JP2005074796A - Method for joining microchip substrate and microchip - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for joining microchip substrates which are formed from a plastic material and have micro-channels in the surfaces without deforming the cross sections of the micro-channels by heat, damaging a bioactive substance fixed to the microchip, blocking the micro-channels by an adhesive, and fouling the inner walls of the micro-channels, and the microchip produced by the method. <P>SOLUTION: In the method for joining the first microchip substrate having the micro-channel on the surface and the second microchip substrate having a surface adhered to the surface having the micro-channel of the first microchip substrate, the first microchip substrate and/or the second microchip substrate are formed from the plastic material, and a process for joining the microchip substrates by laser beam fusion bonding is included. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a method for bonding microchip substrates having microchannels.

  Recently, research on the miniaturization of chemical reactions and separation systems using microfabrication technology called microreactors and microanalysis systems has been actively conducted, such as nucleic acids, proteins, sugar chains, etc. performed on microchips with microchannels. It is expected to be applied to analysis and synthesis, rapid analysis of trace chemical substances, and high-throughput screening of drugs and drugs. As an advantage of such a micro system, it is considered to realize an inexpensive system that can be carried in a small space because the amount of sample and reagent used or the amount of discharged waste liquid is reduced. Further, by increasing the ratio of the surface area to the volume, it is possible to increase the speed of heat transfer and mass transfer. As a result, precise control of reaction and separation, high speed and high efficiency, and suppression of side reactions are expected.

A microchannel is generally manufactured by bonding two members of a microchip substrate having a microfabrication to at least one member. Until now, glass has been mainly used as a substrate material for microchips. In order to form a microchannel using a glass substrate, for example, there is a method in which a metal or a photoresist resin is coated on the substrate, a microchannel pattern is exposed and developed, and then an etching process is performed. Thereafter, the glass substrate is bonded by anodic bonding or the like (Non-Patent Document 1).
However, since very dangerous chemicals such as hydrofluoric acid are used for etching glass, and exposure, development, and etching are performed for each sheet, the efficiency is very low and the cost is high.

  These microchips can be manufactured by injection molding using various plastics. In injection molding, a molten thermoplastic material is introduced into a mold cavity, the cavity is cooled, and the resin is cured, whereby a microchip substrate can be manufactured efficiently and economically, and is suitable for mass production. As a method for laminating the substrates, a heat press, heat fusion using ultrasonic waves, a method using an adhesive, and the like are mainly performed (Patent Document 1). However, in heat fusion using heat press or ultrasonic waves, the plastic resin is excessively melted by heat and the cross section of the microchannel is easily deformed, or the physiologically active substance immobilized on the microchip is easily damaged. There is a high possibility that it will not function or operate normally. In addition, the adhesive is more likely to be surplus than between the substrates, and the microchannel is likely to be blocked and the inner wall is likely to be contaminated.

Also proposed is a method of manufacturing a microchannel by forming at least one of a plastic substrate by casting, releasing the mold in the course of polymerization of a thermosetting plastic, bringing the substrate into close contact, and then completing the polymerization reaction (patent) Reference 2). Thermosetting plastics can be processed at low temperatures, so it is possible to bond microchip substrates without damaging equipment and devices due to heat and vibration, but they are not suitable for mass production due to the long cooling and curing time. In addition, it is difficult to release from the mold cavity and there is a high possibility of damaging the microchannel, and the mold cavity itself is also damaged, so that the mold life is short and not economically suitable.
Nobuaki Honda, Chemical Engineering, Vol. 66, No. 2, P71-74 (2002) JP 2002-139419 A JP 2002-207027 A

  An object of the present invention is to deform a microchip substrate made of a plastic material and having a microchannel on the surface thereof, by deforming the cross section of the microchannel due to heat, damaging a physiologically active substance immobilized on the microchip, The present invention provides a method for bonding without causing blockage and contamination of the inner wall, and a microchip that can be manufactured by this method.

The present invention
(1) A method of bonding a first microchip substrate having a microchannel on a surface thereof and a second microchip substrate having a surface closely attached to a surface of the first microchip substrate having a microchannel, A bonding method of a microchip substrate, wherein the first microchip substrate and / or the second microchip substrate is made of a plastic material and has a step of bonding by laser fusion,
(2) In the step of bonding by laser fusion, using a mask patterned in the same shape as the microchannel, bonding is performed by irradiating only the bonding portion other than the microchannel with laser (1). Method,
(3) The bonding method of the microchip substrate according to (1) or (2), wherein the bonding portion of the microchip substrate is bonded 1 μm to 2000 μm away from the edge portion of the microchannel,
(4) A method of bonding a microchip substrate according to any one of (1) to (3), including a step of immobilizing a physiologically active substance on a part of a microchannel before the step of bonding by laser fusion.
(5) The method for bonding microchip substrates according to (4), wherein the physiologically active substance contains at least one of nucleic acid, protein, sugar chain, and glycoprotein,
(6) The method for joining microchip substrates according to any one of (1) to (5), wherein the plastic material is a saturated cyclic polyolefin,
(7) (1) to (6) a microchip bonded by any one of the microchip substrate bonding methods,
It is.

  By using the bonding method of the present invention, a microchip substrate made of a plastic material and having a microchannel on the surface is deformed by heat, the cross section of the microchannel is deformed, the physiologically active substance immobilized on the microchip is damaged, and the adhesive is used. Bonding can be performed without blocking the microchannel and causing contamination of the inner wall.

Hereinafter, the present invention will be described in detail.
FIG. 1 shows a first macrochip substrate 1 having a microchannel 3 having at least one physiologically active substance immobilized on the surface thereof, and a surface that can substantially adhere to a surface of the first macrochip substrate having a microchannel. 1 shows a microchip in a state where a second macrochip substrate 2 having at least one is finally bonded.
At least one of the first and second microchip substrates used in the present invention is a plastic material. Various plastic materials can be used, but moldability, heat resistance, chemical resistance, adsorption, etc. according to the characteristics of the application, processing, solvent used, bioactive substance, and detection method of the produced macrochip. It is appropriately selected in consideration of the properties and the like.
For example, polystyrene, polyethylene, polyvinyl chloride, polypropylene, polycarbonate, polyester, polymethyl methacrylate, polyvinyl acetate, vinyl-acetate copolymer, styrene-methyl methacrylate copolymer, acrylonitrile-styrene copolymer, acrylonitrile- Examples thereof include butadiene-styrene copolymer, nylon, polymethylpentene, silicone resin, amino resin, polysulfone, polyethersulfone, polyetherimide, fluororesin, saturated cyclic polyolefin, and polyimide.
Among them, a cyclic polyolefin with little autofluorescence is most preferable because the detection method currently used most, including processability and economy, is fluorescence detection. In addition, additives such as pigments, dyes, antioxidants, and flame retardants may be appropriately mixed with these plastic materials.

The material of the other microchip substrate is not particularly limited, such as glass, silicon wafer, and plastic, but it is preferable to use the same material as the other microchip substrate because the same material is more likely to adhere when laser welding. In addition to the characteristics of microchips, materials are selected in consideration of the compatibility of fusion.
In addition, even if the compatibility between the materials is poor, the compatibility of the fusion is improved by carrying out the surface treatment, so that surface modification, such as introduction of functional groups, fixation of functional materials, It is also possible to perform imparting and imparting hydrophobicity.

Since bonding is performed by laser fusion, a microchip substrate using a material with a high laser absorption rate has better fusion bonding efficiency, and can be bonded with less laser power and less time. When it is necessary to fabricate a microchip substrate with a material having a low laser absorption rate, a material that absorbs the laser well between the microchip substrates 1 and 2 and melts and adheres to the bonding surface of the microchip substrate before bonding as a close contact layer. Processing and joining are also possible.
A microchip substrate using a plastic material is preferably used for the microchip substrate 1 for processing a microchannel from the viewpoint of ease of processing. As a method of processing a microchannel on a microchip substrate using a plastic material, injection molding using a microcavity mold cavity is preferable for mass production, but machining such as a drill, processing by hot embossing, laser processing Processing methods such as processing, dry etching pattern processing, and wet etching pattern processing can be selected.

  The width of the microchannel is preferably 1 μm or more and 500 μm or less, more preferably 10 μm or more and 300 μm or less, from the viewpoint of the amount of sample or reagent used or the amount of waste liquid discharged and the speed of heat transfer / mass transfer. The depth is preferably from 1 μm to 500 μm, more preferably from 10 μm to 300 μm. However, the flow path design of these microchannels is not limited to the above because it is appropriately designed in consideration of the detection object and convenience. In addition, the microchannel function is equipped with microdevices, specifically membranes, pumps, valves, sensors, motors, mixers, gears, clutches, microlenses, electrical circuits, etc., or multiple microchannels on the same substrate It can be compounded by processing on top.

  Examples of the physiologically active substance include nucleic acids, proteins, sugar chains, glycoproteins and the like, and an optimal physiologically active substance can be appropriately selected depending on the characteristics of the detection target. A plurality of physiologically active substances may be immobilized on the same channel, or different microchannels may be formed on the same microchip substrate and the physiologically active substances may be separately immobilized.

FIG. 2 shows a view at the time of joining when the laser 5 is used.
The microchip substrate 2 is brought into a close contact state by being sandwiched by a transparent pressing plate that does not absorb laser, for example, a glass plate or the like, and is attached to a close contact portion between the microchip substrate 1 and the microchip substrate 2, that is, a portion other than the microchannel. The heat generated by irradiating the laser melts and adheres to each other. Since the bioactive substance fixed to the microchannel is hardly thermally stressed because it is melt-bonded only at the close contact portions, the microchip can be joined while maintaining its function.

The laser wavelength, laser power, pressing pressure, and irradiation time, which are conditions for laser fusion, are determined by optimizing appropriately according to the materials of the upper and lower microchip substrates. The point of optimization is whether the joint surface is in close contact with the liquid when it flows into the microchannel, does not leak to a place other than the microchannel, or the cross section of the microchannel is not deformed. Etc.
More specifically, the bonding portion of the microchip substrate is preferably bonded to be 1 μm or more and 2000 μm or less away from the edge portion of the microchannel, and more preferably 5 μm or more and 1000 μm or less. If it is less than the lower limit from the edge, the melted part may melt into the microchannel and affect the microchannel. If the upper limit is exceeded, the liquid that should flow through the microchannel will ooze out from the channel.・ In addition to the efficiency of replacement when switching reagents, etc., the detection accuracy also deteriorates.

FIG. 3 shows a view at the time of bonding when the mask 5 patterned in the same shape as the microchannel is used.
Since it is patterned in the same shape as the microchannel, it is the same as in FIG. 2 except that no laser is irradiated onto the microchannel, and the bonding performance is not significantly different from the bonding method of FIG. The advantage of the mask 5 of this head is that, when bonding without using a mask, it is necessary to accurately irradiate a contact portion other than the microchannel with a laser, so that the operations such as alignment are complicated and the accuracy is distorted. In such a case, it is possible to ameliorate the problem of irradiating the microchannel with a laser to cause degradation of the physiologically active substance and the like, resulting in a decrease in sensitivity during detection. In addition, the speed of tact time and the improvement of productivity are mentioned as the advantage that the bonding is completed only by irradiating the microchip substrate once by spreading the laser in a belt shape.

Next, as an actual joining example, an example will be described in which an antibody against a protein in the flow path is immobilized and joined.
A substrate having a size of about 25 mm × 75 mm and a thickness of about 0.5 to 1.5 mm and having a microchannel on the surface as shown in FIG. 4 is formed by injection molding to obtain a main body substrate. A substrate having the same size and no microchannel is formed to form a lid substrate. After subjecting the main body substrate to a hydrophilization treatment using low-temperature oxygen plasma or the like, an aminosilane is reacted with the antibody fixing portion 6 in the microchannel, and an aldehyde group is introduced by further reacting with glutaraldehyde. The antibody is dissolved at an appropriate concentration in a buffer solution such as PBS (−) to prepare an antibody solution. Subsequently, the antibody solution is spotted on the antibody fixing portion with a pin having a diameter of about 40 μm and left to stand. The standing time is about 30 minutes to 2 hours at room temperature and about 6 to 12 hours at 4 ° C. After leaving and immersing in pure water and washing, after immersing in blocking solution in which BSA etc. is dissolved in PBS (-) and blocking, after immersing and washing the substrate in the washing solution, Dry at room temperature. Subsequently, bonding by laser fusion is performed. Finally, ports (7, 8) are provided for solution flow.
As described above, a bonded substrate having a detection antibody immobilized on a microchannel can be obtained.

Example 1
A substrate having a size of 25 mm × 75 mm, a thickness of 0.5 mm, and a microchannel having a width of 100 μm and a depth of 50 μm arranged on the surface as shown in FIG. 4 was molded by injection molding with a saturated cyclic polyolefin resin to obtain a main substrate. . A substrate having the same size and no microchannel was also molded with a saturated cyclic polyolefin resin to form a lid substrate. After subjecting the main body substrate to a hydrophilic treatment by low-temperature oxygen plasma, aminosilane was reacted with the antibody fixing part 6 in the microchannel, and glutaraldehyde was further reacted to introduce an aldehyde group. An anti-rat albumin antibody was dissolved at a concentration of 1 μg / ml in PBS (−) to prepare an antibody solution. Subsequently, the antibody fixing part was spotted with a pin having a diameter of 40 μm. After being left for 30 minutes and then immersed in pure water for cleaning, the substrate was immersed in a blocking solution in which 1% BSA was dissolved in PBS (-) for blocking, and then the substrate was immersed in the cleaning solution for cleaning. And dried at room temperature. Subsequently, after covering with a lid substrate, laser bonding was performed using a mask sandwiched between glass substrates and patterned in the same shape as the microchannel. Ports (7, 8) for circulating the solution were provided and used for a sensitivity evaluation test as a biological substance detection substrate for detecting rat albumin.

(Example 2)
A substrate having a size of 25 mm × 75 mm, a thickness of 0.5 mm, and a microchannel having a width of 100 μm and a depth of 50 μm arranged on the surface as shown in FIG. 4 was molded by injection molding with a saturated cyclic polyolefin resin to obtain a main substrate. . A substrate having the same size and no microchannel was also molded with a saturated cyclic polyolefin resin to form a lid substrate. After subjecting the main body substrate to a hydrophilic treatment by low-temperature oxygen plasma, aminosilane was reacted with the antibody fixing part 6 in the microchannel, and glutaraldehyde was further reacted to introduce an aldehyde group. An anti-rat albumin antibody was dissolved at a concentration of 1 μg / ml in PBS (−) to prepare an antibody solution. Subsequently, the antibody fixing part was spotted with a pin having a diameter of 40 μm. After being left for 30 minutes and then immersed in pure water for cleaning, the substrate was immersed in a blocking solution in which 1% BSA was dissolved in PBS (-) for blocking, and then the substrate was immersed in the cleaning solution for cleaning. And dried at room temperature. Subsequently, after covering with a lid substrate, the glass substrate was sandwiched and laser fusion was performed without using a mask to perform bonding. Ports (7, 8) for circulating the solution were provided and used for a sensitivity evaluation test as a biological substance detection substrate for detecting rat albumin.

(Comparative Example 1)
A substrate having a size of 25 mm × 75 mm, a thickness of 0.5 mm, and a microchannel having a width of 100 μm and a depth of 50 μm arranged on the surface as shown in FIG. 1 is molded by injection molding with a saturated cyclic polyolefin resin to obtain a main body substrate. . A substrate having the same size and no microchannel was also molded with a saturated cyclic polyolefin resin to form a lid substrate. After subjecting the main body substrate to a hydrophilic treatment by low-temperature oxygen plasma, aminosilane was reacted with the antibody fixing part 6 in the microchannel, and glutaraldehyde was further reacted to introduce an aldehyde group. An anti-rat albumin antibody was dissolved at a concentration of 1 μg / ml in PBS (−) to prepare an antibody solution. Subsequently, the antibody fixing part was spotted with a pin having a diameter of 40 μm. After being left for 30 minutes and then immersed in pure water for cleaning, the substrate was immersed in a blocking solution in which 1% BSA was dissolved in PBS (-) for blocking, and then the substrate was immersed in the cleaning solution for cleaning. And dried at room temperature. Next, cover with a lid substrate and heat for 2 minutes at a hot plate temperature of 130 ° C. to join the body substrate and lid substrate, provide ports (7, 8) to distribute the solution, It used for the sensitivity evaluation test as a biological substance detection board | substrate to detect.

(Protein detection test)
Rat albumin was dissolved in PBS (−) to a concentration of 0.1 μg / ml and 0.01 μg / ml to prepare a rat albumin solution. Rat albumin solution of each concentration was allowed to flow from the substrate port for 3 minutes at a feeding speed of 20 μl / min. Next, a washing station containing 0.5% TWEEN 20 was washed by flowing from the port into the flow path for 3 minutes at a liquid feed speed of 20 μl / min. Subsequently, an anti-rat albumin antibody solution labeled with rhodamine (dissolved in PBS (−) at 1 μg / ml) was allowed to flow for 3 minutes at a feeding speed of 20 μl / min, and then the washing station containing 0.5% TWEEN 20 was ported. To the flow path for 3 minutes at a liquid feed speed of 20 μl / min for washing. Finally, ultrapure water was allowed to flow from the port at a liquid feed speed of 20 μl / min for 3 minutes.
The intensity of the fluorescent spot on the antibody fixing part 6 was measured with a confocal laser scanner. Table 1 shows the relative intensities of each concentration of the rat albumin solution, with the spot fluorescence intensity of the substrate of Example 1 being 100. For each concentration of the rat albumin solution, Table 2 shows a comparison of SN ratios with the SN ratio of the fluorescence intensity of the spots measured on the substrate of Example 1 being 100. Here, the S / N ratio was calculated by dividing the fluorescence spot intensity of the antibody immobilization part by the fluorescence intensity other than the antibody immobilization part.

  The bonding method of the present invention can bond a microchip substrate without causing deformation of the cross section of the microchannel, damage to the physiologically active substance immobilized on the microchip, blockage of the microchannel by the adhesive, and contamination of the inner wall. Therefore, it can be applied to microchips for various uses.

It is the cross-sectional schematic which becomes one Example of the microchip of this invention. In the joining method of the microchip board | substrate of this invention, it is a cross-sectional schematic diagram at the time of joining at the time of joining by laser fusion. In the joining method of the microchip board | substrate of this invention, it is the cross-sectional schematic at the time of joining at the time of using the mask patterned by the same shape as a microchannel. FIG. 2 is a schematic plan view showing an embodiment of a macrochip substrate having a microchannel on the surface used in the present invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Microchip board | substrate which has a microchannel 2 Microchip board | substrate which has the surface which closely_contact | adheres to the surface which has the microchannel groove | channel 3 Microchannel 4 Laser 5 Mask 6 patterned in the same shape as a microchannel 6 Antibody immobilization part 7 Injection port 8 Discharge port

Claims (7)

A method of bonding a first microchip substrate having a microchannel on a surface thereof and a second microchip substrate having a surface closely attached to a surface of the first microchip substrate having a microchannel, A method of joining microchip substrates, comprising: a step of joining the microchip substrate and / or the second microchip substrate made of a plastic material by laser fusion. The method of bonding a microchip substrate according to claim 1, wherein, in the step of bonding by laser fusion, only a bonding portion other than the microchannel is irradiated with laser using a mask patterned in the same shape as the microchannel. The method for bonding a microchip substrate according to claim 1 or 2, wherein the bonding portion of the microchip substrate is bonded 1 μm to 2000 μm away from the edge portion of the microchannel. The method for bonding a microchip substrate according to any one of claims 1 to 3, further comprising a step of immobilizing a physiologically active substance in a part of the microchannel before the step of bonding by laser fusion. The method for bonding microchip substrates according to claim 4, wherein the physiologically active substance contains at least one of nucleic acid, protein, sugar chain, and glycoprotein. The method for joining microchip substrates according to claim 1, wherein the plastic material is a saturated cyclic polyolefin. The microchip joined by the joining method of the microchip board | substrate in any one of Claims 1-6.

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