JP5870813B2 - Manufacturing method of micro chemical device - Google Patents

Description

The present invention relates to a manufacturing method of a microchemical device for holding various cells and proteins in a well for inspection and screening, and more specifically, a microchemical device having different surface states on the bottom and side walls of the well . It relates to a manufacturing method .

  When a protein is immobilized on a substrate, the protein cannot be immobilized at a specific position because the protein is nonspecifically bound to the substrate (nonspecific adsorption) by a general method. Therefore, in Non-Patent Document 1, a silicone resin (polydimethylsiloxane: PDMS) is used using MEMS technology, a hole of 10 μm or less is made in a thin PDMS film, and the protein is fixed to the substrate (glass substrate). It is used as a sieve for sieving. In the research described in Non-Patent Document 1, the hole to be opened in the PDMS film is 10 μm or less, and the PDMS film is peeled off from the substrate when the protein is immobilized on the substrate.

  Various prior patents have been filed for well-shaped and channel-shaped devices using PDMS (for example, Patent Document 1). In Patent Document 1, in order to improve the surface properties of PDMS, a polyether-modified surfactant is contained in the sheet, and after the formation of the fine structure, oxygen plasma treatment and organosilane solution treatment are performed to make the surface permanently hydrophilic. It is described that it is possible to manufacture a device made of PDMS having a contact angle evaluation with water of 40 ° or less. The used organosilane is hydrolyzed and the hydroxyl group is substituted on the siloxane skeleton of the main chain, so that PDMS is hydrophilized.

  FIG. 4 shows a conventional highly integrated well device 10. In the conventional method, a plurality of wells 12,... Having a diameter of several tens to several hundreds of μm are formed on the surface of a plastic substrate 11 by injection molding. In the well device 10 thus manufactured, the entire surface of the device including the well 12 including the bottom and side walls of the well 12 is in the same surface state. Then, cells, proteins 13, ... are also adsorbed on the surface between the wells 12,12. In order to prevent this phenomenon, only the inner surface of the well 12 of the well device 10 is subjected to surface treatment, and the surface state between the well 12 and the surface other than the well 12 (in terms of the water droplet contact angle), for example, Even if the water droplet contact angle of water is 40 ° or less and the contact angle other than the well 12 is 80 ° or more, which is the original water droplet contact angle of the polymer, the diameter of the well 12 is several tens to several hundreds of μm. Further, it is very difficult to hydrophilize only the inner surface of the well 12, and if the surface treatment is forcibly performed, there is a high possibility that the treatment cost is expensive and uneven.

JP 2006-181407 A

Kyoko Atsuta et al., “PDMS perforated structure for protein patterning”, Institute of Industrial Science, University of Tokyo, Research Bulletin, Vol. 55, No. 6, p498-501 (2003)

Therefore, in view of the above-described situation, the present invention intends to solve the problem in a microchemical device including a plastic plate in which wells with a diameter of several tens to several hundreds of μm are highly integrated on the surface. A microchemical device that can have a different surface state (except for the degree of hydrophilicity) at the bottom and other than the bottom of the well, thereby preventing cells and proteins from adhering to the plate surface, which is inherently undesirable. It is in the point of providing a manufacturing method .

In order to solve the above-mentioned problems, the present invention includes a step of molding an upper plate made of polydimethylsiloxane (PDMS) having a plurality of through holes having a diameter of several tens to several hundreds of μm at arbitrary intervals, and a planar shape. Forming a lower plate made of plastic other than PDMS having a surface, an oxygen plasma treatment step in which the lower plate is exposed to oxygen plasma to introduce oxygen-containing groups on the surface, and the surface of the lower plate is subjected to oxygen plasma A step of introducing a functional group capable of interacting with the surface of a cell or protein by applying a silane coupling agent having a concentration of 0.05 wt% to 3 wt% after introducing an oxygen-containing group by treatment; and the upper plate Immediately before joining the bottom plate and the lower plate, a process of oxygen plasma treatment, laminating both plates up and down, pressurizing both plates with a predetermined pressure, and joining and integrating, And forming a well bottom having a functional group to which cells and proteins easily adhere on the surface of the lower plate, and forming a well side wall to which cells and proteins are difficult to adhere in the through hole of the upper plate. The manufacturing method of the microchemical device characterized by the above was established .

The manufacturing method of the microchemical device of the invention according to the present invention as described above is a PDMS having a planar surface and an upper plate made of polydimethylsiloxane (PDMS) provided with a plurality of through holes to be wells at arbitrary intervals. A lower plate made of plastic, and before the two plates are stacked and joined together, the lower plate is subjected to oxygen plasma treatment to introduce oxygen-containing groups on the surface, and then the concentration is 0.05 wt. A functional group capable of interacting with the surface of cells and proteins is introduced by applying a 3 to 3 wt% silane coupling agent, and then both plates are subjected to oxygen plasma treatment, and both plates are pressed at a predetermined pressure to be integrated. As a result, a well bottom is formed on the surface of the lower plate, and a well sidewall is formed in the through hole of the upper plate. By making the surface different from the surface of the upper plate (the degree of hydrophilization is different), the surface state of the well bottom and other parts are made different so that cells and proteins adhere to the bottom of the well (adsorption) ), And it can be easily prevented from adhering (adsorbing) to the surface of the other upper plate, and its manufacture is also easy. In addition, the size of the well is determined by the diameter of the through hole formed in the upper plate, and the depth of the well is determined by the thickness of the upper plate, which facilitates design and increases the degree of freedom in design.

  In the present invention, the silane coupling agent treatment is performed after the plasma treatment, but the main purpose of this treatment is not the hydrophilization of the plate (upper plate: PDMS), but the lower plate (eg, polystyrene, acrylic resin, polycarbonate, etc.). And a functional group capable of interacting with cells and proteins on the surface-treated surface of the lower plate (therefore, this functional group exists on the surface of the bottom of the well). Immediately before joining the upper plate and the lower plate, oxygen plasma treatment is performed, the plates are stacked one above the other, and both the plates are pressurized at a predetermined pressure, so that they can be stably joined and integrated. Furthermore, after the silane coupling treatment at a concentration of 0.05 wt% to 3 wt%, the surface of the lower plate, which is the bottom of the well subjected to the oxygen plasma treatment, tends to adhere (adsorb) cells and proteins due to the presence of functional groups. , Can significantly reduce toxicity to cells.

The microchemical device of this invention is shown, (a) is a simple perspective view which shows a structure, (b) A simplified sectional view. It is a simplified sectional view showing the technical idea of the microchemical device of the present invention. It is explanatory drawing which shows the manufacturing method of the microchemical device of this invention. It is a simplified sectional view showing a conventional highly integrated well device.

  Next, the present invention will be described in more detail based on the embodiments shown in the accompanying drawings. The microchemical device of the present invention is classified as a highly integrated well device, in which a large number of wells (bottomed holes) are formed on the surface of a plate. The present invention proposes a device that adsorbs cells and proteins to the well bottom by making the surface state of the well bottom different from the surface other than the well bottom.

  As shown in FIG. 1A, the configuration of the microchemical device 1 of the present invention is composed of a plastic upper plate 2 and a plastic lower plate 3 having a diameter of several tens to several hundreds of μm. The upper plate 2 provided with the through holes 4,... At an arbitrary pitch and the lower plate 3 having a planar surface are created separately. In this case, it is important that the upper plate 2 and the lower plate 3 have different surface states. That is, in order to make the surface states of the upper plate 2 and the lower plate 3 different, the upper plate 2 and the lower plate 3 may be a combination of different materials, or the surface treatment process is changed for each plate. In addition, a technique of changing different materials and surface treatment processes may be used. Then, the upper plate 2 and the lower plate 3 are stacked one above the other, and the two plates are joined and integrated, whereby a well 6 is formed by the through hole 4 of the upper plate 2 and the surface 5 of the lower plate 3. The That is, as shown in FIG. 1B, the bottom of the well 6 is formed on the surface 5 of the lower plate 3, and the side wall of the well 6 is formed in the through hole 4 of the upper plate 2. Reference numeral 7 in the figure denotes cells and proteins.

  Thus, by configuring the microchemical device 1, the surface state of the bottom of the well 6 (surface 5 of the lower plate 3) is different from the surface other than the bottom of the well 6 (well side wall and the surface of the upper plate 2). Can be different. As a result, various cells, protein 7 and the like can be adsorbed only on the bottom of the well 6, and further, cells, protein 7 and the like that have entered the well 6 in the washing process of the cell and protein 7 are difficult to flow out. Can be demonstrated. The cross-sectional shape in the plate thickness direction of the through hole 4 having a diameter of several tens to several hundreds of μm provided in the upper plate 2 is not particularly limited. In general, it is straight or has a cone shape that tapers toward the bottom of the well.

  In more detail, the manufacturing method of the microchemical device 1 of this invention is demonstrated based on FIG.1 and FIG.3. The material of the upper plate 2 is not particularly limited. By combining with the lower plate 3, the through hole 4 provided in the upper plate 2 becomes a well 6 into which cells and proteins 7 are put, and the diameter of the well 6 is several tens. Since the thickness is from μm to several hundred μm, a sheet having a thickness of several hundred μm is required even if it is thick. Therefore, a PDMS sheet is an example of the production of the upper plate 2. This sheet may be produced by injection molding from a liquid silicone resin (for example, KE-1935 (A / B), KE-1950-30 (A / B) manufactured by Shin-Etsu Chemical Co., Ltd.), or a commercially available silicone sheet. May be used. And the through-hole 4 was provided in the PDMS sheet (upper plate 2) with the carbon dioxide laser.

  Specifically, the thickness of the PDMS sheet is 50 μm or 100 μm. The carbon dioxide laser used for punching the PDMS sheet is LP-400 made by Panasonic SUNX. As an example of the laser processing conditions, the laser power is 40% and the scanning speed is 10 mm / sec. Through holes 4 of φ100 μm were formed at regular intervals in the 1.4 × 1.4 mm area of the PDMS sheet, and a plurality of 1.4 × 1.4 mm areas were produced in the PDMS sheet.

  In addition, the hole processing of the upper plate 2 is not limited to the processing by the carbon dioxide laser, and a UV laser, a picosecond laser, a hemtosecond laser, or the like can be used according to the size of the through hole 4. Furthermore, using a lithography technique, a UV resist pattern with a diameter of several tens to several hundreds of μm is produced, and a nickel electroformed plate is produced using this resist pattern as a master mold, and various penetrations are made through this nickel electroformed plate. It is also possible to provide holes 4 in the upper plate 2. When the through hole 4 is provided in the upper plate 2 through such a plate, the shape of the hole is not limited to a circle, and a polygonal shape such as a quadrangle or a hexagon is also possible.

  The material of the lower plate 3 is not particularly limited, and is not particularly limited as long as it is a plastic resin that can be joined to the upper plate 2 and can be manufactured by injection molding or other molding methods. Further, the size of the device is not particularly limited.

  The upper plate 2 and the lower plate 3 are joined as shown in FIG. First, the lower plate 3 is exposed to oxygen plasma to introduce oxygen-containing groups on the surface (oxygen plasma treatment), then immersed in a silane coupling aqueous solution (silane coupling treatment), and dehydrated in a drying furnace. Introduce a functional group on the surface. Thereafter, it is cooled to room temperature and washed. Then, immediately before the upper plate 2 and the lower plate 3 are joined together, an oxygen plasma treatment is performed, the two plates are joined, pressurized at a predetermined pressure, and integrated. At this time, heating may be performed.

  Thus, in the present invention, two types of plastic upper plate 2 and lower plate 3 are accurately aligned and joined and integrated, and a through hole having a diameter of several tens to several hundreds of μm formed in the upper plate 2 is formed. 4, the side wall of the well 6 is formed, and the surface 5 of the lower plate 3 forms the bottom of the well 6. Here, for the positioning of the upper plate 2 and the lower plate 3, a generally used technique can be adopted. For example, through holes having a diameter of 500 μm are provided in the four corners on the upper plate 2 side, and a convex shape (column: diameter 10 to 20 μm smaller than φ500 μm) is provided in the lower plate 3.

  In the present invention, in order to prevent various cells and proteins 7 from adhering to other than the well 6 of the upper plate 2, the surface state of the bottom of the well 6 is different from that of the other part. Therefore, different plate materials are used for the upper plate 2 and the lower plate 3, and the surface treatment method for changing the surface state of each plate is changed. By these techniques, in each well 6 of the highly integrated device, the surface state of the well bottom and the well sidewall excluding the well bottom is different (the contact angle is different when expressed by the water contact angle of water), and various cells and proteins 7 can be prevented from adhering to portions other than the well portion.

  For this purpose, in this embodiment, only the lower plate 3 is subjected to silane coupling treatment. An example of a silane coupling agent is 3-aminopropylmethoxysilane, and an amino group can be introduced on the surface of the lower plate by the treatment. The concentration of the silane coupling agent is preferably about 0.05 wt% to 10 wt%. The presence of this amino group on the surface of the well bottom makes it possible to adsorb cells and proteins to the bottom of the well due to the combined action with cells and proteins. Regarding the bonding between the upper plate 2 and the lower plate 3, the higher the concentration of the silane coupling agent contributes to the stability of the bonding, while the toxicity to cells and proteins also increases.

In this embodiment, although the example which used the silicone resin for the upper plate 2 was shown, polystyrene is mentioned as polymer materials other than a silicone resin. In the case of polystyrene, as in the case of PDMS, it is possible to provide a through hole 4 having a diameter of several tens to several hundreds of μm by laser processing on a sheet having a thickness of 50 μm to several hundreds of μm. In the case of polystyrene, when polystyrene, which is the same material, is used for the lower plate 3, it can be thermally bonded to the upper plate 2 after the oxygen plasma treatment and the silane coupling treatment.
Further, when the cyclic olefin polymer is used for the upper plate 2, the same processing process is possible, and when the same material is used for the lower plate 3, after the oxygen plasma treatment and the silane coupling treatment, It is also possible to join the plate 2 and the lower plate 3 by diffusion bonding. In the case of an olefin resin, diffusion bonding can be performed only by pressurization at room temperature at the time of bonding, but in the case of an acrylic resin (PMMA or the like), heating and pressurization are performed. In any case, it is possible to shorten the bonding time by heating the bonding surface from 50 ° C. to 120 ° C.

  As described above, the silane coupling agent used in the present invention can introduce a functional group capable of interacting with cells and proteins into the surface of the well bottom formed by joining the upper plate and the lower plate. Is the purpose. Therefore, the silane coupling agent to be used is not limited to 3-aminopropylmethoxysilane, and a silane coupling agent capable of introducing a functional group capable of interacting with cells and proteins may be appropriately selected. That is, the chemical structural formula of the silane coupling agent is as follows.

And the following can be used as various functional groups (Y).
In the case of an amino group (—NH ₂ ), 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane, N- (2-aminoethyl) -3-aminopropyltrimethoxysilane, N- (2-amino) Ethyl) -3-aminopropylmethyldimethoxysilane, 3- (N-phenyl) aminopropyltrimethoxysilane, and the like can be used.
In the case of an isocyanate group (—N═C═O), 3-isocyanatepropyltriethoxysilane, 3-isocyanatepropyltrimethoxysilane, or the like can be used.
In the case of a ureido group (—NHCONH ₂ ), 3-ureidopropyltriethoxysilane or the like can be used.
In the case of a mercapto group (—SH), 3-mercaptopropyltrimethoxysilane, 3-mercaptopropyltriethoxysilane, or the like can be used.
In the case of a carboxyl group (—COOH), a carboxylic acid anhydride or the like can be used.

  Such various functional groups can be appropriately used depending on the type of cell and protein. In addition, since it is necessary to dissolve the silane coupling agent in the solvent, it is possible to use alcohols (for example, methanol, ethanol, etc.) in addition to pure water in consideration of the solvent resistance of the device. Yes, a mixed solvent of pure water and alcohol may be used as necessary.

  On the other hand, the plate surface to be surface-treated needs to be plasma-treated because the plate surface needs to be sufficiently wetted with the solution when immersed in the silane coupling agent solution. The plasma treatment is not limited to oxygen plasma treatment, and the device is sufficiently wetted with the silane coupling agent solution used (preferably a water droplet contact angle of 40 ° or less when the solvent is dropped on the device surface). There is no problem if the plasma irradiation conditions are as follows. Also, the concentration of the silane coupling agent is not particularly limited, and the concentration of the silane coupling agent is adjusted to a concentration that is soluble in the solvent in consideration of the types of the upper plate 2 and the lower plate 3 to be joined, the cells to be used, and the type of toxicity to the protein. That's fine. Furthermore, it does not limit to a silane coupling agent, What is necessary is just what hydrolyzes, such as a titanium coupling agent. In general, one having a hydrolyzable organic group can be used, and an OH group needs to be generated.

  The present invention generally relates to an adhesive silicone resin (for example, X-34-1625A / B, KE-2090 manufactured by Shin-Etsu Chemical Co., Ltd.) used for bonding a silicone resin to polycarbonate, polybutylene terephthalate, polyphenylene oxide, polyamide, and the like. -50A / B etc.). Furthermore, it is different from the joining process using various primers without plasma treatment.

  The material of the upper plate is PDMS, and the material of the lower plate is polystyrene. The well diameter is φ100 μm and the well depth is 50 μm.

  Specific examples of the lower plate surface treatment are shown below. Oxygen plasma processing conditions are as follows: the lower plate is placed in a plasma processing apparatus (PDC210, manufactured by Yamato Material Co., Ltd.), the inside of the apparatus is purged with nitrogen gas, the pressure is reduced to 0.01 MPa, 100 cc of oxygen is introduced, RF output Discharge at 300W for 5 to 60 seconds.

  The silane coupling agent treatment was adjusted such that 3-aminopropyltrimethoxysilane had a concentration of 0.05, 0.5, 1, 3, 5, 7, 10, 15 wt% in pure water. The lower plate after the oxygen plasma treatment is immersed in the silane coupling solution of each concentration for 20 to 300 seconds, and the surface of the lower plate is reacted with the silane coupling agent.

  A specific example of the joining process between the upper plate and the lower plate is shown below. Oxygen plasma treatment is performed on the joint surface of both the upper and lower plates. Oxygen plasma treatment conditions were as follows: each plate was placed in a plasma treatment apparatus, the inside of the apparatus was purged with nitrogen gas, the pressure was reduced to 0.01 MPa, 100 cc of oxygen was introduced, and an RF output of 300 W was discharged for 5 seconds to 30 seconds. . Then, join the two plates together with the oxygen plasma surface. In this case, in this case, the bonding can be efficiently performed by pressurizing the bonding surface at a pressure of 0.01 MPa to 0.1 MPa and then heating from 50 ° C. to 120 ° C.

  Toxicity test evaluation of various cells was performed using the thus produced microchemical device. The procedure is shown below.

1. Hybridoma
• 50,000 viable cells are suspended in 1 ml of cell culture medium (RPMI 1640).
・ Put the total volume of 1ml into the test device.
A centrifuge (500 rpm, 3 min as an example of conditions).
-Wash twice with culture medium.
・ Leave for about 1 hour.
-Trihan blue staining to stain dead cells.
・ Measure cells with cell counter.

2. 50,000 Mouse ES cells / viable cells are suspended in 1 ml of cell culture medium (GMEM + 10% FBS).
・ Put the total volume of 1ml into the test device.
A centrifuge (500 rpm, 3 min as an example of conditions).
-Wash twice with culture medium.
・ Leave for about 1 hour.
-Trihan blue stain to stain dead cells.
・ Measure cells with cell counter.

Table 1 shows the results of the evaluation of the cell toxicity and the bonding properties of the upper plate and the lower plate at each concentration of the silane coupling agent treatment.

  As can be seen from this result, when the concentration of the silane coupling agent treatment is 0.05 wt% to 10 wt%, the adhesion of both plates is good, and at the same time, the survival rate of Mouse ES cells is 75 to 100%, Hybridoma The survival rate was 70 to 100%. In particular, when the concentration of the silane coupling agent treatment was 0.05 wt% to 3 wt%, the survival rate of Mouse ES cells was 90 to 100%, and the survival rate of Hybridoma was 80 to 100%.

1 Microchemical device 2 Upper plate 3 Lower plate 4 Through hole 5 Surface of lower plate 6 Well 7 Cell, protein

Claims (1)

Forming an upper plate made of polydimethylsiloxane (PDMS) having a plurality of through holes having a diameter of several tens to several hundreds of μm at an arbitrary interval;
Molding a plastic lower plate other than PDMS having a planar surface;
An oxygen plasma treatment step in which the lower plate is exposed to oxygen plasma to introduce oxygen-containing groups on the surface;
After the surface of the lower plate is subjected to oxygen plasma treatment to introduce oxygen-containing groups, a functional group capable of interacting with the surface of cells or proteins is applied by applying a silane coupling agent having a concentration of 0.05 wt% to 3 wt%. Introducing the process;
Immediately before joining the upper plate and the lower plate, a process of oxygen plasma treatment, laminating both plates up and down, pressurizing both plates with a predetermined pressure and integrating them,
And forming a well bottom having a functional group to which cells and proteins easily adhere on the surface of the lower plate, and forming a well side wall portion to which cells and proteins do not easily adhere in the through hole of the upper plate. A method for producing a microchemical device.

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