JP3746658B2 - Method and apparatus for producing probe array using fine particles - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a probe array used for detection and diagnosis of biological substances such as peptides, proteins, DNA and RNA, and analysis of biological substances such as DNA, and a method and apparatus for producing the same.
[0002]
[Prior art]
DNA analysis, DNA testing or diagnosis requires a method for amplifying a small amount of DNA, a method for separating and detecting the obtained DNA fragment, and the like. PCR (polymerase chain reaction) is widely used for amplification of DNA. According to this, even a very small amount of DNA can be detected by increasing its copy number by several orders of magnitude. On the other hand, DNA sequencers and fragment analyzers that combine gel electrophoresis and fluorescence detection are used for separation and detection of various DNAs. However, when the number of specimens increases or the number of examination items increases, there is a difficulty in the method using electrophoresis, and a simple method using a DNA probe has begun to attract attention. In particular, a DNA chip that produces a probe array in which multiple types of probes are immobilized on a solid surface, and causes hybridization with a specimen to trap and detect only specific DNA on the solid surface is beginning to attract attention (Nature) Medicine 2, 753 (1996)).
On the other hand, probe detection is also used for analysis of proteins and peptides, or various biological substances that interact with these, and peptide chips corresponding to DNA chips have begun to be used.
[0003]
In this way, peptide or DNA is immobilized on a solid surface and hybridization is performed with a sample to separate and detect. The blotting method uses a radioactive label and probes for the presence of target DNA and the like using a probe immobilized on a membrane. Has been used for a long time. However, small areas of solid surfaces such as glass or silicon (1 cm ² The DNA chip on which many probes are fixed has the advantage that the sample can be saved and at the same time it can be tested using a very large number of types of probes. There are roughly two methods for producing these DNA chips. The first is a method of synthesizing a DNA probe into a narrow, solid area (0.05mm-0.2mm square) by photochemical reaction one base at a time using a photomask method used in semiconductors (Science 251, 767 (1991)). The second is a method in which synthesized DNA, PCR-amplified DNA, or DNA obtained by cloning is immobilized in a small compartment 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 with the necessary probes can be made relatively easily, and is used by many venture companies.
[0004]
[Problems to be solved by the invention]
Probe tips for biological materials such as DNA are a promising testing technique, but for practical use 1) To make small quantities of various products at low cost, 2) To have good uniformity of probe fixation, and 3) To reproduce data It must be good in nature, can be used repeatedly, and 4) can be heated to remove non-specific adsorption. However, since the probe is fixed on the solid surface as a droplet, it varies from section to section, and it takes time to make it, so the positioning of the probe and the fixing to the solid surface are performed at the same time. There are problems such as the fact that the probe fixing section cannot be made very fine and the uniformity of the probe is not good. In addition, many of these are fixed by adsorption or the like, have a weak binding force with a solid, and the probe may be peeled off from the surface by heating.
[0005]
[Means for Solving the Problems]
In order to solve the above-mentioned problem, the probe was fixed to the solid surface and the probe arrangement was made a separate process. 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 nonspecific adsorption by heating. Using a fine particle as a solid for fixing the probe, and arranging them, a probe array having a desired compartment size is prepared. The target probe array can be easily produced by exchanging the arranged fine particles with the probe. The method of arranging these fine particles can be arranged by pinching with tweezers when the fine particles are about 0.3 mm in diameter, but it is difficult when the particle size is 0.1 mm or less. Therefore, a method and apparatus for producing 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 provided on a flat plate are provided. As another method, a probe array is formed by flowing fine particles one by one into a liquid flow while receiving them in a capillary. In this specification, “to make an array” means to form a probe array by arranging fine particles with probes in one or two dimensions. In addition, in order to improve the reproducibility of measurement, etc., a plurality of fine particles with probes are arranged for each probe to check inspection variations and so on to obtain highly reliable data.
[0006]
That is, the present invention provides the following (1) to (18).
(1) A probe array, wherein probes are fixed to a plurality of fine particles for each type of probe, and a predetermined number of the fine particles for a plurality of probes are collected to form an array.
[0007]
(2) Probes that immobilize probes on the surface of microparticles and arrange the immobilized microparticles one-dimensionally or two-dimensionally in an order determined for each type of probe to constitute a probe array of biologically relevant substances The probe array according to claim 1, wherein fine particles serving as markers are arranged at regular intervals.
(3) A probe array in which 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 distinguish different types of probes.
[0008]
(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 probe, a sheet having a hole larger than the fine particles, and the sheet is contacted to hold the capillary Or a probe capable of moving relative to a substrate having a groove, controlling the position of fine particles trapped in a hole in a sheet, and sequentially transferring the fine particles to the capillary or groove. Array fabrication method.
[0009]
(5) A step of spraying fine particles on which a single type of probe is immobilized on a holed sheet and dropping it into the hole portion, a step of removing excess fine particles that did not enter the hole, and a hole portion The method for producing a probe array as described in (4) above, wherein the step of transferring fine particles to a capillary or a groove is repeated.
[0010]
(6) A plurality of microparticles each immobilizing a plurality of different probes are held in different holes on the sheet, and transferred to a probe array holder or a capillary connected to the probe array holder in a predetermined order for each type. The method for producing a probe array according to (4) above.
[0011]
(7) In a method in which fine particles with immobilized probes are arranged in a capillary or an optical cell in an arrangement determined according to the type of probe, the fine particles are held in an introduction capillary and are controlled in the solution flow while controlling the fine particles one by one together with the solution. A method of producing a probe array by releasing and introducing into a capillary tube and holding various kinds of microparticles with probes in a predetermined order.
[0012]
(8) By finely holding the fine particles on which the probes are immobilized in different compartments for each type of probe, holding the fine particles in holes in the bottom of the compartments, and dropping them into a groove dug in another substrate plane A method of producing a probe array by arranging fine particles with probes in a predetermined order and then separately transferring them to capillaries, grooves or optical cells and at the same time concentrating the intervals.
[0013]
(9) An apparatus for producing a probe array, comprising independently 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 arrangement determined according to the type of probe, a sheet having a hole larger than the fine particles, and a contact with the sheet The means for holding the capillary or relatively moving the substrate having the groove and the position of the fine particle trapped in the hole of the sheet are controlled and moved, and the fine particle is sequentially transferred to the capillary or the groove. And a probe array manufacturing apparatus.
[0014]
(11) A means for spraying fine particles on which a single type of probe is immobilized on a holed sheet and dropping it into the hole part, a means for removing excess fine particles not entering the hole, and a hole part. The probe array manufacturing apparatus according to (10), further including means for transferring fine particles to a capillary or a groove.
(12) Means for holding a plurality of microparticles each immobilizing a plurality of different probes in different holes on the sheet, and means for transferring them to a probe array holder or a capillary connected to the probe array holder in the order determined for each type The probe array manufacturing apparatus according to (10) above, characterized by comprising:
[0015]
(13) In a probe array manufacturing apparatus in which fine particles with immobilized probes are arranged in a capillary or an optical cell in an array determined according to the type of probe, the fine particles are held in an introduction capillary, and the fine particles are controlled one by one together with the solution. An apparatus for producing a probe array, characterized by comprising means for discharging into a solution flow, means for introducing into a capillary tube, and means for holding the introduced microparticles with various probes arranged in a predetermined order.
[0016]
(14) Means for dividing and holding fine particles on which probes are immobilized in different sections for each type of probe, means for holding fine particles in holes in the bottom surface of the sections, and grooves dug in another substrate plane An apparatus for producing a probe array, comprising: means for arranging fine particles with a probe in a predetermined order by dropping into a tube; and means for densely spacing the gaps while transferring them to a capillary, a groove or an optical cell.
[0017]
(15) A probe that is immobilized on the surface of a fine particle, and the immobilized fine particle is arranged one-dimensionally or two-dimensionally in an order determined for each type of probe to constitute a probe array of biologically relevant substances. An analyzer with an array.
(16) The analyzer according to (15) above, 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.
[0018]
(17) One-dimensional or two-dimensionally distributed cells having holes capable of holding fine particles, walls defining at least each cell, and optically transparent members spaced at a distance smaller than the size of the fine particles Probe array consisting of a fine particle array holder.
(18) An analyzer using the probe array according to any one of (1) to (3) and (17).
[0019]
DETAILED DESCRIPTION OF THE INVENTION
The present invention will be described below with reference to examples. The present invention is common to probe arrays of DNA, RNA, proteins, peptides and other biological substances, but here, DNA will be described as an example.
[0020]
The DNA probe array according to the present invention may be held one-dimensionally in a capillary tube or may be held two-dimensionally in a narrow gap optical cell. This will be described using the example used. Although an example using spherical beads as the fine particles will be described, a rectangular or other shape may be used. The bead size can be used up to 1-300 microns, but here, an example using 20-micron beads will be mainly described. In addition, glass or plastic can be normally used as the material of the beads, but metal such as gold can also be used. Here, plastic was used.
[0021]
[Example 1]
FIG. 1 is an example of a probe array according to the present invention. The probe beads 105 holding the probe have a diameter of 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 arranged inside thereof. Black marker beads 104 were inserted every 10 pieces, and the 100th piece was red. Since the number of marker beads 104 is 99, the number of probe beads 105 is 900. In other words, 900 types of probes can be arranged and inspected. When these are densely packed, they fall within a width of 2 mm, but considering the hybridization reaction, etc., they were held in a region of 5 mm with a somewhat 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, and if it is short, handling is troublesome, and a sample sufficient for sufficient hybridization may not be secured. 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 cleaning liquid are injected from the inlet 101 and discharged from the outlet 102. Even if the number of probes is 10,000, the length of the reaction region may be 20-30 mm, which has the advantage of being compact and easy to handle.
[0022]
The fluorescence detection apparatus used for this is as shown in FIG. 2 and is obtained by relatively scanning the laser beam 206 irradiated from the laser light source 209 via the mirror and lens 205 and the probe array holding capillary 202. Measure fluorescence. The relative movement is performed by adjusting the mirror and / or the probe array moving plate 203, and the detection position 204 is irradiated with the 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 detector controller 211, and displayed on the display device 212. In the present invention, every 10 probe beads 201 contain marker beads, and the type of probe can be easily known according to the order of each bead. The marker beads may be color-coded for easy identification of the number, or the beads with probes instead of the marker beads may be colored so that the color changes every ten. In this case, it is necessary to employ a color having a wavelength that does not hinder fluorescence detection.
[0023]
[Example 2]
The following examples relate to a method and apparatus for arranging beads one by one in a capillary in a defined order. 3 and 4 show an example of a bead array manufacturing method and apparatus. For convenience of explanation, an example in which a bead array is formed on one capillary 306 will be described. However, in practice, a material in which a large number of holes and capillaries on a sheet are arranged is used. Step 1 (FIG. 3-a): Probe beads 304 (first probe beads) with a first probe are attached to a bead capturing hole 305 together with a solvent injected from a solution injection port 302 from a bead supply nozzle 309. It introduce | transduces in the cell which has a sheet | seat which has in a bottom face. When the beads are settled and the liquid is moved back and forth and left and right, one of the beads falls into the bead capturing hole 305. Step 2 (FIG. 3-b): The remaining beads are then removed from the outlet 301 with the solvent and washed. Only the beads 308 that fall into the holes 305 remain in the cell. In this case, the solvent is ejected from the direction perpendicular to the sheet toward the hole 305, the beads near the hole 305 are removed, and the beads taken into the capillaries in step 3 become only the beads 308 that have fallen into the holes. May be. The bottom surface of the hole is covered with the capillary holding substrate 307. An arraying capillary 306 is fixed to the capillary holding substrate 307. However, in steps 1 and 2, the capillary axis and the hole 305 do not coincide with each other, and the beads are held in the hole 305. Step 3 (FIG. 3-c): The capillary holding substrate 307 and the sheet are relatively moved so that the capillary axis and the hole 305 are aligned. The probe bead 1 is introduced into the capillary by applying suction from the other end of the capillary 306 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 pressure transmission element. Step 4 (FIG. 4-d): The capillary holding substrate 307 and the sheet are moved relative to each other so that the capillary axis is displaced from the hole again. Step 5 (FIG. 4-e): The beads with the second probe (second probe beads) are introduced into the cell, and one of the beads is dropped into the hole 305. Step 6 (FIG. 4-f): Excess beads other than the beads in the hole 305 are removed from the cell as in Step 2. Step 7 (FIG. 4-g): The capillary holding substrate 307 and the sheet are moved relative to each other so that the capillary axis matches the hole, and the second probe bead is introduced into the capillary 306. As a result, in the capillary 306, beads with a probe 1 (first probe beads) and beads with a probe 2 (second probe beads) are arranged. Repeat the same process to create a bead array with probes in the desired sequence. Reference numeral 303 denotes a cover plate, and 310 denotes a stopper.
[0024]
The capillary used here may be removed and used as a probe array holder at the time of measurement. Alternatively, a separately prepared probe array holder may be attached to the lower part of the capillary, and the bead array may be transferred there. Examples of the probe array holder include a capillary or a groove. Here, the probe array holder shown in FIG. 5 was used. A sample is injected from the inlet 403 at the left end in the figure. After sufficient hybridization, a washing solution is introduced from the inlet 403 and unreacted sample is removed from the outlet 402. The stopper 406 prevents the beads from being discharged. The fluorescence emitted by irradiating a laser beam to each probe bead 405 that is mounted on the measurement unit and arranged in the groove for bead arrangement 404 is detected. 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. The detection may be anything as long as the presence or absence of hybridization is known.
[0025]
Although an example in which one capillary is fixed to a capillary holding substrate has been described here, a large number of probe arrays can be simultaneously formed using a plurality of capillaries. In this case, needless to say, it is necessary to increase the number of holes provided in the sheet according to the number of capillaries.
[0026]
[Example 3]
In this example, a solution containing probe beads placed in a titer plate is sequentially fed to a site (capturing site) with a sheet with holes in the order determined for each type, and arranged in a groove dug in a capillary or a flat plate. (Fig. 6). The probe beads 508 are sucked from the wells 503 of the titer plate 502 with the pipetter 501 and transferred to the bead holding wells 505 for transfer. The bottom surface of the well 505 has a capturing hole. One of the probe beads 508 injected into the capture site (a plurality when there are a plurality of holes) falls into the hole (509). Optically confirm that the beads have fallen into each hole. Next, a washing solution is poured to recover 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 arranging groove 507 or the capillary, and by moving this, the bead can be moved to the groove 507 or the capillary side. The actual movement uses liquid flow. The bead holder 504 with a hole may be moved so that the hole and the center of the groove 507 or the capillary coincide with each other, and the captured beads may be moved to the groove or the capillary. Move the valve 511 after the bead movement is completed, or shift the hole and capillary so that the bead can be captured in the hole. The next probe-attached bead is introduced into the capture site by the pipetter 501. Thereafter, the same process is repeated to prepare a bead array. The produced bead array is moved as it is or in a state where the arrangement is maintained in another container and used as a probe array. Reference numeral 506 denotes an arrayed probe bead, and 512 denotes a holding substrate.
Such an operation can be performed by a system having a plurality of holes, so that the time required for array fabrication can be shortened or a plurality of identical arrays can be fabricated simultaneously.
[0027]
[Example 4]
In Example 2, one cell was used at a time, and one type of probe bead was handled at a time. However, in this example, a plurality of types of probes are divided and held, and the production efficiency is increased. As shown in FIG. 7, a plurality of rectangular cells 603 are arranged on the rotating disk 601. The bottom surface of each cell 603 is provided with the same sheet with holes as in 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. When the rotating disk 601 is moved so that the hole and the capillary axis coincide with each other, the probe beads are transferred into the capillary as in the previous example. There are as many holes as there are capillaries. The hole arrangement is matched to the capillary arrangement, but in order to prevent deviation during rotational movement, the block with capillary is moved in the direction of the rotation axis 602 of the rotary disk 601 using a tracking technique similar to that of the CD-ROM. A mechanism for adjusting is provided. In the examples, 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 distance of 5 cm from the center. The cell pitch is 2 mm, and about 150 cells can be made on the circumference. A sheet with holes is stretched on the lower surface of the cell, and the pitch of the holes is 2 mm. In this example, a total of 10 holes can be arranged to make a probe bead array on 10 capillaries at a time. Of course, the number of capillaries and the number of probe arrays that can be fabricated at a time can be changed as necessary. Reference numeral 604 denotes a bead holding hole provided in the cell 603.
[0028]
The rotating plate has two modes: a high-speed rotation mode and a mode that rotates slowly but with high accuracy. Place the beads with the solution in the cell. Shake the disc and drain the solution from the hole, dropping the beads into the hole. Next, a high-speed rotation mode is set, and surplus beads are moved to the bead reservoir at the cell tip by centrifugal force and the flow of the solution. The rotation is stopped and the probe bead 1 is set in the high-accuracy rotation mode so that it is in the position of the capillary. The shutter at the bottom is opened, the block holding the capillary tube is brought into contact with the rotating plate, and the beads are transferred into the capillary along with the flow of the solution. Next, the rotating plate is rotated in the high accuracy mode so that the cell including the probe beads 2 comes to the position of the capillary. Thereafter, the beads are sequentially transferred to the capillary, and a probe bead array is prepared in a predetermined order. Many probe beads can be held while being placed in the capillary by exchanging the disk or changing the probe beads in each cell and repeating the same thing. It is convenient to confirm the position of the probe in the produced probe bead array if the color of the beads to be put in the cell is changed every ten.
[0029]
[Example 5]
The following example relates to a method and apparatus for arranging probe beads one by one in a defined order using a liquid flow. FIG. 8 shows this outline. The probe beads 702 are transported from the bead solution reservoir 701 to the capillaries by a pump via the transfer pipe 703 and the sheath flow cell 704. The tip of the capillary is placed in the flow of the transfer liquid 705, and beads are discharged from the tip of the transfer capillary 706 one by one into the liquid flow. The beads are released into the liquid stream at a substantially constant time interval, and in order to stabilize the beads, the capillary 707 holding the beads is ultrasonically applied so that nodes can be formed along the capillary axis. . By controlling conditions such as the intensity of ultrasonic waves, beads are released one by one into the liquid flow at regular intervals. Reference numeral 708 denotes a holding substrate, and 709 denotes a solution discharge pipe.
[0030]
[Example 6]
In the examples so far, one bead is associated with one type of probe, but it is convenient to associate a plurality of beads with one probe in order to see variations in the hybridization reaction and increase detection sensitivity. The number of corresponding beads in each probe is not necessarily the same. In this case, it is necessary to put a sorting bead 803 having a different color or size as a mark between different probe beads 802. FIG. 9 is an example of this. The apparatus required for the production basically matches the above-mentioned one, but the inner diameter of the probe holding capillary 804 is made several times larger than the size of the probe bead 802 so that a plurality of beads are trapped in the hole. . The following operations are the same as before. Reference numeral 801 denotes dummy beads, and 805 denotes a solution discharge pipe.
[0031]
Note that 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 sequentially stored in the capillary. Identify different probes and what type of beads are of interest by injecting colored beads or different size beads 803 as isolators before injecting different types of beads Can do.
[0032]
[Example 7]
The probe array manufacturing method of the previous example was an example of arranging in a capillary, but in FIG. 10, beads are arranged in a groove provided on a plane and the beads are densely used as a probe array. A method and apparatus for moving in and using as a probe array is disclosed. First, a bead array production 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 maintaining the arrangement in the capillaries or the like to produce a probe array. The probe beads 907 arranged in a plurality of grooves 906 are introduced into different capillaries 908, respectively. Used as a probe array. On the bead array production holder 905, a sheet 904 with a hole for trapping and holding the bead in the hole and moving it to the groove 906 is placed. As shown in FIG. 10, the sheet 904 with a hole has a groove (bead reservoir 902) of a narrow groove holding plate 901 made so as to be perpendicular to the groove 906 of the bead array production holder 905 and a bead opened at the bottom thereof. A holding hole 903 is provided. Although there are still a plurality of grooves, beads with different probes are injected into these grooves, and the different probe beads 907 for each groove are held in the holes. These two sheets or flat plates are used in close contact with each other, but can slide on each other. First, the positions of the holes 903 of the sheet 904 with holes and the grooves 906 of the holder 905 for producing the bead array are shifted. The probe beads 907 are replenished in the state where the probe beads 907 are divided into the respective grooves of the sheet 904 with a hole for each type of probe. One probe bead 907 enters the hole, but in this state, the bottom surface is closed and is held here. When the positions of the holes 903 of the sheet 904 with holes and the grooves 906 of the holder 905 for preparing the bead array are aligned, the beads fall into the grooves 906 one by one from the respective holes 903. Since each probe bead 907 falls into each groove 906 one by one, various probe beads 907 are held in each groove 906. The interval at which the beads are placed is substantially the same as the interval between the bead reservoirs 902 of the sheet 907 with a hole, and is 2 mm in this example. The total number of probe beads 907 dropped into the groove 906 of the bead array production holder 905 is 50 in this example. In addition, the number of bead arrays produced at the same time is 10, but can be increased as necessary. When the beads are dropped, the positions of the holes 903 of the sheet 904 with holes and the grooves 906 of the holder 905 for making the bead array are shifted again to make the grooves sealed, and the beads are guided to the capillary 908 together with the solution. When the number of types of probe beads 907 is increased, the above operation is repeated.
In the above embodiment, the probe bead array arranged in one dimension is disclosed, but it goes without saying that a probe array having more kinds of probes can be made by arranging a plurality of these or arranging them in two dimensions. .
[0033]
[Example 8]
In this embodiment, the probe bead array holder uses a cell composed of a plate with a hole and a cover glass distributed one-dimensionally or two-dimensionally. This is similar to a very small titer plate. As shown in FIG. 11, beads are collected from a titer plate containing probe beads 1004 using a small amount of micropipette and transferred to a hole (cell 1003) of the microtiter plate 1001. Reference numeral 1002 denotes a spacer. A bead array similar to a microtiter plate holding probe beads is made by matching the types of probes attached to the beads with the positions of holes on the plate. After the beads are inserted, a cover glass 1005 that is optically transparent and has no hindrance to fluorescence measurement or chemiluminescence measurement is covered to form a cell array. To detect. Between the cover glass 1005 and the wall defining each cell of the microtiter plate cell array, the beads are smaller than the bead size and the beads do not move relative to each other. The reaction solution and the like can freely move from the solution discharge port 1006 and the solution injection port 1007. In use, the cell is inverted so that the glass surface is on the bottom. In this case, regardless of the depth of the cell, the beads on the glass surface are in sufficient contact with the reaction solution, and the probe can hybridize with the target.
[0034]
【The invention's effect】
As described above, according to the present invention, peptide and DNA probe arrays can be easily produced in large quantities. By separating the process of fixing the probe to the solid surface and the process of arranging the probe, each can be optimized. For this reason, it is possible to make a fixed probe that is uniform and difficult to peel off from the solid surface, and it is possible to easily make an array having necessary types of probes by arranging them in a predetermined order. By reducing the size of the beads, it is possible to produce a fine probe array that is difficult to produce by conventional methods. By creating the necessary DNA probe, immobilizing it on the bead surface, and setting it on the production device, a probe array with a new configuration can be produced, so the user's desire can be provided at any time. By arranging a plurality of beads having the same probe, a statistical average can be obtained, and reproducibility, quantitativeness, etc. can be examined together, and a reliable measurement can be performed. In addition, the surface area is large, unlike a probe array using a flat-held DNA chip or the like, and the reaction can be carried out more rapidly and high sensitivity can be obtained. The bead size can be varied from 1 micron to 300 microns, so it can be easily made 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 produce a plurality of arrays having the same probe sequence at the same time, which 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 for measuring a probe bead array held in a capillary or the like.
FIG. 3 is a partial cross-sectional view of a bead arranging device.
a) Conceptual diagram of beads supply in an off-line state.
b) It is a conceptual diagram of the state which trapped the bead in the hole.
c) It is a conceptual diagram of the state which transfers a bead to a capillary etc.
FIG. 4 is a partial cross-sectional view of a bead arranging device.
d) Diagram with one bead held in a capillary
e) Diagram of the second probe bead dropped into the hole to capture it
f) Removal of excess beads
g) Diagram of introducing captured beads into a capillary
FIG. 5 is a conceptual diagram of a groove type bead arranging jig.
a) External view
b) Sectional view
FIG. 6 is a conceptual diagram of a bead array manufacturing method 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 the groove
b) Cross-sectional view of 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 segment beads.
FIG. 10 is a conceptual diagram of a probe bead arranging method using a sheet with holes.
FIG. 11 is a conceptual diagram of a microtiter plate type bead array holder. a) Conceptual sketch
b) Sectional view
c) Conceptual diagram during measurement
[Explanation of symbols]
101: solution and sample inlet, 102: outlet, 103: capillary, 104: marker beads, 105: probe beads, 106: dummy beads
201: Probe beads, 202: Probe array holding capillaries, 203: Probe array moving plate, 204: Detection position, 205: Lens, 206: Irradiation laser, 207: Optical filter, 208: Lens, 209: Laser light source,
210: Detector, 211: Data processing and detector controller, 212: Display device
301: Discharge port 302: Solution injection port 303: Cover plate
304: Probe beads, 305: Bead capture holes, 306: Capillaries for arrangement, 307: Capillary holding substrate, 308: Captured beads,
309: Nozzle for supplying beads, 310: Stopper
401: Probe array holder 402: Discharge port
403: injection port 404: groove for bead arrangement
405: Probe beads, 406: Stopper
407: Upper window
501: Pipetter, 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: Bead holding hole
701: Bead solution reservoir, 702: Probe beads, 703: Transfer tube
704: Sheath flow cell, 705: Transfer liquid, 706: Capillary tube for transfer
707: Capillary, 708: Support substrate
709: Solution discharge pipe
801: Dummy beads, 802: Probe beads, 803: Sorting beads, 804: Probe holding capillaries, 805: Sample flow path
901: Fine groove holding plate, 902: Bead reservoir, 903: Hole
904: Sheet with hole, 905: Holder for preparing bead array, 906: Groove, 907: Probe bead, 908: Capillary
1001: microtiter plate,
1002: Spacer, 1003: Cell, 1004: Probe beads, 1005: Cover glass, 1006: Solution outlet, 1007: Solution inlet,
1008: Laser light, 1009: Lens, 1010: Detector

Claims (9)

  A probe array in which immobilized microparticles having probes immobilized on their surfaces are arranged in a predetermined order for each type of probe, and microparticles serving as markers are arranged at regular intervals between the predetermined orders. .   2. The probe array according to claim 1, wherein the fine particles serving as the marker are colored beads.   A probe array characterized in that a plurality of fine particles having probes immobilized on their surfaces are classified and held for each type of probe using fine particles having different sizes as separation partitions.   Fine particles on which various probes are immobilized are captured using a sheet having holes, the substrate or capillary having grooves and the sheet are relatively moved, and the fine particles captured in the holes are transferred to the grooves or capillaries. A method for producing a probe array, comprising transporting and arranging the fine particles in an array determined according to a type of the probe.   The method includes a step of supplying a plurality of fine particles on which one kind of the probe is immobilized onto the sheet and transferring the fine particles to the hole, and a step of removing the fine particles that have not entered the hole. Item 5. A method for producing a probe array according to Item 4. A step of holding fine particles with immobilized probes in different compartments for each type of probe, a step of holding the fine particles in holes provided in the wall surfaces of the compartments, and providing fine particles held in the holes on a substrate And a step of transferring the fine particles to a groove and arranging the fine particles in a predetermined order. A sheet having a hole for capturing fine particles having various probes immobilized thereon;
A substrate or capillary having a groove for containing the fine particles;
A probe array manufacturing apparatus comprising: a substrate having the groove or means for relatively moving the capillary and the sheet. 8. The method according to claim 7, further comprising: means for supplying a plurality of fine particles on which one kind of the probe is immobilized onto the sheet; and means for removing the fine particles that have not entered the hole. Probe array production equipment. A container provided with a plurality of compartments each having a hole provided in the wall surface and holding the fine particles to which the probe is immobilized for each type of the probe;
A fine particle holding container which is provided with a hole in a wall surface and stores the fine particle transferred from the container;
A substrate or a capillary having a groove for arranging and storing the fine particles in a predetermined order;
The probe array manufacturing apparatus, wherein the holes capture the fine particles, and the fine particles captured in the holes are transferred to the grooves or the capillaries and arranged in the order.

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