JP2000346842A - Method and device for production of probe array using micro particles - Google Patents

Abstract

PROBLEM TO BE SOLVED: To enable a DNA probe to be produced on the surface of a solid material evenly by separating fixation of the probe to the surface of the solid material from arrangement of the probe. SOLUTION: The interval between beads arranged substantially corresponds to an interval between bead receptors 902 of a bored sheet 904 and is about 2 mm. The number of prove beads 907 dropped into grooves 906 of a holder 905 for producing a bead alley is 50 in total in case. Though the number of bead alleys to be produced simultaneously is 10, it can be increased if required. After the beads are dropped thereinto, the positions of holes 903 of the bored sheet 904 and the grooves 906 of the holder 905 are shifted, then the grooves 906 are made airtight, and the beads are lead to capilleries 908 together with a solution. In increasing the kind of the beads 907, the operation is repeated. Though a one-dimensionally arranged prove bead alley is shown in this case, it is possible to produce alleys having many kinds of probes by arranging them in a plural manner or in a two-dimensional manner.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to peptides, proteins,
The present invention relates to a probe array used for detection and diagnosis of biological substances such as DNA and RNA, and analysis of biological substances such as DNA, and a method and an apparatus for producing the same.

[0002]

2. Description of the Related Art A method for amplifying a small amount of DNA and a method for separating and detecting the obtained DNA fragment are required for DNA analysis, DNA test or diagnosis. PCR (pol
ymerase chain reaction) is widely used. According to this, even a very small amount of DNA can be detected by increasing the copy number by several orders of magnitude. On the other hand, for separation and detection of various DNAs, a DNA sequencer or a fragment analyzer that combines gel electrophoresis and fluorescence detection is used. However, when the number of specimens increases or the number of test items increases, the method using electrophoresis has a problem that it is troublesome, and a simple method using a DNA probe is attracting attention. In particular, attention has been drawn to a DNA chip that produces a probe array in which various types of probes are immobilized on a solid surface, causes hybridization with a sample, and traps and detects only specific DNA on the solid surface (Nature Medi
cine 2, 753 (1996)). On the other hand, probe detection is also used in the analysis of proteins and peptides or various biologically relevant substances interacting with them, and peptide chips corresponding to DNA chips have begun to be used.

[0003] As described above, a peptide or DNA is immobilized on a solid surface, and hybridization is performed with a specimen.
The separation and detection method has been used for a long time as a blotting method using a radioactive label and examining the presence of a target DNA or the like with a probe immobilized on a membrane. But,
A DNA chip having a large number of probes immobilized on a small area (1 cm ² ) of a solid surface such as glass or silicon has the advantages that samples can be saved and inspection can be performed using a large number of types of probes. There are roughly two methods for producing these DNA chips. The first is a method in which a DNA probe is synthesized by a photochemical reaction one base at a time in a narrow solid area (0.05 mm-0.2 mm square) using a photomask method used for semiconductors (Science 251, Science 251). 7
67 (1991)). Second is synthesized DNA or PCR amplified D
NA, a method in which DNA obtained by cloning is fixed to small compartments on the solid surface for each probe compartment (Nature
Biotech 16, 27 (1998)). This method has the advantage that a peptide chip or DNA chip having necessary probes can be relatively easily formed, and is adopted by many venture companies.

[0004]

[Problems to be Solved by the Invention] Probe tips for biological substances such as DNA are a promising testing technique, but practical use is as follows:
2) Good probe fixation uniformity, 3) Good data reproducibility, repeated use, 4) Heating to remove non-specific adsorption are required. However, since the probe is immobilized on the solid surface as droplets, there are variations in each section, it takes time to make, and since the position of the probe and fixing to the solid surface are performed at the same time, There are problems that a very fine probe fixing section cannot be formed and the uniformity of the probe is not good. Further, many of them are fixed by adsorption or the like, the bonding force with the solid is weak, and the probe may peel off from the surface by heating.

[0005]

In order to solve the above problems, the fixing of the probe to the solid surface and the arrangement of the probe are performed in separate steps. Thereby, a DNA probe can be produced uniformly on a solid surface. Since the probe can be immobilized by a covalent bond, it is resistant to heat, and is suitable for removing non-specific adsorption by heating. Microparticles are used as solids for immobilizing probes, and they are arranged to produce a probe array with a desired compartment size. The target probe array can be easily prepared by exchanging the probe-attached fine particles. The method of arranging these fine particles can be pinched with tweezers when the fine particles have a diameter of about 0.3 mm, but it is difficult when the fine particles are 0.1 mm or less. Therefore, a method and apparatus for manufacturing a probe array by using a sheet having fine holes, holding and carrying fine particles in the holes, and arranging them in a capillary or a groove formed in a flat plate are provided. As another method, a probe array is formed by pouring fine particles one by one into a liquid flow while controlling them and receiving the fine particles into a capillary. In this specification, the term “array” means that probe-attached fine particles are arranged one-dimensionally or two-dimensionally to form a probe array. Furthermore, in order to improve the reproducibility of measurement, a plurality of fine particles with a plurality of probes are arranged for each probe to check for variations in the inspection and the like so that highly reliable data can be obtained.

That is, the present invention provides the following (1) to (1)
8) is provided. (1) A probe array in which probes are immobilized on a plurality of fine particles for each type of probe, and a predetermined number of the fine particles for the plurality of probes are collected to form an array.

(2) A probe is immobilized on the surface of the fine particles,
A probe array in which the immobilized microparticles are arranged one-dimensionally or two-dimensionally in an order determined for each type of probe to constitute a probe array of a bio-related substance. The above-mentioned probe array, which is arranged at intervals. (3) A probe array characterized in that a plurality of fine particles are held in a probe array holder for each probe, and fine particles having different sizes are used as separation partitions in order to separate different types of probes.

(4) In a method of arranging fine particles on which various probes are immobilized in a capillary or an optical cell in an arrangement determined according to the type of the probe, a sheet having holes larger than the fine particles, Holding the capillary or relatively moving the substrate having a groove, and controlling the position of the fine particles trapped in the hole of the sheet, and sequentially transferring the fine particles to the capillary or groove. A method for producing a probe array.

(5) A step of dispersing fine particles on which a single type of probe is immobilized on a sheet with holes to drop the fine particles into the holes, a step of removing excess fine particles that did not enter the holes, The method of producing a probe array according to the above (4), wherein the step of transferring the held fine particles to a capillary or a groove is repeated.

(6) A plurality of fine particles each having a plurality of different probes immobilized thereon are held in different holes on the sheet,
The method for producing a probe array according to the above (4), wherein the probe array is transferred to a probe array holder or a capillary connected to the probe array holder in an order determined for each type.

(7) In a method of arranging microparticles having a probe immobilized in a capillary or an optical cell in an arrangement determined according to the type of probe, the microparticles are held in an introduction capillary, and the solution is controlled while controlling the microparticles one by one together with the solution. A method of producing a probe array by releasing various kinds of microparticles with probes by releasing them into a flow and introducing them into a capillary tube in a predetermined order.

(8) The fine particles on which the probe is immobilized are divided and held in different sections for each type of probe, and the fine particles are held in holes formed on the bottom surface of the section, and are inserted into grooves dug in another substrate plane. A method of producing a probe array by dropping and arranging probe-attached microparticles in a predetermined order, and then separately transporting the particles to a capillary, a groove, or an optical cell and at the same time densely spacing.

(9) An apparatus for producing a probe array, comprising: means for immobilizing probes on a solid surface; and means for arranging probes in a predetermined order. (10) In a probe array manufacturing apparatus in which fine particles on which various probes are immobilized are arranged in a capillary or an optical cell in an array determined according to the type of the probe, a sheet having a hole larger than the fine particles and a sheet contacting the sheet. Means for holding the capillary or relatively moving the substrate having the groove, and controlling the position of the fine particles trapped in the hole of the sheet, and sequentially transferring the fine particles to the capillary or the groove. Means for producing a probe array.

(11) A means for spraying fine particles having a single type of probe immobilized on a perforated sheet to drop the fine particles into a hole portion, a means for removing excess fine particles not entering the hole, Means for transferring the held fine particles to a capillary or a groove, the apparatus for producing a probe array according to (10) above. (12) A means for holding a plurality of fine particles each having a plurality of different probes immobilized thereon in different holes on a sheet, and a means for transferring to a probe array holder or a capillary connected to the probe array holder in an order determined for each type. Wherein (1)
An apparatus for producing a probe array according to 0).

(13) In an apparatus for producing a probe array in which microparticles on which probes are immobilized are arranged in a capillary or an optical cell in an arrangement determined according to the type of probe, the microparticles are held in an introduction capillary, and the microparticles are added one by one together with a solution. Producing a probe array, comprising: means for releasing into a solution flow while controlling; means for introducing into a capillary tube; and means for holding and arraying the introduced fine particles with various probes in a predetermined order. apparatus.

(14) means for dividing and holding the fine particles on which the probe is immobilized in different sections for each type of probe;
A means for holding fine particles in a hole at the bottom surface of the compartment, a means for arranging fine particles with probes in a predetermined order by dropping the fine particles into a groove dug in another substrate plane, a capillary, a groove or an optical cell And a means for concentrating the intervals at the same time as transferring to a probe array.

(15) The probe is immobilized on the surface of the microparticles, and the immobilized microparticles are arranged one-dimensionally or two-dimensionally in the order determined for each type of probe to constitute a probe array of biologically relevant substances. An analyzer having a probe array. (16) The analyzer according to the above (15), wherein the probe array is fixed to fine particles for each type of probe, and a plurality of the fine particles are collected to form an array.

(17) Distributed in one or two dimensions,
A probe array comprising a fine particle array holder formed of cells having holes capable of holding fine particles, at least a wall defining each cell, and an optically transparent member spaced at a distance smaller than the size of the fine particles. . (18) An analyzer using the probe array according to any one of (1) to (3) and (17).

[0019]

DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will be described below with reference to embodiments. The present invention is common to probe arrays of DNA, RNA, proteins, peptides and other biologically
A will be described as an example.

The DNA probe array according to the present invention may be one-dimensionally held in a capillary tube or two-dimensionally holding a solid probe in an optical cell having a narrow gap. An example using a capillary tube will be described. Although an example using spherical beads as the fine particles will be described, rectangular or other shapes may be used.
The size of the beads can be used up to 1-300 microns. Here, an example using beads of 20 microns will be mainly described. In addition, as the material of the beads, glass or plastic can be generally used, but metal such as gold can also be used. Here, plastic was used.

Embodiment 1 FIG. 1 shows an example of a probe array according to the present invention. The diameter of the probe bead 105 holding the probe is 20 microns, and the capillary 103
Has an inner diameter of 25 microns. In this example, about 20 dummy beads 106 are arranged at both ends, and 999 beads are placed inside thereof.
Arranged. Black marker beads 104 were inserted for every ten, and the 100th one was red. Marker beads 104
Are 99, the number of probe beads 105 is 90
It becomes zero. That is, 900 types of probes can be arranged and tested. When they are densely packed, they fall within a width of 2 mm. However, considering the hybridization reaction and the like, they were kept in a 5 mm area with a rather rough density. The length of the region may be longer or shorter, but if it is too long, a large amount of sample is required. If the region is short, handling is troublesome, and a sample sufficient for sufficient hybridization may not be obtained in some cases. The volume of the reaction zone is about 2.3 nl. Stoppers are placed at both ends to prevent beads from flowing out. The sample and the washing liquid are injected from an inlet 101 and discharged from an outlet 102. Even if the number of probes is 10,000, the length of the reaction region may be 20 to 30 mm, which is advantageous in that it is compact and easy to handle.

The fluorescence detector used for this is as shown in FIG. 2, which scans a laser beam 206 emitted from a laser light source 209 via a mirror and a lens 205 and a probe array holding capillary 202 relatively. The resulting fluorescence is measured. The relative movement is performed by adjusting the mirror and / or moving the probe array moving plate 203 and irradiating the detection position 204 with a laser. Light emitted from the detection position 204 is detected by the detector 210 via the lens 205, the optical filter 207, and the lens 208, processed by the data processing and the detector controller 211, and displayed on the display device 212. In the present invention,
Marker beads are contained every ten probe beads 201, and the type of probe can be easily known in accordance with the order of each bead. The marker beads may be color-coded so that the number of the marker beads can be easily identified. Alternatively, instead of the marker beads, a bead provided with a probe may be colored so that the color changes every ten beads. In this case, it is necessary to adopt a color having a wavelength that does not hinder fluorescence detection.

Example 2 The following example relates to a method and an apparatus for arranging beads one by one in a capillary in a predetermined order. 3 and 4 show an example of a method and an apparatus for producing a bead array. For convenience of explanation, an example in which a bead array is formed in one capillary 306 will be described. However, in practice, a number of such holes and capillaries on a sheet are used. Step 1 (FIG. 3A): From the bead supply nozzle 309, the probe beads 304 with the first probe (first probe beads) are inserted into the bead capturing holes 305 together with the solvent injected from the solution injection port 302. Is introduced into a cell having a bottom surface. When the beads are settled and the liquid is moved back and forth, right and left, one of the beads falls into the bead capturing hole 305. Step 2 (FIG. 3-b): Next, the remaining beads are removed from the outlet 301 together with the solvent and washed. Only the beads 308 that have fallen into the holes 305 remain in the cell. in this case,
The solvent is spouted from the direction perpendicular to the sheet toward the hole 305 to remove beads near the hole 305, and step 3
Alternatively, the beads to be taken into the capillary may be only the beads 308 that have fallen into the holes. The bottom surface of the hole is in a state of being closed by the capillary holding substrate 307. This capillary holding substrate 307 has a capillary 3 for arrangement.
06 is fixed, but in steps 1 and 2, the capillary axis does not match the hole 305, and
In the state of being held. Step 3 (FIG. 3C): The capillary holding substrate 307 and the sheet are relatively moved to align the capillary axis with the hole 305. Capillary 30
The probe beads 1 are introduced into the capillary by applying suction from the other end of the probe 6 or applying pressure from the solution side. In this case, the movement may be about the size of the hole 305, and it is convenient to use a piezoelectric element. Step 4 (FIG. 4D): The capillary holding substrate 307 and the sheet are relatively moved so that the capillary axis is again shifted from the hole. Step 5 (FIG. 4-e): Second
(The second probe bead) is introduced into the cell, and one of the beads is dropped into the hole 305. Step 6 (FIG. 4-f): Hole 305 as in step 2
Excess beads other than the beads in the cell are removed from the cell. Step 7 (FIG. 4-g): Capillary holding substrate 307
And the sheet are moved relatively so that the capillary axis is aligned with the hole, and the second probe bead is introduced into the capillary 306. As a result, beads with the probe 1 (first probe beads) and beads with the probe 2 (second probe beads) are arranged in the capillary 306. Thereafter, the same procedure is repeated to form a probe-attached bead array with a desired sequence. 303 is a cover plate, 3
10 is a stopper.

The capillary used here may be removed and used as a probe array holder for measurement.
A separately prepared probe array holder may be attached to the lower part of the capillary, and the bead array may be transferred there and used. Examples of the probe array holder include a capillary and a groove. Here, the probe array holder shown in FIG. 5 was used. Injection port 403 at the left end in the figure
Inject sample from. After sufficient hybridization, a washing solution is introduced through the inlet 403, and an unreacted sample is removed through the outlet 402. Stopper 4
06 prevents ejection of the beads. Each probe bead 405 mounted on the measurement unit and arranged in the bead arrangement groove 404 is irradiated with laser to detect fluorescence emitted. If the upper window 407 is made of a transparent member, detection is easy. Of course, instead of irradiating a laser to obtain fluorescence, light may be emitted using a chemiluminescent reagent and the light may be received. Any detection can be used as long as the presence or absence of hybridization is known.

Here, an example in which one capillary is fixed to the capillary holding substrate has been described. However, a large number of probe arrays can be simultaneously formed using a plurality of capillaries. In this case, it is needless to say that the number of holes provided in the sheet needs to be increased according to the number of capillaries.

[Embodiment 3] In this embodiment, a solution containing a probe bead placed in a titer plate is sequentially sent to a site (capture site) having a perforated sheet in an order determined for each type, and the solution is formed into a capillary or a flat plate. This is an example of an apparatus for arranging in a dug groove (FIG. 6). Pipettor 5
01 and the probe beads 508
From the well 503 and transferred to the bead holding well 505 for transfer. The bottom of the well 505 has a hole for capturing. One of the probe beads 508 injected into the capture site (if a plurality of holes are provided, a plurality of them) falls into the holes (509). Optically confirm that beads have fallen into each hole. Next, a washing solution is flown to collect or remove excess beads. A valve 511 that can be driven by a piezoelectric element 510 or the like is provided between the bead capturing hole and the bead array arrangement groove 507 or the capillary, and by moving this, the beads can be moved to the groove 507 or the capillary side. The actual movement makes use of the flow of the liquid. The holed bead holder 504 may be moved so that the hole is aligned with the center of the groove 507 or the capillary, and the captured beads may be moved to the groove or the capillary. After the movement of the beads is completed, the valve 511 is moved or the hole and the capillary are shifted so that the beads can be captured in the hole. Pipette 5 beads with the next probe.
01 introduces into the capture site. Hereinafter, the same operation is repeated to produce a bead array. The prepared bead array is used as a probe array by moving as it is or while maintaining the arrangement in another container. Reference numeral 506 denotes an array of probe beads, and 512 denotes a holding substrate. Such an operation is performed by a system having a plurality of holes, so that the time required for manufacturing the array can be shortened, and a plurality of the same arrays can be simultaneously manufactured.

[Embodiment 4] In Embodiment 2, one cell handles one kind of probe bead at a time. However, in this example, a plurality of cells are used to separate and hold a plurality of kinds of probes. This is an example of increasing efficiency. As shown in FIG. 7, a plurality of rectangular cells 603 are arranged on a rotating disk 601. The bottom of each cell 603 is provided with a perforated sheet similar to that of the first embodiment. The lower part of the rotating disk provided with the sheet is in contact with the substrate holding the capillary so that the beads captured in the holes do not fall. Rotating disk 60
When 1 is moved so that the hole and the capillary axis coincide with each other, the probe beads are transferred into the capillary in the same manner as in the previous example. There are as many holes as there are capillaries.
The arrangement of the holes is matched to the arrangement of the capillaries, but in order to prevent deviation during rotation, the block with capillaries is moved in the direction of the rotation axis 602 of the rotation disk 601 using a tracking technology similar to CD-ROM. An adjustment mechanism is provided. In the embodiment, a rotating plate having a diameter of 16 cm was used. A cell having a width of 1 mm and a length of 30 mm is arranged at a position 5 cm from the center. The cell pitch is 2 mm and 1
About 50 cells can be made. A sheet with holes is provided on the lower surface of the cell, and the pitch of the holes is 2 mm. In this example, a probe bead array can be formed on ten capillaries at a time by arranging a total of ten holes. Of course, the number of capillaries and the number of probe arrays that can be prepared at one time can be changed as necessary. 604 is a cell 60
3 is a hole for holding beads.

The rotating plate has two modes: a high-speed rotation mode and a mode in which the plate rotates slowly but with high precision. Put the beads with the solution in the cell. Shake the disc and drain the solution through the hole, dropping the beads into the hole. Next, the mode is set to the high-speed rotation mode, and the excess beads are moved to the bead reservoir at the tip of the cell by the centrifugal force and the flow of the solution. The rotation is stopped, and the probe bead 1 is set in the high-precision rotation mode so as to match the position of the capillary. Open the shutter on the bottom,
The block holding the capillary tube is brought into contact with the rotating plate, and the beads are transferred into the capillary together with the flow of the solution. Next, the rotating plate is rotated in the high-precision mode so that the cell containing the probe beads 2 comes to the position of the capillary. Thereafter, beads are sequentially transferred to a capillary, and a probe bead array is prepared in a predetermined order. By exchanging the disk or exchanging the probe beads to be put in each cell, the same operation can be repeated to hold many probe beads while arranging them in the capillary. It is convenient to change the color of the beads to be inserted into the cell for every ten beads in confirming the position of the probe in the prepared probe bead array.

Example 5 The following example relates to a method and an apparatus for arranging probe beads one by one in a capillary in a predetermined order by using a liquid flow. FIG. 8 shows this outline. Probe beads 702
Is pumped from the bead solution reservoir 701 to the capillary via the transfer tube 703 and the sheath flow cell 704. The distal end of the capillary is placed in the flow of the transfer liquid 705 and the beads are discharged one by one from the distal end of the transfer capillary 706 into the liquid flow. The beads are released into the liquid stream at approximately constant time intervals, and in order to stabilize the beads, the capillary 707 holding the beads is subjected to ultrasonic waves so as to form nodes along the axis of the capillary. . By controlling the conditions such as the intensity of ultrasonic waves, beads are released one by one into the liquid flow at regular intervals. 708 is a holding substrate, and 709 is a solution discharge pipe.

[Example 6] In the above examples, one bead corresponds to one kind of probe. However, in order to check the variation of the hybridization reaction or to increase the detection sensitivity, a plurality of beads per one probe are used. It is convenient to correspond to The number of beads corresponding to each probe does not necessarily need to be the same. In this case, it is necessary to insert a distinction bead 803 having a different color or size between different probe beads 802. FIG. 9 shows this example. The device required for the fabrication basically corresponds to that described above, but the inner diameter of the probe holding capillary 804 is made several times larger than the size of the probe beads 802 so that a plurality of beads are trapped in the holes. . The following operation is the same as before. 801 is a dummy bead,
805 is a solution discharge pipe.

The use of the flow system described in the previous example facilitates the production of a bead array. Pipette a small amount of beads from the bead reservoir and inject into the liquid flow. The number of injected beads cannot be determined, but can be stored sequentially in the capillary. By injecting colored beads or differently sized bead 803 as an isolator before injecting another kind of beads, it is possible to identify the number of beads and what probe they have. Can be.

[Embodiment 7] The method of fabricating the probe array in the previous embodiment is an example in which the beads are arranged in a capillary. In FIG. 10, beads are arranged in grooves provided on a plane, and the beads are densely arranged. Disclosed are methods and apparatus for use as a probe array or for moving into a capillary for use as a probe array. First, a bead array manufacturing holder 905 having a plurality of grooves on a plane is prepared. The probe beads 907 are arranged in the grooves 906, and the beads are moved while keeping the arrangement in a capillary or the like to prepare a probe array. The probe beads 907 arranged and arranged in the plurality of grooves 906 have different capillaries 90, respectively.
8 and used as a probe array. A holed sheet 904 for trapping and holding the beads in the holes and moving them to the grooves 906 is placed on the bead array producing holder 905. This sheet 90 with holes
Reference numeral 4 denotes a groove (bead reservoir 902) of the narrow groove holding plate 901 formed so as to be orthogonal to the groove 906 of the holder 905 for preparing a bead array, and a hole for holding a bead formed in the bottom thereof. 903. Also having a plurality of grooves, beads with different probes are respectively injected into these holes, and different probe beads 90 for each groove are inserted into the holes.
7 is to hold. These two sheets or flat plates are used in close contact but can slide relative to each other. First, the position of the hole 903 of the perforated sheet 904 and the position of the groove 906 of the holder 905 for bead array are shifted. Probe bead 907 is inserted into holed sheet 90
The grooves 4 are supplied in a state of being classified according to the type of the probe. One probe bead 907 enters the hole, but is held here because the bottom surface is closed in this state.
When the position of the hole 903 of the perforated sheet 904 and the position of the groove 906 of the bead array producing holder 905 are aligned, beads fall into the groove 906 one by one from each hole 903. Each probe bead 907 falls into each groove 906 one by one.
6 is held. The spacing between the beads is substantially equal to the spacing between the bead reservoirs 902 of the perforated sheet 907, and is 2 mm in this example. Bead array production holder 90
The number of the probe beads 907 dropped into the five grooves 906 is 50 in this example. The number of bead arrays produced at the same time is ten, but can be increased as necessary. After the beads are dropped, the holes 903 of the perforated sheet 904 and the holder 90 for preparing the bead array are returned.
The position of the groove 906 of No. 5 is shifted so that the groove is sealed, and the beads are guided to the capillary 908 together with the solution. When the number of types of the probe beads 907 is increased, the above operation is repeated to achieve the purpose. In the above embodiment, a one-dimensionally arranged probe bead array is disclosed. However, it is needless to say that a probe array having more types of probes can be made by arranging a plurality of these or two-dimensionally arraying them. .

[Embodiment 8] This embodiment is an example in which a probe bead array holder uses a cell composed of a plate with holes and a cover glass distributed one-dimensionally or two-dimensionally. This is similar to a very small titer plate. As shown in FIG.
Using a micropipette, a small amount of beads are separated from the titer plate containing
Transfer to the first hole (cell 1003). 1002 is a spacer. A bead array resembling a microtiter plate holding probe beads is prepared by associating the type of probe attached to the bead with the position of the hole on the plate. After the beads are inserted, the cell array is covered with a cover glass 1005 that is optically transparent and does not hinder fluorescence measurement or chemiluminescence measurement. The cell array is irradiated with, for example, laser light 1008, and light emission is detected by a detector 1010 via a lens 1009. To detect. The beads are smaller than the bead size and do not move relative to each other between the cover glass 1005 and the wall that covers each cell of the cell array of the microtiter plate shape. The reaction liquid and the like are supplied to the solution outlet 1006 and the solution inlet 1
007 can move freely. In use, the cells are inverted so that the glass surface faces down. In this case, regardless of the cell depth, the beads on the glass surface come into sufficient contact with the reaction solution, and the probe can perform hybridization with the target.

[0034]

As described above, according to the present invention, a large number of peptide and DNA probe arrays can be easily prepared. By separating the process of immobilizing the probe on the solid surface and the process of arranging the probe, each process can be optimized. For this reason, a fixed probe that is uniform and hard to peel off from the solid surface can be manufactured, and an array having required types of probes can be easily manufactured by arranging the probes in a predetermined order. By reducing the size of the beads, a fine probe array, which is difficult to produce by a conventional method, can be produced. Required DN
A probe array with a new configuration can be produced simply by producing the A probe, immobilizing it on the surface of the beads, and setting it in the production device, so that what the user wants can be provided at any time. By arranging a plurality of beads having the same probe, a statistical average can be obtained, the reproducibility, the quantitative property, etc. can be checked together, and reliable measurement can be performed. Also, unlike a probe array using a DNA chip or the like that holds a plane, the surface area is large, so that the reaction can be performed more quickly and high sensitivity can be obtained. Since the size of the beads can be varied from 1 micron to 300 microns, they can be easily made even when a high-density probe array is required. For example, if 6 micron beads are used, 1500 probes can be held per 10 mm capillary, and if a two-dimensional probe array holder is used, 1 million or more probes can be arranged per square centimeter. It is very easy to simultaneously produce a plurality of arrays having the same probe sequence at the time of production, and it is suitable for mass production.

[Brief description of the drawings]

FIG. 1 is a conceptual diagram of a probe array chip composed of probe beads arranged in a capillary.

FIG. 2 is a conceptual diagram of a detection system that measures a probe bead array held in a capillary or the like.

FIG. 3 is a partial cross-sectional view of the bead arrangement device. a) It is a conceptual diagram of supply of beads in an offline state. b) It is a conceptual diagram of a state where beads are trapped in a hole. c) It is a conceptual diagram of a state where beads are transferred to a capillary or the like.

FIG. 4 is a partial cross-sectional view of the bead arrangement device. d) A diagram in which one bead is held in the capillary e) A diagram in which the second probe bead is dropped into a hole for capturing f) Removal of extra beads g) A diagram in which the captured bead is introduced into the capillary

FIG. 5 is a conceptual diagram of a groove type bead arrangement jig. a) External view b) Cross-sectional view

FIG. 6 is a conceptual diagram of a method of manufacturing a bead array using a groove and a movable valve. a) Conceptual diagram of the process of sucking beads from the reservoir of probe beads and arranging them in grooves b) Cross-sectional view of a device that captures beads one by one

FIG. 7 is a conceptual diagram of a disk-type probe bead transfer system.

FIG. 8 is a conceptual diagram of a liquid flow type bead array manufacturing method.

FIG. 9 is a conceptual diagram of a type of bead array in which a large number of probe beads are separated by divided beads.

FIG. 10 is a conceptual diagram of a probe bead arranging method using a perforated sheet.

FIG. 11 is a conceptual diagram of a microtiter plate type bead array holder. a) Conceptual sketch b) Cross-section c) Conceptual diagram at the time of measurement

[Explanation of symbols]

101: solution and sample inlet, 102: outlet, 10
3: Capillary, 104: Marker beads, 105:
Probe bead, 106: dummy bead 201: probe bead, 202: probe array holding capillary, 203: probe array moving plate, 20
4: detection position, 205: lens, 206: irradiation laser, 207: optical filter, 208: lens, 20
9: laser light source, 210: detector, 211: data processing and detector controller, 212: display device 301: outlet, 302: solution inlet, 303: cover plate, 304: probe beads, 305: hole for capturing beads 306: Capillary for arraying, 307: Capillary holding substrate, 308: Captured beads, 30
9: Bead supply nozzle, 310: Stopper 401: Probe array holder, 402: Outlet 403: Injection, 404: Bead array groove 405: Probe beads, 406: Stopper 407: Upper window 501: Pipettor, 502: Titer plate,
503: well, 504: bead holder with hole 505: bead holding well, 506: arranged probe beads, 507: groove for bead array arrangement, 508:
Probe beads, 509: Probe beads captured in holes 510: Piezoelectric element, 511: Valve, 512: Holding substrate 601: Rotating disk, 602: Rotating shaft 603: Cell, 604: Hole for holding beads 701: Bead solution reservoir, 702: probe beads,
703: transfer tube 704: sheath flow cell, 705: transfer liquid, 70
6: Capillary tube for transfer 707: Capillary, 708: Support substrate 709: Solution discharge tube 801: Dummy beads, 802: Probe beads, 80
3: beads for sorting, 804: capillaries for holding probes, 805: sample flow path 901: narrow groove holding plate, 902: bead reservoir, 903:
Hole 904: Sheet with hole, 905: Holder for making bead array, 906: Groove, 907: Probe beads,
908: Capillary 1001: Microtiter plate, 1002: Spacer, 1003: Cell, 100
4: Probe beads, 1005: Cover glass, 10
06: solution outlet, 1007: solution inlet, 100
8: laser beam, 1009: lens, 1010: detector

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/566 G01N 35/02 F 35/02 C12N 15/00 A

Claims (18)

[Claims] 1. A probe array wherein a probe is fixed to a plurality of fine particles for each type of probe, and a predetermined number of the fine particles for the plurality of probes are collected by an array to form an array. 2. The method according to claim 1, wherein a probe is immobilized on the surface of the microparticles, and the immobilized microparticles are arranged one-dimensionally or two-dimensionally in an order determined for each type of the probe to constitute a probe array of a biological substance. The probe array according to claim 1, wherein fine particles serving as markers are arranged at regular intervals. 3. A probe array characterized in that a plurality of fine particles are held in a probe array holder for each probe, and fine particles of different sizes are used as separation partitions in order to separate different types of probes. 4. A method of arranging microparticles on which various probes are immobilized in a capillary or an optical cell in an arrangement determined according to the type of probe, comprising: a sheet having holes larger than the microparticles; Or the substrate having a groove is relatively movable, the position of the fine particles trapped in the hole of the sheet is controlled and moved, and the fine particles are sequentially transferred to the capillary or the groove. , Probe array fabrication method. 5. A step of dispersing fine particles having a single type of probe immobilized on a perforated sheet and dropping them into holes, and a step of removing excess fine particles not entering the holes.
The method for producing a probe array according to claim 4, wherein the step of transferring the fine particles held in the holes to the capillaries or grooves is repeated. 6. A method in which a plurality of fine particles each having a plurality of different probes immobilized thereon are held in different holes on a sheet, and transferred to a probe array holder or a capillary connected to the probe array holder in an order determined for each type. The method for producing a probe array according to claim 4, characterized in that: 7. A method for arranging microparticles having a probe immobilized in a capillary or an optical cell in an arrangement determined according to the type of probe, wherein the microparticles are held in an introduction capillary and the solution flow is controlled while controlling the microparticles one by one together with the solution. A method of producing a probe array by releasing various probe-attached microparticles in a predetermined order by releasing them into a capillary tube. 8. The fine particles on which the probe is immobilized are divided and held in different sections for each type of probe, the fine particles are held in holes at the bottom of the section, and dropped into grooves dug in another substrate plane. In this method, the probe-attached microparticles are arranged in a predetermined order, and then are separately transferred to a capillary, a groove, or an optical cell, and at the same time, the intervals are densely formed to produce a probe array. 9. An apparatus for producing a probe array, comprising: means for immobilizing probes on a solid surface; and means for arranging probes in a predetermined order. 10. A probe array manufacturing apparatus for arranging fine particles on which various probes are immobilized in a capillary or an optical cell in an array determined according to the type of probe, a sheet having a hole larger than the fine particles, A means for contacting and holding the capillary or relatively moving the substrate having the groove, and controlling and moving the position of the fine particle trapped in the hole of the sheet, and sequentially moving the fine particle into the capillary or the groove. And means for transferring the probe array. 11. A means for spraying fine particles on which a single type of probe is immobilized on a sheet with holes to drop the fine particles into the holes, means for removing excess fine particles not entering the holes, and holding the fine particles in the holes. The apparatus for producing a probe array according to claim 10, further comprising means for transferring the fine particles to a capillary or a groove. 12. A means for holding a plurality of fine particles each having a plurality of different probes immobilized thereon in different holes on a sheet, and transferring the particles to a probe array holder or a capillary connected to the probe array holder in an order determined for each type. The apparatus for producing a probe array according to claim 10, further comprising: 13. An apparatus for producing a probe array in which microparticles on which probes are immobilized are arranged in a capillary or an optical cell in an arrangement determined according to the type of the probe, the microparticles are held in an introduction capillary, and the microparticles are controlled one by one together with a solution. A device for producing a probe array, comprising: means for discharging the solution into the solution flow, means for introducing the solution into the capillary tube, and means for arranging and holding the introduced fine particles with various probes in a predetermined order. . 14. A means for dividing and holding fine particles on which a probe is immobilized in different sections for each type of probe, a means for holding fine particles in a hole in the bottom surface of the section, and a means for digging the fine particles into another substrate plane. Means for arranging the probed microparticles in a predetermined order by dropping them into the groove,
Means for transferring to a capillary, a groove, or an optical cell and, at the same time, increasing the distance between the cells. 15. A probe array of biological substances, wherein a probe is immobilized on the surface of a microparticle, and the immobilized microparticle is arranged one-dimensionally or two-dimensionally in an order determined for each type of probe. An analyzer having a probe array that performs the analysis. 16. The analyzer according to claim 15, wherein the probe array is formed by immobilizing fine particles for each type of probe and collecting a plurality of the fine particles to form an array. 17. One or two-dimensionally distributed cells having holes capable of holding fine particles, at least a wall defining each cell, and an optically transparent space smaller than the size of the fine particles. Probe array consisting of a fine particle array holder formed with various members. 18. An analyzer using the probe array according to any one of claims 1 to 3, and 17.