JP4856057B2 - Probe array and probe array manufacturing method - Google Patents

Description

The present invention relates to a probe array holding probes in an array and a method for manufacturing the same, and more particularly to a probe array having a plurality of array regions, a separator therefor, a method for manufacturing a probe array, a nucleic acid hybridization method, and the like.

  In a general DNA microarray using a glass substrate, a plurality of array regions (each array region is a probe fixing region to which a large number of probes are fixed) are formed on one substrate, and multiple samples are simultaneously prepared. Attempts have been made to process. Hybridization is performed by placing a single cover glass on a glass substrate having a plurality of array regions. However, in such a method, there is a problem in that reaction solutions (solutions including targets) in adjacent array regions mix with each other. It can happen. For this reason, for example, in JP-A-2002-65274, while imparting hydrophobicity to the partition wall that partitions the array region, the reaction solution is held in the array region and mixed by making the array region hydrophilic. It is disclosed to avoid this.

  As shown in FIG. 19, in an array in which a large number of probes such as DNA microarrays are held on a substrate, the probe is usually subjected to surface treatment for immobilizing the probe on the substrate surface, and the probe is applied by various methods such as an ink jet method. An immobilization process for fixing the probe to the substrate surface is performed after being supplied to the substrate surface. Printing or chemical treatment is used to form a hydrophobic region for partitioning a plurality of array regions. In order to fix such a hydrophobic region to a substrate, a high temperature of several hundred degrees or a predetermined liquid on the substrate is used. It may be necessary to immerse the sample in biological materials such as nucleic acids and proteins that are used as probes and surface treatment. For this reason, conventionally, as shown in FIG. 19, the hydrophobic region forming step is performed prior to the surface treatment or spotting, and the surface of the substrate on which the hydrophobic region is previously formed is not formed on the surface. Processing is performed, followed by spotting.

  However, according to the study by the present inventors, when a surface treatment is performed on a substrate on which a hydrophobic region has been formed in advance, and a probe is spotted and immobilized, a hybridization reaction in an array region partitioned by the hydrophobic region. It has been found that the uniformity of the glass tends to be lower than before. In addition, the efficiency of the hybridization reaction in the array region partitioned by the hydrophobic region by printing or coating tends to decrease. Upon further investigation, it was found that such a defect in hybridization was caused by the heterogeneity of the chemical properties of the surface of the array region.

  Further, in such an array having a plurality of array areas, contamination of the test liquid between the array areas of the test liquid becomes a big problem. For example, after the test solution is dropped on the array region, contamination is likely to occur with the transfer of the array, such as storing the array in a predetermined reaction chamber or setting it in an incubator under predetermined conditions.

  Furthermore, in an array having a plurality of array regions, it is difficult to identify individual array areas or to identify a specific array.

  Accordingly, an object of the present invention is to provide a probe array having an array region in which the chemical properties of the surface are made uniform and partitioned. Another object of the present invention is to provide a probe array having a partitioned array region that can realize a good reaction between a probe and a test solution such as hybridization. It is another object of the present invention to provide a probe array that can suppress or avoid contamination between array regions. Another object of the present invention is to provide a probe array manufacturing technique capable of forming the above array region in an arbitrary region on a substrate. Furthermore, another object of the present invention is to provide a probe array having excellent array region discrimination.

  As a result of studying the above problems, the present inventors have found that the formation of the hydrophobic region for the partition of the array region performed prior to the surface treatment of the substrate or the presence thereof is a chemical property of the surface of the array region. As a result, the surface treatment becomes non-uniform, and the shape and size of the probe spot and the immobilization amount become non-uniform, resulting in an increase in the variation of interaction reactions such as hybridization. I found out. In particular, it has also been found that such non-uniformity occurs at the edge of the array region close to the hydrophobic region. For this reason, the present inventors have a partition wall that can partition the array region with respect to the substrate subjected to the surface treatment or the like while eliminating the step of forming the hydrophobic region by printing or chemical treatment on the substrate surface. The present invention was completed by obtaining the knowledge that the conventional problems can be solved by mounting the separator. Therefore, according to the present invention, the following means are provided.

  According to one aspect of the present invention, there is provided a probe array including one or more array regions on a substrate that are partitioned and have a large number of probes immobilized thereon, and partition the one or more array regions. There is provided a probe array comprising a separator having a partition mounted on the substrate. In the present invention, the probe is preferably a nucleic acid probe. The probe is also preferably a protein probe. The array region is preferably a plurality.

  In this aspect, the separator may be attached to a surface of a probe immobilization layer to which the probe on the substrate is immobilized, and in this case, the separator is arranged after the probe immobilization. It is preferable to be mounted on the surface.

  The separator is preferably in the form of a sheet and is fixed to the substrate via an adhesive layer. Furthermore, it is preferable that the separator is fixed to the substrate in a separable manner.

  Furthermore, at least a part of the separator may have a hydrophobic region. In this aspect, it is preferable to have the hydrophobic region on the top surface of the partition wall of the separator. The contact angle of water in such a hydrophobic region is preferably 60 ° or more. More preferably, it is 70 ° or more. The hydrophobic region can contain a material selected from the group consisting of polycarbonate, polyolefin, polyamide, polyimide, acrylic resin, and fluorides and halides thereof.

  In addition, an area surrounded by a partition partitioning the array area of the separator can constitute an open cavity for the interaction between the probe and the test sample on the array area.

Furthermore, the design area of the array region defined by the partition walls may be 0.3 mm ² or more and 2000 mm ² or less. Preferably, it may be a 3 mm ² or more 90 mm ² or less. Furthermore, it is possible to provide one or more and 400 or less array regions, preferably one or more and 144 or less.

Furthermore, the design area (Sd) of the array region defined by the partition walls is 90 mm ² or less, and the probing effective area ratio represented by the following formula (1) is 70% or more. it can.
Probing effective area ratio (%) = Se [mm ² ] / Sd [mm ² ] × 100 (1)
However, Se represents the area of a region where the variation coefficient of the signal intensity based on the interaction between the probe and the object is 20% or less.
Note that probing means exploring, detecting, and confirming an object by causing the interaction with the object using a probe that can selectively or specifically interact with the object. It shall be. The signal intensity variation coefficient is a signal variation coefficient based on an interaction obtained by supplying an object to interact with a probe on a probe array (signal intensity detected from a micro area of the array area). Standard deviation / average value of signal intensity detected from a small area of the array area). The variation coefficient of the signal intensity forms an appropriate number of microscopic areas of a single type of probe (for example, spots) in the array area, and supplies an object that interacts with the probe to perform hybridization. And measuring the signals detected from each microregion and obtaining the standard deviation and average value of these signal intensities. In addition, the region where the variation coefficient of signal intensity is 20% or less is an area where there is a minute region where the variation coefficient of signal intensity is 20% or less among an appropriate number of minute regions in the array region. Shall mean. Note that the contour of a region where the variation coefficient of signal intensity is 20% or less can be defined by the minute region located on the outermost side of the minute region group that satisfies the variation coefficient. Further, the number of minute regions for which the variation coefficient of the signal intensity is to be calculated is preferably 3 or more, more preferably 20 or more.

  Moreover, it is also preferable that the array area to be partitioned has a variation coefficient of signal intensity obtained by probing of 20% or less in an area excluding a range within 0.8 mm from the partition in the array area. The array area to be partitioned preferably has a depth of 10 μm or more and 240 μm or less.

Moreover, the ratio [R] represented by the following formula | equation (2) can also be 0.02 or less for the said array area | region divided.
R = d [mm] / Sd [mm ² ] (2)
Here, d represents the depth of the array region, and Sd represents the design area of the array region.

Moreover, in this aspect, the separator can be capable of forming a plurality of partition walls having different heights. Furthermore, the separator may be capable of reducing the height of the partition wall by removing at least a part of the partition wall. In this embodiment, the separator can form a plurality of partition walls having different heights in the range of 10 μm to 1000 μm. Further, the partition wall of the separator has a height of 60 μm or more during a reaction in which a test sample is supplied to the probe for reaction, and has a height of less than 60 μm when a signal of the reaction product after the reaction is detected. It can also be made to have. In this embodiment, the area of the array region can be 0.3 mm ² or more. Furthermore, the separator may have a laminate structure. The separator may include a fragile portion from which at least a part of the partition wall can be removed. The separator may be a laminate, and the fragile portion may be an interface portion of the stacked layers.

  In this aspect, the separator may have an overhanging portion that projects toward the upper side of the array region at least at a part of the upper side of the partition wall. Furthermore, the overhang portion may be provided over the entire periphery of the array region. Furthermore, at least a region of the portion that protrudes from the partition wall above the array region of the overhang portion that can contact the test solution can have a hydrophobic region.

Further, the height of the lowest end of the overhang portion (height from the substrate surface) may be 40 μm or more and 990 μm or less, and the overhang portion does not reach the probe fixed in the array region. You may overhang in the range. Furthermore, the region defined by the inner peripheral surface of the overhang portion may be a region where the probe in the array region is immobilized. Furthermore, at least a part of the overhanging part may be integrated and / or removable with respect to the remaining part of the partition wall, and the entire overhanging part may be integrated with the remaining part of the partition wall. It may also be removable.

  Further, in this aspect, the separator can constitute at least a part of the height of the partition wall that partitions the array region, and the substrate has at least a part of the height of the partition wall that partitions the array region. You may comprise.

  Further, in this aspect, an identification layer having a color and / or an image capable of identifying the one or more array regions can be provided. In this case, the identification layer can be provided on the substrate side of the separator, and preferably, the separator includes the identification layer. The identification layer may be a layer formed by printing. Furthermore, the identification layer may have a color capable of identifying the one or more array areas, and can be selected from letters, numbers, symbols, and figures so that the one or more array areas can be specified. Any one or two or more kinds of labels may be included. Furthermore, you may provide the color and the image.

  According to another aspect of the present invention, there is provided a separator having a partition for partitioning the array region on the substrate on which a plurality of probes form one or more array regions and are immobilized. Is done. In this aspect, it is preferable to have a hydrophobic region on the upper surface of the partition wall of the separator. A plurality of array regions can be provided. The separator may be capable of forming a plurality of partition walls having different heights. In addition, the separator may have an overhanging portion that projects toward the upper side of the array region at least at a part of the upper side of the partition wall.

  The separator may have an identification layer having a color and / or an image so that the one or more array regions can be identified. In this case, the identification layer is preferably provided on the substrate side. Further, the identification layer may be a layer formed by printing.

  The separator may have a laminated structure. Furthermore, it is preferable that the separator is for the probe array described above.

  According to another aspect of the present invention, there is provided a probe array in which a large number of probes are immobilized by forming one or more array regions, and the one or more array regions are formed by the separator. A compartmented probe array is also provided. In addition, the probe array of this aspect is a probe array provided with the array area | region which is not partitioned, The probe array which has the partitioned array area | region is mounted | worn by mounting said separator on the probe array of this aspect. can get.

  According to still another aspect of the present invention, there is provided a method for manufacturing a probe array, the step of immobilizing a plurality of probes on a substrate by forming one or more array regions, and the probes being immobilized. Attaching a separator having a partition wall capable of partitioning the surface to the surface of the substrate, and partitioning the plurality of probes into one or more array regions. The In this embodiment, a plurality of the array regions can be provided.

  According to still another aspect of the present invention, there is provided a nucleic acid hybridization method, wherein a test solution containing a nucleic acid test sample is supplied to the array region of a probe array of nucleic acid probes to hybridize with the probe. There is also provided a hybridization method comprising the steps of performing. In this method, the probe array has an open system cavity for hybridization on the array region, and the hybridization step supplies a test solution to the open system cavity under humidified conditions. It can be carried out.

  According to another aspect of the present invention, there is provided a reaction method using a probe array, wherein a test solution is supplied to the array region of any one of the probe arrays described above comprising a protein probe, and the probe is used. There is provided a reaction method comprising the step of carrying out the reaction with.

According to still another aspect of the present invention, there is provided a reaction method using a probe array,
A test solution is supplied to one or more of the array areas partitioned by a first height partition wall of the probe array that is partitioned and has one or more array areas on which a plurality of probes are immobilized. And a reaction step for reacting and detecting washing or reaction products after the reaction step in a state where the partition wall is absent or the height of the partition wall is reduced to a second height lower than the first height. And a post-process.

In this aspect, prior to the reaction step, a step of forming the first height partition wall on the substrate after fixing the probe to the substrate can be provided. Further, the partition wall having the first height in the reaction step has an overhang portion that projects toward the upper portion of the array region at least at a part above the partition wall, and the overhang portion is removed in the post-process. Thus, the height of the partition wall can be reduced to the second height. Furthermore, the partition may be formed by applying a separator capable of partitioning the array region to the substrate surface, and the separator may be a laminate.

It is a figure which shows an example of a probe array. It is a figure which shows the detail of an example of a probe array. It is a figure which shows an example of the partition form in a separator sheet. It is a figure which shows an example of the partition form in a separator sheet. It is a conceptual diagram of a probing effective area ratio. It is a figure which shows an example of the manufacturing process of a probe array. It is a figure which shows the state in which the array area | region (not divided) of the probe was formed on the board | substrate in the manufacturing process of a probe array. It is a figure which shows the pattern of the array area | region of the nucleic acid probe array of an Example. It is a figure which shows the structure of the separator sheet of an Example. It is a graph which shows the relationship between the distance from a partition, and CV of fluorescence intensity. It is a graph which shows the difference of the hybridization effective area rate of an Example and a comparative example. It is a graph which shows the relationship between the depth of an array area | region, and fluorescence intensity. It is a graph which shows the relationship between water repellency and fluorescence intensity. It is a graph which shows the relationship between ratio R of the depth of an array area | region with respect to the design area of an array area | region, and fluorescence intensity (in the case of 0.25 mm of array area depth). It is a graph which shows the relationship between the ratio R of the depth of an array area | region with respect to the design area of an array area | region, and fluorescence intensity (when the array area depth is 0.1 mm). 6 is a view showing a laminated structure of a separator sheet produced in Example 5. FIG. 6 is a view showing a laminated structure of a separator sheet produced in Example 5. FIG. FIG. 10 is a diagram and a plan view showing a laminated structure of an array created in Example 6. It is a figure which shows an example of the preparation process of the conventional probe array.

  The probe array provided with the array area on which the plurality of partitioned probes are fixed on the substrate according to the present invention is characterized by including a separator having a partition partitioning the array area and its periphery. According to the probe array of the present invention, in the array region, a separator having a partition partitioning the array region and its periphery is mounted on the substrate. That is, the array region is partitioned without forming a hydrophobic region by printing or dipping a water repellent material on the substrate. For this reason, such treatment is performed before the surface treatment, and the chemical properties of the substrate surface are made non-uniform due to the presence of hydrophobic or water-repellent regions on the substrate in the probe array preparation stage. Non-uniformity in various operations such as non-uniformity in probe shape, size, and amount of immobilization can be eliminated. As a result, it is possible to form a partitioned array region in which the chemical properties in the array region are uniform, and to form a partitioned array region in which the probe immobilization form and amount are uniformed and good probing can be performed. it can. As a result, even multi-sample, multi-item probing can be performed efficiently.

  In addition, the separator of the present invention is characterized by having a partition for partitioning the array region on the substrate on which a plurality of probes form one or more array regions and are immobilized. Therefore, the probe array having the partitioned array region of the present invention can be formed by attaching this separator to a probe array in which a large number of probes form one or more array regions and are immobilized. As a result, various advantages similar to those described above can be obtained.

  Furthermore, the method for producing a probe array of the present invention comprises a step of immobilizing a plurality of probes on a substrate in a pattern capable of forming one or more array regions, and a surface of the substrate on which probes are immobilized Mounting a separator having a partition wall whose surface can be partitioned into one or more, and partitioning a plurality of the probes into one or more array regions in the pattern. According to this method, the probes are fixed in a pattern that assumes the partitioning of the array region, and thereafter, a plurality of probes are partitioned into a plurality of array regions by the separator. For this reason, the array region is partitioned without forming a hydrophobic region by printing or dipping the water repellent material on the substrate. Therefore, such a hydrophobic region forming process can be omitted, and a preferable probe array can be manufactured by eliminating various types of non-uniformity due to the presence of such a hydrophobic region. The array area is preferably plural.

  In addition, the nucleic acid hybridization method of the present invention is characterized by comprising a step of supplying a test solution containing a test sample to the array region of the probe array and performing hybridization with the probe. According to this method, various inconveniences resulting from the conventional partitioning method are eliminated in the array region of the probe array, so that good hybridization can be performed.

  The probe array of the present invention can form a plurality of partition walls having different heights as a separator. By doing so, a good reaction can be realized between the probe and the test solution. That is, the array region can be partitioned at a required height according to probe array production, reaction process, etc., such as probe immobilization, blocking, reaction, washing, signal detection and the like. For example, the height of the partition wall is set to a height or zero (no partition wall) at the time of probe immobilization or surface treatment, and avoids or suppresses contamination of the test solution during reaction. It is possible to set the height as high as possible and to make the partition wall low or zero during washing and signal detection. In addition, what is necessary is just to form a separator so that isolation | separation from a board | substrate is possible in order to make a partition height into zero. In addition, by adjusting the height of the partition wall, the degree of freedom in the amount of test solution supplied to the cavity including the array region is improved, and reaction can be easily performed with an appropriate amount of test solution depending on the type of test solution and the type of reaction. Can be made. Furthermore, when the color reaction is performed for each array region after the reaction, the height of the partition wall may be maintained or increased. Otherwise, the partition wall may be removed or the height may be decreased.

  In addition, the probe array of the present invention can also have an overhang portion that projects toward the array region at least at a part above the partition wall. In this way, when the test solution is supplied to the cavity including the array region partitioned by the partition wall, the contamination of the test solution with other array regions can be effectively avoided or suppressed. A good reaction with the liquid can be realized. Furthermore, when the site | part which contacts the test liquid of such an overhang | projection part has a hydrophobic area | region, contamination can be avoided or suppressed more effectively about a hydrophilic test liquid. Moreover, according to such an overhang | projection part, sufficient contamination suppression effect can be acquired, suppressing the height of the partition 14. FIG. It should be noted that, even for the partition wall having such an overhang portion, at least a part of the overhang portion can be integrated with or removed from the remaining portion of the partition wall, thereby hindering the manufacture of the probe array and various operations and processes. It is possible to exhibit the effect of suppressing contamination.

  Furthermore, in the reaction method using the probe array of the present invention, the probe and the test solution are reacted in a state where the array region is partitioned by the partition walls having the first height, and the array region is lower than the first height. Various post-processes such as washing and detection after the reaction are performed in a state of being partitioned at a height of 2 or having no partition walls. By doing so, a good reaction can be realized between the probe and the test solution. That is, as already described, since the characteristics (height, etc.) required for the partition walls that divide the array region differ depending on the manufacturing and reaction process of the array, the partition wall height is high as necessary, that is, during the reaction. By making it lower in the subsequent steps, a preferable reaction can be realized on the probe array.

    Hereinafter, the probe array, separator, probe array production method and nucleic acid hybridization method of the present invention will be described in order.

(Probe array)
One embodiment of the probe array 2 of the present invention is shown in FIG. 1, and a detailed view thereof is shown in FIG. The probe array 2 includes a substrate 4, the substrate 4, and a separator 12. On the substrate 4, spot-like minute regions 8 to which a large number of probes 6 are fixed are provided. These minute regions 8 are aligned to form one array region 10, for example.

(probe)
In the present invention, the probe 6 is not particularly limited, and may interact directly or indirectly as a result of interaction with various test samples in the test solution, such as nucleic acids, proteins, synthetic or natural compounds, on the array region 10. Any signal can be used as long as the signal can be detected. For example, various interactions such as hybridization between nucleic acids based on base pairing, specific binding between a protein and a nucleic acid, a protein and a low molecular compound, and a nucleic acid and a low molecular compound can be used.

Most typically, a nucleic acid can be used as a probe of the probe array of the present invention. The nucleic acid may be any nucleic acid that at least partially hybridizes with other nucleic acids by nucleic acid base pairing, and includes both natural and synthetic nucleotide oligomers and polymers, and further includes DNA such as genomic DNA and cDNA, The concept includes PCR products, RNA such as mRNA, and peptide nucleic acids. A nucleic acid can detect a signal by an interaction by a hybridization reaction by base pairing or an interaction by specific binding with a protein or a low molecular weight compound.

  As proteins, enzymes, antibodies, antigens, receptors, and the like can be used. Since these react specifically with the substrate, antigen, antibody, and nucleic acid, respectively, signals can be detected. Furthermore, other synthetic or natural compounds include various drug candidate compounds synthesized by combinatorial chemistry. In addition, in this invention, it is not limited to these illustrations, If it can detect a target compound, it can be used as the probe 6.

  For example, the probe 6 is present on the substrate 4 as a minute region 8 having a diameter of about 50 μm to 500 μm. In the present invention, the fixing method and fixing form of the probe 6 to the substrate 4 are not particularly limited, and include all forms known at the time of the present application. Therefore, in addition to a probe fixing method using a contact type pin and a probe fixing method using a non-contact type ink jet method, a method of synthesizing the probe 6 on the substrate 4 is also included.

(substrate)
The shape and material of the substrate 4 are not particularly limited, but various materials other than various materials used in conventional DNA chips, DNA microarrays, and the like can be used. For example, ceramics including silicon ceramics such as glass, silicon dioxide, and silicon nitride, resins such as silicone, polymethylmethacrylate, and poly (meth) acrylate, metals such as gold, silver, and copper can be used. Appropriate coats may be applied to impart desired surface properties. Among these, a glass substrate, silicone, and acrylic resin can be used.

  As shown in FIG. 2, the substrate 4 is preferably provided with a probe-immobilized layer 5 that is processed according to the type of the probe 6 in order to fix various probes 6 such as nucleic acids to the substrate 4. . The type of probe immobilization layer 5 is not particularly limited. For example, when nucleic acid is immobilized as probe 6, a positively charged polymer layer such as polylysine, a functional group in nucleic acid, or a reactive group added to nucleic acid can be bound. A polymer layer having a proper aldehyde group or carboxyl group can be provided as the probe immobilization layer 5. In addition, it is preferable that the area | region which should fix the probe 6 in the board | substrate 4 is substantially flat including the case where it has micro three-dimensional shapes, such as being porous.

(Array area)
An array region 10 is provided on the substrate 4. The array region 10 is a region where the micro region 8 is formed. The array region 10 is formed as an aggregate of a large number of minute regions 8. On the substrate 4, one or more, preferably a plurality of array regions 10 are provided. Different groups of probes 6 may be immobilized on different array regions 10. The array region 10 in the probe array 2 of the present invention is at least partitioned from the periphery or partitioned from other array regions 10. In the partitioned array region 10, when a test solution is supplied onto the array region 10, a certain amount of the test solution is retained in the array region 10 and does not flow out to the surrounding or adjacent array region 10. It is preferable that it is compartmentalized. By supplying test solutions containing different test samples to these array regions 10, multiple sample (or different types) test solutions can be assayed simultaneously.

Such partitioning is preferably made to form cavities 7 in the array region 10. Examples of the cavity 7 include an open cavity in which at least a part of the array region 10 is open. Typically, it is a concave cavity open at the top. Further, the cavity 7 may be a closed system cavity that is provided with an opening for opening and closing the test liquid. Such a cavity 7 is formed by a separator 12 described later. Such a cavity 7 is a space for the interaction between the probe 6 and the test sample. When the probe array 2 is a nucleic acid probe array, it becomes a space for a hybridization reaction.

  A part of the depth of the cavity 7 may be formed by a concave portion or a convex portion included in the substrate 4 itself. For example, the substrate 4 itself may have a shallow well corresponding to the array region 10, and conversely, a partition wall having a height corresponding to a part of the height of the partition wall 14 that partitions the flat array region 10. You may have.

  The depth of the cavity 7 (which is synonymous with the height of the partition wall 14 described later) is preferably 10 μm or more and 240 μm or less. Within this range, the thickness of the test liquid held in the cavity 7 can suppress the influence of the partition walls constituting the cavity 7 to ensure the convection of the test liquid and the diffusion of the test sample, and as a result, highly sensitive detection. Is possible. More preferably, it is 150 micrometers or less, More preferably, it is 100 micrometers or less. Note that the depth of the array region 10 can be a height from the surface of the substrate 4 on which the probe is fixed to the top surface of the partition wall 14 as will be described later. Such a distance can be easily measured by, for example, various digital length measuring devices. Such a structure of the cavity 7 is preferable when probing is performed in an aqueous medium such as nucleic acid hybridization.

Furthermore, as for the relationship between the depth of the cavity 7 and the design area of the array region 10, the ratio [R] represented by the following formula (2) is preferably 0.02 or less. When the ratio R is within this range, the influence of the partition wall 14 of the cavity 7 can be suppressed to ensure the convection of the test solution and the diffusion of the test sample, and as a result, highly sensitive detection is possible. Such a structure of the cavity 7 is also preferable when probing is performed in an aqueous medium such as nucleic acid hybridization.
R = d [mm] / Sd [mm ² ] (2)
Here, d represents the depth of the array region, and Sd represents the design area of the array region.

The size of the array region 10, that is, the designed area (hybridization area) of the array region is preferably 0.3 mm ² or more and 2000 mm ² or less. This is because within this range, the variation coefficient of the signal intensity based on the interaction between the probe and the test sample is well suppressed. If it is 0.3 mm ² or less, the convection of the test solution and the diffusion of the test sample are likely to be inhibited by the influence of the partition wall 14, and if it is 2000 mm ² or more, the signal intensity is not sufficient only by the convection of the test solution and the diffusion of the test sample. It becomes difficult to suppress the coefficient of variation. More preferably 3 mm ² or more 90 mm ² or less. The designed area of the array region is an area inside the array region defined by the partition walls 14 of the separator 12 described later. The number of array regions 10 on the substrate 4 is not particularly limited, but preferably 1 or more and 400 or less, and more preferably 1 or more and 144 or less. From the viewpoint of adapting to existing infrastructure, 400 or less is preferable for a size of about 96-well plate, and 144 or less is preferable for a slide glass size.

(separator)
A separator 12 that partitions the array region 10 is provided on the substrate 4. As shown in FIGS. 1 to 4, the separator 12 includes a partition wall 14 that partitions the array region 10. The partition 14 in the separator 12 is for partitioning the array region 10 from the periphery, and corresponds to the size and shape of the array region 10 to be partitioned. In the form shown in FIG. 3, the partition walls 14 intersect in a grid shape so that adjacent array regions 10 can be partitioned. In the form shown in FIG. 4, the staggered partition walls can be partitioned. 14. Note that the partitioned array region 10 is not limited to the form shown in FIGS. 3 and 4, and can be used to partition one or more array regions 10 in any form. Also good. In addition, it is preferable that the partition of the separator 12 becomes the partition 10 of the form which surrounds the whole of each array area | region 10. As shown in FIG.

  The form of the partition 14 of the separator 12 varies depending on the size, number, and shape of the array region 10 to be partitioned, but the separator 12 itself is preferably a sheet-like body. If it is in a sheet state, it is easy to mount it on the substrate 4 using its flat surface. The sheet-like body means the whole shape, and even if the partition wall 14 has a skeleton shape, it may be a sheet shape from the thickness and outer shape (width, length, etc.) of the separator 12.

  The separator 12 may be attached to the substrate 4 so as to partition the array region 10. Accordingly, it is possible to place the separator 12 on the substrate 4 and fix them with a jig or the like, but the separator 12 is fixed to the substrate 4 via the adhesive layer 16 as shown in FIG. It is preferable that By being fixed by the adhesive layer 16, the separator 12 can be easily attached to the substrate 4 after the probe 6 is fixed. Specifically, the adhesive layer 16 may be a so-called strong and long-time adhesive, or may be a pressure-sensitive adhesive that can be easily separated. The mounting using the adhesive layer 16 is a particularly preferable form when the separator 12 is a sheet-like body.

  It is preferable that the height of the separator 12, that is, the height of the partition wall 14 can be changed as necessary. In order to increase or decrease the height, it is preferable that the separator 12 has a laminate structure in which the constituent layers can be integrated and / or removed. That is, different heights can be realized by making the separator 12 a laminate having two or more layers and making the constituent layers removable at at least one interface. On the other hand, a stackable structure including stackable constituent layers may be used. Furthermore, it is good also as a laminated body which can be isolate | separated and integrated.

  In the laminated body, the interface portion is a fragile portion, and it is preferable that this portion is configured to be integrated and / or removable. For example, it may be formed so as to be integrated or removable by a layer such as an adhesive or a pressure-sensitive adhesive, or may be configured to be integrated or removable depending on the adhesiveness or adhesion of the component layer itself. According to the adhesive or the like, the adhesive strength between the constituent layers can be easily adjusted, and the operability during height adjustment can be easily ensured. Further, the constituent layers may be configured to be integrated or separable by mechanical fitting or the like. The interface part which makes a structure layer integral and / or removable can be provided in one place or two places or more as needed.

  Moreover, in order to be able to change the height of the separator 12, it can be set as the structure which has a weak part in a predetermined | prescribed height site | part. Even if it does not depend on a laminated structure, it has a part weak in terms of structure or material, so that a part of the separator 12 can be removed and the height of the part can be reduced.

It is preferable that the height of the separator 12 having the partition wall 14 having such a variable height can be adjusted, for example, in the range of 10 μm to 1000 μm. Within this range, the height of the partition wall 14 that can avoid or suppress contamination of the test solution can be configured during the reaction, and the partition wall can have a height that does not interfere with washing and signal detection after the reaction. More specifically, it is preferably 60 μm or more during the reaction. This is because if it is 60 μm or more, contamination between the array regions at the time of array transfer can be well suppressed while ensuring a sufficient supply amount of the test solution. More preferably, it is 200 μm or more. When it is 200 μm or more, it becomes easy to cause the reaction in the cavity 7 in a state where the level of the test liquid supplied to the cavity 7 including the array region 10 does not exceed the partition wall 14. On the other hand, it is preferably less than 60 μm in subsequent washing, signal detection and the like. This is because if the thickness is less than 60 μm, the cleaning solution can be easily applied to the array area at the time of cleaning, and the signal detection does not hinder the application to a general existing apparatus (infrastructure). More preferably, it is 50 μm or less. In addition, it is preferable that the height of the partition 14 is 40 micrometers or more also in a post process. This is because when the thickness is 40 μm or more, contamination between the array regions 10 of the test solution after the reaction can be suppressed even when the upper portion of the partition wall 14 is removed to reduce the height of the partition wall 14.

  In addition, the separator 12 can include an overhanging portion that extends toward the array region 10 at least at a part above the partition wall 14. The protruding portion may be formed over the entire periphery of the partition wall 14 surrounding the array region 10 or may be formed only in part. As a form that is formed only partially, for example, it may be formed on a pair of partition walls facing each other in the periphery, or a plurality of discontinuous portions that are discontinuously spaced at appropriate intervals over the entire periphery. May be formed.

  Further, the shape of the overhanging portion is not particularly limited. The overhanging portion may be overhanging parallel to the surface of the substrate 4 or may be overhanging obliquely downward. Further, the overhanging portion may have a notch shape such that the inner peripheral surface thereof is tapered upward such as a dome shape with respect to the surface of the substrate 4. The overhang amount of the overhang portion is preferably in a range that does not reach the probes immobilized on the array region 10. This is because if the overhanging portion reaches the probe, a problem such as leaving bubbles on the probe may occur when supplying a test solution including the test sample. In other words, in the case where the overhanging portion is provided, the region defined by the most protruding tip side of the overhanging portion is the region where the probe is immobilized by the array region 10. Therefore, it is preferable that the tip of the overhang portion is within 0.8 mm from the partition wall 14.

  Moreover, it is preferable that the position of the lowest end of an overhang | projection part is 40 micrometers or more and 990 micrometers or less. This is because even when the overhanging portion is bent and removed when the thickness is 40 μm or more, contact and damage between the overhanging portion and the array region can be prevented. More preferably, it is 60 μm or more and 500 μm or less.

  It is preferable that the portion of the overhanging portion that can come into contact with the test solution has a hydrophobic region 18 described later. By providing the hydrophobic area | region 18, the contamination of a test liquid can be avoided or suppressed more effectively. Examples of the portion that can be contacted with the test solution include a lower surface or an inner peripheral surface facing the surface of the substrate 4 of the overhang portion. In the case where the formation position of the overhanging portion of the partition wall 14 is relatively low, for example, the height of the uppermost end of the overhanging portion is less than 60 μm and the overhanging portion is formed on the uppermost portion of the partition wall 14. When the top surface is exposed, it is preferable that the top surface also includes a hydrophobic region 18. When the hydrophobic region 18 is added to the overhanging portion, it is typically preferable that the overhanging portion itself is formed of a hydrophobic material described later.

  The overhanging portion may be provided with respect to the partition wall 14 and the separator 12 in any form. Only the protruding portion can be attached to or removed from the uppermost portion of the partition wall 14 or can be provided in advance on a part of the uppermost portion of the partition wall 14 or the like. The overhanging portion may be a patch-like body that partially blocks the periphery of the opening of the cavity 7 including the array region 10 surrounded by the partition wall 14 (configured by the upper edge of the partition wall 14). Good.

  In addition, contamination with a test liquid or the like can be suppressed while suppressing the height of the partition wall 14 by providing the overhanging portion.

(Identification layer)
The separator 12 can include an identification layer. The identification layer may have a color and / or an image so that one or more (preferably two or more) array regions 10 can be identified. In particular, it is preferable to have a color or the like so that the array region 10 can be identified visually. The identification layer is formed around the array region 10 so that the array region 10 can be identified. Therefore, for example, the identification layer can have a planar form that roughly corresponds to a planar region (hereinafter also referred to as a partition-corresponding region) constituting the partition 14 in the separator 12. The identification layer may not have a color and / or an image over the entire area corresponding to the partition wall. For example, it may be formed in a frame shape surrounding the array region 10 in the vicinity of the periphery of the array region 10, or may be formed in a part of the vicinity of the periphery of the array region 10.

  When the substrate 4 is a transparent substrate, the identification layer provided for identifying the array region 10 preferably has an appropriate color. For example, the array region 10 can be easily visually recognized by adding some color to the partition equivalent region of the separator 12 or a part thereof. Moreover, by having an image composed of numbers, characters, symbols, figures, and combinations thereof that can identify each array region 10 in the vicinity of the peripheral edge of each array region 10 in the partition equivalent region of the separator 12, The array region 10 can be specified and easily identified.

  Such an identification layer may be, for example, a colored film corresponding to the shape of the separator 12, or may be a similar shape and provided with a printing layer or a printing layer. It is preferable that the identification layer includes a printing layer because any color or image can be imparted by printing. The identification layer may not be formed from a single layer. Two or more layers may be laminated. Two or more layers can have different colors and images.

  The identification layer may be provided in any part of the separator 12 as long as the array region 10 can be identified. That is, it may be the outermost surface of the separator 12, but is preferably provided on the inner layer side that is not the outermost surface, that is, on the substrate 4 side. By having the identification layer on the substrate 4 side, it is possible to suppress the reaction between the test liquid and a coloring material such as ink constituting the identification layer.

  Such an identification layer may identify not only the identification and identification of the array region 10 but also the probe array itself. Misidentification can be reduced by identifying the probe array by color or image. Furthermore, any color or image may be given in accordance with the signal detection method or model. In addition, according to such an identification layer, the operation can be performed while confirming the position of the array region 10, so that the reliability of the operation is improved. Furthermore, the front and back of the probe array can be easily determined. In addition, in the case of having a color or image with high light-shielding performance, it is possible to suppress deterioration of the adhesive layer in the lower layer, and to suppress a decrease in adhesion between the separator 12 and the substrate 4.

  The identification layer is not necessarily provided in the separator 12. For example, it may be mounted separately from the separator 12 after the fixing layer 5 is formed on the surface of the substrate 4 and before the separator 12 is mounted. Further, it may be provided on the front surface of the substrate 4 prior to the formation of the immobilization layer 5, or may be provided on the back surface of the substrate 4.

As shown in FIG. 2, the separator 12 is preferably attached to the surface of the probe fixing layer 5 of the substrate 4. Since the separator 12 is mounted after the probe immobilization layer 5 is formed, the probe immobilization layer 5 can be uniformly formed on the substrate regardless of the presence of the conventional hydrophobic region partitioning the array region 10 and the influence of the treatment. 4 is formed on the surface. Further, for this reason, the surface treatment layer such as the probe immobilization layer 5 is uniformly formed and provided in the edge region of the array region 10 adjacent to the partition wall of the separator 12 as well as other portions. In addition, when the board | substrate 4 is provided with the partition of the height corresponding to a part of height of the partition 14, what is necessary is just to mount | wear with the separator 12 so that it may correspond to such a partition. In the case where the substrate 4 includes a part of the partition wall 14, as with the flat substrate 4, the substrate 4 including the partition wall is subjected to surface treatment on the entire surface on which the array region 10 including the partition wall is formed. After the probe fixing layer 5 is formed, the separator 12 is preferably attached. The material of the separator 12 is not particularly limited, and for example, resins such as acrylic resin, thermoplastic elastomer, natural or synthetic rubber, silicone, polyolefin, polyamide, polyimide, vinyl halide, and polycarbonate can be used. Moreover, it is preferable that the separator 12 has flexibility, and may have transparency.

  It is preferable that a hydrophobic region 18 is provided on at least a part of the separator 12. By providing the separator 12 with the hydrophobic region 18, water repellency with respect to the aqueous test solution can be exerted, and contamination between the array regions 10 of the test solution can be suppressed, and as a result, the original capacity of the cavity 7 is exceeded. A large amount of test liquid can be retained in the array region 10. The hydrophobic region in the present invention means a region having at least surface characteristics showing water repellency, and preferably a region having higher water repellency than general sodium silicate glass not subjected to hydrophilic treatment or the like. means.

  Since the hydrophobic region 18 is exposed to the cavity 7, it acts as a driving force that promotes natural convection of the test solution in the cavity 7. For example, the hydrophobic region 18 can cause interaction between substances such as hybridization. It can be expressed well.

  The hydrophobic region 18 is preferably provided on the top or top surface of the partition wall of the separator 12 from the viewpoint of suppressing the outflow and contamination of the test solution and enhancing the ability of the test solution to be retained in the cavity 7. Here, the top portion or the top surface is a portion or a surface of the partition wall 14 opposite to the substrate 4. Further, from the viewpoint of promoting the substance interaction in the cavity 7, the hydrophobic region 18 is preferably formed in the region of the separator 12 exposed in the cavity 7, for example, inside the partition wall 14 (array region). 10 side).

  Such water repellency of the hydrophobic region 18 can be generally expressed by the contact angle of water on a flat surface. In the present invention, the water contact angle of the hydrophobic region is preferably 30 ° or more, more preferably 60 ° or more, and further preferably 70 ° or more. Most preferably, it is 90 ° or more. The contact angle refers to the angle of the portion where the droplet is in contact with the solid when the droplet is placed on a horizontal solid plate. As the contact angle, a static contact angle, a forward contact angle or a receding contact angle as a critical value, and a dynamic contact angle can be used, but a static contact angle measured by a droplet method can be used. preferable.

  The droplet method for measuring the static contact angle includes (1) tangent method, (2) θ / 2 method, and (3) three-point click method. The tangent method in (1) is a method for directly obtaining the contact angle by aligning the cursor with the tangent of the droplet using a reading microscope or the like, and the θ / 2 method in (2) The angle between the straight line connecting the two and the solid surface is doubled to obtain the contact angle. The three-point click method (3) uses two points of contact between the droplet and the solid surface and the vertexes on the computer image or the like. This is a method of clicking and obtaining by image processing. In these droplet methods, the contact angle is preferably obtained by the methods (2) and (3).

  The hydrophobic region 18 can also be configured by using a hydrophobic material as the material of the separator 12 itself, and the hydrophobic material and / or hydrophobicity (water repellency) with respect to the region where the hydrophobic region 18 is to be formed. ) Can also be formed by providing a surface form or layer that exhibits. Examples of the hydrophobic material constituting the hydrophobic region 18 include polyolefins such as polycarbonate, polyethylene, and polypropylene, vinyl halides, polyamides, polyimides, acrylic resins, and fluorides or chlorides of these resins. Moreover, as a surface form which exhibits water repellency, the form roughened so that a contact angle may be set to 60 degrees or more by the chemical modification and mechanical treatment on the surface of various materials can be mentioned, for example.

  Such a separator 12 is mounted on the substrate 4 on which the probes 6 are fixed to constitute the probe array 2. The separator 12 before being attached to the substrate 4 itself independently constitutes one aspect of the present invention. The separator 12 before being mounted on the substrate 4 can be provided with an adhesive layer 16 on the mounting surface to the substrate 4, and the adhesive layer 16 is preferably protected by a release layer until use.

  In such a probe array 2, the hydrophobic region forming process conventionally performed on the surface of the substrate 4 is not performed, and the hydrophobic region does not exist on the substrate 4. The surface treatment is applied uniformly. Further, for this purpose, the probe immobilization layer 5 is uniformly applied to the entire array region 10, and the formation form of the micro region 8 of the probe 6 in the array region 10, the probe immobilization rate, etc. Is uniformized. In other words, the conventional problems such as the distortion of the shape of the minute region 8 of the probe 6 in the edge region of the array region 10 or the variation or low immobilization efficiency are eliminated.

From the above, the probe array 2 of the present invention has an improved probing effective area ratio (%) of the array region 10 as compared with the conventional array. The probing effective area ratio is the ratio of the probing effective area (Se) to the designed area (Sd) of the array region (see the following formula (1)).
Probing effective area ratio (%) = Se [mm ² ] / Sd [mm ² ] × 100 (1)
However, the area (Sd) of the designed array region is the area in the array region 10 surrounded by the partition walls 14, and the probing effective area (Se) is the distance between the probe and the object in the array region 10. The area is such that the interaction can be detected with a certain accuracy or more, and the area of the variation coefficient of the signal intensity based on the interaction is 20% or less. For example, in the case of a hybridization reaction using a nucleic acid probe, the variation coefficient of signal intensity such as fluorescence due to hybridization is 20% or less. A conceptual diagram of the probing effective area ratio is shown in FIG.

The probing effective area ratio varies depending on the designed area (Sd) of the array area of the partitioned array area 10. For example, when Sd is 90 mm ² or less, the probing effective area ratio may be 70% or more. This is a preferred embodiment. In the case of this Sd, the probing effective area ratio does not reach 70% in the conventional array (see Example 1). Preferably, in this Sd, the probing effective area ratio is 80% or more, more preferably 85% or more.

  Further, the segmented array region 10 of the probe array 2 of the present invention has a variation coefficient of signal intensity based on the interaction between the probe 6 and the test sample in a region excluding the range within 0.8 mm from the partition wall 14. It can be 20% or less. In the conventional array, such uniformity cannot be ensured unless about 1 mm is excluded from the hydrophobic region corresponding to the partition 14. Preferably, the variation coefficient is 20% or less in a region excluding the range of 0.6 mm or less, more preferably 0.4 mm or less, and most preferably 0.3 mm or less. In these various array regions, the variation coefficient is more preferably 15% or less, and further preferably 10% or less. Note that such a determination can be made by measuring the signal intensity of the minute region 8 on a line that is a predetermined distance away from the partition wall 14 and calculating the coefficient of variation.

(Probing array manufacturing method)
Next, the manufacturing method of the probe array of this invention is demonstrated. The probe array 2 shown in FIGS. 1 and 2 is exemplified as the probe array, and the manufacturing method of the present invention will be described with reference to FIG.

  The method for manufacturing a probe array of the present invention includes a step of fixing a plurality of probes 6 to a substrate 4 in a pattern capable of forming a plurality of partitioned array regions 10, and the substrate on which the probes 6 are fixed. Mounting a separator 12 having a partition wall 14 capable of partitioning the surface into a plurality of surfaces, and partitioning the plurality of probes 6 into a plurality of array regions 10 in the pattern. Yes.

  In the step of immobilizing the probe 6, the probe 6 is supplied to the substrate 4 including the probe immobilization layer 5 by various methods. The probe 6 is preferably supplied so as to form the minute region 8. The method for supplying the probe 6 is not particularly limited as described above, and the method for synthesizing the probe 6 on the surface of the substrate 4 is not excluded. The probe 6 is supplied in a pattern capable of forming a plurality of array regions 10 in the subsequent stage. For example, as shown in FIG. 7, several array regions 10 in which a large number of minute regions 8 are aligned are formed on the substrate 4. This pattern corresponds to the partition form of the array region 10 defined by the partition walls 14 of the separator 12 mounted in the subsequent stage. The substrate 4 may be provided with a concave portion corresponding to the array region 10 and a convex portion corresponding to at least a part of the height of the partition wall 14. In this case, the probe immobilization layer 5 is similarly applied to these. Is preferably given.

  The substrate 4 on which the pattern of the micro area 8 of the probe 6 is formed is subjected to a treatment corresponding to the type of the probe 6 and the probe immobilization layer 5 such as heat treatment, immersion in a liquid, dehydration and washing. The probe 6 is fixed.

  In this method, prior to the immobilization of the probe 6, the hydrophobic region forming process for the partition is not performed, and as a result, the hydrophobic region is not present on the substrate 4. For this reason, the probe immobilization layer 5 is uniformly applied on the substrate 4, and as a result, the probe 6 is surely immobilized in a better form by the immobilization layer 5. Become.

  In this way, a probe array in which a large number of probes 6 are fixed to the substrate 4 in a pattern capable of forming a plurality of partitioned array regions 10 is obtained. Such a probe array is a probe array including an array region 10 that is partitioned by attaching a separator 12 during use. Therefore, the probe array including the array region 10 that is not mounted with the separator 12 and can be stored as it is, and can be distributed.

  Next, the separator 12 is mounted on the surface of the substrate 4 on which the probe 6 is fixed. Since the outermost layer of the substrate 4 is the probe immobilization layer 5 on which the probe 6 is immobilized, the separator 12 is attached to the probe immobilization layer 5. The separator 12 includes a partition wall 14 that can partition the surface of the substrate 4 into a plurality of parts. By mounting the separator 12 on the surface of the substrate 4, the array region 10 can be partitioned. The mounting method and mounting form of the separator 12 are not particularly limited, and the adhesive layer 16 may be interposed, or the substrate 4 and the separator 12 may be sandwiched by a jig or the like.

  The probe array 6 thus obtained can have the configuration as described above. Therefore, the probe array manufacturing method of the present invention includes various forms of the probe array 6 already described.

(Method of hybridizing nucleic acid)
By using a nucleic acid probe as a probe in the probe array of the present invention, there is provided a nucleic acid hybridization method capable of simultaneously hybridizing and assaying a large number or different types of test solutions including a nucleic acid test sample. That is, the nucleic acid hybridization method includes a step of supplying a test solution containing a nucleic acid test sample to the partitioned array region 10 of the probe array 2 and performing hybridization with the nucleic acid probe 6. In this hybridization method, it is preferable to have an open cavity 7 for hybridization on the array region 10. In the hybridization step, a test solution is supplied to the open cavity 7, under humidified conditions. Hybridization is preferably performed. By doing so, it is possible to simultaneously assay a large number of nucleic acid test solutions while avoiding contamination of the test sample solution.

(Reaction method using probe array)
The reaction method using the probe array of the present invention is a probe array having one or more array regions on which a plurality of probes are immobilized and partitioned, and is partitioned by a partition wall having a first height. The reaction step is performed in a state in which the test solution is supplied to the one or more arrayed regions to be reacted, and there is no partition wall or the height of the partition wall is reduced to a second height lower than the first height. It is preferable to perform subsequent washing or detection of reaction products. Here, the first height is preferably 10 μm or more, for example, and preferably 1000 μm or less. For example, the second height is preferably less than 60 μm, more preferably 50 μm or less. The second height is preferably 40 μm or more. Although the partition walls 14 may be removed, the contamination between the array regions 10 is reduced when the partition wall 14 having the height or higher is left to reduce the height of the partition walls 14 by removing the upper portion of the separator 12. It can be effectively suppressed.

  In the reaction using the probe array having the partitioned array region 10 as described above, in order to change the height of the partition wall 14 as necessary, a separator that forms the partition wall 14 may be mounted each time. Although it is good, it is preferable to use the separator 12 of the present invention capable of changing the height. According to such a separator 12, it is possible to easily construct a partition wall 14 having a height corresponding to various steps from reaction to detection, and supply a sufficient amount of test liquid while effectively suppressing contamination of the test liquid. It is easy to wash and can be made to a thickness suitable for signal detection. Moreover, when the separator 12 has an overhang | projection part, since the contamination of a test liquid can be suppressed effectively, the height of the partition 14 can also be made low.

  Hereinafter, the present invention will be specifically described with reference to examples. In this example, the uniformity of the surface treatment coat of the substrate is compared between the case where the surface is coated after chemical operation (water repellency imparted by printing) and the case where a separator sheet is pasted after the surface coating. In the comparison method, the coefficient of variation (CV: (standard deviation / average value) × 100 (%)) of fluorescence intensity obtained by hybridization between three substrates is calculated for each column along the partition wall, so that the CV is stable. The uniformity of the coat was evaluated from the size of the effective area ratio of hybridization. The hybridization effective area ratio is defined as (area of array region where stable CV was obtained / area of designed array region) × 100 (%).

First, 99 kinds of rat-derived cDNA were spotted on a glass substrate coated with poly-L-lysine as shown in FIG. Then, for this glass substrate, 8
Heat treatment at 0 ° C. for 1 hour, immersion treatment in blocking solution (15 minutes), immersion treatment in boiled sterilized water (3 minutes), dehydration with ethanol, centrifugal drying, and nucleic acid probe array ( Before partitioning). The blocking solution was 70 mM succinic anhydride, 0.1 M sodium borate (pH 8.0), and 1-methyl-2-pyrrolidoneone.

  A separator sheet that is partitioned into a plurality of array regions has the configuration shown in FIG. 9, and was produced as follows. That is, a double-sided adhesive sheet and a single-sided adhesive sheet are laminated on the opposite side of the water-repellent surface of an acrylic sheet (t = 0.037 mm) subjected to silicone-based water repellent treatment (water contact angle 110 °) on one side, This laminate was punched out in a pattern corresponding to a plurality of sections shown in FIG.

  This separator sheet was affixed to the nucleic acid probe array before compartmentalization, and the nucleic acid probe array of the Example was produced.

  The nucleic acid probe array of the comparative example was produced as follows. That is, based on the pattern shown in FIG. 8, a glass substrate coated with poly-L-lysine is prepared after the water-repellent material is preliminarily printed on the glass substrate and the water-repellent material is heated and solidified. Thereafter, heat treatment, blocking, washing and drying were performed, and a nucleic acid probe array of a comparative example was produced without attaching a separator sheet.

  Next, using 1 μg of rat-derived mRNA per substrate, Cell Engineering Vol. No. 18 7 Cy3-labeled cDNA was prepared according to the procedure described in 1999. 8 μl of this test solution was dropped into each array region of the DNA array, and hybridization was performed at a final concentration (5 × SSC, 0.5% SDS). This probe array was placed in a tight box containing 3 ml of sterilized water and allowed to stand for hybridization. Hybridization conditions were 42 ° C., humidity 100% RH, and 16 hours.

  After a predetermined time, the probe array was placed in the following three types of solutions ((2 × SSC, 0.1% SDS) solution, (1 × SSC) solution, (0.1 × SSC) solution) in this order 5 Washed by shaking. After washing, the probe array was dried with a centrifuge (1000 rpm, 3 minutes), and fluorescence was measured with a scanner (Scan Array 4000 manufactured by Packard BioChip Technologies). The measured value of the fluorescence intensity is digitized using numerical analysis software (Gene Pix. Pro manufactured by Axon), and further, the CV between the three substrates in each of the example and the comparative example is calculated using Excel, While determining the distance from the wall surface of the stable spot, the hybridization effective area ratio was calculated from the distance.

  As shown in FIG. 10, the probe array of the example (array with a sheet applied after surface coating and imparted water repellency) is the same as the probe array of the comparative example (array with surface coating after imparting water repellency). In comparison, CV was stable at a short distance from the partition wall. The probe array of the example was 0.21 mm from the partition wall, and the probe array of the comparative example was 0.88 mm.

The hybridization effective area ratio based on the calculation result of the distance was as follows.
Example probe array: CV distance from stable partition wall is 0.21 mm
Hybrid effective area ratio is {(4-0.21 × 2) × (6-0.21 × 2)} / 24 = 83.2%
Probe array of comparative example: distance from the stable partition wall of CV is 0.88 mm
Hybrid effective area ratio is {(4-0.88 × 2) × (6-0.88 × 2)} / 24 = 39.5%

  As shown in FIG. 11, the hybridization effective area ratio of the example was expanded to about 2.1 times the hybridization effective area ratio of the comparative example. From the above, according to the probe array of the present invention, it was found that the coating unevenness around the partition wall can be suppressed, a highly uniform array region is formed, and the nucleic acid probe is effectively fixed.

  In this example, the effect of the depth of the array region on the fluorescence intensity as a result of hybridization is confirmed. In the separator sheet produced in Example 1, the type of the surface water-repellent treatment agent and the thickness of the double-sided adhesive sheet and / or single-sided adhesive sheet were adjusted to provide two types of water repellency (70 ° as the water contact angle). 110 °), separator sheets having various thicknesses were prepared. Except for using such a separator sheet, the same operation as in Example 1 was carried out to prepare the nucleic acid probe array of Example, hybridization was performed, and the fluorescence intensity was measured and digitized. The results are shown in FIG.

  As shown in FIG. 12, it was found that the fluorescence intensity greatly changed when the depth of the array region (also the thickness of the separator sheet) was around 250 μm. From this result, it was found that the depth of the array region is preferably 250 μm or less. Note that no difference in fluorescence intensity was observed due to the difference in water repellency (here, 70 ° and 110 °).

  In this example, the effect of the water repellency of the hydrophobic region on the upper surface of the partition wall of the separator sheet on the fluorescence intensity as a result of hybridization is confirmed. In the separator sheet produced in the examples, various water repellency (water contact angles) separator sheets were prepared by adjusting the type of surface treatment of the acrylic sheet. Except for using separator sheets with different water repellency, the same operation as in Example 1 was carried out to prepare the nucleic acid probe array of the Example, perform hybridization, measure the fluorescence intensity, and quantify it. The results are shown in FIG.

  As shown in FIG. 13, it was found that the fluorescence intensity changed greatly when the contact angle of water was around 60 ° to 70 °. From this result, it was found that the water repellency of the separator sheet was such that the water contact angle was preferably 60 ° or more, and more preferably 70 ° or more.

In this example, the influence of the design area of the array region and the depth of the array region on the fluorescence intensity as a result of hybridization is confirmed. In the separator sheet produced in Example 1, the thickness of the double-sided adhesive sheet and / or single-sided adhesive sheet was adjusted to produce separator sheets having two types of thickness (0.25 mm and 0.1 mm (depth of the array region)). At the same time, a total of seven types of sheets of 1, 4, 9, 16, 25, 36, and 49 mm ² were prepared as punching patterns by the mold. The water repellency by the surface treatment was the same as in Example 1. Except for using these separator sheets, the same procedure as in Example 1 was followed to prepare the probe array of the Example, perform hybridization, measure the fluorescence intensity, and quantify it. The results are shown in FIGS.

FIG. 14 shows the relationship between the design area of the array region and the fluorescence intensity when the depth of the array region (the thickness of the separator sheet) is 0.25 mm. As is clear from this figure, The fluorescence intensity changed greatly and became a substantially constant value when the design area of the array region was 15 mm ² or more and less than that. When the design area of the array region is 15 mm ² , the ratio R of the array region depth (mm) to the design area (mm ² ) of the array region (see the right vertical axis in FIG. 14) is 0.02. As the design area of the array region increases, the ratio R becomes smaller.

FIG. 15 shows the relationship between the design area of the array region and the fluorescence intensity when the depth of the array region is 0.1 mm. When the design area of the array region is 5 mm ² or more, FIG. Compared with the case of less than that, the fluorescence intensity greatly changed and became a substantially constant value. The ratio R at this time is 0.02 (see the right vertical axis in FIG. 15), and the ratio R becomes smaller as the design area of the array region increases.

From the above, it has been found that the ratio of the array area depth (mm) to the design area (mm ² ) of the array area is preferably 0.02 or less.

  This example is an example of preparing a separator sheet that can constitute a partition wall having a variable height and a separator sheet that further includes an overhang portion. In this example, a hybridizing reaction and a washing step were performed in the same manner as in Example 1 except that the separator sheet was prepared as follows.

  The separator sheet produced in this example is shown in FIGS. The separator sheet 90 shown in FIG. 16 is made of a polyethylene (PE) double-sided adhesive sheet 100 having an acrylic double-sided adhesive layer on the upper and lower surfaces, and a 38 μm thick polyethylene terephthalate (PET) coated with silicon on the upper surface. A sheet 101, a polyethylene sheet 102 having a thickness of 90 μm having a silicon-based adhesive layer on the lower surface, the polyethylene double-sided adhesive sheet 101 and a PET sheet 103 having a film thickness of 145 μm are stacked and punched in a necessary pattern. . Here, the PE layer 102, the double-sided adhesive sheet 101, and the PET sheet 103 on the upper layer side have a film thickness of 300 μm as a whole. Moreover, in this separator sheet, since the interface between the PET sheet 101 and the PE sheet 102 is made of a silicon-based material, it is the most fragile interface part in the entire layer thickness of the sheet.

  The separator sheet 110 shown in FIG. 17 is obtained by stacking a polycarbonate sheet 104 (film thickness: 145 μm) instead of the PET sheet 103, and the polycarbonate sheet 104 is overhanging in parallel to the inner side of the array region. It has the same configuration as the separator sheet 90 shown in FIG. The separator sheet shown in FIG. 17 is aligned after the PE double-sided adhesive sheet 100, the PET sheet 101, the PE sheet 102, the PE double-sided adhesive sheet 100, and the PC sheet 104 are punched separately. And integrated.

  Each of these separator sheets is attached to a glass substrate having an array area prepared, and 8 μl of a test solution is dropped into a cavity corresponding to each formed array area. The array is then stored in a sealed box for hybridization. In addition, contamination of the test solution could be avoided in the operation of installing this sealed box in the oven. In the substrate on which the separator sheet having a PC overhang portion shown in FIG. 17 was attached, the liquid was more stably held in the cavity. In addition, in any of the substrates to which the separator sheet was attached, after the hybridization reaction, the laminate portion having an upper layer thickness of 300 μm could be easily removed at the interface between the PET sheet 101 and the PE sheet 102. . Moreover, the preferable reactivity was acquired by wash | cleaning in the state which removed the upper layer part and reduced the partition height.

  In this example, a separator sheet having an identification layer was prepared, and a probe array provided with this separator sheet was produced. In this example, the test solution was injected into the array region in the same manner as in Example 1 except that the separator sheet was prepared as follows.

  The separator sheet 200 and the probe array 300 produced in this example are shown in FIG. As shown in FIG. 18 (a), the separator sheet 200 includes, from the substrate side, a polyethylene (PE) double-sided adhesive sheet 202 having an acrylic double-sided adhesive layer, a silicon coating layer on the top surface, and an identification layer 206 on the bottom surface. A 38 μm-thick polyethylene terephthalate (PET) sheet 204 having a thickness of 38 μm was superposed and punched out in a necessary pattern. The identification layer 206 is printed entirely with white ink, and numbers 1 to 24 are printed on the side visible from the surface at positions corresponding to the periphery of the array area corresponding to the individual array areas.

  A probe array 300 having 24 array regions is obtained by pasting this separator sheet 200 through a double-sided adhesive sheet 202 on a glass substrate on which probes are spotted in 24 sections by operating in the same manner as in Example 1. Produced. As shown in FIG. 18B, in this probe array 300, 24 transparent array areas are clearly shown on a white background, and individual array areas can be easily identified by numerals 1 to 24. It was. When 8 μl of the test solution was dropped into the formed cavity of each array region, the individual array regions could be easily visually confirmed, and the test solution could be reliably injected into the array region.

The present invention relates to Japanese Patent Application No. 2005-089403 filed on March 25, 2005, Japanese Patent Application No. 2005-315262 filed on October 28, 2005, January 31, 2006. The international application PCT / JP2006 / 301565 filed and US provisional application 60 / 778,100 filed on March 2, 2006 are based on priority claims, the entire contents of which are incorporated by reference.

INDUSTRIAL APPLICABILITY The present invention can be used in the manufacture of detection devices for nucleic acids, proteins, synthetic or natural compounds in samples, and industries that use the detection results.

Claims (61)

A probe array comprising one or more array regions on a substrate that are partitioned and have multiple probes immobilized thereon,
A separator having partition walls defining one or more array regions is mounted on the substrate ;
The probe array wherein the design area (Sd) of the array region defined by the partition walls is 90 mm ² or less, and the probing effective area ratio represented by the following formula (1) is 70% or more.
Probing effective area ratio (%) = Se [mm ² ] / Sd [mm ² ] × 100 (1)
However, Se represents the area of a region where the variation coefficient of the signal intensity based on the interaction between the probe and the test sample is 20% or less. A probe array comprising one or more array regions on a substrate that are partitioned and have multiple probes immobilized thereon,
A separator having partition walls defining one or more array regions is mounted on the substrate ;
The array area to be partitioned is a probe array in which a variation coefficient of signal intensity obtained by probing is 20% or less in an area excluding a range within 0.8 mm from the partition in the array area. A probe array comprising one or more array regions on a substrate that are partitioned and have multiple probes immobilized thereon,
A separator having partition walls defining one or more array regions is mounted on the substrate ;
The array area to be partitioned is a probe array in which the ratio [R] represented by the following formula (2) is 0.02 or less.
R = d [mm] / Sd [mm ² ] (2)
Here, d represents the depth of the array region, and Sd represents the design area of the array region. A probe array comprising one or more array regions on a substrate that are partitioned and have multiple probes immobilized thereon,
A separator having partition walls defining one or more array regions is mounted on the substrate ;
The separator can form a plurality of partition walls having different heights. The probe array according to claim 4 , wherein the separator can form a plurality of partition walls having different heights in a range of 10 μm to 1000 μm. A probe array comprising one or more array regions on a substrate that are partitioned and have multiple probes immobilized thereon,
A separator having partition walls defining one or more array regions is mounted on the substrate ;
The separator is a probe array in which at least a part of the partition walls can be removed to reduce the height of the partition walls. The partition wall of the separator has a height of 60 μm or more during a reaction in which a test sample is supplied to the probe and reacted, and has a height of less than 60 μm when a signal of the reaction product after the reaction is detected. The probe array according to any one of claims 4 to 6 . The probe array according to any one of claims 4 to 7 , wherein an area of the array region is 0.3 mm ² or more. The probe array according to any one of claims 4 to 8 , wherein the separator has a laminated structure. The probe array according to any one of claims 4 to 9 , wherein the separator includes a fragile portion capable of removing at least a part of the partition wall. The probe array according to claim 10 , wherein the separator is a laminate, and the fragile portion is an interface portion of the laminated layers. A probe array comprising one or more array regions on a substrate that are partitioned and have multiple probes immobilized thereon,
E Bei a separator having a partition wall for partitioning one or more of said array region and mounted on the substrate,
The separator is a probe array, wherein the separator has a projecting portion that projects toward an upper part of the array region in at least a part of the partition. The probe array according to claim 12 , wherein the protruding portion is provided over the entire circumference of the array region. At least a region contactable with the test solution has a hydrophobic region, a probe array of claim 12 or 13 of the projecting portion from the partition wall in the array region over the overhang. The probe array according to any one of claims 12 to 14 , wherein a height of a lowermost end of the overhang portion is 40 µm or more and 990 µm or less. The probe array according to any one of claims 12 to 15 , wherein the projecting portion projects in a range that does not reach the probe fixed in the array region. The probe array according to any one of claims 12 to 16 , wherein the region defined by the inner peripheral surface of the overhang portion is a region where the probe in the array region is fixed. The probe array according to any one of claims 12 to 17, wherein a portion including the overhang portion can be integrated and / or removed with respect to a remaining portion of the partition wall. The probe array according to claim 18 , wherein the entire overhanging part can be integrated and / or removed with respect to the remaining part of the partition wall. Said array region is plural, the probe array according to any one of claims 1 to 19. The probe array according to any one of claims 1 to 20, wherein the separator is attached to a surface of a probe fixing layer on which the probe on the substrate is fixed. The probe array according to claim 21 , wherein the separator is attached to a surface of the probe immobilization layer after the probe immobilization. The probe array according to any one of claims 1 to 22 , wherein the separator has a sheet shape and is fixed to the substrate via an adhesive layer. The probe array according to any one of claims 1 to 23 , wherein the separator is detachably fixed to the substrate. The probe array according to any one of claims 1 to 24 , which has a hydrophobic region in at least a part of the separator. The probe array according to claim 25 , wherein the hydrophobic region is provided on a top surface of the partition wall of the separator. The probe array according to claim 25 or 26 , wherein a contact angle of water in the hydrophobic region is 60 ° or more. The probe array according to any one of claims 25 to 27 , wherein the hydrophobic region contains a material selected from the group consisting of polycarbonate, polyolefin, polyamide, polyimide, acrylic resin, and fluorides and halides thereof. Areas surrounded by the partition wall partitioning the array region of the separator constitute an open system cavity for interaction with the probe and the test sample on the array region, according to any one of claims 1 to 28 Probe array. The probe array according to any one of claims 1 to 29 , wherein a design area of the array region defined by the partition walls is 0.3 mm ² or more and 2000 mm ² or less. The probe array according to any one of claims 1 to 30 , comprising one or more and 400 or less of the array regions. The probe array according to any one of claims 1 to 31 , wherein the array region to be partitioned has a depth (d) of 10 µm or more and 240 µm or less. The probe array according to any one of claims 1 to 32 , wherein the separator constitutes at least a part of a height of a partition wall that partitions the array region. The probe array according to any one of claims 1 to 33 , wherein the substrate constitutes at least a part of a height of a partition wall that partitions the array region. The probe array according to any one of claims 1 to 34 , further comprising an identification layer having a color and / or an image capable of identifying the one or more array regions. 36. The probe array according to claim 35 , wherein the identification layer is provided on the substrate side of the separator. 37. A probe array according to claim 35 or 36 , wherein the separator comprises the identification layer. The probe array according to any one of claims 35 to 37 , wherein the identification layer is a layer formed by printing. The probe array according to any one of claims 35 to 38 , wherein the identification layer has a color capable of identifying the one or more array regions. 40. The identification layer according to any one of claims 35 to 39 , wherein the identification layer includes any one or two or more kinds of labels selected from letters, numbers, symbols, and figures so that the one or more array regions can be specified. The probe array described in 1. The probe is a nucleic acid probe, the probe array according to any one of claims 1 to 40. 41. The probe array according to any one of claims 1 to 40 , wherein the probe is a protein probe. Plurality of probes have a partition wall for partitioning the array regions on a substrate which is immobilized by forming one or more array regions can be formed a plurality of different heights of the partition wall, the separator. Plurality of probes have a partition wall for partitioning the array regions on a substrate which is immobilized by forming one or more array regions, having a laminate structure, the separator. 45. The separator according to claim 43 or 44 , wherein the array region is plural. The separator according to any of claims 43 to 45, wherein the separator has a hydrophobic region on the top surface of the partition wall. 47. The separator according to any one of claims 43 to 46 , wherein the separator has an overhanging portion projecting toward the upper part of the array region in at least a part of the upper part of the partition wall. The separator according to any one of claims 43 to 47 , further comprising an identification layer having a color and / or an image so that the one or more array regions can be identified. The separator according to claim 48 , comprising the identification layer on the substrate side. The separator according to claim 48 or 49 , wherein the identification layer is a layer formed by printing. The separator according to any one of claims 43 to 50 , which is for the probe array according to any one of claims 1 to 42 . A probe array,
52. A probe array in which a large number of probes are immobilized by forming one or more array regions, and the one or more array regions are partitioned by the separator according to any one of claims 43 to 51 . 53. The probe array according to claim 52 , wherein the array region is plural. A nucleic acid hybridization method comprising:
42. A hybridization method comprising a step of supplying a test solution containing a nucleic acid test sample to the array region of the probe array according to claim 41 and performing hybridization with the probe. The probe array has an open cavity for hybridization on the array region,
55. The hybridization method according to claim 54 , wherein the hybridization step is performed under humidified conditions by supplying a test solution to the open system cavity. A reaction method using a probe array,
43. A reaction method comprising a step of supplying a test solution to the array region of the probe array according to claim 42 and performing a reaction with the probe. A reaction method using a probe array,
A test solution is supplied to one or more of the array regions partitioned by a partition wall having a first height of the probe array having one or more array regions on which a plurality of probes are immobilized and partitioned. A reaction step of reacting,
A post-process for performing washing after the reaction step or detecting a reaction product in a state where the partition is absent or the height of the partition is reduced to a second height lower than the first height;
A reaction method comprising: 58. The reaction method according to claim 57 , further comprising a step of forming the first height partition wall on the substrate after the probe is immobilized on the substrate prior to the reaction step. The partition wall having the first height in the reaction step has an overhanging portion protruding toward the upper side of the array region at least at a part above the partition wall,
59. The reaction method according to claim 57 or 58 , wherein in the post-process, the overhanging portion is removed to reduce the partition wall height to the second height. The reaction method according to any one of claims 57 to 59 , wherein the partition wall is formed by applying a separator capable of partitioning the array region to the substrate surface. The reaction method according to claim 60 , wherein the separator is a laminate.

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