KR101033617B1 - Method for Fabricating of Microfluidic Device for Screening a Treating Agent of Neurodegenarative Diseases - Google Patents

Abstract

The present invention relates to a method for manufacturing a microfluidic device for screening a degenerative neurological disease therapeutic agent and a method for screening a degenerative neurological disease therapeutic agent using the microfluidic device, and more particularly, to a small amount in a single device having a plurality of microchannels. The present invention relates to a method for manufacturing a microfluidic device capable of rapidly and sensitively screening a therapeutic agent for neurodegenerative diseases by simultaneously generating amyloid precipitates, which are common to pathologies of neurodegenerative diseases.

According to the present invention, it is possible to obtain a microchannel surface activated uniformly without clogging upon microchannel activation, and to use this to screen an effective neurodegenerative agent that inhibits the production of amyloid deposits, thereby effectively reducing neurodegenerative diseases. It can be expected to be applicable to research and therapeutic development.

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

Description

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

The present invention relates to a method for manufacturing a microfluidic device for screening a degenerative neurological disease therapeutic agent and a method for screening a degenerative neurological disease therapeutic agent using the microfluidic device, and more particularly, to a small amount in a single device having a plurality of microchannels. The present invention relates to a method for manufacturing a microfluidic device capable of rapidly and sensitively screening a therapeutic agent for neurodegenerative diseases by simultaneously generating amyloid precipitates, which are common to pathologies of neurodegenerative diseases.

Amyloid is a protein combination with a linear structure of nanoscale, which is found in the form of plaques accumulated in the brain tissue of patients with various degenerative diseases such as Alzheimer's disease, Parkinson's disease, Huntington's disease and mad cow disease. Amyloid is formed by self-assembly of various kinds of precursor proteins, and they commonly have an aligned secondary structure of cross-beta sheets. Amyloid was first discovered in the brain tissues of Alzheimer's disease patients in Germany about 100 years ago, but the cause and mechanism of self-assembly of amyloid proteins in the form of normal proteins due to environmental changes are still unknown. , 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; Kisilevsky R. et al . , J Struct Biol . 130 : 99-108, 2000; John Hardy. et al. , Science : 353-356, 2002). In addition, the socioeconomic wavelengths of neurodegenerative diseases closely related to amyloid are very large. For example, Alzheimer's disease currently affects more than four million people, about 10% of adults age 60 and older in the United States alone, and there is still no clear treatment.

Although neurodegenerative diseases and amyloid deposits are known to be closely related, limitations such as effective analytical methods and uncertainties in amyloid precipitation mechanisms have led to very slow screening and development of therapeutic candidates. In addition, the screening of candidate amyloid therapeutics is complicated by various environmental conditions affecting amyloid precipitation such as temperature, pH, shear flow, charge amount, surface properties, and metal ions. For example, it is known that the type and rate of precipitation of amyloid deposits are greatly affected by the type and concentration of metal ions.

It is important to develop a high-efficiency analysis system that can screen for effective treatment of neurodegenerative diseases. Successful analytical systems must meet conditions such as low reagent consumption, fast and easy result evaluation and low labor. Recently, screening methods such as capillary electrophoresis, mass spectrometry, enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance spectrometry, and cell-based assays have been developed. However, these analytical methods do not satisfy all of the above conditions.

Therefore, the present inventors compare and analyze the production effect of amyloid precipitate by using a small amount of amyloid precursor and various inhibitory candidates in one microfluidic device having a plurality of microchannels, and decomposing various decomposition into amyloid precipitates generated in the microfluidic device. The present invention has been accomplished by confirming that it can provide a method for rapidly and sensitively screening and analyzing the effect of decomposing the generated amyloid precipitate of candidates.

An object of the present invention is to provide a method for manufacturing a microfluidic device for screening a neurodegenerative disease treatment.

Another object of the present invention to provide a method for screening a neurodegenerative disease treatment agent using the microfluidic device.

The present invention to achieve the above object, (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; (i) silanizing the microchannel surface to form vinyl groups; (ii) oxidizing the vinyl group to a carboxyl group; And (iii) activating the surface of the microchannels comprising treating the crosslinker; And (e) provides a method for manufacturing a microfluidic device for screening degenerative neurological disease therapeutic agent comprising the step of forming an amyloid precipitate in the activated microchannel.

The present invention also provides a method for screening a neurodegenerative agent for treating neurodegenerative diseases using a microfluidic device for screening a neurodegenerative agent for treating a neurodegenerative disease, wherein the amyloid protein is injected into an activated microchannel alone, and The present invention provides a method for screening a neurodegenerative agent for neurodegenerative diseases by comparing the degree of formation of amyloid deposits when the protein and the candidate agent for treating the neurodegenerative disease are injected together.

According to the present invention, it is possible to obtain a uniformly activated microchannel surface without clogging during microchannel activation, and to rapidly generate amyloid precipitate with a small amount of reagent in such a miniaturized microfluidic device, and by using the amyloid Effective degenerative neurodegenerative agents can be screened for inhibiting the formation of sediment, and therefore, the present invention can be expected to be effective for the effective research and development of therapeutic agents for degenerative neurological diseases.

The present invention relates to a method for producing a microfluidic device for screening a therapeutic agent for degenerative neurological diseases by generating amyloid precipitate, which is a common cause of degenerative neurological diseases.

Specifically, the present invention provides a microfluidic device for screening a therapeutic agent for neurodegenerative diseases, but injecting the amyloid and the neurodegenerative agent candidates related to the neurodegenerative disease together into a microchannel of the microfluidic device to produce an amyloid precipitate. By comparing, the therapeutic agent for neurodegenerative diseases can be screened.

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) manufacturing a master (master) including 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; (i) silanizing the microchannel surface to form vinyl groups; (ii) oxidizing the vinyl group to a carboxyl group; And (iii) activating the surface of the microchannels comprising treating the crosslinker; And (e) relates to a method for manufacturing a microfluidic device for screening degenerative neurological disease therapeutic agent comprising the step of forming an amyloid precipitate in the activated microchannel.

In the present invention, the amyloid is 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.

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 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. The chemical bonding may be performed by vacuum treating the upper substrate and the lower substrate after UV ozone surface treatment or oxygen plasma treatment. Here, the UV ozone surface treatment is performed using a treatment apparatus, and the treatment time is not particularly limited, but may be, for example, 20 minutes to 2 hours, preferably 20 to 40 minutes. Oxygen plasma treatment is also not limited to the treatment time, it can be treated for 30 seconds to 5 minutes, preferably 30 seconds to 2 minutes. Oxygen plasma and UV ozone surface treatment both remove organic materials from the surface of PDMS and glass, and make OH groups to enable chemical bonding between Si of PDMS and Si of glass by covalent bonds.

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, step (d) is a step of activating the formed microchannel, (i) silanizing the surface of the microchannel to form a vinyl group; (ii) oxidizing the vinyl group to a carboxyl group; And (iii) treating the crosslinker.

In the step (d), (i) silanizing the surface of the microchannel to form a vinyl group, containing one selected from the group consisting of octenyl trichlorosilane, vinyltriactosilane and vinyltrimethylsilane The organic solvent may be treated and silanized to form vinyl groups. Here, the organic solvent may be toluene, chloroform, chlorobenzene, xylene, methylene chloride, benzene and the like.

Through the activation process as described above, the surface of the microchannel may be uniformly activated without clogging.

In addition, it may be characterized in that the curing at 70 to 90 ℃ after the silanization.

In the step (d), (ii) oxidizing the vinyl group to a carboxyl group may include at least one compound selected from the group consisting of KMnO ₄ , K ₂ CO ₃ , NaIO ₄ , RuO ₄ , H ₂ O ₂ and OsO ₄ . It may be characterized by using, in particular, KMnO ₄ may be preferably used, RuO ₄ , H ₂ O ₂ or OsO ₄ And the like will also be suitable compounds for carboxyl group oxidation.

In the step (d), the crosslinking agent in step (iii) is used to activate the microchannels so that the amyloid protein or the additionally seeded amyloid monomer can be covalently bonded, and the crosslinking agent may use ECD / NHS. 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 using a syringe, an amyloid seed is formed inside the microchannel, and then the activated microchannel remaining without the amyloid seed is formed. Blocking the surface removes the activated surface of the portion other than the seed, and incubates by continuously injecting amyloid monomer at a constant rate, and then forms an amyloid precipitate inside the microchannel. Such seed formation in vitro (in In vitro ), the experiment is to create an environment similar to the phenomenon occurring in vivo, to obtain more reliable results.

In the present invention, the step (e) may be characterized by injecting different neurodegenerative agent candidates with amyloid protein into each of the plurality of microchannels, to generate amyloid precipitate.

In the present invention, the neurodegenerative disease treatment agent refers to a substance that inhibits the production of amyloid deposits associated with neurodegenerative disease, and as candidates for the treatment of neurodegenerative disease, for example, 1) the natural structure of proteins and peptides. Natural osmolytes, such as ectoine, hydroxyectoine, or trehalose, which stabilize the composition, and 2) structural of Congo red that specifically binds to amyloid precipitates. Analogues such as Congo red, chrysamine G, curcumine, or rosmarinic acid, and 3) drugs known in the art that are known to lower the risk of Alzheimer's disease in long-term use Such as ibuprofen (nonsteroidal anti-inflammatory agent, NSAID), or simvastatin (cardiovascular drug), and the like; 4) amyloid precipitation Other materials known to interfere with, for example, the melatonin and nicotine, or the like hemin (hemin).

In another aspect, the present invention provides a method for screening a degenerative neurological disease therapeutic agent using a microfluidic device for screening a degenerative neurological disease therapeutic agent produced by the above method, wherein the amyloid protein is injected into an activated microchannel alone. The present invention relates to a method for screening a therapeutic agent for neurodegenerative diseases by comparing the degree of formation of amyloid deposits when the amyloid protein and the candidate agent for treating neurodegenerative diseases are injected together.

In the present invention, a different neurodegenerative agent candidate is injected into the plurality of microchannels of the microfluidic device, respectively, along with amyloid protein, and the relative growth of amyloid precipitates is compared to screen for the neurodegenerative disease agent. Can be.

In addition, when screening the amyloid precipitate using the microfluidic device according to the present invention, due to the use of a plurality of microchannels, various conditions, temperature, injection of amyloid monomer solution such as protein concentration, buffer type, pH, salt concentration, etc. After various growth conditions such as speed and time are differently applied to each microchannel, amyloid precipitates may be simultaneously generated in each microchannel. For example, by injecting various degenerative neuropathic drug candidates 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.

Comparing the degree of formation of the amyloid precipitate, a variety of conventionally known detection methods can be applied, as in the following examples, by using a fluorescence microscope, atomic force microscope, etc., by measuring the degree of amyloid precipitation formation can be compared the inhibitory effect have.

Hereinafter, the present invention will be described in more detail by way of 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 100μm, or 50μm in width and height, respectively, was 10mm 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 16 microchannels each having an inlet at both ends were formed on 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 using an atomic force microscope, after cutting the glass substrate into a size of 12mm × 8mm, the polydimethylsiloxane substrate prepared in 1-2 and the glass substrate physically by 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, thereby physically bonding the polydimethylsiloxane substrate and the glass substrate on which the microchannels are formed. I was.

(2) chemical bonding of upper and lower substrates

In order to be able to observe the amyloid precipitate using an optical microscope or a fluorescence microscope, UV ozone treatment of the polydimethylsiloxane substrate and the glass substrate prepared in the above 1-2 was performed for about 30 minutes using a device. After the treatment, the microchannels were fixed to the organic substrate, thereby chemically bonding the polydimethylsiloxane substrate and the glass substrate.

1-4. Activation of Microchannels

To activate the inside of the microchannels using NHS functional groups to covalently bind amyloid seeds into the microchannels, toluene containing octenyl trichlorosilane (7-Octenyltrichlorosilane, 1 mM) was injected with a syringe (11 Plus syringe pump, Harvard Apparatus, Holliston, MA) was injected at a rate of 5ml / h and the microchannels were silanized. After 10 minutes the remaining solution with toluene was removed and cured at 80 ° C., and the vinyl groups formed in the microchannels were KMnO ₄ (0.5 mM), K ₂ CO ₃ (1.8 mM), and NaIO ₄ (19.5). mM) was injected and immediately oxidized to carboxyl groups. After 1 hour, the microchannels were washed by injecting 0.1 M NaHSO ₃ , water, 0.1 N HCl, and water in that order. Finally, 0.1 M 1-ethyl-3- (3-dimethylaminopropyl-) carbodiimide hydrochloride ( EDC) and 0.025 M N- hydroxysuccinimide (NHS) in a 1: 1 solution mixed at room temperature for 20 minutes to chemically activate the inside of the microchannel (FIG. 1).

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 200μL / h to wash or inject. In this case, a solution containing 10 mM of Aβ 42 in 10 mM phosphate buffer was used as the fluid sample.

In the chemically activated microfluidic device in 1-4, a solution containing 10 mM of Aβ42 in 10mM phosphate buffer was flowed at 5μL / h for 5 minutes to immobilize 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 (Seed).

Through the sample inlet of the microfluidic device to which the amyloid protein is immobilized, a solution containing 10 mM of Aβ42 in a 10mM phosphate buffer into a microchannel and a solution containing a candidate agent for treating a neurodegenerative disease at 5 μL / h are continuously added. Flow to form an amyloid precipitate in the microchannels. Candidates for the treatment of degenerative neurological diseases, using HSA, NaCl as a control group, and ectoine, trehalose, congo red, curcumin, rosemarine acid, ibuprofen, simvastatin, hemin, melatonin and nicotine as experimental groups, A microchannel device was fabricated to analyze the effects of candidate drug for neurodegenerative diseases on amyloid precipitation.

Example  2: Highly Efficient Analysis for Screening Drugs for Degenerative Neurological Diseases

The microchannels injected with the amyloid prepared in Example 1 and the candidate amyloid precipitation inhibitor which can be used as a therapeutic agent for neurodegenerative diseases were observed with an atomic force microscope and a fluorescence microscope, and then fluorinated with thioflavin S. Observed by.

As a result, in Fig. 2 (A and B), the atomic force microscope shows the degree of amyloid precipitation according to the fluorescence intensity, and the fluorescence intensity when incubated for about 12 hours is about half that of incubation for 24 hours. FIG. 3 shows the result of measuring the degree of precipitation formation by fluorescence microscopy using a case of injecting a solution containing only amyloid Aβ 42 as a control.

For example, as shown in Figure 3, when injected with melatonin showed a fluorescence intensity of about half, in this case it can be seen that the precipitate was formed in the same state as in the photographic results at 12 hours as shown in Figure 2A.

In addition, in FIG. 4, the degree of inhibition of precipitation formation in each experimental group is shown as a bar graph on the basis of a control group containing only amyloid Aβ 42. As shown in the graph, as a main component of blood, the positive control group NaCl and HSA showed little inhibitory effect on the formation of sediment, and amyloid sediment formation effect was different depending on the type of therapeutic agent for neurodegenerative diseases injected into a plurality of microchannels. It was found to be different. For example, curcumin, ibuprofen, simvastatin and the like are known to be effective in treating degenerative neurological diseases, and these results show excellent amyloid precipitation inhibitory effects through the present experimental results, and screening for the treatment of degenerative neurological diseases through the microfluidic device. Confirmed that it can.

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 precipitates according to the present invention (Aβ 42: amyloid; Inhibitor: degenerative neurological disease treatment agent that is an inhibitor of amyloid precipitation).

Figure 2 is a graph showing the photographs and fluorescence intensity of the amyloid precipitate formed in the microchannel of the microfluidic device according to the present invention by fluorescence microscope and atomic force microscope.

Figure 3 shows the effect of each drug candidate by injecting a candidate agent for the treatment of degenerative neurological disease, which is an amyloid and amyloid deposit formation inhibitor in the microchannel of the microfluidic device according to the present invention with a fluorescence microscope.

4 is a bar graph showing the effect of inhibiting amyloid precipitation formation of candidate drugs for treating neurodegenerative diseases that are amyloid precipitation formation inhibitors in the microchannels of the microfluidic device according to the present invention.

Claims (13)

Method for manufacturing a microfluidic device for screening degenerative neurological disease treatment comprising the following steps: (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; (i) silanizing the microchannel surface to form vinyl groups; (ii) oxidizing the vinyl group to a carboxyl group; And (iii) activating the surface of the microchannels comprising treating the crosslinker; And (e) forming an amyloid precipitate in the activated microchannel. According to claim 1, wherein the amyloid is beta-amyloid of Alzheimer's disease (β-amyloid), alpha-synuclein of Parkinsonism (α-synuclein), Huntington's and Prion of Huntington's disease (Prion) A method characterized in that the protein or a fragment thereof selected from the group consisting of prions of related diseases. The method of claim 1, wherein the bonding of step (c) is characterized in that the bonding by physical compression or chemical bonding. The method of claim 3, wherein the physical pressing is to combine the upper substrate and the lower substrate by using a holder. The method of claim 3, wherein the chemical bonding is using UV Ozone surface treatment. The method of claim 1, wherein step (d) comprises silanization of the microchannels using an organic solvent containing one selected from the group consisting of octenyl trichlorosilane, vinyltriectosilane and vinyltrimethylsilane to form a vinyl group. Characterized in that the method. The method of claim 1, wherein step (d) comprises curing at 70 to 90 ° C after the silanization. The method of claim 1, wherein step (d) comprises oxidizing a vinyl group to a carboxyl group using at least one compound selected from the group consisting of KMnO ₄ , K ₂ CO ₃ , NaIO ₄ , RuO ₄ , H ₂ O ₂ and OsO ₄ . Characterized in that the method. The method of claim 1, wherein the crosslinking agent in step (d) is EDC / NHS. The method of claim 1, wherein the step (e) comprises injecting amyloid into a plurality of microchannels to seed and block the microchannel surface, thereby blocking amyloid precipitates by covalently binding to the microchannel surface. How to. The method of claim 1, wherein the step (e) comprises injecting different degenerative neuropathy drug candidates together with amyloid protein into each of the plurality of microchannels to generate amyloid precipitates. 12. A method for screening a neurodegenerative agent for treating neurodegenerative diseases using a microfluidic device for screening a neurodegenerative agent for treating a neurodegenerative disease prepared by any one of claims 1 to 11, wherein the amyloid protein is injected alone into an activated microchannel. And screening the therapeutic agent for neurodegenerative diseases by comparing the degree of formation of amyloid deposits when the amyloid protein and the candidate agent for treating neurodegenerative diseases are injected together. The method of claim 12, wherein a plurality of microchannels of the microfluidic device are injected with different neurodegenerative agent candidates, each with a amyloid protein, to compare the relative growth of amyloid deposits to screen for the neurodegenerative agent treatment How to.

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