JP5569122B2 - Biomolecule detection device - Google Patents

Description

  The present invention relates to a biomolecule detection device capable of efficiently contacting and reacting a biomolecule (target molecule) to be detected with a biomolecule (probe) for specifically detecting it, and a method for producing the same. About.

The nucleic acid array has a large number of nucleic acid probes mounted on a carrier at a high density so that they are not mixed with each other. The nucleic acid probe mounted on the nucleic acid array serves as a sensor for capturing a nucleic acid molecule constituted by a sequence complementary to the base sequence by hybridization.
Conventionally, as nucleic acid arrays, those in which a nucleic acid probe is mounted on a surface-treated glass or silicon carrier have been used, but in recent years, the carrier has been modified by other methods such as a gel carrier. It has been broken.

As a method for producing a nucleic acid array, a method of directly synthesizing a nucleic acid probe on a substrate and a method of fixing a nucleic acid probe prepared in advance on a substrate such as a slide glass or silicon are known.
For example, according to the photolithographic method of directly synthesizing a nucleic acid probe on a substrate, a substance having a protecting group that is selectively removed by light irradiation is used, and a combination of photolithography technology and solid-phase synthesis technology is used. A nucleic acid array can be prepared by selectively synthesizing DNA in a predetermined region (reaction site) of the matrix (Non-patent Document 1).
Moreover, as a method for mounting a nucleic acid probe prepared in advance, a spotting method is mainly cited (Non-patent Document 2). In this method, a solution containing nucleic acid probes prepared in advance by PCR or artificial synthesis is arranged on the chip surface in a minute volume of several nl to several pl using a special device such as a spotter or an arrayer, and specified on the substrate. This technology is installed in the area.
In addition to the above method, a method for producing a microarray using a hollow fiber array has been developed. In this method, the through-hole substrate is manufactured using a hollow fiber array in which a plurality of hollow fibers made of a synthetic polymer are regularly arranged in the fiber axis direction. One feature of this production method is that a nucleic acid probe is immobilized in each hollow fiber hollow portion of the hollow fiber array, and the hollow fiber array is sliced by a method perpendicular to the fiber axis direction, so that This is the point that microarray products having the same specifications can be manufactured in large quantities (Patent Document 1).

  A nucleic acid detection method using a nucleic acid array is a method in which a nucleic acid sample to be tested is hybridized to a nucleic acid probe mounted on the nucleic acid array, and a hybrid formed in a sequence-specific manner is detected by a fluorescent substance or the like. It is. In this method, for a plurality of nucleic acid probes, a nucleic acid molecule containing a base sequence complementary to the base sequence can be quantitatively or qualitatively examined, and the expression level for a plurality of genes or the sequence of a specific nucleic acid base sequence Used to analyze itself.

However, since a special apparatus such as a spotter is required for the production of the array, the operation is fine and complicated. In addition, once the array is manufactured, the mounted probe cannot be changed, and therefore an unnecessary probe unrelated to the detection target is mounted, which may be economically undesirable. Furthermore, in arraying, the number of probes is limited according to the size of the array.
Although devices that are not arrayed are known, in which probes are immobilized on beads, etc., even before this, beads are immobilized before sample hybridization, and the probes mounted on each bead are revealed. (Patent Document 2). Furthermore, in order to check the mounted probe, it may be performed simultaneously with detection using a different type of fluorescence from the detection target. However, since it is necessary to use multiple types of fluorescence, the detection apparatus becomes large (patented) Reference 3).
On the other hand, composite arrays using hybridization chambers and microspheres, optical sensors using fibers, and the like are known (Patent Documents 4 to 11).

JP 2000-245460 A JP-T-2004-502138 Japanese translation of PCT publication No. 2002-501184 International Publication No. 98/40726 Pamphlet WO99 / 18434 pamphlet International Publication No. 00/16101 Pamphlet WO99 / 67641 pamphlet WO99 / 045357 pamphlet U.S. Pat.No. 6023540 U.S. Pat. U.S. Pat.No. 6327410

Science 251,767-773 (1991) Science 270, 467-470 (1995)

  An object of the present invention is to provide a detection device that can detect a target molecule simply and accurately, and a detection method thereof.

As a result of earnest research to solve the above problems, the present inventor found that the above problems can be solved by giving a predetermined shape to the carrier on which the probe is immobilized and identifying the shape, The present invention has been completed.
That is, the present invention is as follows.
(1) A target molecule detection device including a probe-immobilized carrier formed by immobilizing probes on a plurality of carriers processed so as to be distinguishable from each other according to shape.

(2) Target molecule detection method including the following steps:
(A) contacting the target molecule with the device according to (1),
(B) identifying the probe-immobilized carrier by shape,
(C) detecting a signal derived from the target molecule bound to the identified probe-immobilized carrier.

(3) Furthermore, this invention provides the manufacturing method of the device for target molecule detection which can be mutually distinguished according to a shape including the process of the following (3-1) or the process of (3-2).
(3-1)
(a) a step of arranging a plurality of hollow fibers so that each fiber axis is in the same direction to form a hollow fiber bundle, and fixing this with a resin to obtain a fiber array;
(b) filling the hollow portion of the hollow fiber with a gel precursor polymerizable solution containing a probe, and gelling the solution;
(c) cutting the hollow fiber array holding the gel in a direction perpendicular to the longitudinal direction of the hollow fiber to produce a flake, and
(d) A step of cutting the obtained thin piece so as to include a cut surface of one hollow fiber and shaping the resin portion of the cut piece.
(3-2)
(a) applying a predetermined shape processing to each of the plurality of hollow fibers in the direction of each fiber axis;
(b) filling the hollow portion of the hollow fiber with a gel precursor polymerizable solution containing a probe, and gelling the solution; and
(c) A step of cutting the hollow fiber holding the gel in a direction perpendicular to the longitudinal direction of the hollow fiber.

  In the detection method of the present invention, it is preferable that step b and step c are performed simultaneously. Moreover, in this invention, it can carry out, without immobilizing a probe fixed support | carrier.

The present invention provides a target molecule detection device including a plurality of probe-immobilized carriers in which probes are immobilized on a carrier that can be identified by shape. The device of the present invention has the following advantages because the carrier can be identified by shape.
(i) Since it is not necessary to make an array, manufacturing is easy.
(ii) Since each probe is scattered, only necessary probes can be selected and used for hybridization. That is, it is economical because it is not necessary to prepare a probe unnecessary for analysis.
(iii) The number of probes is not limited.
(iv) Since the mounted probe can be determined by shape detection at the time of detection, it is not necessary to be immobilized at the time of detection, and decoding processing is unnecessary.
(v) Since the probe type can be easily visually confirmed, the detection device does not become large.

It is explanatory drawing which observed the support | carrier of this invention from the direction perpendicular | vertical to a detection surface, and demonstrated various variations. It is explanatory drawing which illustrates roughly the support | carrier of this invention which has a predetermined shape. It is explanatory drawing which illustrates roughly the support | carrier of this invention which has a predetermined shape. It is sectional drawing which cut | disconnected the support | carrier of this invention by the plane perpendicular | vertical to a detection surface. It is the perspective view explaining the support | carrier shape | molded into various shapes by making various notches. It is the schematic explaining roughly the manufacturing method which cuts out the some support | carrier of this invention from one array. It is explanatory drawing explaining simply the manufacturing method of an array. It is explanatory drawing explaining another manufacturing method of the device of this invention. It is the perspective view shown about the device of this invention which has a predetermined shape. It is explanatory drawing which shows the result of the fluorescence detection using the device of this invention. It is explanatory drawing shown about the detection result of a comparative test.

2, 4, 6, 8, 10, 12, 20, 30-32, 40-42: carrier 22: opening,
35 to 37: notches 45 to 47: gel 50: reading device 59: holder 60: thin piece 62: cutting line 65: cutting piece 70: perforated plate 72: hole 74: hollow fiber 76: plate-like product 80: hollow fiber bundle

Hereinafter, the present invention will be described in detail.
1. Outline The present invention is a target molecule detection device including a carrier on which a probe is immobilized (hereinafter also referred to as “probe-immobilized carrier”), and the shape of the carrier is processed so as to be identifiable. The inventors cut a part of the spot of the microarray to which the probe is fixed, and then processed a part of the cut piece so that it can be distinguished by the shape. As a result, it was possible to identify which probe was fixed according to the shape, and the probe spot could obtain a sufficiently detectable signal. The present invention has been completed based on these findings.
Therefore, the device of the present invention is characterized in that the carrier on which each probe is fixed is independent, and is not fixed to a flat substrate (not immobilized). is there.

In the present invention, the carrier means a support for supporting the probe, and the carriers are processed so as to be distinguishable from each other depending on the shape. By immobilizing a probe on a carrier, individual biomolecules can exist independently without being mixed with other biomolecules.
In general, in a microarray, what type of probe is fixed at which position can be known from the position coordinates on the array where the probes are fixed and the type of probe at those coordinates. In the present invention, the shape of the carrier is used as an index for knowing which type of probe is fixed to which carrier.

2. A plurality of carriers processed so as to be distinguishable from each other by shape. The probe-immobilized carrier may have any shape as long as it can be distinguished from each other by shape.
In the present invention, a probe can be fixed to a flat substrate generally used for a microarray, and then each section of the substrate can be processed into a predetermined shape.
A planar substrate is a plate whose surface is substantially planar. Examples of the substrate include a slide glass, a silicon substrate, a resin substrate, and a substrate made of gel.

As shown in FIGS. 1A to 1D, when any one of the four corners of the rectangular carrier is cut, it can be distinguished into several types of shapes. For example, the carrier 2 cut in all four directions (FIG. 1A), the carrier 4 cut only one corner (FIG. 1B), and the carrier 6 cut only two corners (FIG. 1C) ), And the carrier 8 (FIG. 1 (d)) whose corners are not cut.
Then, four types of probes can be fixed by four types of carriers. By diversifying the shape of the shape of the carrier so that many combinations can be formed, a large number of probes corresponding to the number of combinations can be fixed. For example, if there are 1000 types of carriers, 1000 types of probes can be fixed according to the shape, similar to a microarray in which 1000 types of probes are fixed. Moreover, since the carriers are physically separated from each other, only necessary carriers can be freely combined and used. As a result, it is possible to eliminate waste caused by fixing unnecessary probes as in a normal microarray.
The combination of shapes is arbitrary and is not particularly limited in the present invention. For example, FIG. 2 is a front view of a carrier that is shaped so as to cut off a pair of opposite corners of a square carrier.
FIG. 3 is a front view of a carrier that has been shaped so that one side of the square is cut into a chevron instead of cutting off square corners.

The device of the present invention will be described in more detail using the carrier illustrated in FIG.
As shown in FIG. 4, the carrier 20 is provided with an opening 22, whereby the carrier 20 has a frame shape. A gel containing a probe that can bind to a biopolymer such as a nucleic acid, protein, or sugar chain is disposed in the opening 22 of the carrier 20.

  The carrier 20 is formed in a unique shape, so that a carrier having another shape can be identified. The types of probes arranged in the opening 22 also correspond to the difference in shape of the carrier 20, the same probe between the carriers 20 having the same shape, and different probes arranged in the opening 22 between the carriers 20 having different shapes. Has been.

  As described above, the probe carrier 4 is constituted by the carrier 20 having a unique shape and the unique probe provided on the carrier 20, so that a plurality of shapes can be obtained based on the shape of the carrier 20 formed in a unique shape. The probes immobilized on the carrier can be identified, and the user can select a carrier based on the shape of the carrier, thereby providing a highly convenient probe carrier.

In the above, an embodiment has been described in which several dozen types of probes can be identified mainly on the assumption that they are identified directly visually. However, in the present invention, more types of probes can be identified. It can also be configured as follows.
For example, a carrier having a unique shape can be formed by making a unique notch in the edge of the carrier so that at least several hundred kinds of probes can be detected. As shown in FIG. 5, each of the carriers 30 to 32 can be identified by making unique cuts 35 to 37 at the edges of the carriers 30 to 32 having a rectangular outer shape. . For the recognition of the cuts 35 to 37, the reading device 50 provided with a photo sensor, a line sensor, an image sensor, or the like can be used to reliably read even a fine cut. Gels 45 to 47 containing different probes are arranged on the respective carriers 30 to 32, whereby the probes can be identified based on the shapes of the carriers 30 to 32.

  The shapes of the carriers 30 to 32 can be made unique by changing the number of cuts 35 to 37, the width, the cut interval, and the cut depth. By changing the width and arrangement of the grooves in the cuts, for example, the cuts can be shown in the form of barcodes, and more identification is possible. The photo sensor, line sensor, or image sensor mounted on the reading device 50 outputs a light reception signal according to the shape of each of the notches 35 to 37, and an identification circuit mounted on the reading device 50 is based on this light reception signal. The difference between the carriers 40 to 42 is identified. The cutting position may be anywhere as long as it is the edge of the carriers 30 to 32. Therefore, it is possible to easily form a large number of carriers having a unique shape, and it is used when identifying a large number of probes of hundreds or more. can do. In addition, by making it possible to identify the probe carrier through such a reader, the variety of shape processing of the carrier can be dramatically increased.

  The holder 59 can also be used so that the carriers holding different probes can be handled collectively. A plurality of carriers may be placed on such a holder 59, and the carriers may be identified using the reading device 50 from above the holder 59. According to such an embodiment, even if the carrier is subjected to shape processing that is difficult to visually identify, the user can select whether to use the carrier in response to the reading result of the reading device 50. .

  In the present invention, the probe-immobilized carrier is a carrier on which a biomolecule is immobilized. The probe-immobilized carrier is used as a target molecule detection device. An example of such a device is one in which sections such as a DNA array, protein array, antibody array, and lectin array are processed into a predetermined shape, and the respective sections are distinguished from each other by their shapes. In the device of the present invention, since the entire section is not immobilized on the substrate, the probe-immobilized carrier can be freely moved without being immobilized. Such a device can minimize the amount of specimen used for the reaction and perform detection at high speed. Moreover, it is not necessary to arrange the carriers at high density.

  In the present invention, the material used as the carrier is a resin, gel, glass, silicon or the like used for general microarrays. The gel is a hydrophilic gel and may be any hydrophilic gel as long as it can immobilize a desired biomolecule.

  Examples of the hydrophilic gel that can be used in the present invention include acrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, N-acryloylaminoethoxyethanol, N-acryloylaminopropanol, N-methylolacrylamide, and N-vinylpyrrolidone. , One or more monomers such as hydroxyethyl methacrylate, (meth) acrylic acid, and allyl dextrin, and polyfunctional monomers such as methylene bis (meth) acrylamide and polyethylene glycol di (meth) acrylate A copolymerized gel may be mentioned. Other examples of the gel that can be used in the present invention include gels such as agarose, alginic acid, dextran, polyvinyl alcohol, and polyethylene glycol, and gels obtained by crosslinking these.

  In the present invention, the fixation of the probe is a treatment for preventing the probe from being released from the carrier. In the present invention, the probe may be immobilized on the gel as it is, or a derivative obtained by chemically modifying the probe, or a probe denatured as necessary may be immobilized. For immobilization of the probe on the carrier, a method of physically enclosing the probe in the carrier may be used, or direct binding to the constituent components of the carrier may be used. Alternatively, the probe may be once bound to a polymer or inorganic particle by a covalent bond or a non-covalent bond, and they may be comprehensively immobilized on a carrier. For example, when a nucleic acid is used as a probe and a gel is used as a carrier, a vinyl group can be introduced into the terminal group of the nucleic acid (WO98 / 39351) and copolymerized with a gel component such as acrylamide.

  In the copolymerization, there are a method of copolymerizing with a monomer, a polyfunctional monomer and a polymerization initiator, a method of copolymerizing with a monomer and a polymerization initiator, and then gelling with a crosslinking agent. Alternatively, agarose may be converted to imide carbonate by the cyanogen bromide method and bonded to the amino group of the terminally aminated nucleic acid before gelation. In this case, the gel may be a mixed gel of agarose with immobilized nucleic acid and another gel (for example, acrylamide gel).

3. Biomolecule In the present invention, the biomolecule is not limited as long as it can be used in the present invention as a probe or target molecule. For example, nucleic acid (DNA, RNA, etc.), protein, peptide, antibody, sugar Examples include chains and lectins.

  In the present invention, a probe is a biomolecule for specifically capturing a target molecule contained in a specimen, and refers to a molecule having a structure corresponding to each target molecule. The probe may be any kind of molecular species as long as it can specifically recognize a predetermined target molecule, and may be a natural product or an artificially synthesized one. . Examples of such biomolecular probes include nucleic acid probes, protein probes, peptide probes, antibody probes, sugar chain probes, lectin probes, and the like. The target molecule refers to an arbitrary biomolecule to be analyzed.

  In the present invention, when a nucleic acid such as a certain kind of DNA or RNA is a target molecule, it has a sequence complementary to the base sequence of the target molecule, and can specifically recognize and capture these base sequences. Nucleic acids can be used as probes. Moreover, when protein or peptide is a target molecule, an antibody that specifically recognizes them as an antigen can be used as a probe. Conversely, proteins and peptides that serve as antigens and target antigens can be used as probes. Furthermore, when a sugar chain is used as a target molecule, a lectin or the like can be used as a probe. That is, when a specific molecule (A) has a property of specifically binding to a specific molecule (B), if A is a detection target as a target molecule, B is a probe, and conversely, B is If it becomes a target molecule, A becomes a probe.

4). Method for Manufacturing Target Molecule Detection Device The target molecule detection device of the present invention is a target molecule detection device that can be distinguished from each other by shape. The present invention provides a method for manufacturing such a device (referred to as “method 1”), and includes the following steps.

<Method 1>
(a) a step of arranging a plurality of hollow fibers so that each fiber axis is in the same direction to form a hollow fiber bundle, and fixing this with a resin to obtain a fiber array;
(b) filling the hollow portion of the hollow fiber with a gel precursor polymerizable solution containing a probe, and gelling the solution;
(c) cutting the hollow fiber array holding the gel in a direction perpendicular to the longitudinal direction of the hollow fiber to produce a flake, and
(d) A step of cutting the obtained thin piece so as to include a cut surface of one hollow fiber and shaping the resin portion of the cut piece.

  In the above method, first, hollow fibers are bundled and fixed with a resin, and sliced in a direction perpendicular to the longitudinal direction of the hollow fibers. Thereby, the flakes in which the hollow fiber portions are regularly arranged can be obtained. These slices are then cut so that the hollow fiber portions are individually separated, i.e. including the cut surface of one hollow fiber. For example, as shown in FIG. 6A, when the thin piece 60 is cut in the vertical direction and the horizontal direction along the cutting line 62, a rectangular cut piece 65 including one hollow fiber portion is obtained. Furthermore, the device of the present invention can be obtained by processing the obtained cut pieces so as to be identifiable. For example, by processing a rectangular cut piece as shown in FIG. 6B, three types of processed devices can be obtained.

The step (a) is a step of producing a fiber array by arranging a plurality of hollow fibers so that the respective fiber axes are in the same direction to form hollow fibers and fixing them with a resin or the like. The hollow fiber bundle is not limited as long as a plurality of hollow fibers are arranged and fixed so that the fiber axes are in the same direction. Then, the hollow fiber bundle is hardened with a resin or the like to obtain a fiber array.
The production of the hollow fiber bundle in the step (a) can be performed, for example, using an array fixing device shown in FIG. Specifically, first, the two porous plates 70 are overlapped so that the holes 72 coincide with each other, the hollow fibers 74 are inserted into the holes, the interval between the porous plates 70 is increased after the insertion, and the space between the porous plates 70 is increased. A container is manufactured by surrounding the three surfaces of the plate with a plate-like object 76. Next, a resin raw material is poured into the container, the resin is cured, and the porous plate 70 and the plate-like object 76 are removed. Thereby, the hollow fiber bundle 80 in which the plurality of hollow fibers 74 are arranged and fixed in three dimensions can be manufactured. When fixing the hollow fiber, in order to facilitate the filling of the gel precursor polymerizable solution in step (b), instead of fixing the entire length of the hollow fiber, it may be a free end without fixing one end. preferable.

  Examples of the raw material of the hollow fiber include polyamide-based hollow fibers such as nylon 6, nylon 66 and aromatic polyamide, polyester-based hollow fibers such as polyethylene terephthalate, polybutylene terephthalate, polylactic acid, polyglycolic acid and polycarbonate, and polyacrylonitrile. Acrylic hollow fiber, polyolefin hollow fiber such as polyethylene and polypropylene, polymethacrylate hollow fiber such as polymethyl methacrylate, polyvinyl alcohol hollow fiber, polyvinylidene chloride hollow fiber, polyvinyl chloride hollow fiber, polyurethane Examples include hollow fibers, phenolic hollow fibers, fluorine hollow fibers made of polyvinylidene fluoride and polytetrafluoroethylene, polyalkylene paraoxybenzoate hollow fibers, and the like.

The step (b) is a step of filling the hollow portion of the hollow fiber with a gel precursor polymerizable solution containing a probe and gelling the solution.
When hollow fiber bundles are used in the method of the present invention, gel precursor polymerizable solutions containing different types of probes for each hollow fiber can be filled. It is easy to fix to the hollow part of the fiber.

  In the present invention, the “gel precursor polymerizable solution” includes a probe, a monomer, a polyfunctional monomer, a polymerization initiator, water, and the like. Examples of the monomer include acrylamide, N, N-dimethylacrylamide, N-isopropylacrylamide, N-acryloylaminoethoxyethanol, N-acryloylaminopropanol, N-methylolacrylamide, N-vinylpyrrolidone, hydroxyethyl methacrylate, hydroxyethyl. One or more monomers such as methyl methacrylate, methyl pyrrolidone, (meth) acrylic acid, and allyl dextrin, and polyfunctional monomers such as methylene bis (meth) acrylamide and polyethylene glycol di (meth) acrylate Can be mentioned. Examples of the polymerization initiator include redox compounds such as persulfate, 2,2′-azobis [2- (2-imidazolin-2-yl) propane] dihydrochloride, 2,2′-azobis (4- And azo initiators such as cyanopentylcarboxylic acid) and 2,2′-azobis (2-methylpropionamidine) dihydrochloride.

  The gel precursor polymerizable solution can be filled, for example, by the following method. First, a gel precursor polymerizable solution and a hollow fiber bundle that have been stored at a low temperature are placed in a desiccator. The gel precursor polymerizable solution is sufficiently deaerated beforehand. Here, an inert gas such as nitrogen or helium is introduced, and the inside of the desiccator is replaced with the gas. Replacement with the inert gas is carried out in 10 to 20 hours. Next, the inside of the desiccator is brought into a reduced pressure state, and the free end where the hollow fibers of the hollow fiber bundle are not fixed is immersed in the polymerizable solution. Next, an inert gas is blown into the desiccator to bring it into a pressurized state, and the hollow portion of each hollow fiber is filled with the solution.

  In the present invention, “gelation” is carried out by filling a hollow portion of each hollow fiber with a gel precursor polymerizable solution, then heating the hollow fiber bundle, and performing a polymerization reaction. The polymerization temperature is appropriately selected depending on the monomer used. By this operation, the gel is held in the hollow fiber bundle.

The step (c) is a step of manufacturing the thin piece 60 by cutting the hollow fiber bundle holding the gel in a direction perpendicular to the longitudinal direction of the hollow fiber. In the manufacture of the flake 60, a microtome, a laser, or the like can be used. The thickness of the thin piece 60 is preferably 1 μm or more and 5,000 μm or less, and more preferably 10 μm or more and 500 μm or less.
In the step (d), the obtained thin piece 60 is cut so that each hollow fiber portion is individually included, and the resin portion of the cut piece is shaped. The thin piece 60 is in a state in which hollow fibers 74 in which probes are fixed in the hollow portions are arranged. Therefore, as shown by a broken line in FIG. 6, individual cut pieces can be obtained by cutting in the vertical and horizontal directions. By performing predetermined shape processing on the cut pieces, it is possible to obtain sections that can be identified by their shapes.

  Here, in method 1, the order of steps (a) and (b) may be reversed. Accordingly, the hollow portion of the hollow fiber 74 is filled with a gel precursor polymerizable solution containing a probe, the solution is gelled, the fibers holding the gel are then focused, and the bundle of fibers bundled is elongated. You may slice in the direction orthogonal to the direction (fiber direction).

  Thus, by processing each section so as to be identifiable, the biomolecule-immobilized carrier can be produced so as to be identifiable by shape. Furthermore, since the hollow fiber bundle 80 can be easily produced in a large amount, it is effective in that a biomolecule-immobilized array can be produced easily and inexpensively in a short time.

<Method 2>
Moreover, in the method of this invention, the device which can be identified with a shape can be manufactured, without processing the part fixed with the said resin by giving shape processing to fiber itself. That is, as another aspect of the present invention, a method (method 2) including the following steps is provided.
(a) applying a predetermined shape processing to each of the plurality of hollow fibers in the direction of each fiber axis;
(b) a step of obtaining a hollow fiber bundle by arranging the plurality of hollow fibers having the shape processed so that each fiber axis is in the same direction;
(c) filling the hollow portion of the hollow fiber with a gel precursor polymerizable solution containing a probe, and gelling the solution; and
(d) A step of cutting the hollow fiber bundle holding the gel in a direction perpendicular to the longitudinal direction of the hollow fiber.

  In the step (a), each fiber of the plurality of hollow fibers is subjected to a predetermined shape processing in the longitudinal direction of the fiber. For example, as shown in FIG. 8 (a), when one groove along the longitudinal direction P is formed in the outer peripheral portion of the hollow fiber, in the step (d), in a direction orthogonal to the longitudinal direction P of the hollow fiber. When cut, a mark by one groove is formed as shown in FIG. Further, if two grooves along the longitudinal direction P are formed on the outer peripheral portion of the hollow fiber (FIG. 8B), after the step (d), marks by the two grooves can be formed (FIG. 9). (B)). Therefore, both can be identified by the mark by the one groove and the mark by the two grooves. By increasing the number of grooves and changing the groove pattern (groove shape, width, spacing, number, etc.), various types of landmarks can be formed, and each can be identified by its shape.

Moreover, although the groove | channel was formed along the longitudinal direction P above, you may form a plane substantially parallel to the longitudinal direction P along the longitudinal direction P (FIG.8 (c)). Thereby, after the step (d), a mark by a plane can be formed. Further, even when all other carriers are processed in shape, by using a carrier that is not subjected to shape processing (FIG. 9D), this carrier can also be distinguished from the other carriers.
As described above, by slicing a fibrous body having four cross-sectional shapes in a direction perpendicular to the longitudinal direction, four probe carriers having unique outer shapes can be efficiently produced.

5. Detection In the present invention, the target molecule detection method includes the following steps.
(A) contacting the target molecule with the device of the present invention,
(B) identifying the probe-immobilized carrier by shape,
(C) detecting a signal derived from the target molecule bound to the identified probe-immobilized carrier.

In the detection method of the present invention, the method for performing the step (a) is not particularly limited as long as the device of the present invention and the target molecule can contact each other. For example, a method in which a solution containing the target molecule is sprinkled on the device, a method in which the solution containing the target molecule and the device are mixed in the same container, and the like can be mentioned.
Step (b) is a step of identifying the probe-immobilized carrier by its shape. The step of identifying by shape includes a method of identifying the carrier by observing with the naked eye, a method of identifying the carrier by observing with a microscope, a method of identifying the carrier by a shape sensor (photo sensor, line sensor, image sensor, etc.), etc. There is. The shape sensor can automatically identify the shape of the carrier in advance by storing the shape of the carrier in the sensor program.

In step (c), a signal emitted from the target is detected by contact between the target molecule and the probe.
When the target molecule is a nucleic acid, the target nucleic acid can be detected by labeling the target nucleic acid with a fluorescent label, a radiolabel, etc., and detecting the signal with a detection device or visually. By quantifying or qualifying the hybridization between the probe nucleic acid and the target nucleic acid using a signal derived from a substance previously labeled with the target nucleic acid, such as fluorescence, chemiluminescence, or radioisotope, the target nucleic acid corresponding to the probe nucleic acid The existence can be confirmed. Further, the detection method is not limited to the above. For example, it is possible to label the blocking nucleic acid and confirm the loss of the blocking nucleic acid due to the hybridization of the target nucleic acid with a decrease in signal.

The above “labeling” method is not particularly limited as long as hybridization of nucleic acid can be detected. Examples of labeling substances used for labeling include fluorescent substances such as Cy3, Cy5 and Alexa Fluor, enzymes or proteins for utilizing chemiluminescence accompanying substrate degradation using alkaline phosphatase or horseradish peroxidase, γ- ³² P And radioisotopes such as α- ³² P are preferable, but it is preferable to use a fluorescent substance because it is simple.

  When incorporating these labeling substances into nucleic acid molecules, direct, indirect, physical, or chemical binding is possible as long as the labeling method is stable and does not inhibit specific hybridization with the probe nucleic acid. Regardless, any method can be used. As a method for chemical modification, for example, a method in which a base previously labeled with a fluorescent substance is incorporated during the reaction, an analog base modified with biotin or an aminoallyl group is incorporated during the reaction, and the labeling substance is incorporated through this. The method of making it. In addition, SYBR Green, acridine orange, SYBR Gold, etc. are used to intercalate the labeling substance to the complementary nucleic acid molecule, and the labeling substance is bound to the complementary nucleic acid molecule via platinum, such as ULYSIS. You can also

  In the present invention, since the carrier is not fixed (immobilized) to the substrate, shape identification and label detection can be performed simultaneously. The shape of the probe can be identified by the shape, and the target molecule bound to the probe can be detected by the labeling substance.

In the above description, nucleic acid is exemplified as the target molecule, but protein or sugar chain may be used as the target molecule. In this case, it is necessary to change the probe for binding the target molecule, but the embodiment of the shape processing of the carrier can be applied.
For example, a probe carrier is prepared in which a gel containing an antibody that specifically binds to an antigen is placed in the opening of the carrier. At this time, a gel containing the same probe is placed in the opening for those having the same carrier shape, and a gel containing a different probe is placed in the opening for those having a different carrier shape.
As a result, the type of probe can be identified from the shape of the carrier, and a highly convenient device can be provided.

  Hereinafter, the present invention will be described more specifically with reference to examples. However, the present invention is not limited to these examples.

Examples (1) Synthesis of Probes Oligonucleotides represented by SEQ ID NO: 1 to SEQ ID NO: 3 shown in Table 1 were synthesized. In the final stage of synthesis, aminolink ™ (manufactured by PE Biosystems) was reacted with the oligonucleotide and then subjected to deprotection, whereby an aminohexyl group was introduced at the end of each oligonucleotide 5 ′. -O-aminohexyl oligonucleotide was prepared. Next, methacrylic anhydride was reacted with the 5′-O-oligonucleotide to prepare a 5′-terminal vinylated oligonucleotide, which was used as a probe.

(2) Production of Hollow Fiber Bundle Next, a hollow fiber bundle was produced using the array fixing device shown in FIG. In FIG. 7, x, y, and z are orthogonal three-dimensional axes, and the x-axis coincides with the longitudinal direction of the fiber. First, two perforated plates 70 each having a thickness of 0.32 mm, each having a total of nine holes 3 in a row and 3 in a row, each having a hole 72 having a diameter of 0.32 mm and a center distance of 0.42 mm, were prepared. . These perforated plates 70 were overlapped, and polycarbonate hollow fibers 74 (addition of 1% by mass of carbon black manufactured by Mitsubishi Engineering Plastics) having an inner diameter of approximately 0.18 mm were passed through all the holes 72 one by one. The position of the two perforated plates 70 is moved in a state in which a tension of 0.1 N is applied to each fiber in the X-axis direction, so that the position is 20 mm and 100 mm from one end of the hollow fiber 74. Fixed. That is, the interval between the two perforated plates 70 was 80 mm. Next, the three surrounding surfaces of the space between the perforated plates 70 were surrounded by a plate-like object 76. In this way, a container having an open top only was obtained. The resin raw material was poured into the container from the upper part of the container. As resin, what added 2.5 mass% carbon black was used with respect to the total weight of a polyurethane resin adhesive (Nippon Polyurethane Industry Co., Ltd. Nippon Run 4276, Coronate 4403). The resin was cured by standing at 25 ° C. for 1 week. Next, the porous plate 70 and the plate-like material 76 were removed, and a hollow fiber bundle 80 was obtained.

Next, a gel precursor polymerizable solution containing a monomer and an initiator mixed at a mass ratio shown in Table 2 was prepared for each probe.

  The gel precursor polymerizable solutions containing the oligonucleotides represented by SEQ ID NO: 1 to SEQ ID NO: 3 prepared above are shown in FIG. 6 (the numbers in FIG. 6 indicate the sequence numbers of the base sequences of the probes). The hollow portion of the hollow fiber 74 in the hollow fiber bundle 80 was filled such that B represents a blank portion not containing an oligonucleotide. Specifically, first, the container containing the polymerizable solution and the hollow fiber bundle 80 produced above were installed in a desiccator so as to have the arrangement shown in FIG. After the inside of the desiccator was decompressed, the unsealed end of each yarn of the hollow fiber bundle 80 was immersed in a container containing the predetermined polymerizable solution. Nitrogen gas was sealed in the desiccator, and a gel precursor polymerizable solution containing a probe was introduced into the hollow portion of the hollow fiber 74. Subsequently, the inside of a container was 70 degreeC and the polymerization reaction was performed over 3 hours. In this way, a hollow fiber bundle 80 was obtained in which the probe was held in the hollow portion of the hollow fiber 74 via the gel-like material.

  Next, the obtained hollow fiber bundle 80 is sliced in a direction orthogonal to the longitudinal direction of the fiber using a microtome, and a plurality of thin sheet 60 (through-hole type biomolecule-immobilized array) having a thickness of 0.25 mm is obtained. Obtained. The probe was immobilized with the arrangement shown in FIG. The numbers “1”, “2”, and “3” in the openings in FIG. 6 indicate the sequence numbers in the sequence listing, and “B” indicates a blank. In the prepared through-hole type biomolecule-immobilized array, probes having the base sequences indicated by the respective sequence numbers are arranged so as to correspond to the sequence numbers shown in FIG.

(3) Subdivision of probe-immobilized carrier (FIG. 6)
In the obtained through-hole type biomolecule-immobilized array, the matrix portion is cut along the shape shown by the broken line in FIG. 6 (a) so that each probe is separated, and the probe is fixed so that the cut surface becomes a square. A modified carrier was obtained (FIG. 6B). The same treatment was performed on 10 through-hole type biomolecule-immobilized arrays, and the obtained probe-immobilized carriers were stored separately in 2 × SSC solution so as not to be confused.

(4) Characterization of probe-immobilized carrier Subsequently, characterization according to the shape of the probe-immobilized carrier was performed as follows. For the carrier on which the probes of SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3 are immobilized, a part of the probe-immobilized carrier (broken line portion) is cut as shown in FIGS. By doing so, it was characterized by shape. The carrier on which the probe was not immobilized had a shape as shown in FIG. 1 (d) because no special treatment was performed.
Each probe-immobilized carrier was collected in one tube and stored in a 2 × SSC solution to obtain a probe-immobilized carrier group.

(5) Preparation of model specimen A model specimen for evaluating the probe-immobilized carrier group was prepared as follows. First, oligonucleotides represented by SEQ ID NO: 4 to SEQ ID NO: 6 described in Table 3 were synthesized. Among these oligonucleotides, those having a sequence complementary to the oligonucleotide serving as a probe are included.

All oligonucleotides listed in Table 3 were prepared to 100 pmol / μl. Each of these oligonucleotides was used in an amount of 2 μl, and water was added thereto to prepare 500 μl of each oligonucleotide mixed solution. Using 5 μl of this oligonucleotide mixed solution, fluorescent labeling was performed using a labeling reagent PlatinumBright ™ Nucleic Acid Labeling Kit (model number: GLK-003) (manufactured by Kreatech Biotechnology). Specifically, first, a labeling agent and a buffer attached to the kit were mixed so that the composition shown in Table 4 was obtained, to obtain a reaction solution.

  Next, the reaction solution was heated on a heat block at 85 ° C. for 30 minutes and then cooled to 4 ° C. The reaction solution after cooling was purified according to the protocol attached to the kit using the purification column attached to the kit to obtain about 20 μl of a labeled oligonucleotide mixed solution (model sample).

(6) Hybridization In contacting and reacting the model specimen with the probe-immobilized carrier group, first, a hybridization solution having the composition shown in Table 5 was prepared. This hybridization solution was brought into contact with the probe-immobilized carrier group prepared above to perform hybridization. Hybridization was performed by bringing the probe-immobilized carrier group and 250 μl of the hybridization solution into contact with each other at 50 ° C. for 16 hours in a sealed state in a plastic tube.

(7) Washing Each 10 ml of 2 × SSC / 0.2% SDS solution as a washing solution and 2 × SSC as a stock solution were prepared. After the hybridization solution was extracted from the plastic tube after the completion of hybridization, the probe-immobilized carrier group was immersed in 250 μl of a washing solution kept at 50 ° C. for 20 minutes to wash away excess hybridization solution. After the same operation was repeated once more, the probe-immobilized carrier group was immersed in 250 μl of a preservation solution kept at 50 ° C. for 10 minutes, and then cooled at room temperature.

(8) Fluorescence detection The detection operation is carried out using a cooled CCD camera-type nucleic acid array automatic detection apparatus, after the cover glass is covered with the probe-immobilized carrier group immersed in the above storage solution, and then labeled nucleic acid sample molecules Fluorescence images from were taken. The detected image is shown in FIG. Here, it can be seen from the shape of each carrier that the probe 1 is fixed by the triangle, the probe 2 is fixed by the circle, and the probe 3 is fixed by the rectangle. In addition, it can be seen that the probe is not immobilized on the carrier without enclosure. Since it is possible to calculate the fluorescence signal value for each probe-immobilized carrier, it can be said that the shape of each carrier and the signal intensity derived from hybridization could be detected simultaneously.

Comparative example It carried out like the Example except not characterizing a probe fixed carrier. Since each carrier has the same shape, it is unclear whether the carrier is immobilizing the probe even when fluorescence is detected, and which carrier is immobilizing which type of probe. (FIG. 11).

  The device of the present invention is used in a method for detecting a target molecule such as a biological sample easily and accurately.

Sequence number 1-6: Synthetic DNA

Claims (2)

A method for producing a target molecule detection device, in which a plurality of carriers have different shapes, and can be distinguished from each other according to the shape, comprising the following steps:
(a) a step of arranging a plurality of hollow fibers so that each fiber axis is in the same direction to form a hollow fiber bundle, and fixing this with a resin to obtain a fiber array;
(b) filling the hollow portion of the hollow fiber with a gel precursor polymerizable solution containing a probe, and gelling the solution;
(c) cutting the hollow fiber array holding the gel in a direction perpendicular to the longitudinal direction of the hollow fiber to produce a flake, and
(d) The method comprising the steps of cutting the resulting flakes so as to include a cut surface of a single hollow fiber and shaping the resin portion of the cut pieces. A method for producing a target molecule detection device, in which a plurality of carriers have different shapes, and can be distinguished from each other according to the shape, comprising the following steps:
(a) applying a predetermined shape processing to each of the plurality of hollow fibers in the direction of each fiber axis;
(b) filling the hollow portion of the hollow fiber with a gel precursor polymerizable solution containing a probe, and gelling the solution; and
(c) The said method including the process of cut | disconnecting the hollow fiber holding the said gel in the direction orthogonal to the longitudinal direction of a hollow fiber.