JP3969651B2 - Plastic substrate for microarray - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention mainly relates to a microarray substrate used for immobilizing biological materials such as DNA and proteins on the surface and used for measurement of these biological materials, particularly a microarray substrate.
[0002]
[Prior art]
As the method of genome analysis advances and the expression status of genes can be observed at once, DNA fragments are spotted at high density on a glass slide substrate, and the DNA fragments and DNA fragments from the specimen are hybridized to obtain a gene sequence. DNA microarrays that observe expression have come to be used.
[0003]
As an operation for observing gene expression with a DNA microarray, fluorescent labeling was applied to the sample DNA, this DNA solution was brought into contact with the DNA microarray, hybridized with the DNA strand immobilized on the DNA microarray, and fluorescently labeled. By observing the DNA hybridization status, an operation is performed in which the expression status of each gene is observed. What is necessary for the measurement in this fluorescence is to reduce the measurement error in the operation. Currently, one of the causes of variation in the measurement results in the measurement of gene expression status in a DNA microarray is due to variation in hybridization. Variations in hybridization are often due to operational ineligibility. What is important in the hybridizing operation is to bring a DNA solution as a specimen uniformly into contact with an area where DNA is fixed. For that purpose, it is necessary to clearly know the area where the DNA is fixed. Therefore, many DNA microarrays having a frame printed on the substrate are commercially available so that the DNA fixing area on the substrate can be easily identified.
[0004]
A major factor of variation in hybridization is due to non-uniformity of the DNA solution serving as a sample. The non-uniformity is the non-uniformity in the thickness of the DNA solution serving as a specimen, and the non-uniformity in concentration. Hybridization with a DNA microarray is performed by dropping a sample DNA solution into an area in which DNA is immobilized and covering the cover glass to cover the entire area where DNA is immobilized. To spread. At this time, the thickness of the DNA solution is biased, and the amount of DNA hybridization reaction is biased in the DNA area. In order to make the thickness of such a DNA solution uniform, a cover glass for hybridization is commercially available in which a protrusion is provided on the cover glass like a clog tooth and the gap between the DNA microarray and the DNA immobilization surface is constant. Has been. However, even when such a cover glass is used, there arises a disadvantage that the cover glass is displaced during the operation.
Moreover, such a cover glass is produced by bonding glass to a cover glass in the shape of a clog tooth. Glass is easy to break, and the work of attaching glass to a thin cover glass is a laborious work.
[0005]
Therefore, it is conceivable to make such a cover by molding plastic, but the plastic is light and the cover floats on the DNA solution, so the cover is easily displaced, and the plastic cover for hybridization used in the DNA microarray The realization of was difficult.
[0006]
[Problems to be solved by the invention]
The problem to be solved by the present invention is to solve the non-uniformity of the DNA solution thickness in the hybridization, which is seen in the conventional glass microarray substrate, to ensure the reliability and reproducibility in the hybridization, and to make it plastic. Thus, such a problem is to be realized at low cost.
[0007]
[Means for Solving the Problems]
As a result of intensive studies, the inventors have made the hybridization cover and the microarray main body fit together, thereby preventing the hybridization cover from shifting even if it is made of plastic, eliminating the imbalance in the thickness of the solution, In addition, the present inventors have found that the evaporation of the solution can be prevented and completed the present invention.
That is, the present invention
(1) Forming a specimen capturing part in which a substance that captures biological substances is spot-fixed within a predetermined range of the substrate surface, bringing the specimen capturing part into contact with the specimen capturing part, and capturing the biological substances to be detected A plastic substrate used to make a microarray for determining the presence or absence of a substrate, comprising a substrate body and a cover, the substrate body having a frame formed by convex portions protruding from the substrate surface, and the cover having a surface edge The convex part of the cover has a fixed part, and the convex part of the cover and the convex part of the cover form a fixed gap between the surface of the specimen capturing part of the substrate body and the cover. Plastic substrate,
(2) The plastic substrate for microarray according to (1), wherein a part of the convex part of the frame of the substrate body and a part of the convex part of the cover have a defective part to form a solution injection port and / or a deaeration port. ,
(3) The plastic substrate for microarray as set forth in (2), wherein an injection port and a deaeration port are formed and the injection port and the deaeration port face each other.
(4) The plastic substrate for microarrays according to any one of (1) to (3), which has fine ground glass-like irregularities on the cover surface,
(5) The plastic substrate for microarray as set forth in any one of (1) to (3), wherein the cover surface has a plurality of fine grooves.
(6) The plastic substrate for microarray according to (4) or (5), wherein the height difference of the unevenness on the cover surface is 1 to 10 μm,
(7) The plastic substrate for microarray according to any one of (1) to (6), wherein the cover material is a transparent resin;
It is.
[0008]
DETAILED DESCRIPTION OF THE INVENTION
Hereinafter, the present invention will be described in detail.
First of all, it is a plastic material used for the substrate body for the microarray of the present invention. However, it is mainly used for the detection method of the specimen including the DNA microarray, and when the substrate itself emits fluorescence, it is detected. In view of this, it is preferable not to emit autofluorescence. Further, in the operation with the DNA microarray, since there is a heating operation such as immersion in boiling water, a resin having heat resistance is preferable. Examples of such resins include cyclic polyolefin resins and fluorine resins such as ETFE.
[0009]
On the other hand, although it is a plastic material used for the cover of the present invention, it is not necessary to consider autofluorescence at the time of detection because this cover is removed at the time of detection. However, since it is necessary to confirm the state of the solution in the inside, it is preferable that it is transparent. Examples of the plastic that satisfies such required characteristics include polypropylene, polyethylene, polystyrene, cyclic polyolefin, and polycarbonate. In hybridization with a DNA microarray, it is necessary to heat at about 60 ° C. In such a case, it is preferable to use polypropylene, polycarbonate, cyclic polyolefin or the like.
[0010]
Next, the overall shape of the plastic substrate body for microarray and the cover of the present invention will be described. Although it is a microarray main body, the same shape as a slide glass is suitable as a form suitable for the scanner currently widely used for reading of a DNA microarray.
The cover is not particularly defined, but since the microarray body to be fitted is a slide glass shape, a shape suitable for this shape is suitable, and the shape is preferably a square shape.
[0011]
Next, a more detailed description will be given with reference to the drawings.
FIG. 1 is a schematic diagram showing a state in which a main body and a cover of a plastic microarray plastic substrate according to one embodiment of the present invention are set, and FIG. 2 is a schematic diagram showing a microarray substrate main body according to one embodiment of the present invention. FIG. 3 is a schematic view showing a cover according to an embodiment of the present invention, and FIG. 4 is a cross-sectional view showing a state where the microarray substrate body and the cover are engaged according to an embodiment of the present invention.
The plastic substrate body (1) for microarray has a portion (11) for fixing DNA or the like to the substrate. The DNA is fixed by attaching a DNA solution to the tip of a thin pin having a diameter of 50 to 500 μm and spotting it. The range to be spotted is displayed by a frame (7). The frame is formed by projecting from the substrate surface. A conventional microarray substrate is made of glass, and the display of the frame is performed by printing or roughening in a wrinkled shape. However, with plastic, it is possible to form the protrusions by injection molding.
[0012]
The shape of the frame (7) is preferably square and may be closed. However, an inlet (3) for injecting a sample solution and a deaeration port (in the case of injecting the solution) (described later) In order to form 4), it is better to provide partially missing portions (5) and (6).
[0013]
The cover (2) is provided with a convex portion (8) at the edge. Although this convex part (8) may be formed in a state of being closed at the edge part of the cover, the defective part (9) is formed in order to form the inlet (3) and the deaeration port (4) as in the case of the main body. And (10) should be provided.
[0014]
The size of the gap (12) between the surface of the spot area of the microarray main body (1) and the cover (2) is such that the frame (7) of the microarray substrate main body (1) and the projection (8) of the cover (2). Adjusted by height. The size of the gap is preferably 15 to 200 μm, and if it is less than 15 μm, the amount of the sample solution is small and sufficient hybridization cannot be achieved, and if it exceeds 200 μm, the amount of the sample solution increases and more samples are contained. Preparation is required.
[0015]
By providing a concave and convex portion on the side of the cover that comes into contact with the solution, it is possible to easily recognize the state of filling of the solution. In a state where the solution is not filled, the embossed portion remains, and when the solution is filled, the embossed portion disappears and the entire cover becomes transparent, so that the recognition can be easily performed.
Further, by providing a fine groove in the direction from the solution inlet to the deaeration port, the solution flow can be improved and the solution can be filled smoothly.
[0016]
The method of contacting the plastic substrate for microarray of the present invention with a sample solution will be described by taking hybridization with a DNA microarray as an example.
First, the frame of the substrate body and the convex part of the cover are fitted together. Next, the solution is injected from the solution injection port (3) formed from the defect portion of the frame of the substrate body and the defect portion of the convex portion of the cover. The solution is filled from the injection port to the deaeration port (4). When the solution is filled up to the deaeration port, the solution injection is stopped, and the substrate is placed in the hybridization case and left to perform the hybridization. . In the present invention, since the solution can be reliably filled and the cover is not displaced, the hybridization can be performed reliably.
After the hybridization is completed, remove the cover, perform washing, etc., and check the DNA capture status with a scanning device such as a scanner.
[0017]
【The invention's effect】
According to the present invention, the contact operation with the sample solution in the microarray, particularly the contact with the substrate of the DNA solution serving as the sample in the hybridization in the DNA microarray can be performed uniformly and reliably, so that DNA in the hybridization can be obtained. The reaction can be performed uniformly, and a highly reproducible detection result can be obtained. In addition, plasticization is possible, and supply at a lower cost is possible compared to conventional glass substrates and covers.
[Brief description of the drawings]
FIG. 1 is a schematic diagram showing a state in which a main body and a cover of a plastic substrate for a microarray made of plastic according to an embodiment of the present invention are set.
FIG. 2 is a schematic diagram showing a microarray substrate body according to an embodiment of the present invention.
FIG. 3 is a schematic diagram showing a cover according to an embodiment of the present invention.
FIG. 4 is a cross-sectional view showing a state in which a microarray substrate body and a cover are engaged according to an embodiment of the present invention.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 Board | substrate body 2 Cover 3 Inlet 4 Deaeration port 5 The 1st defect | deletion part 6 of a board | substrate body The 2nd defect | deletion part 7 of a board | substrate body 8 The convex part 9 The 1st defect | deletion part 10 of a cover The 2nd defect | deletion of a cover Part 11 Specimen capture part 12 Gap

Claims (7)

Forming a specimen capture part in which a substance that captures biological substances is spot-fixed to a predetermined range on the surface of the substrate, contacting the specimen capture part with the sample solution, and capturing the biological substance targeted for detection A substrate made of plastic used to manufacture a microarray, comprising a substrate main body and a cover, the substrate main body having a frame formed by convex portions protruding from the substrate surface, and the cover protruding to the edge of the surface A convex portion forming a frame of the substrate body and the convex portion of the cover are fitted with a gap of a certain size between the surface of the specimen capturing portion of the substrate body and the cover. Plastic substrate for microarray. 2. The plastic substrate for microarray according to claim 1, wherein a part of the convex part of the frame of the substrate body and a part of the convex part of the cover have a defective part to form a solution injection port and / or a deaeration port. 3. The plastic substrate for microarray according to claim 2, wherein an injection port and a deaeration port are formed, and the injection port and the deaeration port are arranged to face each other. The plastic substrate for a microarray according to any one of claims 1 to 3, wherein the cover surface has fine ground-like irregularities. The plastic substrate for microarray according to any one of claims 1 to 3, wherein the cover surface has a plurality of fine grooves. The plastic substrate for microarray according to claim 4 or 5, wherein the height difference of the unevenness on the cover surface is 1 to 10 µm. The plastic substrate for microarray according to any one of claims 1 to 6, wherein a material of the cover is a resin having transparency.

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