KR101597210B1 - Method for forming microchannels of lab-on-a-chip - Google Patents

Method for forming microchannels of lab-on-a-chip Download PDF

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Publication number
KR101597210B1
KR101597210B1 KR1020150039965A KR20150039965A KR101597210B1 KR 101597210 B1 KR101597210 B1 KR 101597210B1 KR 1020150039965 A KR1020150039965 A KR 1020150039965A KR 20150039965 A KR20150039965 A KR 20150039965A KR 101597210 B1 KR101597210 B1 KR 101597210B1
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South Korea
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substrate
channel pattern
microchannel
polydimethylsiloxane
thermoplastic plastic
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KR1020150039965A
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Korean (ko)
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이내윤
장민정
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가천대학교 산학협력단
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
    • B81C1/00—Manufacture or treatment of devices or systems in or on a substrate
    • B81C1/00436—Shaping materials, i.e. techniques for structuring the substrate or the layers on the substrate
    • B81C1/00444—Surface micromachining, i.e. structuring layers on the substrate
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
    • B81C1/00—Manufacture or treatment of devices or systems in or on a substrate
    • B81C1/00015—Manufacture or treatment of devices or systems in or on a substrate for manufacturing microsystems
    • B81C1/00023—Manufacture or treatment of devices or systems in or on a substrate for manufacturing microsystems without movable or flexible elements
    • B81C1/00055—Grooves
    • B81C1/00063—Trenches
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
    • B81C1/00—Manufacture or treatment of devices or systems in or on a substrate
    • B81C1/00436—Shaping materials, i.e. techniques for structuring the substrate or the layers on the substrate
    • B81C1/00444—Surface micromachining, i.e. structuring layers on the substrate
    • B81C1/0046—Surface micromachining, i.e. structuring layers on the substrate using stamping, e.g. imprinting
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B81—MICROSTRUCTURAL TECHNOLOGY
    • B81C—PROCESSES OR APPARATUS SPECIALLY ADAPTED FOR THE MANUFACTURE OR TREATMENT OF MICROSTRUCTURAL DEVICES OR SYSTEMS
    • B81C3/00—Assembling of devices or systems from individually processed components
    • B81C3/001—Bonding of two components

Abstract

A method to form a microchannel for a lab-on-a-chip without photolithography is disclosed. The method to form a microchannel for a lab-on-a-chip without photolithography according to an embodiment of the present invention comprises: a step a) of forming a first channel pattern having a semicircular cross section on a first thermoplastic substrate to be engraved; a step b) of stacking a second flat thermoplastic substrate on the first thermoplastic substrate and forms the first channel pattern on the surface of the second thermoplastic substrate to be embossed through the hot-embossing of the stacked body; a step c) of dismantling the stacked body after the stacked body is cooled and transcribing the embossed first channel pattern formed by using the second thermoplastic substrate as a mold into a polydimethylsiloxane substrate; and a step d) of bonding a pair of polydimethylsiloxane substrates obtained by repetitively performing the step c) to each other to allow the surfaces of the polydimethylsiloxane substrates where the first channel patterns are formed to face each other.

Description

[0001] METHOD FOR FORMING MICROCHANNELS OF LAB-ON-A-CHIP [0002]

The present invention relates to a microchannel forming method, and more particularly, to a microchannel forming method for forming a microchannel for a lab-on-a-chip without using photolithography.

In addition to microfluidics, lab-on-a-chip systems have led to significant advances in biological analysis methods such as PCR (polymerase chain reaction), cell manipulation, and tissue engineering. PDMS (polydimethylsiloxane) is the most widely used material in the lab-on-a-chip system because of easiness of duplicate molding of PDMS, easy surface modification, easy joining and excellent biocompatibility.

On the other hand, most of microdevices using PDMS are usually fabricated by photolithography. However, despite the advantages of the photolithography method, several disadvantages are pointed out. The first is high cost. This is because the photolithography method uses expensive equipment and greatly increases the manufacturing cost. The second is that the master mold produced by the photolithography method has a micro-pattern of a rectangular cross-sectional shape. Therefore, there is a limit in that it is not suitable for the production of a micro pattern requiring a circular cross section. That is, micro-channels having a circular cross section (so-called cylindrical micro-channels) are required or photolithography methods are not suitable for competitive micro vascular system manufacturing, cell capture, uniform inner wall coating, and valve application channels.

The same is true of microchannels having a multi-layered structure (or a multi-layered structure). In a lab-on-a-chip device, microchannels having a multi-layer structure are utilized in the micro-bead and cell self-sorting, hemocyte separation, microvascular imitation, and the like in fluid migration technology. Conventionally, a microchannel having a multilayer structure has been formed by repeating a photolithography method (or using an electron beam or an ion beam), but it has a disadvantage that it takes a long time and involves high cost and is not suitable for manufacturing a complicated structure. Many researchers have attempted to develop a method for fabricating a microstructure that is not based on a photolithography method (see Non-Patent Documents 1 and 2).

 Zhoa et al, Lab Chip, 2003, 3, 93-99.  Wilson et al, Lab Chip, 2011, 11, 1550-1555.

The present invention seeks to provide a method of forming a microchannel for a lab-on-a-chip having a cylindrical or multi-layered structure with ease and high reproducibility without using a photolithography process.

According to an aspect of the present invention, there is provided a method of manufacturing a semiconductor device, comprising the steps of: a) forming a first channel pattern having a semicircular cross section on a first thermoplastic plastic substrate at an oblique angle; b) forming a first channel pattern on the surface of the second thermoplastic plastic substrate by embossing the laminated body after laminating a flat second thermoplastic plastic on the first thermoplastic plastic substrate; c) dissolving the laminate after cooling, and transferring the first channel pattern formed in a relief with the second thermoplastic plastic as a mold onto a polydimethylsiloxane substrate; And d) bonding a pair of the polydimethylsiloxane substrates obtained by repeating the step c) so as to face the side on which the first channel pattern is formed facing each other. have.

According to another aspect of the present invention, there is provided a method of manufacturing a semiconductor device, comprising the steps of: a) forming a multi-step second channel pattern having a plurality of rectangular cross sections at different depths on a first thermoplastic plastic substrate; b) forming a second channel pattern on the surface of the second thermoplastic plastic by embossing the laminated body after laminating a flat second thermoplastic plastic on the first thermoplastic plastic substrate; c) dissolving the laminated body after cooling, and transferring the second channel pattern formed with the second thermoplastic plastic as a mold onto the polydimethylsiloxane substrate; And d) bonding another polydimethylsiloxane substrate flat on said polydimethylsiloxane substrate. ≪ Desc / Clms Page number 7 >

The first thermoplastic plastic substrate and the second thermoplastic plastic substrate have different glass transition temperatures, and the hot embossing process in the step (b) may be performed in such a manner that the glass transition temperature of the first thermoplastic plastic substrate and the glass transition temperature of the second thermoplastic plastic substrate RTI ID = 0.0 > glass transition temperature. ≪ / RTI >

In addition, the first thermoplastic plastic substrate may be a polymethyl methacrylate substrate, and the second thermoplastic plastic substrate may be a polyethylene terephthalate substrate.

In addition, the channel pattern in the step a) may be formed by using CNC milling (computer numerical control milling).

In addition, the multi-stage second channel pattern may be formed to have an aspect ratio of 1 or less.

According to embodiments of the present invention, a microchannel for a lab-on-a-chip having a cylindrical or multi-layered structure is formed to have a reproducibility that is faster, simpler, and less expensive than a method of forming a microchannel using a photolithography process can do.

Furthermore, since the microchannel can be formed using a relatively inexpensive plastic material, the design of the microstructure can be more flexibly modified.

1 is a schematic view sequentially illustrating a method of forming a cylindrical microchannel according to an embodiment of the present invention.
2 is a schematic view sequentially illustrating a method of forming a multi-layer microchannel according to an embodiment of the present invention.
3 is a cross-sectional image of a microchannel formed in accordance with an embodiment of the present invention.
4 is a digital camera image and an optical microscope image of a cylindrical microchannel according to an embodiment of the present invention, which simulates a human vascular network filled with red ink.
Figure 5 shows a multilayer microchannel according to an embodiment of the present invention that simulates human sinusoidal structures on a chip.

Hereinafter, the present invention will be described in detail with reference to the accompanying drawings.

1 is a schematic view sequentially illustrating a method of forming a cylindrical microchannel according to an embodiment of the present invention. In the present specification, the cylindrical microchannel means a microchannel having a circular cross section.

Referring to FIG. 1, a first channel pattern S1 having a semicircular cross section may be formed on the first thermoplastic plastic substrate 110 at a recessed angle (see FIG. 1A). At this time, the first channel pattern may be formed using CNC milling (computer numerical control milling). CNC milling is one of the milling cutting methods using computer numerical control. The CNC milling machine can precisely perform flat cutting, surface cutting, grooving, cutting, etc. of workpieces. Since the CNC milling is commonly known in the cutting field, a detailed description thereof will be omitted.

The first channel pattern S1 is semi-circular in cross section, and it is sufficient if it has a semicylindrical shape as a whole, and the diameter and the like are not limited.

Next, a flat second thermoplastic plastic substrate 120 is laminated on the first thermoplastic plastic substrate 110 (see FIG. 1B), and hot embossing is performed on the laminated body to form a second thermoplastic plastic substrate 120 A first channel pattern S1 is formed on the surface of the first channel pattern S1 at an angle (denoted by S2 in Fig. 1C).

Hot embossing is one of the pattern forming techniques, and is a technique of copying a pattern without going through an exposure or etching process. That is, the polymer substrate is heated to the vicinity of the glass transition temperature of the polymer by a contact pattern forming technique, not by the optical pattern forming technique, and then the pattern on the stamp is imprinted by pressing and pressing on the desired substrate using the master stamp on which the pattern is formed Or transcription. The shape of the polymer is deformed by the master stamp, and after a certain time, the temperature of the polymer is lowered to the glass transition temperature or lower, whereby the pattern of the stamp is replicated by hardening of the polymer.

In the present embodiment, the first thermoplastic plastic substrate 110 having the first channel pattern S1 formed at an oblique angle serves as a master stamp in the hot embossing process, and the second thermoplastic plastic substrate 120 serves as a master stamp, And the target substrate S1 is imprinted / transferred.

Such hot embossing may be accomplished using a pneumatic press machine. The temperature during the hot embossing process may be performed at a temperature between the glass transition temperature of the first thermoplastic plastic substrate 110 and the glass transition temperature of the second thermoplastic plastic substrate 120, It does not.

Accordingly, the first thermoplastic plastic substrate 110 and the second thermoplastic plastic substrate 120 may be different kinds of thermoplastic plastic substrates, and may be materials having different glass transition temperatures.

For example, the first thermoplastic plastic substrate 110 and the second thermoplastic plastic substrate 120 may be formed of a material selected from the group consisting of polycarbonate (PC), polyethylene terephthalate (PET), polyvinyl chloride (PVC), polypropylene (PP), polystyrene (PE), amorphous polyethylene terephthalate (APET), polypropylene terephthalate (PPT), polynaphthalene terephthalate (PEN), polyethylene terephthalate glycerol (PETG), polycyclohexylenedimethylene terephthalate (PCTG) (CPA), polymethylmethacrylate (PMMA), polyisobutylene (PMMA), polyacrylonitrile (PMMA), polydimethylsiloxane, polydimethylsiloxane, polydimethylsiloxane, modified triacetyl cellulose (TAC), cycloolefin polymer (PE), a silicone resin, a fluororesin, a polyamide (PA), a modified epoxy resin, and the like. May each be selected. For example, the first thermoplastic plastic substrate 110 may be a polymethylmethacrylate substrate, and the second thermoplastic plastic substrate 120 may be a polyethylene terephthalate substrate.

On the other hand, the greater the difference between the glass transition temperature of the first thermoplastic plastic substrate 110 and the glass transition temperature of the second thermoplastic plastic substrate 120, the easier the hot embossing process, and the imprinting / transferring of the channel pattern can be accurately performed.

Next, the laminated body of the first thermoplastic plastic substrate 110 and the second thermoplastic plastic substrate 120 is cooled and then physically separated (see Fig. 1C). A channel pattern S2 having a first channel pattern S1 formed on the first thermoplastic plastic substrate 110 is formed on the separated second thermoplastic plastic substrate 120. [

Next, the first channel pattern S2 formed with a positive angle using the second thermoplastic plastic substrate 120 as a mold is transferred onto the polydimethylsiloxane substrate 131. Then, Specifically, after the second thermoplastic plastic substrate 120 is disposed so that the first channel pattern S2 faces upward, the polydimethylsiloxane prepolymer mixture and the curing agent are poured on the second thermoplastic plastic substrate 120, So that the first channel pattern S2 can be transferred. When the cured polydimethylsiloxane substrate 131 is separated, a first channel pattern S3 is formed on the polydimethylsiloxane substrate 131 at an oblique angle (see FIG. 1F).

In this specification, the first channel pattern formed on the first thermoplastic plastic substrate 110 is denoted by S1, the first channel pattern formed on the second thermoplastic plastic substrate 120 is denoted by S2, the polymethylsiloxane substrate The first channel pattern formed on the substrate 131 is denoted by S3, but S1 to S3 are all channel patterns of the same type, and are formed only on the substrate by embossing or embossing. In other words, it can be seen that the first channel pattern formed on the first thermoplastic plastic substrate 110 is formed on the polydimethylsiloxane substrate 131 through two transfers.

Next, another polydimethylsiloxane substrate 132 is subjected to the same process as above to form a first channel pattern S3 on its surface. I.e., a pair of identical polydimethylsiloxane substrates 132 through one second thermoplastic plastic substrate 120. [

Next, the pair of polydimethylsiloxane substrates 132 manufactured as described above are bonded to each other with the face on which the first channel pattern S3 is formed faced (the bonded body is denoted by 100). The bonding can be done by conventional plasma bonding and heat treatment and the process conditions are not specified (see FIG. 1g). Since the first channel pattern S3 formed on the polydimethylsiloxane substrates 131 and 132 at an obtuse angle is semicircular in cross section, the pair of polydimethylsiloxane substrates 131 and 132 are bonded so as to face the face on which the first channel pattern S3 is formed A microchannel having a completely circular cross section can be formed. That is, the microchannel has a cylindrical shape.

2 is a schematic view sequentially illustrating a method of forming a multi-layer microchannel according to an embodiment of the present invention. In the present specification, a multi-layered microchannel refers to a microchannel having a plurality of rectangular cross sections having different depths. Hereinafter, the same or similar parts as those in the above-described embodiments will be described with respect to different parts by omitting redundant explanations.

Referring to FIG. 2, a second channel pattern m1 having a plurality of rectangular cross-sections having different depths may be formed on the first thermoplastic plastic substrate 210 at a negative angle (see FIG. 2a). At this time, the second channel pattern m1 may be formed using CNC milling. Since CNC milling has been described above, redundant description is omitted.

The second channel pattern m1 has a rectangular cross section, and may have a plurality of rectangular cross sections, and the square cross sections may have the same or different depths. The width and depth are not limited. 2A, the second channel pattern m1 is formed with three grooves (rectangular in cross section) and the grooves on both sides of the grooves are formed to have a larger depth than the grooves in the center. However, the present invention is not limited thereto and may be formed to have various aspect ratios .

Next, a flat second thermoplastic plastic substrate 220 is laminated on the first thermoplastic plastic substrate 210 (see FIG. 2B), and the laminated body is hot-embossed to form a laminate on the surface of the second thermoplastic plastic substrate 220 The second channel pattern m1 is formed with a relief (expressed as m2 in Fig. 2C). Since the hot embossing process has been described above, redundant description is omitted.

The first thermoplastic plastic substrate 210 in which the second channel pattern m1 is formed at an oblique angle serves as a master stamp in the hot embossing process and the second thermoplastic plastic substrate 220 serves as a master stamp in the second channel pattern m1, and serves as a target substrate on which the substrate m1 is imprinted / transferred.

The first thermoplastic plastic substrate 210 and the second thermoplastic plastic substrate 220 may be different kinds of thermoplastic plastic substrates and may have different glass transition temperatures as in the above embodiments. For example, the first thermoplastic plastic substrate 110 may be a polymethyl methacrylate substrate, and the second thermoplastic plastic substrate 120 may be a polyethylene terephthalate substrate.

Next, the laminate of the first thermoplastic plastic substrate 210 and the second thermoplastic plastic substrate 220 is cooled and then physically separated (see FIG. 2C). A channel pattern m2 having a second channel pattern m1 formed on the first thermoplastic plastic substrate 210 is formed on the separated second thermoplastic plastic substrate 220.

Next, the second channel pattern m2 formed by embossing with the second thermoplastic plastic substrate 220 as a mold is transferred onto the polydimethylsiloxane substrate 231. Then, Since the transfer method has been described above, redundant description is omitted. When the cured polydimethylsiloxane substrate 231 is separated, a second channel pattern m3 is formed on the polydimethylsiloxane substrate 231 at a negative angle (see FIG. 2F).

The second channel pattern formed on the first thermoplastic plastic substrate 210 is denoted by m1 and the second channel pattern formed on the second thermoplastic plastic substrate 220 is denoted by m2 as in the above- And the second channel pattern formed on the polydimethylsiloxane substrate 231 is denoted by m3. However, m1 to m3 are all channel patterns of the same type and are formed only on the substrate at a negative or positive angle. In other words, it can be seen that the second channel pattern formed on the first thermoplastic plastic substrate 210 is formed on the polydimethylsiloxane substrate 231 through two transfers.

Next, another flat polydimethylsiloxane substrate 232 is bonded on the polydimethylsiloxane substrate 231 (the second channel pattern is formed at a recessed angle) prepared above (the assembly is denoted 200). The bonding can be done through conventional plasma bonding and heat treatment and the process conditions are not specified (see FIG. 2g). Accordingly, a multi-layered (multi-layered) microchannel can be formed inside the bonded body 200.

As described above, according to the embodiments of the present invention, it is possible to form the microchannel having the cylindrical or multilayer structure faster, simpler, less expensive, and higher in reproducibility without using the photolithography process . In the case of using the photolithography process, it is difficult to form the cylindrical microchannel as in the embodiment of the present invention. In the case of the multilayer microchannel, it is possible to form the cylindrical microchannel. However, There is a problem in that the cost is greatly increased.

However, in the embodiments of the present invention, since microchannels are formed using a thermoplastic plastic material as a mold, microchannels can be formed quickly and at low cost, and channels are formed using CNC milling, The channel also has the advantage that it can be formed in a single process.

Hereinafter, the present invention will be described in more detail with reference to examples and test examples of the present invention. However, it is apparent that the following examples and test examples do not limit the present invention.

1. Example

A polymethylmethacrylate substrate (thickness: 2 mm, Goodfellow, hereinafter referred to as PMMA substrate) was prepared for forming microchannels (cylinder type, multilayer type) according to the embodiments of the present invention, and CNC milling machine TinyCNC-SC, TINYROBO), microchannels having a semicircular shape (Example 1) and a multistage shape (Example 2) were formed on the PMMA substrate, respectively. The ball mill used for the CNC milling machine had R values of 0.1, 0.15, 0.25, and 0.5 mm and D values of 0.2, 0.3, 0.5, and 1.0 mm for the end mill. The spindle speed (spindle speed) of the CNC milling machine was 13,500 rpm and the feed speed was 30 mm / s.

Next, a flat polyethylene terephthalate substrate (thickness 2 mm, Goodfellow, hereinafter referred to as PET substrate) was laminated on the PMMA substrate on which the pattern was formed, and the laminate was hot-embossed. The hot embossing was carried out using a custom pneumatic press machine at 85 DEG C and 0.1 MPa for 30 minutes. And the 85 ℃ corresponds to a temperature value between the PMMA (T g = 105 ℃) and PET (T g = 65 ~ 80 ℃) a glass transition temperature value (close to the intermediate value). The larger the glass transition temperature difference between the two substrates, the more successful the transfer of the channel pattern of the PMMA substrate onto the PET (the pattern does not melt or deform).

After cooling the laminate, the laminate was physically disassembled, and the pattern was transferred onto a polydimethylsiloxane (hereinafter referred to as a PDMS) substrate using a PET substrate having the pattern formed in an embossed form as a mold PDMS substrate production). Specifically, a 10: 1 (w / w) PDMS prepolymer mixture and a curing agent (Dow corning) were degassed, poured onto the PET substrate, and then cured at 80 ° C for 30 minutes. Next, the inlet and outlet ports for fluid introduction were formed by punching, and then the two PDMS substrates were subjected to oxygen plasma treatment (50 W, 0.1 Torr). The two PDMS substrates were then aligned and bonded under a microscope with the channels facing each other. The bonding was carried out at 80 DEG C for 30 minutes.

2. Analysis of microstructures and surface profiles

Microstructures and sections were observed using an inverted microscope (model IX71, Olympus). The ink flow phenomenon and packing of the micro-beads were also observed using the above-mentioned inverted microscope.

3 is a cross-sectional image of a microchannel according to an embodiment of the present invention. As described above, according to the embodiment of the present invention, the cylindrical microchannel having a semicircular cross section and the multi-layer microchannel having a cross-section are formed. 3A and 3B, the scale bar is 300 mu m, and the left image of each image is a PMMA substrate (the channel is formed at a recessed angle). And the image on the right side of each image is a PET substrate (the channel is embossed).

In the case of a multi-layer microchannel having a multi-layered cross-section, end mills were used for pattern formation with a CNC milling machine, and the diameters of the flat mills were 0.2, 0.3, 0.5 and 1.0 mm. As a result, a channel having various depth ratios ranging from 0.0625 (1:16) to 2 (2: 1) was formed.

Referring to FIG. 3A, it was confirmed that as the aspect ratio of the pattern is relatively low, the relief pattern formation on the PET substrate is more accurate. That is, as the aspect ratio increases, it becomes difficult for the PET substrate to accurately reproduce the engraved pattern of the PMMA substrate because the partially melted PET can not completely fill the engraved pattern on the PMMA substrate. As a result, it was confirmed that a rounded shape was formed at the end of the bump pattern formed on the PET substrate. This can be seen in the aspect ratio 1: 2 image when the D is 0.2 mm in FIG. 3A and the aspect ratio 2: 1 image and when D is 1.0 mm. From the above results, it was confirmed that when the multilayer microchannel is formed, the pattern can be successfully replicated (pattern embossing) on the PET substrate when the aspect ratio is 1 or less, The size of the flat mill used in the CNC milling machine was confirmed to be less than 0.5 mm in diameter.

Referring to FIG. 3B, in the case of a cylindrical microchannel having a semicircular cross section, a ball-mill was used for pattern formation with a CNC milling machine, and the radius of the ball mill was 0.1, 0.15, 0.25 and 0.5 mm .

Unlike the multi-layer microchannel, the aspect ratio is fixed at 1 when the cylindrical microchannel is formed, because a complete circular cross-section can be formed when the cylindrical microchannel is formed when the aspect ratio is 1 Optimal value). Also, referring to FIG. 3B, it was confirmed that an accurate emboss pattern was formed on the PET substrate regardless of the radius of the ball mill used.

3. Cell culture in microchannel

HUVECs (human umbilical vein endothelial cells, umbilical vein endothelial cells) were cultured in F-12K medium and then incubated at 37 ° C in a 5% CO 2 incubator. The F-12K medium contained 10% FBS (fetal bovine serum, fetal bovine serum), 0.1 mg mL -1 heparin, and 0.05 mg mL -1 ECGS (endothelial cell growth supplement).

After the incubation, the cells were treated with 0.25% trypsin-EDTA (Gibco) for 2 minutes for cell desorption and then centrifuged for enrichment and re-growth in complete medium (supplemented F-12K medium). The PDMS microchannel was sterilized with 70% ethanol and exposed to UV overnight. The interior of the microchannel was coated with an aqueous solution of 1 mg mL -1 fibronectin (extracted from human plasma) and reacted at 37 ° C for 2 hours. After the fibronectin coating, the microchannels were washed with DPBS (Dulbecco's phosphate buffered saline, Sigma-Aldrich) and the HUVECs were introduced into the microchannels. After incubation in a 5% CO 2 incubator at 37 ° C for 20 minutes for cell desorption, the microchannels were reprocessed with HUVECs and incubated and then rotated at 90 degrees. This step was repeated until the cells were attached onto the entire surface of the microchannel. The microchannels were then incubated overnight. After incubation, the cells were placed in 5% formalin solution (neutral) for 15 minutes and treated with 0.1% Triton X-100 for 10 minutes. Cells were stained with fluorescein isothiocyanate (FITC) -conjugated paloidin (Enzo Life Science) for 40 min (cell staining for F-actin).

4. Cylindrical microchannels simulating microvascular networks

4 is a digital camera image and an optical microscope image of a cylindrical microchannel according to an embodiment of the present invention, which simulates a human vascular network filled with red ink. 4A is a digital camera image, and Figs. 4B to 4E are optical microscope images corresponding to (1) to (4) shown in Fig. 4A, respectively. In Fig. 4, the scale bar represents 500 mu m.

Referring to FIG. 4, the PDMS laminate was cut in the vertical direction to confirm the cross section, and as a result, a cylindrical microchannel having a complete circular cross section having a diameter ranging from 200 μm to 1 mm was formed to simulate the microvascular network (Fig. 4B to Fig. 4E). The size corresponds to the diameter of arteries and veins (250 [mu] m < d < 2 mm) as well as arterioles and venules (8 [ Meanwhile, red ink was introduced into the channel to confirm successful channel formation and bonding performance, and it was confirmed that a reliable channel was formed as shown in FIG. 4A.

5. Human body Sinusoid ( human liver sinusoid ) Simulated  Multilayer microchannel

Figure 5 shows a multilayer microchannel according to an embodiment of the present invention that simulates human sinusoidal structures on a chip. Such a multi-layer microchannel is utilized in the micro-bead and cell self-sorting and hemocyte separation in the fluid migration technique. The multi-layer microchannel can be easily formed by a CNC milling machine like a cylindrical microchannel.

5A shows a filtration apparatus including a multi-layered microchannel according to an embodiment of the present invention. First, a PMMA substrate on which a multi-stage channel pattern having a plurality of rectangular cross sections with different depths was formed was produced. The depth is 100 mu m at the inflow portion of the microchannel into which the fluid is introduced (the center portion of the filtration apparatus shown in Fig. 5A), the outflow portion of the microchannel through which the fluid flows out (the both side portions of the filtration apparatus shown in Fig. 150 mu m, and in the microfilter portion of the microchannel connecting the inlet portion and the outlet portion was 40 mu m. The width of the microchannel was 1 mm at the inlet and outlet, and 200 m at the microfilter.

5B is a digital camera image after red ink introduction. As can be seen in Figure 5b, there was no leakage in the channel and red ink was found to flow successfully in all channels.

Fig. 5C is an optical microscope image of the portion (1) shown in Fig. 5B. Chelex 100 Molecular Biology Grade Resin (D = 75-150 mu m beads, Bio-Rad Laboratories) was introduced into the microchannel inlet and flow was confirmed without leakage. The width of the microfilter portion (200 탆) was somewhat larger than the diameter of the Chelex resin, but the Chelex resin did not leak due to physical agglomeration, and only the solution was filtered.

FIGS. 5D to 5F are optical microscope images of microchannels formed on the PMMA, PET and PDMS substrates respectively in the multilayer microchannel according to the embodiment of the present invention, and FIGS. 5G to 5I are optical microscope images of the PMMA, (Corresponding to (2) shown in Fig. 5B). 5d to 5i, it can be seen that the multi-layer microchannel according to the embodiment of the present invention has been successfully formed to have high pattern accuracy. The channel pattern finally formed in the PDMS is the same as the channel pattern formed in the PMMA, and despite the two-time pattern duplication (PMMA? PET? PDMS), the fine difference between the patterns was negligible.

5J shows a cross-sectional image of the bonded PDMS substrate. As described above, it can be seen that the micro-filter part is clearly formed between the inflow part (center part) and the outflow part (both side parts) of the microchannel.

FIG. 5K is a table in which the heights of the microchannels shown in FIGS. 5D to 5F are measured, and is represented by CV values (coefficients of variation) obtained by measurement in 15 areas of the microchannels. The mean heights of the microchannels formed on PMMA, PET and PDMS were 200.0 ± 3.1, 200.2 ± 3.3, and 199.6 ± 2.0 μm, respectively, and CV values of 1.5%, 1.7%, and 1.0% Were at the same level.

The embodiments of the present invention have been described above. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the spirit or scope of the inventive concept as defined by the appended claims. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the present invention.

110, 210: a first thermoplastic plastic substrate
120, 220: a second thermoplastic plastic substrate
S1, S2, S3: First channel pattern
m1, m2, m3: second channel pattern

Claims (6)

A method of forming a microchannel for a non-photolithography based lab-on-a-
a) forming a first channel pattern having a semicircular cross section on the polymethylmethacrylate substrate at an oblique angle;
b) laminating a flat polyethylene terephthalate substrate on the polymethylmethacrylate substrate, and hot-embossing the laminate to form the first channel pattern on the surface of the polyethylene terephthalate substrate in an embossed state;
c) cooling the laminate and disassembling the laminate, and transferring the first channel pattern formed on the polyethyleneterephthalate substrate as a mold to the polydimethylsiloxane substrate; And
d) bonding a pair of the polydimethylsiloxane substrates obtained by repeating the above step c) so as to face the surface on which the first channel pattern is formed. A method of forming a microchannel for a non-photolithography based lab-on-a-
a) forming a multi-stage second channel pattern having a plurality of rectangular cross sections different in depth on the polymethyl methacrylate substrate at an engraved angle;
b) laminating a flat polyethylene terephthalate substrate on the polymethylmethacrylate substrate, and hot-embossing the laminate to form the second channel pattern on the surface of the polyethylene terephthalate substrate in an embossed form;
c) cooling and dissolving the laminate, and transferring the second channel pattern formed on the poly (ethylene terephthalate) substrate as a mold to a polydimethylsiloxane substrate; And
d) bonding another polydimethylsiloxane substrate flat on said polydimethylsiloxane substrate. &lt; RTI ID = 0.0 &gt; 8. &lt; / RTI &gt; The method according to claim 1 or 2,
Wherein the hot embossing treatment in step b) is carried out at a temperature between the glass transition temperature of the polymethyl methacrylate substrate and the glass transition temperature of the polyethylene terephthalate substrate, . delete The method according to claim 1 or 2,
Wherein the channel pattern of step a) is formed using CNC (computer numerical control milling). The method of claim 2,
Wherein the multi-stage second channel pattern is formed to have an aspect ratio of 1 or less.
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