seal with a depression. In a glass or silicon wafer-like substrate, for example, grooves and through holes can be formed by a technique such as photolithography-etching. In the case of plastic resin or rubber, the grooves and through holes can be formed by machining or molding.

  Furthermore, the probe-immobilized reaction array of the present invention is produced by pasting the substrate 102 having the linear probe so as to intersect the partition partitions of the partition partition base material 103 having a groove structure. By pasting the substrate 102 having a linear probe so as to intersect the partition partition of the partition partition base material 103 having a groove structure, the intersection of the linear probe and the partition partition groove structure of the partition partition base material 103 is obtained. Cause it to occur. A probe spot generated at the intersection functions as a detection spot (probe) 105. Then, hybridization in the probe-immobilized reaction array of the present invention produced as described above is performed in a partitioned space (groove) 104 formed by the groove of the substrate 102 and the partitioned partition base material 103. Of the linear probe, only the portion corresponding to the flow path is used as the detection spot (probe) 105.

  The probe-immobilized reaction array of the present invention does not spot probes one by one at “points”, but “spots” so that a large number of specimens can be seen simultaneously on one array. It is possible to provide a biochip that can reduce the labor, cost, time, and resources required for chip production as a whole, rather than arranging partitioning so as to intersect with each other and performing detection using a probe at the intersection. .

  Further, the two substrates may be joined by means known per se before or after the probe is fixed. For example, when the nucleic acid probe is joined before immobilization, it is preferable to immobilize the nucleic acid probe by applying a photo-solid phase method (see, for example, JP 6-504997). For example, in the case of silicon-quartz glass, an adhesive may be printed by a screen printer and bonded. For example, in the case of silicon-pyrex (registered trademark) glass, bonding may be performed under high temperature and high voltage by an anodic bonding method used in a semiconductor process.

  In the above example, the groove is formed in the substrate 102. However, the partition partition base material 103 may be used, or the groove may be formed in both the substrate 102 and the partition partition base material 103. In addition, as a base material, a glass substrate was used for the substrate 102 on which the probe was solid-phased, and a PDMS material was used for the partition-separating base material 103 for forming the groove, but this is not a limitation. Alternatively, a silicon substrate may be used as a substrate used as a lid, and a glass substrate may be used as a substrate for forming a groove. Further, the two substrates used may be made of the same material. Moreover, you may determine the member to be used so that a transparent member may be arrange | positioned in the direction of observation. Alternatively, a base made of plastic resin or rubber may be used. Moreover, you may use combining the base material formed with materials, such as these materials, glass, a silicon | silicone, a plastic resin, and rubber | gum. In the above example, a plate-like substrate is used as a base, but the present invention is not limited to this.

  Hybridization is performed using the thus formed probe-immobilized reaction array under reaction conditions suitable for the probe provided in each reaction section. As the hybridization condition, for example, temperature control may be performed for each capillary. Moreover, all reaction parts may be maintained at a constant temperature to obtain optimum reaction conditions based on the ion concentration and salt concentration.

  The probe-immobilized reaction array of the present invention can be used in the same manner as Affymetrix and Stanford biochips. For example, the probe-immobilized reaction array of the present invention can be used to detect hybridization as shown in the following examples.

  Examples of the method for producing a probe-immobilized reaction array of the present invention and examples of its use will be described below with reference to examples.

Preparation of probe-immobilized reaction array
Use Hubble Slide Glass (TaKaRa) as the substrate of the oligo DNA microarray, and dilute the aminated oligo (probe oligo) (SIGMA Genosis) to the appropriate concentration using Solution I (TaKaRa), the recommended spot buffer for this substrate. Went spot. Details of the composition of the spot solution are shown below.

Spot solution
5'-aminated oligo (23mer, 100μM) 1μl (final conc. 10μM)
Solution I (× 4) 2.5μl
MilliQ 6.5μl
total 10μl
As a spotting method, spotting was performed by including the spot solution in a commercially available thread picker and pressing it onto a glass substrate. Humidity was about 40%, and spotting was performed quickly so that the spot solution contained did not dry.

Post-spot post-treatment followed the standard protocol of Hubble Slide Glass (TaKaRa). The following are some of the standard protocols for Hubble Slide Glass.
Hubble Slide Glass-Example of DNA immobilization
1. Spot the DNA with an amino group on the glass slide using a chip preparation device.
2. Wash slides with 0.2% SDS for 2 minutes.
3. Wash twice with Milli-Q water.
4. Wash with 0.3N NaOH for 5 minutes.
5. Wash twice with Milli-Q water.
6. Boil in boiling water (Milli-Q water) for 2 minutes.
7. Immerse in cold ethanol (4 ° C) for 3 minutes.
8. Remove moisture with centrifuge or compressed nitrogen gas and dry.

  The size of TaKaRa-Hubble Slide Glass is 1 inch x 3 inch (25.4mm x 76.2mm), which is the standard size of slide glass, and the thickness is 1.1mm ± 0.1mm. It can be used with a general glass array processing apparatus. The spottable area on the slide glass is shown in Fig. 6 (partly quoted from the TaKaRa website).

  A capillary rubber (see Fig. 3; see Fig. 7 for detailed dimensions. OLYMPUS) is pasted on the microarray after the post-treatment so that it crosses the spot line (see Fig. 1), and the hybridization solution is passed through the microarray. And hybridization was performed. The microarray is heated (50 ° C.) in advance, and the hybridization solution is also heated (50 ° C.). The incubation conditions for hybridization were 50 ° C., 3 hours or 1 hour, and the incubation was carried out in a sealable container together with moisture-absorbing paper containing water to prevent drying. The detailed composition of the hybridization solution is as follows.

Hybridization solution
5'-Cy3 modified oligo (46mer, 10nM) 1μl (10fmol)
5'-Cy5 modified oligo (23mer, 10nM) 1μl (10fmol)
2 × SSC, 0. 4% SDS Buffer 10 μl
MilliQW 9μl
total 20μl
The Cy3 and Cy5 modified oligos are target oligos that hybridize specifically with spotted probe oligos.

  After the hybridization reaction, the hybridization solution is removed by aspiration, the capillary rubber is peeled off, and it is immediately immersed in a large excess of washing solution that has been warmed to 50 ° C., and then gently rinsed for 5 minutes. The composition of the cleaning solution is as follows.

Cleaning solution
0.1 × SSC, 0.1% SDS Buffer Large excess (50 ° C).

  It was then rinsed gently for 1 minute in a large excess of MilliQW (room temperature) to remove the wash solution. After removing from the MilliQW, the water was removed by centrifugation or compressed nitrogen gas. After drying, detection was performed using a microarray scanner (GenePix 4000B (AXON)). The PMT range was set to 700 V at an excitation wavelength of 635 nm and 600 V at an excitation wavelength of 532 nm.

  FIG. 4 shows the results of using an 8-unit capillary rubber, and FIG. 5 shows the results of using a 20-unit capillary rubber for the same type of chip. The capillary rubber may be properly used according to the number of specimens.

The figure which shows the one aspect | mode of the probe solidification reaction array of this invention. FIG. 2 is a diagram showing a cross section of the probe-immobilized reaction array of FIG. The figure which shows the one aspect | mode of the manufacturing method of the probe solidification reaction array of this invention. The figure which shows the one aspect | mode of the manufacturing method of the probe solidification reaction array of this invention. The figure which shows the one aspect | mode of the manufacturing method of the probe solidification reaction array of this invention. The figure which shows the one aspect | mode of the manufacturing method of the probe solidification reaction array of this invention. One embodiment of the method for producing a probe-immobilized reaction array of the present invention is a 20-row capillary array. The figure which shows the result of the Example which applied 8 continuous capillary rubber to the linear spot chip | tip. The figure which shows the result of the Example which applied the 20 continuous capillary rubber to the linear spot chip | tip. The figure which shows the spot area of TaKaRa-Hubble Slide Glass.

Explanation of symbols

101 ... Linear probe
102, 201 ... Substrate
103 ・ ・ ・ Subdivision substrate
104 ・ ・ ・ Division space (groove)
105, 107, ... Detection spot (probe)
106 ・ ・ ・ Solution gateway
202 ・ ・ ・ Optical mirror
203 ・ ・ ・ Translucent window
204 ・ ・ ・ Laser / PMT
205 ・ ・ ・ Air cylinder
206 ・ ・ ・ Glass particles
207 ・ ・ ・ SiO ₂ coating
208 ・ ・ ・ Solution gateway
202 ・ ・ ・ Optical mirror
203 ・ ・ ・ Translucent window
204 ・ ・ ・ Laser / PMT
205 ・ ・ ・ Air cylinder
206 ・ ・ ・ Glass particles
207 ・ ・ ・ SiO ₂ coating
208

Claims (16)

A probe-immobilized reaction array,
Probes linearly immobilized on the base of the array;
A linear flow path formed in the array;
A probe-immobilized reaction array, wherein the linearly immobilized probe and the linear flow path are arranged so as to intersect each other.   2. The probe-immobilized reaction array according to claim 1, wherein the linearly immobilized probe has a plurality of different linear probes arranged in parallel with each other. Solid phase reaction array.   3. The probe-immobilized reaction array according to claim 1, wherein the linear flow path formed in the array is formed by a partition-separating base material in which linear grooves are formed. Probe immobilized reaction array.   4. The probe-immobilized reaction array according to claim 1, wherein the probe is directly immobilized on a substrate.   The probe-immobilized reaction array according to any one of claims 1 to 4, wherein the probe is indirectly immobilized on a substrate.   6. The probe-immobilized reaction array according to claim 1, wherein the linearly immobilized probe and the linear flow path are arranged orthogonally. Probe immobilized reaction array. The probe-immobilized reaction array according to any one of claims 1 to 6, wherein the probe is one or more probes selected from a probe group consisting of a nucleic acid, a protein, an antibody, or an antigen. A probe-immobilized reaction array.   The probe-immobilized reaction array according to any one of claims 1 to 7, wherein the probe is a nucleic acid probe. A method for producing a probe-immobilized reaction array comprising:
Linearly immobilizing probes on the base of the array;
Forming a flow path in the array so as to intersect with the solid-phased linear probe;
Including methods. A method according to claim 9, wherein
The probe comprises a plurality of different linear probes arranged in parallel with each other, and a probe-immobilized reaction array.   11. The method according to claim 9, wherein the linear flow path formed in the array is formed by a partition-separating base material in which a linear groove is formed.   12. The method according to any one of claims 9 to 11, wherein the probe is directly immobilized on a substrate.   The method according to any one of claims 9 to 12, wherein the probe is indirectly immobilized on a substrate.   14. The method according to any one of claims 9 to 13, wherein the linearly immobilized probe and the linear flow path are formed in an orthogonal arrangement.   15. The method according to any one of claims 9 to 14, wherein the probe is one or more probes selected from a probe group consisting of a nucleic acid, protein, antibody or antigen.   16. The method according to any one of claims 9 to 15, wherein the probe is a nucleic acid probe.

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