KR101507757B1 - Method for fabricating hydrophobic coating layer on micropatterned hydrophilic substrate and micropatterned hydrophilic substrate including hydrophobic coating layer fabricated thereby - Google Patents

Abstract

(A) contacting a micropatterned hydrophilic polymer substrate with a substrate comprising a siloxane compound, and then irradiating light to transfer the siloxane compound onto the micropatterned hydrophilic polymer substrate ; And (b) separating the substrate comprising the siloxane compound from the micropatterned hydrophilic polymer substrate onto which the siloxane compound has been transferred, to form a hydrophobic coating layer selectively on the micropatterned hydrophilic polymer substrate, and And a hydrophobic coating layer formed thereby. According to the present invention, in the method of selectively forming a hydrophobic coating layer on a micropatterned hydrophilic polymer substrate according to the present invention, a micropatterned hydrophilic polymer substrate on which microchannels and / or microwells are formed is treated with a siloxane compound A coating layer having hydrophobicity selectively by a siloxane functional group can be easily formed on a fine patterned hydrophilic polymer substrate even under atmospheric pressure and room temperature conditions by performing photocuring after conformal contact with a stamp containing a siloxane functional group. According to the above method, the hydrophobic coating layer can be selectively formed without restriction depending on the area of the fine patterned hydrophilic polymer substrate or the shape and size of the pattern formed on the substrate, and the fine The patterned hydrophilic polymer substrate can selectively immobilize a biomolecule and the like on a non-parallel surface such as a microchannel, a microwell, etc. in a high-resolution and high-fidelity manner at the same time in a hydrophilic region and a hydrophobic region , As well as. The coating layer can be maintained in a very solid state even after a long time has elapsed since the formation of the hydrophobic coating layer, so that it has excellent durability at the same time.

Description

TECHNICAL FIELD [0001] The present invention relates to a method for forming a hydrophobic coating layer selectively on a micropatterned hydrophilic polymer substrate and a micropatterned hydrophobic polymer substrate having a hydrophobic coating layer formed thereby thereby}

The present invention relates to a method for selectively forming a hydrophobic coating layer on a micropatterned hydrophilic polymer substrate and a micropatterned hydrophilic polymer substrate having a hydrophobic coating layer formed thereby.

Surface functionalization is currently the area of greatest interest for microarray technology as well as targeted patterning of biomolecules such as numerous biomarkers for cells, proteins, antibodies and cancers, and disease detection and diagnosis. Microcontact printing (μCP), ink jet printing, dip-pen lithography, etc. have been used to pattern and anchor biomolecules at predetermined positions on a planar surface. Have been developed.

However, in some cases, cells, antibodies and sensing receptors need to be immobilized on nonplanar surfaces such as microchannels or spatially separated microscale or submicroscale cavities inside have.

For this reason, researchers have developed a method for selectively immobilizing biomolecules inside a three-dimensional structure or a spatially separated non-planar surface.

In this regard, Foley et al. Introduced various methods for selectively printing biomolecules in contact with the interior of concave regions of microchannels using a PDMS (poly (dimethylsiloxane)) stamp, but using soft PDMS stamps In order to obtain reproducible results at the time of contact printing, there is a problem that precise pressure control is required [Non-Patent Document 0001].

Choi et al. Prepared concave PDMS microwell arrays to cultivate embryonic stem (ES) cells in concave microwells. However, there was a further need for a post-suction process to remove cells that did not settle in the microwells (Non-Patent Document 0002).

Lee et al. Used a hydrophilic composite mold as a stamp and a polymer electrolyte as an ink to transfer a chargeable functional polymer onto an elevated area around a micro-reservoir to form an antibody only inside the micro- And was selectively immobilized. However, repeated contact printing of a polymer electrolyte is inevitable in order to vary the wettability (Non-Patent Documents 0003 and 0004).

Therefore, there is a desperate need for a new method of manufacturing a microarray substrate capable of selectively immobilizing biomolecules or the like on a non-parallel surface such as a microchannel or a microwell by a simple process.

 J. Foley, H. Schmid, R. Stutz, E. Delamarche, Langmuir 21 (2005) 11296.  Y.Y. Choi, B.G. Chung, D.H. Lee, A. Khademhosseini, J.-H. Kim, S.H. Lee, Bioma-terials 31 (2010) 4296.  N.Y. Lee, J.R. Lim, M.J. Lee, S. Park, Y.S. Kim, Langmuir 22 (2006) 7689.  N.Y. Lee, J.R. Lim, M.J. Lee, J.B. Kim, S.J. Jo, H.K. Baik, Y.S. Kim, Langmuir 22 (2006) 9018.

Disclosure of Invention Technical Problem [8] Accordingly, the present invention has been made in view of the above problems, and it is an object of the present invention to provide a biosensor having a hydrophilic region and a hydrophobic region at the same time, , A method of selectively forming a hydrophobic coating layer on a fine patterned hydrophilic polymer substrate through a simple process and a finely patterned polymer substrate produced thereby.

(A) contacting a micropatterned hydrophilic polymer substrate with a substrate comprising a siloxane compound, and irradiating light to irradiate the siloxane compound with the fine patterned Transferring the hydrophilic polymer onto the hydrophilic polymer substrate; And (b) separating the substrate comprising the siloxane compound from the micropatterned hydrophilic polymer substrate onto which the siloxane compound has been transferred. The method of forming a hydrophobic coating layer on a micropatterned hydrophilic polymer substrate, I suggest.

The method of forming a hydrophobic coating layer on a fine patterned hydrophilic polymer substrate, wherein the fine patterned hydrophilic polymer substrate of step (a) comprises a partially cured photocurable polymer.

Also, the photocurable polymer is NOA (Norland Optical Adhesive), which is a method of selectively forming a hydrophobic coating layer on a micropatterned hydrophilic polymer substrate.

The substrate comprising the siloxane compound may also be a substrate comprising (i) a substrate consisting of one layer comprising a siloxane compound or (ii) a coating comprising a polymeric substrate and a siloxane compound formed on the polymeric substrate, Wherein the hydrophobic coating layer is selectively formed on the fine patterned hydrophilic polymer substrate.

Also, the siloxane compound is a linear siloxane, a copolymer thereof, or a cyclic organosiloxane compound, and the hydrophobic coating layer is selectively formed on the fine patterned hydrophilic polymer substrate.

Also, the siloxane compound is PDMS (poly (dimethylsiloxane)), which is a method of selectively forming a hydrophobic coating layer on a micropatterned hydrophilic polymer substrate.

The present invention also provides a micropatterned hydrophilic polymer substrate having a hydrophobic coating layer formed by the above method.

According to the method of selectively forming a hydrophobic coating layer on a micropatterned hydrophilic polymer substrate according to the present invention, a micropatterned hydrophilic polymer substrate on which microchannels and / or microwells are formed is coated with a stamp containing a siloxane compound such as PDMS By carrying out the photocuring after conformal contact, a coating layer having a hydrophobic property selectively by the siloxane functional group can be easily formed on the fine patterned hydrophilic polymer substrate even under atmospheric pressure and room temperature conditions. According to the above method, the hydrophobic coating layer can be selectively formed without restriction depending on the area of the fine patterned hydrophilic polymer substrate or the shape and size of the pattern formed on the substrate.

In addition, the micropatterned hydrophilic polymer substrate having the hydrophobic coating layer formed by the above-described method has a high hydrophilicity and a hydrophobic region, and is capable of forming biomolecules and the like on a microchannel with high-resolution and high-fidelity, Microwells, and the like. In addition, since the coating layer can be maintained in a very solid state even after a long time has elapsed since the formation of the hydrophobic coating layer, it has excellent durability at the same time.

1 is a conceptual diagram showing an example of a method of preparing a micropatterned hydrophilic polymer substrate provided in step (a) of a method for selectively forming a hydrophobic coating layer on a micropatterned substrate according to the present invention.
Fig. 2 is a conceptual diagram showing an embodiment using an example of a stamp containing a siloxane compound provided in step (a) of a method for selectively forming a hydrophobic coating layer on a micropatterned substrate according to the present invention.
3 is a conceptual diagram showing an embodiment using another example of the stamp including the siloxane compound provided in step (a) of the method for selectively forming the hydrophobic coating layer on the micropatterned substrate according to the present invention.
FIG. 4 is a conceptual diagram showing a process of performing target immobilization of beads when a solution containing functional beads such as polystyrene (PS) beads is applied to a micropatterned substrate.
5 is an SEM image showing selective immobilization of a PS fluorescent sphere into a microwell in the microwell array substrate manufactured in Example 1 of the present application.
6 is an optical and fluorescence microscope image showing the selective immobilization of a PS fluorescent sphere into a microwell in the microwell array substrate manufactured in Example 1 of the present application.
Figs. 7 (a) and 7 (b) show the measurement results of changes in the water contact angle with time in the microwell array substrate manufactured in Examples 1 and 2. Fig.

Hereinafter, the present invention will be described in detail.

A method for selectively forming a hydrophobic coating layer on a micropatterned hydrophilic polymer substrate according to the present invention includes the steps of: (a) contacting a micropatterned hydrophilic polymer substrate with a stamp comprising a siloxane compound; transferring the siloxane compound onto the fine patterned hydrophilic polymer substrate by irradiating light onto the fine patterned hydrophilic polymer substrate; And (b) separating the substrate comprising the siloxane compound from the micropatterned hydrophilic polymer substrate onto which the siloxane compound has been transferred, each of which will be described in detail below.

A method of selectively forming a hydrophobic coating layer on a micropatterned substrate according to the present invention, said step (a) comprising contacting a micropatterned hydrophilic polymer substrate with a stamp comprising a siloxane compound And then transferring the siloxane compound onto the micropatterned hydrophilic polymer substrate through microcontact printing and photo polymerization by irradiating the substrate-stamp assembly obtained therefrom with a light (hydrophobic) hydrophobic coating layer.

In this step (a), the micropatterned hydrophilic polymer substrate is patterned with microstructures such as microchannels, microwells, etc., and all or a part of the surface of the micropatterned hydrophilic polymer substrate is hydrophilic (-OH), an amine group (-NH ₂ ), an aldehyde group (-COH), a carboxyl group (-COOH) or the like, or hydrophilicity by introducing the hydrophilic functional group artificially into all or a part of the surface.

The finely patterned hydrophilic polymer substrate may be manufactured by a known method such as soft lithography, soft lithography, or E-beam lithography and may be provided for this step. For example, , A photo curable polymer resin is coated on a hard mold made of Si or the like by spin coating or the like as shown in FIG. 1, and then light such as UV light ), Curing it, and then separating from the hard mold. At this time, it is possible to obtain a fully cured substrate or a partially cured substrate through control of irradiation energy and time at the time of curing, but in the present invention, it is preferable to use a partially cured substrate, When uncured monomers or oligomers are present, they are photo polymerized with the siloxane groups in the light irradiation step, which is carried out after the contact between the micro patterned substrate and the substrate containing the siloxane compound, which will be described later, Is more effectively transferred.

Meanwhile, the photo-curable polymer resin may include known photo-curable oligomers, photo-curable monomers and photoinitiators known before the application of the present invention. Cross-linking polymerization with a siloxane group included in a siloxane compound, which will be described later, The type thereof is not particularly limited.

Specific examples of the photo-curable oligomer include epoxy acrylates, urethane acrylates, polyester acrylates, acryl acrylates, polybutadiene acrylates, silicone acrylates, melamine acrylates, epoxies and the like But is not limited thereto. Specific examples of the photocurable monomer include (meth) acrylate monomers having at least one functional group, but are not limited thereto. As the photopolymerization initiator, any radical photopolymerization initiator known in the art may be used.

In addition to the above-mentioned photocurable polymer resins, Norland Optical Adhesives (NOA) of Norland Co., which is a photo-curable polymer resin based on thiol-ene reaction, can be used. Unlike the above-mentioned photocurable polymer resins, Is not required and curing is performed remarkably quickly, which is more preferable. For reference, the NOA exhibits hydrophilicity through ultraviolet curing during the patterning of the microstructure by including thiol-ene and an acrylic component.

In the step (a), the stamp containing the siloxane compound may be provided in the present step by mixing the siloxane compound with a curing agent and then curing the mixture by thermosetting or photo-curing. At this time, the siloxane compound and the curing agent The compounding ratio may be 10: 1 to 30: 1 on a weight basis.

On the other hand, the stamp containing the siloxane compound may be formed of only one layer including a siloxane compound in its laminated structure as shown in FIG. 2, or may be formed of a poly (ethylene terephthalate) (PET) And a coating layer comprising a polymer substrate and a siloxane compound formed on the polymer substrate.

Specific examples of the material of the polymer substrate include poly (methyl methacrylate) (PMMA), polycarbonate (PC), and polystyrene (PS) in addition to the poly (ethylene terephthalate) , But is not limited thereto.

On the other hand, the siloxane compound may be a linear siloxane or copolymer thereof such as polydimethylsiloxane, polymethylethylsiloxane, polymethylphenylsiloxane, polymethylhydroxysiloxane, polymethylpropylsiloxane, polydiphenylsiloxane and polymethylbutylsiloxane, Specific examples thereof include cyclic organosiloxane compounds such as dimethylsiloxane, cyclic polymethylphenylsiloxane, cyclic polymethylhydroxysiloxane, cyclic polymethylethylsiloxane, cyclic polymethylpropylsiloxane, and cyclic polymethylbutylsiloxane. , But is not limited thereto.

A method for selectively forming a hydrophobic coating layer on a micropatterned substrate according to the present invention, said step (b) comprising: separating the stamp from the substrate-stamp assembly obtained through step (a) And a hydrophobic coating layer containing a compound is selectively formed on the hydrophilic polymer substrate.

A specific method for separating the stamp from the substrate-stamp assembly in performing this step is not particularly limited, and a known method such as a physical separation method or a chemical separation method may be used to remove the damaged hydrophobic coating layer transferred onto the micropatterned hydrophilic polymer substrate This step can be performed by selecting a method that can be minimized.

According to the method of selectively forming the hydrophobic coating layer on the micropatterned hydrophilic polymer substrate according to the present invention described above, the micropatterned hydrophilic polymer substrate on which the microchannel and / or the microwell is formed can be treated with a siloxane compound such as PDMS A coating layer having hydrophobicity selectively by a siloxane functional group can be easily formed on the fine patterned hydrophilic polymer substrate even at atmospheric pressure and at room temperature by performing the photocuring after conformal contact with the stamp including the hydrophilic polymer. According to the method, the hydrophobic coating layer can be selectively formed without restriction depending on the area of the fine patterned hydrophilic polymer substrate or the shape and size of the pattern formed on the substrate.

In addition, the micropatterned hydrophilic polymer substrate having the hydrophobic coating layer formed by the above method has the following problems. The technical problem to be solved by the present invention is to provide a high-resolution and high- biomolecules and the like can be selectively immobilized on non-planar surfaces such as microchannels, microwells, and the like, and the coating layer can be maintained in a very rigid state even after prolonged time after formation of the hydrophobic coating layer.

Hereinafter, the present invention will be described in detail on the basis of embodiments. The presented embodiments are illustrative and are not intended to limit the scope of the invention.

< Example  1>

NOA (Norland Optical Adhesives) 61, a photocurable polymer, was coated on the master Si substrate by spin coating and irradiated with ultraviolet light (UV) at a wavelength of 235 nm for 20 minutes at an irradiation energy of 135 mW / cm ² Si substrate (master) to prepare a microwell array substrate.

PDMS and a curing agent were mixed at a ratio of 10: 0.5 (w / w), followed by thermosetting to prepare a block-shaped stamp.

Then, the stamp prepared above was contact-printed on the microwell array substrate manufactured above, irradiated with ultraviolet rays for 3 hours, and the stamp was separated from the microwell array substrate.

< Example  2>

A microwell array substrate having a PDMS coating layer formed on the periphery of the microwell was manufactured in the same manner as in Example 1, except that PDMS and a curing agent were mixed at a ratio of 10: 1 (w / w) at the time of stamp production.

< Example  3>

NOA (Norland Optical Adhesives) 61, a photocurable polymer, was coated on the master Si substrate by spin coating and irradiated with ultraviolet light (UV) at a wavelength of 235 nm for 20 minutes at an irradiation energy of 135 mW / cm ² Si substrate (master) to prepare a microwell array substrate.

A polyethylene terephthalate (PET) substrate treated with an oxygen plasma was immersed in an aqueous 3-aminopropyltriethoxysilane (APTES) solution (1 wt%) and taken out, washed with distilled water, and dried. , A low molecular weight PDMS (~ 65 cSt) solution having an epoxy functional group at the end was coated on the PET substrate by spin coating to prepare a PDMS monolayer coated stamp.

For reference, the contact angle of the PET substrate before coating was 67.0 ± 1.9 ° (n = 5), but the contact angle after coating the surface with the PDMS monolayer on PET substrate increased to 94.6 ± 1.5 ° (n = 5) This confirmed the successful coating of the PDMS monolayer.

Then, the stamp prepared above was contact-printed on the microwell array substrate manufactured above, irradiated with ultraviolet rays for 3 hours, and the stamp was separated from the microwell array substrate.

In order to confirm the successful transfer of PDMS after separation, the contact angles of the separated stamps were measured to be 76.0 ± 0.8 ° (n = 5) compared with 94.6 ± 1.6 ° before transfer, (67.0 ± 1.9 °) of the untreated PET substrate, which is presumably due to the PDMS remaining on the PET substrate without being transferred to the microwell array substrate.

< Comparative Example >

NOA (Norland Optical Adhesives) 61, a photocurable polymer, was coated on the master Si substrate by spin coating and irradiated with ultraviolet light (UV) at a wavelength of 235 nm for 20 minutes at an irradiation energy of 135 mW / cm ² Si substrate (master) to prepare a microwell array substrate.

< Experimental Example  1> Example  1-2 and In the comparative example  Manufactured Microwell  Polystyrene on array substrate ( PS Selective immobilization experiments and analysis of fluorescent spheres

The microwell array substrate prepared in Example 1 and Comparative Example was polystyrene (PS) yellow functionalized with an amine group having a larger affinity to the hydrophilic surface than the hydrophobic surface as shown in the conceptual diagram of FIG. 4 -green fluorosphere) [diameter 1 μm, excitation 505 nm, emission 515 nm] for 10 minutes, washed with distilled water and dried, and then immobilized on a microwell array substrate Respectively.

2-1. Scanning Electron Microscope ( SEM ) analysis

The surface of the microwell array substrate thus prepared was observed through a long-field scanning electron microscope (FE-SEM), and the results are shown in FIG.

From FIG. 5, it can be seen that the PS fluorescent spheres were selectively filled in microwells having widths, lengths, and depths of 5, 5, and 3 μm, respectively. Specifically, adsorption to the protruding region around the microwell is not achieved except for a very small number of areas in which some PS fluorescent spheres are nonspecifically adsorbed, resulting in surface hydrophobization around the microwell It can be confirmed that selective immobilization of the PS fluorescent sphere into the microwell has been successfully performed.

On the other hand, theoretically, in a single microwell, up to three layers can be filled up to five in each of the width direction and the length direction, but none of the microwells is actually completely filled. This is probably due to the surface tension of the air or PS fluorophore solution trapped inside the microwell. This problem is caused by the degassing of the substrate during the immobilization of functionalized beads or biomolecules, Or by reducing the surface tension of the bead or biomolecule solution through addition of a surfactant.

On the other hand, in the case of the microwell array substrate manufactured in the comparative example, the PS fluorescent spheres are uniformly adsorbed to the surface of the protruding area around the microwell as well as inside the microwell (not shown in the SEM image) .

2-2. Neon( 여광체 ) analysis

The surface of the microwell array substrate thus prepared was observed through an optical microscope and a fluorescence microscope, and the results are shown in FIG.

The optical microscope image on the left in FIG. 6 supports the fact that the fluorescence appearing in the fluorescence microscope image on the right side is due to the PS fluorescent sphere selectively immobilized in the microwell but not due to the background fluorescence, The fluorescence microscope images of the PS fluorescence were selectively filled in the microwells.

< Experimental Example  2> Example  1 and 2 Microwell  Over time for the array substrate Contact angle  Change measurement and analysis

The water contact angle was measured over a period of 350 days to confirm the durability and robustness of the selectively hydrophobic surface of the microwell array substrate prepared in Example 1 and Example 2 , And the results are shown in Figs. 7 (a) and 7 (b).

As can be seen from FIG. 7 (b), which is an enlarged view of a rectangular area indicated by a dotted line in FIGS. 7 (a) and 7 (a), the contact angle of the microwell array substrate manufactured in Example 1 and Example 2 (N = 5) and 96.7 ± 1.8 ° (n = 5) at the initial measurement, respectively, and decreased gradually with time to 85.2 ± 2.3 ° (n = 5) and 85.1 ± 1.2 ° (n = 5). The contact angle shows a tendency to decrease overall while repeating the inflow during the measurement period. However, even at the lowest value, the NOA 61 coating layer, which is above 80 °, is not coated with a hydrophobic coating, for example, on a PET substrate Compared with the initial measured contact angle (61.3 ± 3.0 ° (n = 5)) measured on the surface of NOA 61 coating layer obtained by curing by irradiating UV rays for 2 hours after spin coating NOA 61.

From the results of the contact angle measurement, it was shown that the substrate prepared in Example 1, which had a smaller amount of the curing agent than the PDMS prepolymer in Example 2, exhibited higher hydrophobicity, as the amount of the curing agent added was lower, This is because more cross-linking occurs with the free thiolene group on the partially cured microwell array substrate surface.

Although there is a slight difference in the degree of hydrophobicity depending on the difference in the content of the curing agent as described above, the microwell array substrate manufactured in Examples 1 and 2 still maintains a high contact angle even after a long period of time, It was found that the formed hydrophobic coating layer had sustainability.

Claims (7)

(a) contacting a micropatterned hydrophilic polymer substrate comprising a partially cured photocurable polymer with a stamp comprising a siloxane compound, and then irradiating light with the siloxane compound to form a siloxane compound Transferring onto a fine patterned hydrophilic polymer substrate; And
(b) separating the siloxane compound-containing stamp from the micropatterned hydrophilic polymer substrate onto which the siloxane compound has been transferred. &lt; RTI ID = 0.0 &gt; 11. &lt; / RTI &gt; delete delete The method of claim 1, wherein the stamp comprising the siloxane compound comprises a coating comprising (i) a one-layer stamp comprising a siloxane compound or (ii) a polymeric substrate and a siloxane compound formed on the polymeric substrate, Wherein the hydrophobic coating layer is formed on the hydrophilic polymer substrate. The method according to claim 1, wherein the siloxane compound is a linear siloxane, a copolymer thereof, or a cyclic organosiloxane compound. 2. The method of claim 1, wherein the siloxane compound is a linear siloxane, a copolymer thereof, or a cyclic organosiloxane compound. 6. The method of claim 5, wherein the siloxane compound is poly (dimethylsiloxane) (PDMS). A micropatterned hydrophilic polymer substrate having a hydrophobic coating layer formed by the method according to any one of claims 1 to 6.

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