KR100952080B1 - Microfluidic Device for Screening a Treating Agent of Neurodegenarative Diseases and Method for Fabricating Thereof - Google Patents

Abstract

The present invention relates to a microfluidic device for screening a degenerative neurological disease therapeutic agent, a method for manufacturing the same, and a method for screening a therapeutic agent for a degenerative nephropathy disease using the microfluidic device, and more specifically, an amyloid precipitate which is a pathological common point of a degenerative neurological disease. The present invention relates to a microfluidic device capable of rapidly generating and rapidly and sensitively screening amyloid precipitates of different conditions with a small amount of reagents in one device having a plurality of microchannels, and a method of manufacturing the same.

According to the present invention, a microfluidic device having the effect of rapidly producing amyloid precipitates of different conditions at the same time in a miniaturized microfluidic device and rapidly and sensitively screening a therapeutic agent for degenerative nephropathy with a small amount of reagents and its fabrication By providing a method, it can be expected to be applied to the efficient research and treatment of degenerative neurological diseases.

Amyloid, degenerative, neurological disease, microfluidic device, microchannel, Alzheimer's, Parkinson's, prion, new drug development

Description

Microfluidic Device for Screening a Treating Agent of Neurodegenarative Diseases and Method for Fabricating Thereof}

The present invention relates to a microfluidic device for screening a degenerative neurological disease therapeutic agent, a method for manufacturing the same, and a method for screening a therapeutic agent for a degenerative nephropathy disease using the microfluidic device, and more specifically, an amyloid precipitate which is a pathological common point of a degenerative neurological disease. The present invention relates to a microfluidic device capable of rapidly generating and rapidly and sensitively screening amyloid precipitates of different conditions with a small amount of reagents in one device having a plurality of microchannels, and a method of manufacturing the same.

Formation of amyloid deposits in brain tissue is a common pathological feature of degenerative neurological disorders such as Alzheimer's disease, Parkinson's disease, Huntington's disease and mad cow disease (Jin, L.-W. et. al . , Proc. Natl . Acad . Sci ., 100 : 15294-15298, 2003; JWKelly et al . , Curr . Opin . Struct. Biol ., 6 : 11-17 , 1996; Regina M. et al . , Annual Review of Biomedical Engineering., 4 : 155-174, 2002). However, no mechanism has yet been identified for incorporating steady-state soluble proteins into insoluble precipitates (Kisilevsky R. et. al . , J Struct Biol . 130 : 99-108, 2000; John Hardy. et al . , Science : 353-356, 2002).

In The study of amyloid production in vivo requires very long time, high cost and high labor, so its efficiency is very low. To overcome this shortcoming, most researchers form amyloid precipitates in solution under in vitro conditions (Oh, YB et al . , Neurobiol . Dis . , 16:21, 2004; Perez, M. et al . , Biochem . J. , 335 : 369, 1998; Kanapathipillai, M. et al . , FEBS Letters 579: 4775, 2005). In this case, the amyloid precipitate produced in the solution may have a different degree of precipitation. Therefore, there is a need for a method of producing an amyloid precipitate that is similar to an in vivo environment and can control the degree of precipitation.

On the other hand, the deposition of amyloid on the substrate plays an important role in the study of amyloid-related diseases. However, the use of natural amyloid templates in the study of amyloid formation is very difficult and cumbersome because of the need to separate them from tissue plaques. So many researchers have developed artificial amyloid templates.

As a representative prior art of artificial amyloid templates, artificial amyloid templates, which physically deposit amyloid deposits by placing amyloid protein solutions in 96-well assay plates, screening the production of amyloid deposits using a radioactive isotope for identification. That's how. However, since this method cannot physically observe amyloid deposits and uses physical adsorption, it is weakly bonded to the substrate, thereby making the device less stable for amyloid related research. There is also the hassle of using radioactive isotopes for identification before culturing amyloid precipitates (Esler, WP et al . , Nat Biotechnol ., 15 : 258-263, 1997).

In addition, Ha & Park has co-bonded amyloid seeds and incubated amyloids by N-hydroxysuccinimide-activated surface treatment on glass substrates to overcome device stability. To precipitate amyloid. However, since this experiment is performed on a large substrate using a large amount of amyloid solution, it is not only cumbersome to deal with the solution during the precipitation process, but also a large amount of sample is consumed, and no uniform amyloid deposit is formed on the substrate. There is a problem that it is difficult to screen (Ha, C. et al., Biotechnol . Bioeng ., 90 : 848-855, 2005).

Accordingly, the present inventors have fabricated a microfluidic device having a plurality of microchannels, and various conditions, temperature, injection rate, and the like of a protein monomer solution such as different concentrations, pHs, buffer types, and salt concentrations in the microchannels of the microfluidic device. After generating amyloid precipitate under various growth conditions such as time, and injecting drugs for treating neurodegenerative diseases, it was confirmed that one device can be used to compare and screen the relative growth of amyloid precipitate under different conditions. The present invention has been completed.

Disclosure of Invention An object of the present invention is to provide a microfluidic device for screening a degenerative neurological disease therapeutic agent and a method of manufacturing the same for use in the study of degenerative neurological disease.

Another object of the present invention to provide a screening method for treating neurodegenerative diseases using the microfluidic device.

The present invention to achieve the above object is (a) an upper substrate and a lower substrate combined with the upper substrate; (b) a plurality of microchannels formed on the bottom surface of the upper substrate; (c) amyloid precipitated inside the microchannel; And (d) provides a microfluidic device for screening degenerative neurological disease therapeutic agent comprising a sample inlet formed at both ends of the microchannel.

The invention also comprises the steps of (a) manufacturing a master (master) comprising a plurality of microchannel shape; (b) forming a plurality of microchannels including a sample inlet on the bottom surface of the upper substrate using the master; (c) coupling the upper substrate and the lower substrate; (d) activating the microchannels; And (e) provides a method for manufacturing the microfluidic device comprising the step of forming an amyloid precipitate in the microchannel.

The present invention also provides a method for screening a therapeutic agent for neurodegenerative diseases, characterized by using the microfluidic device.

According to the present invention, a microfluidic device capable of quickly and sensitively screening a degenerative neurological disease therapeutic agent with a small amount of reagents and then rapidly generating amyloid precipitates of different conditions simultaneously in a miniaturized microfluidic device, and the method By providing a method for screening a therapeutic agent for neurodegenerative diseases using a microfluidic device, an effect that can be applied to the efficient research and development of a therapeutic agent for the neurodegenerative disease can be expected.

The present invention relates to a microfluidic device for generating and screening amyloid precipitate, which is a common cause of degenerative neurological diseases, and applying it to future research and development of therapeutic agents for the treatment of degenerative neurological diseases.

Specifically, the present invention provides a microfluidic device for screening a therapeutic agent for neurodegenerative diseases, wherein the amyloid related to the degenerative neurological disease is subjected to various conditions (eg, different temperature, pressure, pH conditions) on the upper and lower substrates of the microfluidic device. Can be used to study the mechanisms of neurodegenerative diseases, as well as to screen for therapeutic agents related to neurodegenerative diseases.

As used herein, the term 'amyloid precipitate' refers to a protein precipitate that causes a disease by changing a secondary or tertiary structure from a random coil or an alpha-helix structure to a beta-sheet by abnormal folding of the protein. The amyloid monomer refers to proteins before structural changes occur.

The term 'microchannel' used in the present invention refers to a cuboid-shaped passage formed on a substrate, and the plurality of microchannels formed in the microfluidic device according to the present invention depend on the size of the substrate and the size of the microchannel to be formed. The number can be arbitrarily determined.

The present invention in one aspect (a) the upper substrate and the lower substrate coupled to the upper substrate; (b) a plurality of microchannels formed on the bottom surface of the upper substrate; (c) amyloid precipitated inside the microchannel; And (d) relates to a microfluidic device for screening degenerative neurological disease therapeutic agent comprising a sample inlet formed at both ends of the microchannel.

In the present invention, the amyloid is a beta-amyloid of Alzheimer's disease (β-amyloid), Parkinson's disease (alpha-synuclein), Huntington's disease (Huntington's disease) and prion (Huntington's disease) Prion) may be a protein or a fragment thereof selected from the group consisting of prions of related diseases.

The diseases listed above are all related to protein misfolding disorders, and beta-amyloid, alpha-synuclein, Huntington and prion, etc. It is a protein involved in the invention. For example, if you look at the brain of a person with Alzheimer's disease, you'll see a deposit of beta amyloid.

In the present invention, the material of the upper substrate may be characterized in that the polydimethylsiloxane (polydimethylsiloxane) or polymethylmethacrylate (polymethylmethacrylate), the material of the lower substrate is characterized in that the glass or polymethyl methacrylate You can do

The upper substrate and the lower substrate of the microfluidic device according to the present invention may be a transparent polymer material or a glass material, respectively, in the process of generating amyloid deposits using atomic force microscope (AFM), optical microscope, and fluorescence microscope. This is to confirm that the amyloid monomer is uniformly injected into the microchannel without clogging in the channel and to screen the amyloid precipitate formed inside the microfluidic device more easily.

On the other hand, the present invention provides a microfluidic device for screening degenerative neurological disease treatment agent by combining the upper substrate and the lower substrate, by forming a microchannel on the bottom surface of the upper substrate, when the amyloid is injected into the microchannel, both upper and lower portions of the channel Since amyloid precipitates can be generated, the sensitivity of the sensing can be increased due to the amyloid precipitates present in both upper and lower portions of the channel during screening.

According to another aspect of the present invention, there is provided a method including: (a) manufacturing a master including a plurality of microchannel shapes; (b) forming a plurality of microchannels including a sample inlet on the bottom surface of the upper substrate using the master; (c) coupling the upper substrate and the lower substrate; (d) activating the microchannels; And (e) forming an amyloid precipitate in the microchannels.

In the present invention, the coupling of the step (c) may be characterized in that the bonding or chemical bonding by physical compression, the physical compression may be performed using a holder of aluminum alloy material. In addition, the chemical bonding is performed by performing an oxygen plasma treatment on the upper substrate and the lower substrate, followed by vacuum treatment.

Meanwhile, when fabricating the microfluidic device according to the present invention, before forming the amyloid precipitate in the microchannels, the microchannels must be chemically activated. The lower substrate is dried in a piranha solution and dried. In addition, the dried lower substrate may be bonded and cured with the upper substrate to chemically activate the microchannel (see Example 2).

In the present invention, the activation of the step (d) is a method of immersing the microfluidic device in the sub-pyran solution, plasma treatment method and aminopropyl triethoxysilane ((3-aminopropyl) triethoxysilane) or N, N'- It may be characterized in that it is carried out by a method selected from the group consisting of reacting with disuccinimidyl carbonate (N, N'-disuccinimidyl carbonate).

In the present invention, in order to form a microchannel in the microfluidic device, a master is used, and the master is manufactured by using a silicon wafer, the size of which is 80-120μm, 80-120μm and 10mm, respectively Since a small mask is used, a small microfluidic device included in the size range can be manufactured.

In addition, in the manufacture of the microfluidic device, the selection of one of the coupling methods as described above for the upper substrate and the lower substrate is determined in accordance with the observation means of the amyloid precipitate formed in the microchannel of the microfluidic device. That is, when the amyloid precipitate is observed using an atomic force microscope, the upper substrate and the lower substrate can be combined by physical adsorption because only the lower substrate of the two-dimensional plane can be observed by the atomic force microscope. In addition, when the amyloid precipitate is observed using an optical microscope or a fluorescence microscope, since the amyloid precipitate formed on the upper substrate and the lower substrate in the microfluidic device can be observed, the upper substrate and the lower substrate are coupled by chemical bonding. Can be.

In the present invention, the step (e) is characterized in that to inject amyloid into a plurality of microchannels by seeding on the surface of the microchannels to block (blocking), and then to inject and grow amyloid again to generate an amyloid precipitate. You can do

Specifically, when the amyloid monomer is injected into the plurality of microchannels by using a syringe, the amyloid seed is formed inside the microchannel, and when the microchannel on which the amyloid seed is formed is blocked, the remaining portions of the non-seed portion remain. After the water is removed, the amyloid monomer is continuously infused at a constant rate, followed by incubation, to form an amyloid precipitate inside the microchannel.

In the present invention, step (e) may be characterized in that the amyloid precipitate is produced by injecting and growing amyloid of different conditions into each of the plurality of microchannels in different ways.

In the present invention, the conditions may be characterized in that it is selected from the group consisting of temperature, concentration and pH, the different method may be characterized in that the treatment of the injection rate or reaction time differently.

In the present invention, the coupling between the upper substrate and the lower substrate of the microfluidic device is a bond by physical adsorption, and the amyloid precipitate formed in the microchannels can be observed using an atomic force microscope. The coupling between the upper substrate and the lower substrate of the microfluidic device is a chemical bond, and the amyloid precipitate formed in the microchannels can be observed using an optical microscope or a fluorescence microscope.

In still another aspect, the present invention relates to a method for screening a therapeutic agent for neurodegenerative diseases, characterized by using the microfluidic device.

In the present invention, by applying a different amyloid monomer solution and various growth conditions to each of the plurality of microchannels using the microfluidic device, the sample is injected into the amyloid precipitates simultaneously generated in the plurality of microchannels, and then the relative growth thereof. It can be characterized by comparing by screening.

When screening amyloid precipitates using the microfluidic device according to the present invention, due to the use of a plurality of microchannels, various conditions, temperature, injection rate of amyloid monomer solution such as protein concentration, buffer type, pH, salt concentration, etc. After varying the growth conditions, such as time, different for each microchannel, it is possible to generate amyloid precipitate in each microchannel at the same time.

In addition, as described above, by injecting a drug into a plurality of microchannels in which amyloid precipitates are produced under different conditions, the relative growth of amyloid precipitates generated in each microchannel can be compared and screened.

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are intended to illustrate the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example  1: Manufacture of Microfluidic Devices

Using a polydimethylsiloxane substrate as the upper substrate and a glass substrate as the lower substrate, a microfluidic device was manufactured by the following method.

1-1. Master Fabrication with Multiple Microchannel Shapes

After spin-coating a photoresist (SU-8, MicroChem, Newton, MA) on a silicon wafer, the wafer was baked at 95 ° C. for 10 minutes. After applying a photomask to the spin-coated wafer for 150 seconds to ultraviolet light, only a channel portion of the master was left. After exposure, the masters were baked at 95 ° C. for 10 minutes and developed with developer (SU-8 developer, MicroChem, Newton, MA) for 15 minutes. At this time, the master was 100 μm or 50 μm in length and width, respectively, and 10 mm in length.

1-2. Microchannel formation on the upper substrate

The polydimethylsiloxane prepolymer and the curing agent (PDMS, PDMS curing reagent, Dow Corning, Midland, MI) are mixed in a ratio of 10: 1, poured into the master to cure, and punched 1.5 mm with holes at both ends of the microchannel. By forming a sample inlet, a polydimethylsiloxane substrate was produced, and four microchannels formed at both ends of the polydimethylsiloxane substrate were formed at the bottom of the polydimethylsiloxane substrate.

1-3. Combination of upper board and lower board

(1) physical coupling of upper and lower substrates

In order to observe the amyloid precipitate by using an atomic force microscope, the glass substrate was cut into a size of 12 mm x 8 mm, and then the polydimethylsiloxane substrate prepared in 1-2 and the glass substrate were physically removed using a holder. Combined. At this time, the holder used is made of an aluminum alloy, and a hole is formed in the corners of the polydimethylsiloxane substrate and the glass substrate, and then fixed using a bolt to physically fix the polydimethylsiloxane substrate and the glass substrate on which the microchannels are formed. Combined.

(2) chemical bonding of upper and lower substrates

In order to observe the amyloid precipitate by using an optical microscope or a fluorescence microscope, the polydimethylsiloxane substrate and the glass substrate prepared in 1-2 were subjected to oxygen plasma treatment, and then placed in a vacuum for 45 seconds. Next, by fixing the microchannel on the organic substrate, the polydimethylsiloxane substrate and the glass substrate were chemically bonded (FIG. 1).

1-4. Activation of Microchannels

The glass substrates of the polydimethylmethylsiloxane substrate and the glass substrate bonded in the above 1-3 were immersed in a Piranha solution in which 70% sulfuric acid and 30% hydrogen peroxide were mixed at a volume ratio of 7: 3 at 60 ° C. for 15 minutes. Thereafter, the glass substrate was washed with distilled water until neutral, and then dried using nitrogen gas.

After combining the dried glass substrate with a polydimethylsiloxane microchannel subjected to oxygen plasma treatment, 95% ethanol containing 35% of 3-aminopropyl triethoxysilane was added for 1 hour. After flowing for a while, washed again with 100% ethanol and cured at 100 ° C. for one hour. After cooling the microfluidic device, a 50 mM sodium bicarbonate buffer (pH 8.5) containing 20 mM N, N'-disuccinimidyl carbonate (N, N'-disuccinimidyl carbonate) was kept at room temperature for 3 hours. At the flow through the microfluidic device at chemically activated inside the microchannel and dried.

1-5. Formation of Amyloid Precipitates in Microchannels

The rate at which the fluid sample is flowed into the microchannel using a syringe pump and a syringe is as follows. That is, it flowed at 5μL / h to chemically react the fluid sample in the microchannel of the microfluidic device, and 150μL / h to wash or inject. Here, a solution containing 1 mg of bovine insulin in 1 mL of 40 mM hydrochloric acid was used as the fluid sample.

In the chemically activated microfluidic device in 1-4, a solution containing 1 mg of bovine insulin in 1 mL of 40 mM hydrochloric acid was flowed at 5 μL / h for 5 minutes to fix the amyloid protein on the wall in the microchannel. The immobilized amyloid monomer serves as a seed for the amyloid precipitate to be produced in the microchannel.

At this time, in order to suppress further reaction by the remaining functional groups, 50 mM phosphate buffer (pH 7.5) containing 0.1% bovine Serum Albumin was flowed at 150 μL / h for 45 minutes, and a 50 mM phosphate buffer ( pH 7.5) and distilled water for 30 minutes to prevent the production of amyloid precipitate other than the Seed.

Through the sample inlet of the microfluidic device to which the amyloid protein was immobilized, a solution containing 1 mg of bovine insulin in 1 mL of 40 mM hydrochloric acid was continuously flowed at 5 μL / h into a microchannel to generate an amyloid precipitate in the microchannel. Let.

As a result, a microchannel device in which amyloid precipitates were formed in a plurality of microchannels was produced.

Example  2: Observation of Amyloid Precipitate Formed in Microchannels

The microchannel in which the amyloid precipitate prepared in Example 1 was produced was dried with nitrogen gas, and then observed with an optical microscope (FIG. 2), an atomic force microscope (FIG. 3), and a fluorescence microscope (FIG. 4), followed by thioflavin ( Thioflavin S) was flowed, and after 3 minutes, the thioflavin S remaining in the microchannels was removed as a tissue, dried with nitrogen gas, and observed under a fluorescence microscope.

As a result, as shown in Figures 2, 3 and 4, it was confirmed that the amyloid precipitate is produced in proportion to the reaction time and the injection speed.

Example  3: High Efficiency Assay for Screening Drugs for Degenerative Neuropathy

Inject various therapeutic agents for neurodegenerative diseases such as inhibitors or disintegrators of amyloid deposits into a plurality of microchannels in which amyloid deposits have grown, and the effects of the therapeutic agents on amyloid deposits over time through atomic force microscope, optical microscope, and light microscope Analyzed

As a result, as shown in Figure 5, it can be seen that different results appear depending on the type of treatment for neurodegenerative diseases injected into a plurality of microchannels, it was confirmed that the treatment for the treatment of degenerative neurological diseases based on these results. It was.

While the present invention has been particularly shown and described with reference to specific embodiments thereof, those skilled in the art will appreciate that such specific embodiments are merely preferred embodiments and that the scope of the present invention is not limited thereby. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Figure 1 shows the manufacturing process of the microfluidic device for the production and screening of amyloid precipitate in accordance with the present invention.

Figure 2 shows a photograph of observation of the amyloid precipitate formed in the microchannel of the microfluidic device according to the present invention with an optical microscope.

Figure 3 shows a photograph of the amyloid precipitate formed in the microchannel of the microfluidic device according to the present invention observed by atomic force microscope.

Figure 4 shows a photograph of the amyloid precipitate formed in the microchannel of the microfluidic device according to the present invention observed with a fluorescence microscope.

5 is a schematic diagram showing a method of applying various degenerative neurological diseases to the amyloid precipitate formed in the microchannel of the microfluidic device according to the present invention, and analyzing the effect with high efficiency.

Claims (16)

delete delete delete delete delete An upper substrate and a lower substrate coupled with the upper substrate, comprising the following steps; A plurality of microchannels formed on a bottom surface of the upper substrate; Amyloid precipitated inside the microchannel; And Method for manufacturing a microfluidic device for screening (degenerative) neurodegenerative agent screening comprising a sample inlet formed at both ends of the microchannel: (a) manufacturing a master including a plurality of microchannel shapes; (b) forming a plurality of microchannels including a sample inlet on the bottom surface of the upper substrate using the master; (c) coupling the upper substrate and the lower substrate; (d) activating the microchannels; And (e) forming an amyloid precipitate in the microchannel. The method of claim 6, wherein the binding of step (c) is characterized in that the bonding by physical compression or chemical bonding. The method of claim 7, wherein the physical pressing is to combine the upper substrate and the lower substrate using a holder. 8. The method of claim 7, wherein said chemical bonding is using oxygen plasma treatment. The method of claim 6, wherein the activation of the step (d) is performed by immersing the microfluidic device in a sub-pyran solution, by plasma treatment, and by (3-aminopropyl) triethoxysilane or N, N '. A process selected from the group consisting of reacting with disuccinimidyl carbonate (N, N'-disuccinimidyl carbonate). The method of claim 6, wherein the step (e) injects amyloid into a plurality of microchannels to seed and block the microchannel surface, and then injects and grows amyloid to generate amyloid precipitates. How to feature. The method of claim 6, wherein the step (e) comprises injecting and growing amyloid with different conditions into each of the plurality of microchannels in different ways to produce amyloid precipitates. 13. The method of claim 12, wherein said condition is selected from the group consisting of temperature, concentration and pH. 13. The method of claim 12, wherein said different method treats different injection rates or reaction times differently. Screening method for treating neurodegenerative disease, characterized in that using the microfluidic device produced by the method of claim 6. 16. The method of claim 15, wherein the microfluidic device of the microfluidic device injects and grows amyloids having different conditions in different ways, and injects a sample into the amyloid deposits simultaneously generated in the plurality of microchannels, and then grows the relative growth. By comparing the screening method for treating neurodegenerative diseases.

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