JP2003294743A - Flexible array, manufacturing method of the flexible array, hybridization method of the flexible array, and measuring method of the flexible array - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a flexible array having a plurality of ligand specimens immobilized thereon, being very convenient in handling such as hybridization, that is, capable of micronizing the reaction capacity, and capable of simplifying, mass-producing, and further cost-reducing consumables. <P>SOLUTION: This flexible array 1 is characterized in that a plurality of ligand specimens 2a to 2f are aligned/immobilized in the longitudinal direction of an oblong carrier 6. <P>COPYRIGHT: (C)2004,JPO

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to an array in which a ligand sample that reacts with a biological sample such as DNA is fixed, and more particularly to a flexible structure in which a plurality of ligand samples are linearly fixed on a ribbon-shaped or thread-shaped carrier. The invention relates to arrays and methods of making and using the flexible arrays.

[0002]

2. Description of the Related Art Conventionally, in the case of detecting a DNA sample or an RNA sample, a DNA array to which an appropriate number of ligand DNAs that selectively hybridize with these samples are attached is prepared in advance, and a DNA as a test subject is prepared. Sample or RNA
The sample is labeled with a fluorescent substance in advance, the labeled DNA sample is hybridized with the ligand DNA on the DNA array, and the presence or absence of the binding of the DNA sample or the RNA sample to each ligand DNA is checked for each ligand. It was measured by observing the amount of fluorescent light of the fluorescent substance under excitation light with a fluorescence detection device.

The DNA array used for such fluorescence detection is conventionally composed of an array made of a flat glass plate. Also, considering the manufacture of the array, the flat plate-shaped D
The NA array has a structure in which a plurality of ligand DNAs are fixed at specific positions on a glass plate. At present, in order to realize such a structure, a plurality of types of ligand DNA solutions are sequentially dropped one by one at a specific position on a flat plate to manufacture an array.

[0004]

However, in such a plate-shaped array, the ligand DNA on the DNA array and the DNA sample in the solution must be hybridized on a plane, and a special solution for reaction is required. However, there is a drawback that various devices are required.

Further, in order to react the liquid droplets on a flat surface, a considerable time is required for the reaction, and it usually takes about 10 hours for the hybridization to occur, which is a time defect.

Further, when mass-producing a DNA array, it is necessary to repeat the dropping operation on a glass plate for the number of arrays produced, which requires a lot of labor and cost to make the array and is suitable for mass production. There was a problem of not having. In recent years, as a method for mass-producing arrays, a method of synthesizing a ligand DNA on a glass plate using a photolithography technique has been proposed and put into practical use, but the manufacturing facility becomes large-scale and complicated, and the manufacturing cost of the DNA array is reduced. The current situation is that it has not arrived.

Furthermore, in future medical diagnostic applications, D
It is predicted that the required number of NA measurement points will be several hundreds or less, and in some cases about 10 points, and it may be possible to meet the demand for miniaturization of the array accompanying the decrease in measurement points. Is desired. For example, it is desired in the future that the length of the chip of the DNA array is set to about several mm or less (width of 1 mm or less) and the amount of sample solution required for the reaction is set to about several μl.

Therefore, the present invention has been made in order to solve the problems of the above-mentioned conventional techniques, and handling such as hybridization is very convenient, that is, the reaction volume can be made small, and consumables can be used. The main object of the present invention is to provide a flexible array on which a plurality of ligand samples are immobilized, which can be simplified, mass-produced, and further reduced in cost.

[0009]

In order to achieve the above object, the flexible array according to claim 1 comprises a plurality of ligand samples aligned and immobilized in the longitudinal direction of an elongated carrier. Characterize. With such a configuration, the array can be miniaturized freely according to the application and the array can be easily handled.

The carrier is preferably ribbon-shaped or thread-shaped. At that time, the ribbon-shaped flexible array has a width direction length of 5 mm or less and a thickness of 0.3 m.
and the thread-like flexible array has a thread diameter of 0.5 or less.
It is preferable that it is less than or equal to mm. With such a configuration, it is easy to reduce the capacity for various treatments while maintaining the performance of the array on which the ligand sample is immobilized.

It is desirable that each of the plurality of ligand samples can be discriminated by a distance from a predetermined portion in the longitudinal direction of the carrier. With such a configuration, it becomes easy to identify individual ligand samples during a predetermined measurement.

[0012] According to another aspect of the present invention, the method for producing a flexible array according to the present invention is such that a plurality of ligand samples are aligned and immobilized in the longitudinal direction of an elongated carrier. A step of immobilizing a plurality of ligand samples in a linear manner on a planar carrier in order to manufacture an array, and cutting the planar carrier into a plurality of rows in a direction orthogonal to the ligand sample rows. It is characterized by consisting of. With such a configuration, it is possible to easily manufacture a plurality of flexible arrays having the above-described effects, and mass production and cost reduction can be achieved.

According to still another aspect of the present invention,
A method for hybridizing a flexible array according to the present invention is characterized by comprising the steps of introducing the above-mentioned flexible array and a solution containing a biological sample into a container and performing hybridization in the container. To do. With such a configuration, the flexible array can be brought into contact with the solution in the container. As a result, the fluidity of the solution around the array can be increased as compared with the flat array, and this can be further promoted by externally vibrating the container. By these, the hybridization reaction can be promoted, and the time can be shortened and the cost can be reduced.

The container is a test tube, and it is also preferable that the container further comprises a step of reducing the capacity of the flexible array before introducing the flexible array into the test tube.
It is preferable to reduce the capacity of the flexible array by processing the flexible array into a spiral or coil shape, or by folding the flexible array. With such a configuration, the flexible array can be hybridized with the solution by an experimental method using a general test tube, and the handling becomes easy.

The container is a tubular container or a groove-shaped channel, and it is also preferable that the container further comprises a step of allowing the flexible array to remain in the tubular container or the groove-shaped channel in an extended state. With such a configuration, even if the reaction is performed in the stretched state, the treatment can be performed in a short time.

[0016] According to still another aspect of the present invention, the flexible array measuring method according to the present invention hybridizes the above-mentioned flexible array, and the hybridized flexible array. Is sequentially measured from the longitudinal end of the flexible array.
In such a configuration, by using the above-mentioned flexible array, the pre-processing before the fluorescence measurement processing can be simplified in a very short time, which leads to the shortening of the entire process time and the cost reduction. .

It is preferable that the method further includes the step of linearly extending the hybridized flexible array before performing fluorescence measurement on the flexible array. With such a configuration, it is possible to improve the measurement performance when using a flexible array having a small capacity and hybridized in a short time.

[0018]

BEST MODE FOR CARRYING OUT THE INVENTION Next, a preferred embodiment of the present invention will be described with reference to FIG. FIG. 1 is a schematic diagram showing the entire DNA array 1 which is an embodiment of a flexible array. In this embodiment, the DNA array 1
Is a flexible carrier 6 in the form of an elongated ribbon,
Ligand DNAs 2a as a plurality of ligand samples arrayed and fixed in a line along the longitudinal direction of the carrier 6
2f (six in this embodiment). The shape of the ribbon-shaped DNA array 1 is 5 m in the width direction.
m or less and the thickness thereof is 0.3 mm or less. It is also possible to make the length of the DNA array several millimeters or less and the width 1 mm or less. With such a size and shape, the volume of the entire DNA array 1 can be reduced while holding the immobilized ligand DNA, as described later. The ligand sample refers to a substance that adsorbs a biological sample, which is a test subject, contained in a solution or reacts with the biological sample, as described later.

A reference point 4 for discriminating the individual ligand DNAs 2a to 2f is provided inside a predetermined portion in the longitudinal direction of the DNA array 1. Here, the “inside” refers to a place other than the place where the ligand DNA is fixed on the ribbon-shaped carrier. In addition, the marking on the reference point 4 can be performed, for example, by applying a fluorescent dye to a certain place inside, and in this case, in order to easily distinguish from the fluorescent signal of the DNA bound to the ligand. It is desirable to change the amount of adhesion to the reference point, the shape of adhesion, or the fluorescent color. As still another method, for example, by measuring the background fluorescence of the ribbon-shaped carrier, or by measuring the absorption by the ribbon-shaped carrier at the same time as the fluorescence, to detect the end of the ribbon-shaped carrier. , This can also be used as a reference point.

In this embodiment, the reference point 4 is D
It is provided between the end 1a on one side in the longitudinal direction of the NA array 1 and the ligand DNA 2a. By providing the reference point 4, the individual ligand DNAs 2a to
Since the distance up to 2f can be measured, the ligand DNA can be discriminated according to the distance. If the individual ligand DNAs 2a to 2f can be discriminated, the DNA array 1
The ligand can be easily identified when various measurements are performed using. The reference point is, as described above, the DNA
It can also be provided at the end 1a on one side in the longitudinal direction of the array 1. Thus, when the end portion 1a of the DNA array 1 is used as the reference for measuring the distance, the end portion 1a itself may be used as a reference for distance measurement without providing a special reference point. Can be simple.

In the present embodiment, the DNA array is formed into a ribbon shape.
The NA array can also be formed into a thread shape. When making a DNA array into a thread, the thread diameter is 0.5 mm.
The following is preferable.

[Manufacturing Method of Flexible Array] Next, a manufacturing method of a DNA array in which a plurality of ligand DNAs are linearly immobilized on a ribbon-shaped carrier will be described in detail with reference to FIG. First, a flat and rectangular carrier 60 is prepared, and continuous ligand DNA is provided in a direction parallel to one side of the rectangle.
2 is fixed linearly on the carrier 60 along the virtual straight line L1a. The linear immobilization of the ligand DNA2 is sequentially performed in a plurality of lines from the virtual straight line L1b to the virtual straight line L1f. After that, the planar carrier 60 is cut into an elongated shape along a virtual straight line L2a positioned in a direction orthogonal to the virtual straight line L1 on which the ligand DNA2 is immobilized. This cutting is repeated a plurality of times from the virtual straight line L2b to the virtual straight line L2f. In this way, the individual elongated carriers 6 that have been cut have the same structure as in FIG.
As shown in FIG. 3, a plurality of ligand DNAs 2a to 2f are immobilized along the longitudinal direction thereof, which constitutes the DNA array 1. According to this manufacturing method, a simplified flexible array can be easily mass-produced, leading to cost reduction. To fix the ligand DNA linearly, a technique such as pen writing, fine spraying, or printing can be applied.

[Hybridization Method of Flexible Array] Next, a hybridization method as a method of using a flexible DNA array will be described in detail with reference to FIGS. 3 and 4. Figure 3 shows DNA in a typical test tube.
It shows the state of hybridizing the array,
FIG. 4 shows a state in which the DNA array is processed into a spiral shape, a coil shape, or a folding process before being put into a test tube to reduce the capacity.

First, a general test tube 10 as a container
A DNA array 1 having a reduced volume and a solution 12 containing a biological sample as a subject, for example, a DNA sample, are put into the container. Then, the lid 14 is airtightly attached to the test tube 10,
Prevent solution 12 from leaking. Then, test tube 10
Within a predetermined period of time within the DNA array 1 and the solution 12
Allow hybridization with NA sample. As described above, by reducing the capacity of the DNA array 1, hybridization can be easily performed in a general test tube 10. Further, since it is possible to infiltrate and diffuse the solution in the solution instead of dropping the droplet onto the glass plate for the reaction as in the conventional case, the time required for the reaction becomes extremely short. Further, since the DNA array has a three-dimensional shape by reducing its capacity, the liquid can easily penetrate into the inside of the DNA array.

The term "reduction in capacity" of the DNA array means
It means to reduce the volume or capacity without changing the total volume of the DNA array itself. For example, as shown in FIG.
It can be performed by (b) coiling or (c) folding. Like this
Since the capacity of the NA array itself can be reduced, the reaction capacity can be reduced. For example, the amount of sample liquid required for the reaction can be set to several μl.

Next, another embodiment of the hybridization method will be described in detail with reference to FIG. Figure 5 (a)
FIG. 5 is a diagram in which a thread-shaped DNA array is inserted into a tubular container, and FIG. 5B is a diagram in which a ribbon-shaped DNA array is inserted into a groove-shaped channel. Referring to FIG. 5 (a), first, a tubular container 20 as a container is prepared, and the filamentous DNA array 1 ′ is retained therein in a linear shape. Then, the solution 12 containing the DNA sample inside the tubular container 20
To allow the DNA array 1'to infiltrate into the solution 12 so that hybridization between the two is performed inside the tubular container 20.

Further, referring to FIG. 5 (b), first, a groove-shaped channel 30 as a container is prepared, and the ribbon-shaped DNA array 1 is retained therein in a linear shape. Then, the solution 12 containing the DNA sample is caused to flow into the groove-shaped flow channel 30 to infiltrate the DNA array 1 into the solution 12, so that hybridization between the both is performed inside the groove-shaped container 30. . Since each DNA array is flexible and elongated, it can be easily inserted into a duct or a channel and can be easily infiltrated in a solution, so that the reaction time of hybridization can be shortened. it can. Further, since the DNA array and the DNA sample are reacted in the stretched state, the subsequent handling of the DNA array becomes easy.

[Method of Measuring Flexible Array] Finally, a method of measuring the flexible array will be described. First, the DNA array 1 as shown in FIG. 1 is hybridized by a predetermined method. Then, this hybridized DNA array 1 is sequentially subjected to fluorescence measurement along the longitudinal direction of the array in which the ligand DNAs 2a to 2f are aligned from the end 1a side by an existing general fluorescence detection device, and individual ligand DNAs 2 are measured.
Observe whether a DNA sample or RNA sample was hybridized to a to 2f.

As a method of sequentially measuring fluorescence along the longitudinal direction of the DNA array 1, for example, as shown in FIG. 6, a method of moving the optical system 40 in the arrow direction with respect to the DNA array 1 and a method of FIG. 2, there is a method of moving the DNA array 1 in the direction of the arrow with respect to the optical system 40, and a method of moving the DNA array and the optical system relative to each other (not shown).

In FIG. 6A, the ligand DN is composed of an excitation light source 41, a fluorescence detection device 42, and a spectroscopic means 43.
An optical system 40 for measuring fluorescence by measuring the intensity of the reflected light from A2a is shown. In FIG. 6 (b), an excitation light source 41 and a fluorescence detection device 42 are provided.
An optical system 40 for directly measuring the fluorescence intensity at the ligand DNA 2a is shown. However, the optical system 40 for performing fluorescence measurement is not limited to these, and any existing device can be used.

FIG. 7 shows an example of a method for moving the DNA array 1 with respect to the optical system 40 (only the excitation light source 41 is shown in this figure, and other components are omitted), as described above. However, in (a), by placing the DNA array 1 on the carrier 50 and moving the carrier 50, DN can be obtained.
A method of moving the A array in the arrow direction is shown.
In (b), a liquid is caused to flow in a channel 70 having a cross-sectional area through which the DNA array 1 can pass, and the flow of this liquid causes DN to flow.
A method of moving the A array 1 in the arrow direction is shown. However, the method for moving the DNA array 1 is not limited to these, and any existing means can be used.

FIG. 8 shows a graph showing an example of the measurement result of the fluorescence intensity. As can be seen from this figure, first, the signals corresponding to the reference points 4 which are clearly different from the fluorescence intensities of the individual ligand DNAs 2a to 2f are measured, and then the individual ligand DNAs 2a to 2f are measured.
Fluorescence intensity is sequentially measured. At the time of such measurement, the scanning speed of the optical system 40 or the moving speed of the DNA array is measured and compared with the change with time of the fluorescence intensity, so that the fluorescence at an arbitrary distance from the reference point 4 of the DNA array 1 is detected. The strength can be discriminated. As a result, the fluorescence intensity of each ligand DNA portion can be known.

As described above, since the DNA array 1 according to the present invention is provided with the reference points 4, it is possible to accurately perform multipoint measurement. In the case of using a DNA array whose capacity has been reduced by using the hybridization method of the present application, it is necessary to extend this DNA array again into a linear ribbon or thread before measurement. Further, although the case where the flexible array according to the present invention is subjected to fluorescence measurement has been described above, the present invention is not limited to this, and the flexible array according to the present invention is a conventional array. It can be appropriately used for the measuring method.

In the above description, the ligand DNA is used as the ligand sample, and the DNA array on which the ligand DNA is immobilized has been described, but the present invention is not limited to this, and the flexibility according to the present invention is described. As a substance that can be immobilized on the array as a ligand sample, a protein such as an antibody or an enzyme, a biological substance such as DNA, or various substances that selectively adsorb other substances or react with an object can be considered. The sample of the biological sample attached to these ligand samples can be used to identify a specific component of DNA, detect hybridization of protein or nucleic acid, antibody assay, DNA chip, RNA chip and the like.

[0035]

EFFECTS OF THE INVENTION The flexible array according to the present invention comprises a plurality of ligand samples aligned and immobilized in the longitudinal direction of an elongated carrier, which facilitates the subsequent handling of the flexible array, and The time for hybridization with the sample can be greatly shortened, and simple and mass production and further cost reduction are possible.

[Brief description of drawings]

FIG. 1 is a schematic diagram showing the entire DNA array according to the present invention.

FIG. 2 is a schematic view showing a method for producing a DNA array in which a plurality of ligand DNAs are linearly immobilized on a ribbon-shaped carrier.

FIG. 3 is a schematic diagram showing a state in which a DNA array is hybridized in a general test tube.

FIG. 4 is a schematic view showing a state in which a DNA array is processed into a spiral shape, a coil shape, or a folding process before being put into a test tube to reduce the capacity.

FIG. 5 is a diagram showing another embodiment of hybridization.

FIG. 6: Moving an optical system with respect to a DNA array,
It is a conceptual diagram which shows the measuring method of a DNA array.

FIG. 7: Moving the DNA array with respect to the optical system,
It is a conceptual diagram which shows the measuring method of a DNA array.

FIG. 8 is a graph showing an example of measurement results of fluorescence intensity.

[Explanation of symbols]

1, 1 '... DNA array (flexible array), 2, 2a-
2f ... Ligand DNA (ligand sample), 4 ... Reference point,
6 ... Carrier, 10 ... Test tube, 12 ... Solution, 14 ... Lid, 20
... tubular container, 30 ... groove-shaped channel, 40 ... optical system, 41 ... excitation light source, 42 ... fluorescence detection device, 43 ... spectroscopic means, 50 ...
Carrier, 60 ... Carrier, 70 ... Flow path, L1a to L1f, L
2a to L2f ... Virtual straight line.

   ─────────────────────────────────────────────────── ─── Continued front page    (72) Inventor Jun Utsumi             1-8 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa               Mitsubishi Heavy Industries, Ltd. Basic Technology Research Center (72) Inventor Satoshi Tahara             1-8 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa               Mitsubishi Heavy Industries, Ltd. Basic Technology Research Center (72) Takuma Sakai             1-8 Koura, Kanazawa-ku, Yokohama-shi, Kanagawa               Mitsubishi Heavy Industries, Ltd. Basic Technology Research Center F-term (reference) 4B024 AA11 CA01 CA09 CA11 HA14                 4B029 AA07 AA21 BB20 CC03 CC11                       FA15                 4B063 QA01 QA18 QQ42 QQ52 QR08                       QR32 QR38 QR41 QR55 QR84                       QS12 QS25 QS34 QS39 QX02

Claims (12)

[Claims] 1. A flexible array in which a plurality of ligand samples are aligned and immobilized in the longitudinal direction of an elongated carrier. 2. The flexible array according to claim 1, wherein the carrier has a ribbon shape or a thread shape. 3. The flexible array according to claim 2, wherein the ribbon-shaped flexible array has a widthwise length of 5 mm or less and a thickness of 0.3 mm or less. 4. The flexible array according to claim 2, wherein the thread-shaped flexible array has a thread diameter of 0.5 mm or less. 5. The method according to claim 1, wherein each of the plurality of ligand samples is configured to be discriminable according to a distance from a predetermined portion in the longitudinal direction of the carrier. Flexible array. 6. In order to manufacture a flexible array in which a plurality of ligand samples are aligned and fixed in the longitudinal direction of an elongated carrier, a plurality of ligand samples are linearly formed on a planar carrier. A method of manufacturing a flexible array, comprising a step of immobilizing a plurality of rows and cutting the planar carrier into a plurality of rows in a direction orthogonal to the rows of the ligand sample. 7. A step of introducing the flexible array according to any one of claims 1 to 5 and a solution containing a biological sample into a container and performing hybridization in the container. Hybrid array hybridization method. 8. The container according to claim 7, further comprising a step of reducing the capacity of the flexible array before introducing the flexible array into the test tube. Flexible array hybridization method. 9. The capacity reduction of the flexible array is performed by processing the flexible array into a spiral or coil shape.
Alternatively, the flexible array hybridization method according to claim 8, which is performed by folding the flexible array. 10. The container is a tubular container or a channel having a groove shape, and the method further comprises the step of allowing the flexible array to remain in the tubular container or the channel in an extended state. 7. The method for hybridizing a flexible array according to 7. 11. A flexible array according to any one of claims 1 to 5 is hybridized and the hybridized flexible array is provided with longitudinal ends of the flexible array. A method for measuring a flexible array, which comprises the steps of sequentially measuring fluorescence. 12. The method for measuring a flexible array according to claim 11, further comprising the step of linearly extending the hybridized flexible array before performing fluorescence measurement on the flexible array.