JP4431724B2 - Microchannel bead array device and manufacturing method thereof - Google Patents

Description

  The present invention relates to an analyzer using beads arranged in a microchannel and a method for producing the same.

  Biological microarray analysis technology is a means for conducting comprehensive biological tests such as typing of gene polymorphisms. In this technique, analysis is performed using a chip in which a large number of different biologically active substances (probes) are arranged in a spatially addressable form on a two-dimensional solid support, but are arranged on a single chip. Since the number of bioactive probes typically ranges from 1000 to 100,000, a chip with probes arranged in a spatially addressable manner is inherently expensive regardless of the manufacturing method. An alternative approach to avoid this is the idea of generating biologically multiplexed arrays using microspheres incorporating fluorescent dyes. Here, addressing of each microsphere is performed by detecting a fluorescent signal from each microsphere. By arranging microspheres two-dimensionally and using them as a microarray, a high-density array can be manufactured without using an expensive technique such as lithography. As such a high-density bead array, there is a method (Science, 287, 451-452 (2000)) that forms a bead array by inserting beads into a microwell array obtained by etching the end face of an imaging fiber. Proposed.

In general, it is difficult to perform an analysis process in a short time in an analysis apparatus that detects a phenomenon in which a biologically active probe fixed on a substrate surface, such as a biological microarray analysis technique, captures an analysis target substance in a solution. As a method for shortening the analysis time, a technique for performing a chemical reaction or the like in a minute space is being developed. In the minute space, the time required for the analyte to be analyzed present in the solution to reach the surface on which the bioactive probe is immobilized by diffusion is overwhelmingly short. For beads placed in a minute space, beads are placed in a capillary (through JP-A-2000-346842), microwell (through-hole) array prepared by anisotropic etching of silicon. (Analytical Chemistry, 75, 4732-4739 (2003)) has been proposed.
JP 2000-346842 A Science, 287, 451-452 (2000) Analytical Chemistry, 75, 4732-4739 (2003)

  Currently used bioarray analysis chips are problematic in that they are expensive and time consuming to analyze, and there is a need for new devices that are less expensive and can reduce analysis time.

Microbead array is a technology that attracts attention from the viewpoint of cost reduction of analysis chips, but when using microbead arrays fabricated on the substrate surface, analysis time is the same as for conventional bioarray analysis chips. This is a problem. In the method of filling microbeads in microchannels such as capillaries, the beads are fixed in contact with each other. When analyzing signals from each spot based on the fluorescence microscope image of the aggregate of beads, it is difficult to separate the signals from each spot when the microbeads are in contact with each other. There is a problem of lowering reliability. In addition, when the beads are not regularly arranged, it is impossible to perform repetitive analysis work by the same scheme, and there is a problem that efficiency of the analysis work is lowered. In the case where beads are arranged in a microwell (through-hole) array produced by anisotropic etching of silicon, the beads are fixed in a non-contact state, but the size is the size of the opening on the bottom of the microwell. Therefore, it is difficult to increase the density. In fact, only beads with a diameter of 80 micrometers or more are used. A substrate having a microwell has a structure in which a large number of through holes are formed on an extremely thin substrate, and there is a problem in strength. Since this substrate needs to be held in the microgap, it is expected that the substrate will be easily damaged, especially when the substrate area is increased to secure a large number of beads. In addition, it is necessary to separately form not only a substrate having a microwell but also two substrates having a microgap, and the production process is complicated.

  Moreover, when using beads in the microchannel, the pressure loss increases by arranging the beads, and the internal pressure of the liquid during liquid feeding increases. As a result, since “leakage” is likely to occur, inter-substrate bonding using the adhesiveness of polydimethylsiloxane (PDMS) widely used for forming microchannels cannot be used. On the other hand, a strong joining method using a thermal technique cannot be used because it destroys a bioactive probe that is weak against heat. When irreversible substrate-to-substrate bonding or bead fixation is performed, the produced chip becomes disposable, which requires analysis costs. There is also a problem that the set of bioactive probes on the chip cannot be easily changed.

  An object of this invention is to provide the microchannel bead array device which can solve these problems, and its manufacturing method.

  When using an array produced on the substrate surface, it takes time to analyze because the substance to be analyzed present in the solution must move to the substrate surface by a diffusion phenomenon. In order to solve this, it is effective to shorten the movement distance due to the diffusion phenomenon by arranging the microbeads used for the array in the microchannel. By using a two-dimensional microstructure pattern in which microbeads are placed in a microchannel that connects bead fixing sites with microgrooves that prevent movement of beads as beads, microbeads are regularly placed in a non-contact state. It becomes possible to arrange in two dimensions. In the present invention, beads are not limited to microspheres. If the object has a size corresponding to a microsphere having a diameter of 1 to 100 micrometers and can be arranged one by one at the bead fixing site, it is produced by polyhedron, rod, or grinding. , Including those in which individual particles have an irregular shape.

  A microchannel is formed by bonding a hard substrate with a fine structure and a rubber elastic substrate. At this time, a microfluidic device that does not leak is formed by appropriately controlling the load with which the rubber elastic substrate is pressed against the hard substrate. Since the above bonding is a reversible inter-substrate bonding, it can be easily disassembled and reconfigured. At that time, the set of bioactive probes on the chip can be changed by replacing the beads on the chip.

That is, the microchannel bead array device according to the present invention has a plurality of two-dimensionally regularly arranged bead fixing portions each accommodating a single bead, and a fine groove for connecting the bead fixing portions to each other. A hard substrate having a fine structure pattern formed thereon, an elastic substrate, and means for bringing the elastic substrate into contact with and pressurizing the surface of the hard substrate having the fine structure pattern formed thereon. Hard substrates include quartz glass, general glass, calcium fluoride, silicon carbide, acrylic, PMMA, sapphire, alumina, crystal, diamond, and other transparent materials, various plastics, silicon and other semiconductors, ceramics, aluminum, and stainless steel An opaque hard material including a metal such as can be used. As the elastic substrate, a rubber material such as silicone resin including polydimethylsiloxane (PDMS), natural rubber, nitrile rubber, and fluorine rubber can be used. These substrates need to have a smooth surface in order to be used together, and at least one of a hard substrate and an elastic substrate needs to be a transparent substrate in order to perform fluorescence observation with a microscope. The hard substrate and the elastic substrate are bonded together without bonding.

  As an example, in a plurality of bead fixing sites, a plurality of beads having biologically active probe molecules bound to the surface are accommodated in a non-contact state. The beads are preferably 1-100 micrometers in diameter. More preferably, 100 beads or more per millimeter square are arranged by using beads having a diameter of 66 micrometers or less. The plurality of bead fixing portions have an internal volume in which two or more beads cannot enter, and the fine groove has a width smaller than the bead diameter. The elastic substrate can be a silicone resin such as polydimethylsiloxane. Moreover, the inclined structure site | part for detecting the load which presses an elastic board | substrate to the said hard board | substrate can be provided in the surface of a hard board | substrate.

  The hard substrate has a through hole connected to the sample solution introduction end of the fine structure pattern and a through hole connected to the sample solution discharge end of the fine structure pattern. The sample solution forms the fine structure pattern of the hard substrate. The sample solution introduced from the surface opposite to the formed surface into the fine structure pattern through the through hole, and the sample solution flowing through the fine structure pattern is on the opposite side of the surface on which the fine structure pattern of the hard substrate is formed through the through hole. Discharged from the surface. By adopting such a structure, it is possible to independently control the pressure for laminating the elastic substrate and the hard substrate and the pressure required when connecting the tube for flowing in and out of the fluid.

  As an example of the fine structure pattern, there is a pattern in which a plurality of bead fixing portions are arranged in a two-dimensional lattice, and the bead fixing portions in each column of the two-dimensional lattice are communicated by the fine grooves. As another example of the fine structure pattern, a reticulated flow path is formed by a plurality of bead fixing portions and fine grooves, a bead fixing portion is set at the confluence of the flow passages, and the flow passage portion connecting the confluence and the confluence There is a pattern in which is a fine groove.

  A method of manufacturing a microchannel bead array device according to the present invention includes a step of forming a microstructural pattern composed of bead fixing portions regularly arranged two-dimensionally on a hard substrate and microgrooves that connect the bead fixing portions, A step of placing beads at the bead fixing site in the structure pattern and a step of forming a micro flow path in which the beads are fixed by pressurizing and bonding the elastic substrate to the fine structure pattern forming surface of the hard substrate. Including. A step of observing the degree of penetration of the elastic substrate into the inclined structure portion provided in the groove structure on the hard substrate and controlling the load for pressing the elastic substrate against the hard substrate may be included.

  The (two-dimensional) array structure using beads arranged in a microchannel is used for biological microarray analysis, which is a comprehensive detection method for biomolecules including nucleic acids (DNA). In addition, this device is manufactured using a microreactor, chemical chip, piochip, Lab-on-a-chip, nanochip, etc. that are used to perform many reactions, separations, analyzes, etc. with a very small amount of sample. It is used as a method for manufacturing a microbead device.

  According to the present invention, an inexpensive device with a short analysis time and a high analysis accuracy can be provided.

Embodiments of the present invention will be described below with reference to the drawings.
The microchannel bead array device of the present invention was manufactured through the following four steps. (1) Fabrication of micro-channel on hard substrate (including micro-pattern for immobilizing beads, wide channel for liquid feeding, and fabrication of through-hole), (2) To bead-fixed site in micro-channel (3) Bonding in a state in which the load of the hard substrate and the rubber elastic substrate is controlled, and (4) bonding of a liquid feeding tube for introducing and discharging the sample solution. Hereinafter, the above four-step process will be described.
(1) Fabrication of micro-channels on a hard substrate (including fabrication of bead-fixed microstructure pattern, liquid-feeding wide channel, and through-hole)

  As the hard substrate, a quartz substrate having excellent chemical stability and transparency was used. A quartz substrate having a square with a side of 2 cm and a thickness of 2 mm was used. Microstructure pattern for immobilizing beads on the quartz substrate surface is prepared by laser induced back surface wet processing (LIBWE method) (Patent No. 3012926 and J. Wang, H. Niino, A. Yabe, Appl. Phys. A, 68, (1999), pp 111-113. With this processing method, a fine structure pattern was formed in a lmm square area at the center of the quartz substrate by reducing the exposure of the laser beam patterned through the mask to 1/8 times.

  FIG. 1 is a plan view showing an example of a fine structure pattern for fixing beads. 1 (a) to 1 (c) show an example of a structure in which bead fixing parts are linearly connected in parallel, and FIG. 1 (d) shows the bead fixing part two-dimensionally. The example of the planar structure connected is shown. The produced bead-fixing fine structure pattern has a structure in which the bead-fixing portions 1 regularly arranged two-dimensionally are joined by a micro-groove 2 having a small cross-sectional area and incapable of moving beads. The fine structure pattern shown in FIG. 1A is obtained by connecting each row of a plurality of bead fixing portions 1 arranged in a lattice pattern by linear micro grooves 2 that serve as sample solution flow paths. . In the fine structure pattern shown in FIG. 1B, each row of a plurality of bead fixing portions 1 arranged in a lattice pattern is connected by a micro groove 2 so that the flow path including the bead fixing portions has a zigzag shape. Is. The fine structure pattern shown in FIG. 1C is provided with a plurality of linear microgrooves 2 serving as a sample solution flow path in parallel, and a bead fixing portion 1 having a trapezoidal planar shape with respect to each microgroove 2. A plurality are arranged. In the fine structure pattern shown in FIG. 1 (d), the flow path is formed in a net-like shape, the confluence of the flow path is the bead fixing portion 1, and the cross-sectional area is small in the flow path connecting the confluence and the confluence. The minute groove 2 cannot be formed.

  The shape of these fine structure patterns can be freely selected by exchanging masks used during processing. The left and right ends of the bead-fixing fine structure pattern shown in the figure are used as a binding site when a plurality of bead-fixing micro-patterns are arranged side by side or as a binding site to a wide flow channel for liquid feeding To do.

  When the bead size is reduced, the width of the microgroove 2 that hinders the movement is significantly reduced, and as a result, the circulation of the solution becomes extremely difficult. Therefore, it is preferable to use beads having a diameter of 1 micrometer or more. . Further, from the viewpoint of producing a higher density array than a DNA array by spotting, the diameter of the beads is preferably 100 micrometers or less. It is preferable to arrange beads of this size at intervals of 1.5 times the diameter or more so that signals from each bead can be easily separated. Under these conditions, the maximum bead arrangement density per millimeter square is 443556 for a diameter of 1 micrometer and 36 for a diameter of 100 micrometers. Further, in order to arrange 100 or more beads per 1 mm square, the bead diameter needs to be 66 micrometers or less.

  The structure produced as an example is for arranging beads having a diameter of 10 micrometers. For example, in the structure shown in FIGS. 1A and 1B, the bead fixing part 1 is a square having a side of 15 micrometers. The depth is 15 micrometers, and the microgrooves 2 having a width of 5 micrometers are coupled to the fixed portion.

FIG. 2 shows a state in which beads are arranged in each fine structure pattern shown in FIG. A flow path is formed by placing the beads 3 on the fine-structured bead fixing portion 1 formed on the surface of the quartz substrate 21 and bonding the beads 3 to the PDMS plate 25. 2A, 2B, and 2C correspond to FIG. FIG. 2B and FIG. 2C are a cross-sectional view of a bead fixing portion and a cross-sectional view of a minute groove after the PDMS plate 25 is attached to the quartz substrate 21. 2B corresponds to the AA ′ cross section of FIG. 2A, and FIG. 2C corresponds to the BB ′ cross section of FIG. FIGS. 2D, 2E, and 2F correspond to FIGS. 1B, 1C, and 1D, respectively.

In the actually produced pattern of FIG. 2 (a), 32 flow paths capable of arranging 30 beads are arranged in a 1 mm square area, and 960 beads can be arranged per 1 mm ² . In the pattern of FIG. 2 (a), the sample solution mainly circulates on one side surface of the bead, whereas in the pattern of FIG. 2 (d), the sample solution circulates on both side surfaces of the bead. In the pattern of FIG. 2E, the wall of the portion where the beads are fixed is slanted, so that the sample solution can easily go around the side surface of the bead (the side surface drawn in the lower side in the figure). In this pattern, there are 50 beads per row, and 1600 beads can be arranged per 1 mm ² . In the pattern of FIG. 2 (f), each bead fixing site is two-dimensionally connected by four fine grooves with a width of 6.2 micrometers, and the cross-sectional area of the entire flow path is large, so that the solution flows. Becomes easier. 2709 beads can be arranged per 1 mm ² .

  In the laser induced back surface wet processing method (LIBWE method), such a fine structure pattern can be formed in a 1 mm square region by batch processing. It is possible to increase the number of beads that can be fixed by arranging 1 mm square fine structure patterns as necessary. In the example of FIG. 3 to be described later, 2 × 2 = 4 1 mm square bead fixing fine structure patterns are arranged. Here, the fabrication of the fine structure pattern for fixing beads is carried out by a laser induced back surface wet processing method (LIBWE method), but other fine processing methods such as reactive ion etching may be used. Moreover, it is also possible to produce by a hot embossing method using the produced fine structure as a mold.

  FIG. 3 is an overall view of a quartz substrate on which a fine structure pattern for fixing beads is formed. 3A is a plan view of the substrate surface, FIG. 3B is a cross-sectional view taken along line AA ′ in FIG. 3A, and FIG. 3C is a cross-sectional view taken along line BB ′ in FIG.

  As shown in the figure, four 1 mm-square bead fixing microstructure patterns 15 produced by the LIBWE method are arranged in the center of the quartz substrate. Wide channels 12 and 13 were formed at both ends of the bead fixing fine structure pattern 15 in order to facilitate connection to the outside. The channels 12 and 13 were formed by using both laser induced back surface wet processing (LIBWE method) and ultrasonic processing. The bead-fixing fine structure pattern 15 was coupled to the wide flow path 13 produced by the LIBWE method having a width of 1 millimeter and a depth of 40 micrometers. The flow path 13 is formed by moving a processing area of 1 mm square while irradiating a laser, and an inclined structure portion 14 exists at the end of the wide flow path 13. The inclined structure portion 14 can be used for detecting an appropriate load when the rubber elastic substrate is bonded to the quartz substrate. The flow path 13 is connected to the through-hole 11 having a diameter of 1 mm through a flow path 12 having a width of 1 mm and a depth of 200 micrometers manufactured by an ultrasonic machine. The flow paths 12 and 13 guide the sample solution introduced from the through-hole 11 to the bead array portion 15, and the width and depth can be freely selected in the range of 10 micrometers to 1 millimeter. It is also possible to use processing methods. Moreover, you may increase the inlet of a solution to two or more as needed.

(2) Filling of beads Uniform beads having a carboxyl group on the surface can be purchased from Bangs Laboratories, Polysciences, etc. Fluorescently labeled beads are prepared with a fluorescent dye that is insoluble in water. For example, the beads are suspended in each of the Eu complex having a tetrahydrofuran solution concentration of 0.001 M to 1 M, and the dye is impregnated into the beads by stirring for about 2 hours. This method is disclosed in Analytical Chemistry, 22, 5618-5624 (2000). By impregnating two or more dyes at different concentration ratios, a large number of beads can be identified. This method is described in Japanese Patent Publication No. 10-500951 and Japanese Patent Publication No. 2002-524739. Also, fluorescent semiconductor nanoparticles (Nature Biotechnology, 19, 631-635 (2001)) can be used instead of the dye molecules.

  After fluorescent labeling, surface immobilization reaction of biologically active probes such as oligonucleotides was performed on the beads by the method described below. This step can be performed based on procedures known in the art (Bangs Laboratories TechNOTE # 205 and Bangs Laboratories TechNOTE # 302).

The beads were washed twice with imidazole buffer having a pH of 7.0 and a concentration of 100 mM. To 1 milligram of beads, 20 micrograms of an oligonucleotide whose 5 ′ end was modified with an amino group was added and incubated at 50 ° C. for 3 hours with stirring. After the reaction, the collected beads were washed three times with SSC buffer containing 0.5% SDS at room temperature and twice with water containing no RNase. The beads obtained by the above procedure were resuspended in PBS buffer containing 0.2% NaN ₃ and stored at 4 ° C. Alternatively, avidin or streptavidin may be immobilized in advance on the bead surface, and an oligonucleotide whose 5 ′ end is modified with a biotin group may be immobilized by the avidin-biotin method.

After dropping an aqueous solution containing beads having a diameter of 10 micrometers onto the fine structure pattern 15 for fixing beads on the quartz substrate, the solution is covered with a cover glass, and the cover glass is lightly pressed and moved back and forth, and left and right. The inside beads were placed at the bead fixing site inside the fine structure pattern for fixing beads. The beads can be placed on the bead fixing site by placing powdered beads on the fine structure pattern and rubbing with a cloth or the like. J.
In A. Ferguson, J. Steemers, DR Walt, Analytical Chemistry, 72, (2000) pp 5618-5624., An aqueous solution of beads is added dropwise, the solvent is allowed to evaporate, and then an antistatic swab is used. A method of wiping off excess beads is disclosed.

  Since beads remaining on the surface other than the fixed part such as the surface cause leakage, it is necessary to completely remove the beads before attaching the elastic substrate. For removal of the beads, a flat plate of polydimethylsiloxane (PDMS) having an adhesive property (manufactured by Dow Corning, Sylpod 184) was used. After the surface of the PDMS plate larger than the quartz glass substrate was washed with ethanol, it was pressed against the entire surface having the fine structure pattern on which the beads were arranged. When the PDMS plate was peeled off after pressing for about 5 seconds, all the beads remaining on the surface of the quartz glass substrate were removed in a form adhered to the surface of the PDMS plate. Beads placed at the bead fixing site are not removed by this treatment. The beads copied on the surface of the PDMS plate are removed by washing with ethanol.

(3) Bonding with controlled load on rubber elastic substrate A device was assembled by bonding a quartz substrate with beads on the rubber elastic substrate. As the rubber elastic substrate, a plate of polydimethylsiloxane (PDMS) (manufactured by Dow Corning, Sylpod 184) having adhesiveness and excellent transparency was used. PDMS plates with thicknesses of 1 and 2 millimeters were made by curing the Sylpod 184 between a 1 millimeter or 2 millimeter gap made of an acrylic plate and cut into suitable shapes for use.

  Device assembly will be described with reference to FIG. 4A is a diagram for explaining the formation of the flow path by attaching the quartz substrate 21 and the PDMS plate 25, and FIG. 4B is a mechanism for controlling the load on the PDMS plate 25, and a solution input / output port. It is explanatory drawing of attachment.

  As shown in FIG. 4 (a), a quartz substrate 21 on which beads are arranged is placed on a substrate holder 22 made of aluminum or stainless steel, and is sandwiched between press plates 23 made of acrylic having a thickness of 5 millimeters, and screws 24 (four) are used. It was completely fixed by tightening. A PDMS plate 25 having a thickness of 2 mm matched to the size of the opening (16 mm square) is attached to the surface of the quartz substrate 21 from the lower surface opening of the substrate holder 22 with care so that bubbles do not enter. Combined. Further, an acrylic spacer 26 having a 2 cm square and a thickness of 3 mm was installed under the PDMS plate 25.

  As shown in FIG. 4B, the lower surface of the PDMS plate 25 is below the lower surface of the quartz substrate holding portion of the substrate holder 22, and the upper surface of the spacer 26 is not in contact with the substrate holder 22. It is in contact with only the PDMS plate 25. After the assembly, the lower surface of the spacer 26 comes out from the substrate holder 22. Next, an acrylic pressing plate 27 having a thickness of 3 millimeters was fixed with screws 29 (four) through which springs 28 were passed. The pressing plate 27 is not in contact with the substrate holder 22 but is in contact with only the spacer 26, and the pressing plate 27 presses the PDMS plate 25 against the quartz substrate 21 through the spacer 26. By adjusting the screwing amount of the screw 29, the load for pressing the PDMS plate 25 against the quartz substrate 21 is controlled.

(4) Joining of liquid feeding tube for introduction and discharge of sample solution As shown in FIG. 4B, on the through hole 11 on the surface opposite to the surface of the quartz substrate 21 where the fine structure pattern is formed. A PDMS plate 30 cut into an annular shape was placed and used as a sealing material. The tube connector 31 was connected through this sealing material, and a PEEK tube having an outer diameter of 1/32 inch was connected. These tubes make it possible to introduce and discharge liquid. Liquid feeding by a syringe pump was performed through this tube.

  In the manufactured device, when the load for pressing the PDMS plate 25 against the quartz substrate 21 is too large, PDMS enters and blocks the microchannel portion, whereas when the load is too weak, the fluid pressure in the microchannel increases. Cause leaks. When a pressure gauge that measures the fluid pressure is installed between the syringe pump and the micro flow device to monitor the fluid pressure and the fluid is fed without applying a load, a leak occurs when the fluid pressure reaches 14 kPa. did. By applying an appropriate weight to the PDMS plate, it is possible to form a leak-free microchannel without blocking the channel.

  In the present invention, an appropriate weight can be detected by observing the contact state with PDMS that has entered the channel of the inclined structure portion 14 in the wide channel on the quartz substrate. This will be described with reference to FIG. FIG. 5 is a schematic cross-sectional view showing a flow channel formed by bonding a quartz substrate 21 having a groove structure and a PDMS plate 25 together. 5A shows a case where no load is applied to the PDMS plate 25, FIG. 5B shows a case where an appropriate load is applied to the PDMS plate 25, and FIG. 5C shows an excessive load applied to the PDMS plate 25. Shows the case. When no load is applied to the PDMS plate 25, PDMS does not enter the groove structure 13 formed on the quartz substrate, but leakage of the liquid flowing through the groove structure 13 occurs. On the other hand, if an excessive load is applied to the PDMS plate 25, the PDMS 32 that has entered the groove structure 13 blocks the flow path.

5D is a view of the inclined structure portion 14 in the wide flow path on the quartz substrate 21 observed from the upper surface (or the lower surface), FIG. 5E is an AA ′ cross-sectional view thereof, and FIG. It is a cross-sectional view. The degree of penetration of the PDMS into the flow path by weighting is observed as the lengths of L1 and L2 in FIG. 5D, and these values are proportional to the magnitude of the weighting. Here, L1 is the length that the contact portion 33 between the PDMS and the inclined structure portion 14 that has entered the flow path due to the load covers the entire width of the inclined structure portion 14, and L2 is the length to the tip of the contact portion 33. is there. In a state where the weight is 13 kPa, the value of L2 indicates that there is an intrusion of PDMS of about 10 micrometers in the central portion of the flow path. In this device, when a liquid supply of 0.03 mL per hour was performed, a stable distribution state was obtained at a liquid pressure of about 25 kPa. This hydraulic pressure is 11 kPa higher than the hydraulic pressure at which leakage occurred in a state where no load is applied. In order to minimize the penetration of the PDMS into the flow path, it is desirable that the weight is as small as possible. In this device, the proper weight is preferably in the range of 12 kPa to 16 kPa. The appropriate weight value may vary depending on conditions such as the number of continuous microstructure patterns in the device and the length of the flow path.

  FIG. 6 is an explanatory diagram of an analysis operation using the assembled microchannel bead array device of the present invention.

  The manufactured device 43 is set on the stage 42 of the microscope 41 in a state where it is connected to the syringe pump 46, and the sample solution is introduced and the reaction process is performed on the stage 42 of the microscope. All this process can be observed by fluorescence microscope images. A pressure gauge for measuring the hydraulic pressure is installed in the middle of the PEEK tube connecting the syringe pump 46 and the device 43. Images are recorded by a CCD camera 44 equipped with a filter system, and numerical data can be extracted immediately by performing image processing by a computer. Four kinds of interference filters are set in the optical path of the excitation light and the observation light for fluorescence observation, and fluorescent signals from different fluorescent dyes can be identified by switching them when necessary. Image observation can be performed collectively on a 1 mm square fine structure pattern, and signals from all beads can be detected by moving the stage as necessary. The operation of these stages, filters, and CCD camera can all be controlled by a computer, so that the analysis operation can be automated.

  FIG. 7 shows a fluorescence microscope image of the fluorescent dye-containing beads having a diameter of 10 micrometers arranged in the pattern of FIG. It can be seen that the individual beads are regularly arranged in a non-contact state. This bead array is a high-density array in which 1600 beads per millimeter square are arranged, and each bead is fixed in an independent state. Therefore, an analysis operation for separating and detecting signals from each bead. It becomes easy to automate.

  The beads entering the bead fixing site can be removed by washing the quartz substrate on which the beads are placed with an ultrasonic washing machine. Since another group of beads can be placed on the quartz substrate from which the beads have been removed and assembled again as a device and used for analysis operations, according to the present invention, the substrate on which the microstructure has been fabricated can be reused without being disposable. Is possible.

The top view which shows the example of the fine structure pattern for bead fixation. The figure which shows a mode that the bead is arrange | positioned to the fine structure pattern for bead fixation. The whole figure of the quartz substrate in which the fine structure pattern for bead fixation was formed. The figure explaining the assembly of a device. Explanatory drawing of the method of detecting the appropriate weight to the PDMS board using the inclination structure site | part. Explanatory drawing of analysis operation. The fluorescence microscope image of the device which has arrange | positioned fluorescent dye containing bead.

Explanation of symbols

1: bead fixing part, 2: fine groove, 3: bead, 11: through hole, 12: flow path, 13: flow path, 14: inclined structure part, 15: fine structure pattern for arranging beads, 21: quartz substrate, 22: Substrate holder, 23: Presser plate, 24: Screw, 25: PDMS plate, 26: Spacer, 27: Presser plate, 28: Spring, 29: Screw, 30: O-ring made of PDMS, 31: Tube connector, 32 : PDMS entering the flow path, 33: Contact portion between the PDMS entering the flow path and the inclined structure part, 41: Microscope, 42: XY stage, 43: Microbead array device, 44: CCD camera, 45: Pressure gauge for liquid pressure measurement, 46: Syringe pump

Claims (10)

A hard substrate having a fine structure pattern formed on the surface thereof, each of which has a plurality of two-dimensionally regularly arranged bead fixing portions each accommodating a single bead and a fine groove for connecting the bead fixing portions to each other; ,
An elastic substrate;
A microchannel bead array device, comprising: means for bringing the elastic substrate into contact with and pressurizing the surface of the hard substrate on which the microstructure pattern is formed.   2. The microchannel bead array device according to claim 1, wherein a plurality of beads having bioactive probe molecules bound to the surface are accommodated in the plurality of bead fixing sites in a non-contact state with each other. Microchannel bead array device.   3. The microchannel bead array device according to claim 2, wherein the beads have a diameter of 1 to 100 micrometers.   The microchannel bead array device according to any one of claims 1 to 3, wherein the plurality of bead fixing portions have an internal volume in which two or more beads cannot enter, and the fine groove is a bead A microchannel bead array device having a width smaller than the diameter of the microchannel bead array device.   5. The microchannel bead array device according to claim 1, further comprising an inclined structure portion on the surface of the hard substrate for detecting a load that presses the elastic substrate against the hard substrate. A microchannel bead array device.   The microchannel bead array device according to any one of claims 1 to 5, wherein the hard substrate includes a through-hole connected to a sample solution introduction end of the fine structure pattern and a sample solution discharge of the fine structure pattern. A sample solution is introduced into the microstructure pattern from the surface of the hard substrate opposite to the surface on which the microstructure pattern is formed, and is introduced into the microstructure pattern through the through hole. The microfluidic bead array device, wherein the sample solution that circulates the structural pattern is discharged from the surface of the hard substrate opposite to the surface on which the fine structure pattern is formed, through the through hole.   The microchannel bead array device according to any one of claims 1 to 6, wherein the plurality of bead fixing portions are arranged in a two-dimensional lattice shape, and the bead fixing portions in each row of the two-dimensional lattice are formed by the fine grooves. A microchannel bead array device characterized by being connected.   The microchannel bead array device according to any one of claims 1 to 6, wherein a net-like channel is formed by the plurality of bead fixing sites and the fine groove, and the bead fixing site is located at a junction of the channels. A microchannel bead array device, characterized in that the channel portion that is set and connects the junction is a fine groove. Forming a fine structure pattern composed of bead fixing portions regularly arranged two-dimensionally on a hard substrate and fine grooves connecting the same;
Placing beads at a bead fixing site in the microstructure pattern;
Forming a microchannel having beads fixed therein by pressurizing and bonding a transparent elastic substrate to the surface of the hard substrate on which the fine structure pattern is formed. Device fabrication method.   10. The method of manufacturing a microchannel bead array device according to claim 9, wherein the weight of pressing the elastic substrate against the hard substrate is observed by observing the degree of penetration of the elastic substrate into the inclined structure portion provided in the groove structure on the hard substrate. A method for producing a microchannel bead array device, comprising the step of controlling

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