CN1245520C - Method for producing probe arrays for biological materials using fine particles - Google Patents

Abstract

The use of probe arrays, in which probes of various biological substances, such as DNA, are immobilized on a solid surface, is gaining acceptance as an efficient tool for high-speed screening. Instead of immobilizing a single solid surface, different types of probes, such as DNA, are immobilized on the surface of a large number of independently processable fine particles, such as beads, and the resulting beads are arranged in a specified order within a capillary or unit. The size of the area where a probe is immobilized is reduced. This bead probe array is characterized by the use of a sheet with cavities, in which small beads are arranged one after another in a specified order, and one or more beads are contained within these cavities, and subsequently transported to a probe array device such as a capillary.

Description

Method for producing probe arrays of biological materials using fine particles

Background of the invention

field of invention

The present invention relates to probe arrays for use in detection of peptides, proteins and DNA, diagnosis and analysis of biological materials including DNA; and methods and apparatus for producing the same probe arrays.

Description of related technologies

For DNA analysis or DNA testing or diagnosis, amplification of small amounts of DNA, isolation and identification of amplified DNA fragments, and other processes are necessary. PCR (Polymerase Chain Reaction) is widely used for DNA amplification, in which extremely small amounts of DNA can be amplified by several orders of magnitude so that they can be detected. On the other hand, for the separation and detection of various DNAs, among other methods, a DNA sequencer and a fragment analyzer are used in which gel electrophoresis and fluorescence detection are combined. However, electrophoresis becomes very labor intensive as the number of samples or test items increases. Therefore, a simple method using DNA probes is attracting attention, especially a DNA chip in which many kinds of probes are immobilized on a solid surface to create a method for hybridization with a sample, after which only specific The DNA is captured on the surface of this solid and detected by the probe array (Nature Medicine 2,753,1996).

This probe detection method is also used for the analysis of proteins or polypeptides or various biomaterials interacting with them, and a peptide chip corresponding to a DNA chip is now being used. The method of separation and detection, in which a peptide or DNA is immobilized on a solid surface and hybridization is performed between the peptide or DNA and a sample, has long been called a blotting method, in which the target DNA or the like is detected using a radiolabeled probe that is immobilized on the membrane. However, a large number of probes can be immobilized on a small area (1 cm ² ) on the surface of a solid such as glass or siloxane on a DNA chip, which requires only a small amount of sample and can be simultaneously Advantages of using a large number of different probes. Methods for producing DNA chips are roughly divided into two groups. In the first group, DNA probes are synthesized on a substrate, each time by a photochemical reaction on small segments (0.05 mm ² to 0.2 mm ² ) of a solid, using the same or something of the like synthesized by optical masking techniques (Science 251, 767, 1991). In the second group, for each segment of individual probes, a synthetic DNA, PCR-amplified DNA, or DNA obtained by cloning was immobilized on a small segment of a solid surface ( Nature Biotech 16, 27, 1998). The latter has the advantage that peptide chips or DNA chips with the required probes can be fabricated relatively easily and is the method of choice for many start-up companies.

Summary of the invention

A probe chip of biological materials, including DNA, is highly expected to be used as a testing tool. However, in practical terms, the following conditions must be satisfied: (A) small quantities of a large number of different chips can be fabricated at low cost, (B) a probe can be uniformly immobilized; (C) the data is highly reproducible , and the chip is reusable; and (D) the chip can be heated to remove non-specifically adsorbed substances. However, problems remain: for example, (a) probes are inconsistent from segment to segment, (b) production is labor intensive, (c) immobilization of very fine segments is impossible, and (d) the probes are not uniform; because (i) they are immobilized as droplets on a solid surface, and (ii) the probes are positioned and immobilized at the same time. Also, (d) is weakly bound to the surface of this solid and can be removed once heated, because (iii) many probe chips are fixed by adsorption or the like.

In order to solve the above-mentioned problems, the immobilization of probes on the solid surface and the arrangement of these probes can be divided into two or more different steps, thereby enabling the production of uniform DNA probes on the solid surface. Probes can be immobilized via thermally stable covalent bonds, and thus, non-specifically adsorbed substances can be properly removed by heating. Fine particles of the solid used as immobilization probes are arranged to produce an array of probes with appropriately sized segments. Any desired array of probes can be easily produced by exchanging the aligned fine particles with these probes. Tweezers can be used to align fine particles with a diameter of about 0.3 mm, but this method will be difficult for particles with a diameter of less than 0.1 mm. Accordingly, in one embodiment, the present invention provides a method and an apparatus for producing an array of probes, each of which is contained in fine particles in a fine cavity above a sheet It's transported and aligned in a capillary, a groove in a plate, or something like that. In an alternative approach, fine particles are controlled to flow as individual particles into a liquid for transport into a capillary to produce a probe array. Also, in order to improve the repeatability of measurement, a plurality of fine particles with a plurality of probes are arrayed for each probe, thereby checking any variation of test results to obtain highly reliable data.

For purposes of summarizing the invention and the advantages obtained over the prior art, certain objects and advantages of the invention have been described above. It is to be understood, of course, that not necessarily all such objects or advantages may be achieved in accordance with any particular embodiment of the invention. Thus, for example, those skilled in the art will recognize that the present invention can be embodied or practiced in the sense of obtaining or optimizing an advantage or collection of advantages as taught herein without necessarily obtaining Other purposes or advantages taught or implied.

Further aspects, features and advantages of the invention will become apparent from the following detailed description of the preferred embodiments.

Brief description of the drawings

These and other features of the invention will now be described with reference to the accompanying drawings which are intended to illustrate rather than limit preferred embodiments of the invention.

FIG. 1 is a theoretical diagram of a probe array chip including beads with probes arranged in a capillary.

Figure 2 is a theoretical diagram of a detection system for measuring an array of beads with probes held in a capillary or the like.

Figures 3a-3g are fragmentary cross-sectional views of a device for arraying beads. Figure 3a is a theoretical diagram of the delivery of beads in an offline state. Figure 3b is a theoretical diagram in which a bead is trapped in a cavity. Figure 3c is a theoretical diagram in which a bead is migrating into a capillary or the like. Figures 3d-3g illustrate this sequential step.

Figures 4a and 4b are theoretical diagrams of a groove-type arrangement of beads. Figure 4a is a perspective view. Figure 4b is a cross-sectional view.

Figures 5a, 5b and 5c are theoretical diagrams of a method of producing a bead array using grooves and a movable valve. Figure 5c is a partial view in cross section.

Fig. 6 is a theoretical diagram of a disc-shaped transport probe bead system.

Fig. 7 is a theoretical diagram of a flow-type bead array production method.

Figure 8 is a theoretical diagram of a bead array in which marker beads are used to separate a large number of beads.

Figures 9a and 9b are theoretical diagrams of a method using a sheet-arranged probe bead with cavities.

Figures 10a, 10b and 10c are theoretical diagrams of a microtiter plate type bead array array.

Figure 10a is a general view. Figure 10b is a cross-sectional view. Figure 10c is a theoretical diagram of the measurement.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention includes several aspects and embodiments. In one aspect, a method for producing a probe array comprises the steps of: (a) selecting several types of probes of interest; (b) fixing the several types of probes on the surfaces of different solid objects, respectively and (c) arranging the probe-immobilized solid objects in a specified order, thereby obtaining a probe array for analyzing a sample solution passing therethrough. In the above, a probe may be a polynucleotide, a peptide or a protein. In one embodiment, these solid objects are beads which may be fine particles. Further, the arrangement of these solid objects may be a one-dimensional arrangement or a two-dimensional arrangement. In another embodiment, the method further includes placing solid objects as markers in the arrangement at specified intervals. These markers may be of different dimensions than the solid object with the probes. In one embodiment, each solid object has one type of probe affixed thereto, and a designated number of solid objects is prepared for each type of probe. Furthermore, the alignment of these solid objects can be performed in an array selected from capillaries, grooves and optical elements.

In one embodiment of the method, the arrangement of the solid objects may be carried out as follows: (i) placing the solid objects with the probes immobilized on a sheet of cavities through which a solid object may pass, The sheet is placed on a movable substrate with a through hole leading to the interior of the array, the movable substrate is placed between the hole of the sheet and the movable substrate (ii) capture one of these solid objects in the cavity of the sheet; (iii) remove the remaining solid object from the sheet; (iii) move the movable substrate to a place where the cavity of the sheet communicates with the through-hole of the movable substrate; (iv) transporting the trapped solid object to the array via the through-hole; and (v) repeating steps ( i) to (iv) until the solid objects with fixed probes are arranged in the array in the specified order.

In another embodiment of the method, alignment can be performed as follows: (i) placing the solid object immobilized with probes on a sheet having cavities through which a solid object can pass, so Said cavity leads to the inside of this array, and said cavity is closed by a valve; (ii) captures one of these solid objects in the cavity of the sheet; (iii) opens the valve so that transporting the trapped solid objects to the array; and (iv) repeating steps (i) to (iii) until the probe-immobilized solid objects are arranged in the capillaries, grooves or optical cells in the specified order.

In yet another embodiment of the method, the alignment may be performed by: (i) placing the probe-immobilized solid objects in wells, each well containing a single type of probe-immobilized solid object, each well having a cavity through which a solid object can pass, said cavity being closed; (ii) trapping one of these solid objects in each cavity of each hole; (iii) in accordance with a specified sequentially moving the holes followed by opening and closing each cavity, thereby transferring each trapped solid object into one array; (iv) moving the holes so that the probe-immobilized solids are aligned in the next array objects; and (v) repeating steps (i) through (iv) until a specified number of arrays are filled with probe-fixed solid objects arranged therein.

In yet another embodiment of the method, the arrangement can be performed by (i) placing the solid object holding the probe in a narrow tube; (ii) using a solution that flows along the narrow tube moving the solid objects one by one, thereby transferring the solid objects into the array, and (iii) repeating steps (i) and (ii) until the solid objects with fixed probes are arranged in a specified order in this array.

Additionally, in one embodiment, the arrangement can be performed by: (i) placing the probe-immobilized solid objects in segments, each segment containing a single type of probe-immobilized solid object, each segment having a cavities through which a solid object passes, said cavities being closed; (ii) trapping one of these solid objects in each cavity of each segment; (iii) moving these solid objects in a specified order Open and close each cavity after the section, thereby transporting the trapped solid objects into a kind of groove; (iv) Repeat steps (i) to (iii) until the solid objects immobilized with probes are arranged in order in the groove; and (v) transporting the aligned probe-mounted solid objects into an array in which the solid objects are closely packed together.

In the above, each embodiment can exhibit at least one of the above-mentioned advantageous effects.

The invention can be applied to other aspects including a probe array for analyzing a sample solution passing therethrough, and various devices for producing a probe array.

The invention will be illustrated by the following examples. A probe array of the invention can generally be described in terms of DNA, protein, peptide or other biological material. Therefore, DNA is used for illustration in the following examples.

In a DNA probe array according to the present invention, solid probes are housed one-dimensionally in a capillary, or two-dimensionally in a small area of an optical unit. For convenience of description, capillary tubes are mainly used in the examples. Although in the embodiment, round beads are used as the fine particles, any particles having a cubic or other shape may be used. Beads having a diameter of 1-300 micrometers can be used; however, beads having a diameter of 20 micrometers are mainly used in the examples. Further, glass and plastic beads are commonly used; however, metallic materials such as gold may also be used. Use plastic beads here.

[Example 1]

Fig. 1 illustrates an example of a kind of probe array according to the present invention, wherein numeral 101 is the inlet of a solution and sample, 102 is an outlet, 103 is a capillary tube that accommodates probe array, 104 is label beads , 105 is a bead with a probe, and 106 is a dummy bead. The diameter of the bead with the probe immobilized is 20 microns, and the inner diameter of the capillary 103 is 25 microns. In this embodiment, about 20 dummy beads 106 are arranged at both ends, and 999 beads 105 are arranged between them. For a total of 99 marker beads and 900 probe beads, every 10th bead is a black bead 104, and every 100th bead is a red bead, i.e., 900 different kinds of probes can be used for testing at the same time . If densely packed, the beads could be arranged within a 2mm length; however, in this example, for hybridization and other considerations, the beads are more loosely packed and accommodated within a 5mm length. The retention length can be longer or shorter than the ranges described above (eg, in the range of 2-10 mm per 1000 beads). However, while a length that is too long increases the amount of sample required, a length that is too short causes handling problems. Also, the hybridization of the sample may not be sufficient. The volume of the reaction area is approximately 2.3 nL. Stoppers are placed at both ends, thereby preventing flow of the beads. Samples and washes are introduced and discharged via inlet 101 and outlet 102 . Since up to 10,000 probes can be accommodated in an area with a length of 20-30mm, it is advantageous that the probe array is compact and easy to handle.

The illuminating laser beam 206 and the probe-containing capillary 202 are scanned relative to each other, and the resulting fluorescence is measured using a fluorescence detection device, for example, as illustrated in FIG. 2 . In Fig. 2, numeral 201 is a kind of bead with probe, 202 is a capillary tube containing probe array, 203 is a plate for moving probe array, 204 is an irradiation and emission point, 205 is a lens, 206 is an illuminating laser beam, 207 is a filter, 208 is a lens, 209 is a laser light source, 210 is a detector, 211 is a data processing and detector controller, and 212 is an indicator . Different probes can be easily identified by putting one marker bead every 10 beads 201 . The labeled beads can be dyed different colors to identify different probes, or alternatively, each set of 10 beads with probes can be dyed a different color. Of course, in this case the color will be chosen so that it does not have a wavelength that interferes with fluorescence detection.

[Example 2]

This embodiment relates to a method and an apparatus in which beads are arranged in a capillary in a predetermined order. Figures 3a-3g illustrate an example of an apparatus for making such bead arrays. In these figures, numeral 301 is a solution and bead outlet, 302 is a solution inlet, 303 is a cover plate, 304 is a bead with a probe, 305 is a capture bead, 306 is a capillary for arraying beads, 307 is a base supporting the capillary, 308 is a cavity for trapping beads, 309 is a nozzle for supplying beads, and 310 is a stopper. For ease of illustration, the beads are arranged in one capillary in this example; however, for practical use, multiple cavities and multiple capillaries on one sheet are used. Step 1 (FIG. 3a): Beads with the first probe (probe bead #1, 304) are introduced with a solvent into the cover plate 303, which has a holed sheet 311 at the bottom. The beads are precipitated and the solvent is moved back and forth and side to side so that one of the capture beads 305 falls into this cavity. Step 2 (FIG. 3b): Use this solvent 302 to remove remaining beads through outlet 301 and wash them. Only beads that fall into this cavity remain in this cell. In this case, the solvent can be blown out of the channel opening at right angles to the sheet, thereby removing the beads close to the channel opening and leaving one bead in the cavity of the sheet to be introduced into the capillary in step 3. The bottom of this cavity is blocked by the base 307 supporting the capillary. The bead-aligning capillary is fixed to this slider, but in steps 1 and 2, the bead-aligning capillary 306 and the cavity are not aligned so that the bead 305 is retained in the cavity. Step 3 (Fig. 3c): The substrate 307 and the sheet 311 supporting the capillaries are moved relative to each other so as to align the shafts and cavities of the capillaries. Beads (305) are introduced into this capillary by suction from the other end or by applying pressure from the solution injection side. In this case, the relative motion of the slider to the sheet was of the order of about the same as the diameter of the cavity, for which a piezoelectric element was successfully used. Step 4 (FIG. 3d): The capillary-supporting substrate 307 and the sheet 311 are relatively moved so that the bead-arranged capillary 306 and the bead-capturing cavity 308 are out of line again. Step 5 (Fig. 3e): A bead with a second type of probe (probe bead #2, 320) is introduced into the cover plate 303 and one of 321 falls into this cavity. Step 6 (FIG. 3f): In the same manner as Step 2, remove excess beads from the cell except for the beads in this cavity. Step 7 (FIG. 3g): The slider and the sheet are moved relative to align the axis of the capillary with the cavity so that beads (321) can be introduced into the capillary. As a result, the beads with probe 1 (probe bead 1) and the beads with probe 2 (probe bead 2) were aligned in the capillary. By repeating these steps, a bead array with probes in the desired order can be produced.

During the measurement, the capillary used here can be removed and used as a probe array container, or a probe container to which the bead array is transported can be separately fabricated and attached to the bottom of the capillary . In this embodiment, the probe array array illustrated in Figures 4a and 4b is used. In these figures, numeral 401 is a substrate having grooves for accommodating a bead array, 402 is a solution outlet capillary, 403 is a bead and various solution inlets, 404 is a groove for arranging beads, 405 is A bead with a probe, 406 is a plug and 407 is an upper window. A sample solution is injected into the inlet (403) of the beads and various solutions in this figure, and after sufficient hybridization, a washing liquid is injected from the solution outlet capillary (402) to remove unreacted sample parts. After assembling the probe array array into a measurement device, each bead is illuminated with a laser beam and the emitted fluorescence is detected. Of course, in addition to the fluorescence emitted by laser beam irradiation, it is also possible to detect the emitted light generated by the chemiluminescence reagent. Any detection method capable of detecting the presence or absence of hybridization can be used.

In this example, only one capillary attached to the slider is used to illustrate the invention; however, multiple capillaries can be used simultaneously to produce a large number of probe arrays. In this case, it is naturally understood that the number of cavities on the sheet must increase along with the number of capillaries.

[Example 3]

This embodiment is to illustrate a kind of device, wherein bead delivery device 504 has the cavity (or hole) that holds each kind of bead respectively, thus they are transported on the supporting substrate 512 that has groove 507 on the support base 512 of arrayed bead, or Transfer to a capillary that arranges the beads into a probe bead array according to a predetermined sequence. First, a solution containing different kinds of probe beads placed in the wells of a microtiter plate is transferred one by one to the designated wells (cavities) of the bead delivery device in a predetermined order, thereby delivering the beads Arranged in a groove or in a capillary produced in a plate (Fig. 5a, 5b, 5c). In these figures, numeral 501 is a pipette/syringe, 502 is a titer plate with wells 503 for receiving probe beads, 504 is a bead delivery device with cavities, and 505 is a delivery device for receiving probe beads. Cavities of probe beads to a groove, 506 is aligned probe beads, 507 is a groove in which various probe beads are aligned, 508 is a probe bead, 509 is captured A probe bead in a cavity, 510 is a piezoelectric element, 511 is a movable valve, and 512 is a supporting substrate. The beads are aspirated from the wells in the titer plate 502 using a pipette 501 and moved into a transfer well 505 . The cavity 520 where the beads are captured is open at the bottom of the well. One of the beads 509 injected into the hole 505 (the beads, if multiple cavities are provided) falls into the cavity 520 and the presence of the dropped bead is optically confirmed. Thereafter, excess beads are recovered or the beads are removed from the wells by rinsing with wash solution. A valve 511 or the like, which may be actuated by a piezoelectric element 510, is placed between the cavity 520 where the beads are captured and the groove 507 or this capillary. By moving this valve, a bead can be transported to the side of the groove or capillary. The actual bead movement is controlled by a fluid flow. Of course, it is also possible to transport the beads through the bead delivery device 504 so that the cavity is aligned with the center of the groove or the capillary. Once the bead is fully transported, the valve is moved back, or the relative position of the cavity and capillary is changed, thereby trapping the bead in the cavity. Use a pipette to introduce the bead with the next probe into the capture site. Repeat the above steps to produce a bead array. The resulting arrayed probe beads 506 are used as-is, or are transported to another container while maintaining the alignment and used as a probe array.

The above steps can be implemented in one system with multiple cavities, saving time in array production, or producing multiple identical arrays at the same time.

[Example 4]

In Example 2, one probe bead was arrayed at a time using a bead delivery device having one cavity. In this embodiment, multiple kinds of probe beads are accommodated in sections using multiple wells in one bead delivery device, thereby improving productivity. As shown in FIG. 6 , a plurality of rectangular holes 603 are placed on a rotating disk 601 . In FIG. 6, numeral 601 is a disc-shaped bead-accommodating plate for delivery of beads, 602 is a rotating shaft, 603 is a groove for accommodating beads, and 604 is a cavity for accommodating beads. As described at the end of Example 1, the bottom of each hole is fitted with a sheet having a cavity. The lower part of the rotating disc with these sheets is in contact with a kind of slide that supports the capillaries so that the beads trapped in the cavities do not fall down. When the rotating disc is moved and the cavities and the axes of the capillaries are aligned, the probe beads are transported into the capillaries in the same manner as described in the previous examples. The number of these cavities corresponds to the number of these capillaries. The cavities and the capillaries are positioned correspondingly; however, to prevent twisting once rotated, a control mechanism is provided using a positioning technique similar to that used for CD-ROMs, according to which disc Move the slider with the capillary in the direction of the axis 602 . In this example, a rotating plate having a diameter of 16 cm was used. Holes 603 (1 mm wide, 30 mm long) were positioned 5 cm from the axis of the disc. The pitch of the holes is 2 mm, and about 150 holes can be arranged radially on the disk. A sheet with cavities spread under these holes, and the spacing of the cavities was 2 mm. In this example, a total of 10 cavities were arrayed so that a probe bead array could be fabricated in 10 capillaries. Of course, the number of capillaries and the number of probe arrays that can be produced at one time can be varied as required.

The rotating plate rotates in two modes of rotation; a high speed rotation mode and a low speed but highly precise rotation mode. A solution is used to introduce the beads into the wells. The beads are made to fall into the cavities by moving the disc and letting the solution flow out of the cavities. In the next step, this disk is spun in a high-speed spin mode, and excess beads are moved by centrifugal force and by water flow into bead containers located at the ends of the wells. The disc was stopped, after which the disc rotation was set to a highly precise pattern, thereby aligning the capillaries and probe bead #1. A shutter at the bottom of the disc is opened and the slider holding the capillaries is brought into contact with the rotating plate, thereby moving the well carrying probe bead #2 to the position of the capillaries. The beads are sequentially transported into the capillaries to produce probe bead arrays in a specified order. By exchanging the disk or the probe beads to be placed in the wells, and repeating the steps described above, a large number of probe beads can be arrayed and accommodated in the capillary. The position of a particular probe within a generated probe bead array can be routinely determined by changing the color of the beads in the array every 10th bead.

[Example 5]

This embodiment relates to a method and an apparatus for arranging probe beads one after the other in a specified sequence into a capillary using a liquid flow. Figure 7 shows a theoretical diagram of this embodiment. In this figure, numeral 701 is a bead solution reservoir, 702 is a bead with a probe, 703 is a transfer tube, 704 is a sheath flow cell, 705 is a transfer liquid, 706 is a 707 is a capillary for bead array arrangement; 708 is a support matrix, and 709 is a solution outlet tube. Beads 702 with probes are pumped into transfer capillary 707 . The end of the capillary is inserted into the flow formed in the sheath flow cell 704 with the transfer liquid 705, and the beads are released into the flow one by one with a substantially constant interval. However, to stabilize the release, ultrasound was applied to the portion of the capillary containing these beads, forming nodules along the axis of the capillary. These beads are released into the liquid stream one by one at specified intervals by controlling conditions such as the intensity of ultrasound.

[Example 6]

In the above embodiments, one bead corresponds to one probe. However, to check the uniformity of the hybridization reaction or to improve detection sensitivity, multiple beads may be used for one probe. It is not necessary, however, to use an equal number of beads for all probes. However, if the number of probes used to make the array in the capillary is different, colored beads or beads of different sizes must be inserted between sets of beads with different probes as labels. This embodiment is shown in FIG. 8 . In this figure, numeral 801 is a large size dummy bead, 802 is a probe bead, 803 is a large size marker bead, 804 is a capillary for accommodating the probe, and 805 is a sample flow path. The equipment used for production is basically the same as above except that the size of the hole is several times larger than that of the bead 802 so that multiple beads 802 can be trapped in the hole. Subsequent steps are the same as above.

Further, if the liquid flow system described in Example 5 is used, the bead array in this example can be easily fabricated. A small amount of beads is aspirated from a bead reservoir with a pipette and injected into the stream. Although this number cannot be verified, the injected beads can be placed into this capillary 804 sequentially. A colored bead or a bead of a different size (801) is injected as a marker before another bead is injected so that the position of the individual bead and the type of probe can be determined.

[Example 7]

The foregoing embodiment is a method of producing a probe array in which probe beads are arranged in a capillary. As illustrated in Figures 9a and 9b, this embodiment discloses a method and a device in which the beads are first arranged in a groove created on the surface of a flat plate and thereafter assembled into a Probe array or transport it into a capillary to produce a probe array. In Figures 9a and 9b, numeral 901 is a plate with holes and 902 is a hole of a bead reservoir. 903 is a cavity for receiving beads, 904 is a sheet with cavities and is usually connected to the plate 901, 905 is a basic array production support with grooves for arranging beads, 906 is a support for arranging probe beads Fine groove, 907 is a bead with a probe, 908 is a capillary of a bead array. First, a bead array production support 905 having a plurality of grooves 906 on the surface of a flat plate is prepared. Align beads with probes 907 in each groove and transport them into a capillary 908 or the like while maintaining their alignment, after which beads arrayed in multiple grooves are introduced into different capillaries and used as a probe array. A plate is placed on top of the bead array production support joined together with a sheet having cavities (901, 904) in which the beads are trapped and transported into the above in the groove described. As illustrated in Figure 9, this plate with one sheet has holes (bead reservoirs 902) orthogonal to the grooves on the bead array production support, and the holes 903 for receiving the beads are through holes, and open to this fine groove. The device does have multiple grooves, but beads with different probes are injected into different wells of the plate and accommodated in different cavities. Using this plate joined together with a sheet and this plate with grooves, they are in close contact but can slide over each other. At the beginning, the cavities 903 of the sheet and the grooves 906 of the bead array production support are not aligned. Beads with probes are supplied into different wells 902 of the plate above the sheet with holes for each probe. A bead falls into a cavity and remains there because in this state the bottom of the cavity is closed. When the cavities of the sheet and the grooves of the bead array production support are aligned, beads drop from the individual cavities into the grooves 906 one by one. Since different bead arrays are dropped into a groove from different positions, the various beads are retained in a groove. In fact the beads are placed on the sheet with the voids at the same intervals as those in the bead reservoir 902 . In this embodiment, this spacing is 2mm. In this example, a total of 50 beads were dropped into each groove of the bead array production support. Again, in this example 10 bead arrays can be produced simultaneously, but this number can be increased as desired. After dropping the beads, the position of the sheet with cavities 903 and the grooves 906 of the bead array production support are moved to seal the grooves, after which the beads are introduced into the capillary 908 with a liquid flow . By repeating the steps described above, the number of different kinds of probes can be aligned.

In this example, a one-dimensional array of probe beads is disclosed; however, naturally, by arranging multiple numbers of these arrays or by two-dimensionally arranging these arrays, it is possible to produce probe array.

[Example 8]

In this embodiment, a probe bead array support includes these units consisting of a flat plate having holes distributed one-dimensionally or two-dimensionally and a cover glass. In Fig. 10a, 10b and 10c, numeral 1001 is a microtiter plate type bead array holder, 1002 is a spacer, 1003 is a cavity for making a bead array unit with a cover glass, 1004 is a bead with a probe, 1005 is a cover glass, 1006 is a solution outlet, 1007 is a solution inlet, 1008 is a laser beam, 1009 is a lens, and 1010 is a detector. This is similar to a microtiter plate. A small amount of beads is aspirated from a titer plate containing probe beads therein, and they are dispensed into wells (cells) 1003 of plate 1001 . Beads 1004 are dispensed into cavities at designated positions according to the type of probe, thereby producing a microtiter plate type bead array with probe beads. After the beads are dispensed, a coverslip 1005 that is optically transparent and does not interfere with the measurement of fluorescence or chemiluminescence is placed on top, thereby producing an array of cells. The spacing between the coverslip and the wall dividing the cells of the microtiter plate-type cell array is smaller than the size of the beads so that the beads cannot migrate outward. Reaction solutions or the like can freely flow through the cells. To use, turn these units upside down so that the glass side is down. In this case, the beads on the glass surface are in full contact with the reaction solution independent of the depth of the cells, while the probes hybridize to the target.

[Invention effect]

As described above, according to the present invention, a large amount of peptide or DNA probe arrays can be produced by a simple procedure. The process of immobilizing the probes on a solid surface and the process of arranging the probes are separated so that both processes can be optimized. As a result, immobilized probes that are uniform and not easily removed from the solid surface can be produced, and thereafter, by arranging beads in a prescribed order, an array having desired kinds of probes can be easily produced. Likewise, an array of fine probes—which is difficult to fabricate by conventional methods—could be produced by reducing the size of these beads. A probe array with a new composition can be produced simply by preparing the desired DNA probes, immobilizing them on the surface of beads, and mounting these probe beads on a production device, such that any Time can provide the array requested by the user. By arranging multiple numbers of beads carrying the same probe, statistical averages can be obtained to analyze repeatability and quantification, and reliable measurements can be performed. Furthermore, the reaction is fast and highly sensitive because the surface area of the reaction is larger than that of a conventional DNA chip or the like held on a plate. The size of these beads can be varied from 1 micron to 30 microns, allowing easy production of high density probe arrays if desired. For example, by using 6-micron beads, 1,500 probes can be arrayed on a 10-mm length in one capillary, or more than 1,000,000 probes can be retained if a two-dimensional probe array support is used. on a ^1cm2 area.

By means of an extremely simple process, multiple numbers of arrays with the same probe arrangement can be produced, so that these arrays are also suitable for mass production.

Those skilled in the art will appreciate that many and various modifications can be made without departing from the spirit of the invention. Therefore, it should be clearly understood that the various forms of the present invention are illustrative only and are not intended to limit the scope of the present invention.

Claims (1)

1. a method of producing a kind of probe arrays comprises the steps:

Select polytype interested probe;

With this polytype probe stationary on different solid articles surfaces; With

This solid articles of having fixed probe is arranged by named order, thereby is obtained the probe arrays of a kind of analysis by its sample solution,

Wherein each solid articles have one type probe stationary thereon, and for the probe of each type prepare specified quantity solid articles and

Wherein arrange and carry out as follows: the solid articles that (i) will fix probe is placed in the hole, fixing of single type contained in each hole solid articles of probe, each hole has one can be a hole that solid articles passed through, and said hole is closed; (ii) in each hole in each hole, capture in these solid articles; (iii) move after these holes, open and close each hole, thereby each captive solid articles is transported in the array according to specified order; (iv) move these holes, thereby in next array, arrange the solid articles of having fixed probe; And (v) repeating step (i) is to (iv), up to the array of having filled a specified quantity with the solid articles of wherein having fixed probe.

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