KR101772766B1

KR101772766B1 - Microchip and method for producing the same, system and method for detecting enzyme using the same

Info

Publication number: KR101772766B1
Application number: KR1020150155591A
Authority: KR
Inventors: 고원건; 한상원; 김민수
Original assignee: 연세대학교 산학협력단
Priority date: 2015-11-06
Filing date: 2015-11-06
Publication date: 2017-08-30
Also published as: KR20170053288A

Abstract

In the present invention, nanofibers made by electrospinning are patterned with a hydrogel to form a support of nanofibers, and a peptide having a fluorescent dye that is specifically decomposed to an enzyme such as MMP is immobilized on the exposed nanofibers. A microchip inserted into a chip structure fabricated by PDMS or the like, a method of manufacturing the same, and an enzyme detection system and method using a microchip are provided. The present invention overcomes the difficulty of combining nanofibers with microchip technology by using hydrogel patterning, and has the advantage that it can detect more rapidly and accurately than other existing enzyme detection methods.

Description

TECHNICAL FIELD The present invention relates to a microchip, a method for producing the same, an enzyme detection system and method using the same,

The present invention relates to a microchip, a method of manufacturing the same, and an enzyme detection system and method using the microchip. More particularly, the present invention relates to a microchip having a sheet of a nanofiber patterned with a hydrogel, And more particularly to a system and method for effectively detecting an enzyme using a chip.

Nanofibers fabricated by electrospinning have been expected to have many applications in immunoassay and proteolytic enzyme detection because the surface area to immobilize proteins and enzymes to be immobilized is very wide. However, due to the low mechanical strength of the nanofibers, And thus it is difficult to carry out a series of processes for attaching proteins. On the other hand, since the microfluidic system can reduce the analysis time, minimize the use of the sample, and help the binding with the analyte, a lot of research has been conducted in the immunoassay and the proteolytic enzyme detection. As described above, in order to fix the nanofibers that can be easily detected in the microfluidic system, a microchip is formed on the nanofiber sheet to form nanofibers at the bottom of the channels, And a microchip is formed by inserting it directly into a microchip. However, when nanofibers are formed at the bottom of the channel, there is a disadvantage in that the nanofibers can not be effectively fixed to the entire channel. When the nanofibers are inserted directly into the microchip, it is difficult to control the amount of nanofibers to be inserted The application to the sensor is limited.

Matrix metalloprotease (MMP) is known to be involved in various pathological processes such as neurogenesis, organogenesis, bone formation, wound healing, angiogenesis, as well as normal biological processes such as cancer, cardiovascular disease, leukemia and liver cirrhosis. Among them, MMP is known to be deeply involved in cancer growth, metastasis, and necrosis. Therefore, a technique for accurately analyzing the concentration of MMP in the body in various fields is required. Immunoassay, gelatin zymography and the like have been mainly used for detecting MMP. Immunoassays are costly and limited in their application to MMP-specific antibodies, and require at least 20 hours of gelatin zymography, which makes it difficult to perform rapid analysis.

It is an object of the present invention to provide a method of immobilizing nanofibers which are difficult to manipulate by forming a support using hydrogel lithography and then fixing the nanofibers in the microchip, And a method of manufacturing the same.

It is another object of the present invention to provide an enzyme detection system and method using a microchip.

In order to achieve the above-mentioned object, the present invention provides, as a flat plate-like layer, a flat plate-shaped layer having a first flow passage connected to an inlet, a first suction inlet for vacuum suction, layer; A layer in the form of a flat plate disposed at the bottom of the first layer, the layer being in the form of a first chamber connected to the first flow path, a second inlet connected to the first inlet, a second flow path connected to the second inlet, A second layer having a third flow path and a second discharge port connected to the first discharge port; A layer in the form of a thin film disposed at a lower portion of the second layer, the third layer having a second chamber connected to the first chamber and a third outlet connected to the second outlet; A fourth channel connected to the second chamber, a separator having a width smaller than that of the third channel and disposed at a lower portion of the third channel and spaced apart from the fourth channel through the separator, A fourth layer having a fourth outlet connected to the fifth flow path, the fifth flow path and the third outlet, respectively; And a nanofiber sheet provided in the first chamber and the second chamber, the nanofiber sheet being patterned with a hydrogel and having an enzyme detection peptide immobilized thereon.

In the present invention, the fluid may flow into the first chamber and the second chamber through the inlet and the first flow path, and may be discharged through the fourth outlet and the first outlet through the fourth flow path, the third flow path, and the fifth flow path.

In the present invention, the third layer is brought into close contact with the separating portion to block the flow of the fluid from the fourth flow path to the fifth flow path, and by the vacuum suction through the first suction port and the second suction port, The fluid is separated from the third layer and separated from the third layer so that the fluid can pass from the fourth flow path to the fifth flow path through the third flow path.

In the present invention, the microchip may be formed of a siloxane-based polymer.

In the present invention, the nanofiber may be selected from the group consisting of polystyrene / poly (styrene-co-maleic anhydride) (PS / PSMA), chitosan, elastin, hyaluronic acid, alginate, gelatin, collagen, cellulose, polyethylene glycol (PEG), polyethylene oxide (PCA), polylactic acid (PLA), polyglycolic acid (PGA), poly (lactic-co- (glycolic acid)) (PLGA), poly [(3-hydroxybutyrate) 3-hydroxyvalerate) (PHBV), polydioxanone (PDO), poly [(L-lactide) -co- (caprolactone)], poly (ester urethane) (PEUU) (PVA), polyacrylic acid (PAA), polyvinyl alcohol (PVA), polyvinyl pyrrolidone (PVP), polyvinyl pyrrolidone Polystyrene (PS), and polyaniline (PAN).

In the present invention, the hydrogel may contain at least one selected from the group consisting of polyethylene glycol-diacrylate (PEG-DA), polyethylene glycol (PEG), polyethylene oxide (PEO), polyhydroxyethyl methacrylate (PHEMA), polyacrylic acid (PVA), poly (N-isopropylacrylamide) (PNIPAM), polyvinylpyrrolidone (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), gelatin, alginate, It may be formed of at least one selected from carrageenan, chitosan, hydroxyalkylcellulose, alkylcellulose, silicone, rubber, agar, carboxyvinyl copolymer, polydioxolane, polyacrylic acetate, polyvinyl chloride, maleic anhydride / vinyl ether have.

In the present invention, the enzyme may be a protease, and the protease may be a matrix metalloprotease (MMP).

In the present invention, the peptide may have an amino acid sequence of Arg-Ser-Trp-Met-Gly-Leu-Pro-Gly.

In the present invention, a fluorescent dye may be attached to the end of the peptide.

The present invention also provides a method of fabricating a nanofiber scaffold, comprising: forming a nanofiber scaffold; Patterning a nanofiber scaffold with a hydrogel to form a nanofiber sheet; Immobilizing the peptide in the exposed region of the nanofiber sheet; Forming the first to fourth layers as described above; And inserting a nanofiber sheet on which the peptide is immobilized in the first chamber and the second chamber.

In the present invention, nanofiber scaffolds can be formed through electrospinning.

The method of manufacturing a microchip according to the present invention may further include a step of forming a nanofiber scaffold and then performing an oxygen plasma treatment.

In the present invention, the hydrogel patterning and the formation of the first to fourth layers may be performed using photolithography or soft lithography.

The present invention also relates to the above-mentioned microchip; And an enzyme detection system including a fluorescence measurement device.

The enzyme detection system according to the present invention may further include a vacuum pump.

The present invention also provides an enzyme detection method characterized by using the enzyme detection system described above.

In the enzyme detection method according to the present invention, the concentration of the enzyme can be analyzed by measuring the fluorescence intensity of the sample discharged from the enzyme detection system.

The present invention enables faster and more accurate detection than conventional enzyme (MMP, etc.) detection methods using microchips based on nanofibers patterned with hydrogel. In addition, by controlling the shape of the hydrogel pattern, it is possible to design an effective enzyme detection system by controlling the reaction area of nanofibers. It is also possible to manufacture microchips capable of simultaneously detecting different types of antibodies or peptides on different nanofiber sheets and simultaneously inserting them into microchips to detect various kinds of detection substances simultaneously.

1 is an exploded perspective view of a microchip according to the present invention.
2 is a plan view of each layer of the microchip according to the present invention.
3 is a side sectional view of the microchip according to the present invention.
4 is an enlarged cross-sectional view showing passage of fluid at boundary portions of the second to fourth layers of the microchip according to the present invention.
FIG. 5 shows that a specific site of a peptide attached to a nanofiber is broken by MMP according to the present invention.
6 is a photograph of a nanofiber sheet patterned with a hydrogel according to the present invention.
7 is a photograph of a nanofiber sheet on which a peptide is immobilized according to the present invention.
8 is a photograph of a microchip fabricated according to the present invention.
9 is a graph showing fluorescence intensities measured according to MMP concentration according to the present invention.

Hereinafter, the present invention will be described in detail.

2 is a plan view of a microchip according to an embodiment of the present invention. FIG. 3 is a cross-sectional view of the microchip according to the present invention. And FIG. 4 is an enlarged cross-sectional view illustrating a process of passing a fluid through the boundary between the second layer and the fourth layer serving as a valve in the microchip according to the present invention.

1 to 3, a microchip according to the present invention includes a first layer 10, a second layer 20, a third layer 30, a fourth layer 40, a nanofiber sheet 50, . The microchip is a combination of multi-layer structures, the four-layer structure being illustrated in the figure, but more or fewer layers may be present.

The first layer 10 is a layer in the form of a flat plate (first flat plate) and has an inlet 11 into which the fluid flows, a first channel 12 connected to the inlet 11, a first suction inlet 13 for vacuum suction, And a first outlet 14 through which the fluid is discharged.

The inlet 11 is where the fluid containing the enzyme to be detected enters, and the fluid may be in the form of, for example, a solution. In the figure, the inlet 11 is located at the left upper end and is shaped like a hole penetrating the flat plate in the vertical direction. Alternatively, the inlet 11 may be inclined at a predetermined angle, Its position is also not particularly limited and can be formed at an appropriate position.

One end of the first flow path 12 is connected to the inlet 11 and the other end of the first flow path 12 is connected to the first chamber 21 to connect the inlet 11 and the first chamber 21. In the drawing, the first flow path 12 is illustrated as a groove formed in the right side in the horizontal direction on the lower surface (bottom surface) of the flat plate. Alternatively, the first flow path 12 may be formed in a hole shape and its direction may be set differently.

The first suction port 13 is formed for vacuum suction. For example, a tube connected to a vacuum pump may be inserted into the first suction port 13 and vacuum suction may be performed. In the drawing, the first suction port 13 is located at the right upper end and is illustrated as a hole passing through the flat plate in the vertical direction. Alternatively, the first suction port 13 may be inclined and its position is not particularly limited.

The first outlet 14 is where the fluid is discharged. In the figure, the first outlet 14 is located at the right lower end and is illustrated as a hole through which the flat plate penetrates in the vertical direction. Alternatively, the first outlet 14 may be inclined and its position is not particularly limited.

The second layer 20 is a layer in the form of a flat plate (second flat plate) disposed under the first layer 10 and includes a first chamber 21 connected to the first flow path 12, A second flow path 23 connected to the second suction port 22, a third flow path 24 connected to the second flow path 23, a first discharge port 14, And a second outlet 25 connected thereto.

The first chamber 21 provides a space into which the nanofiber sheet 50 is inserted. The first chamber 21 is connected to the first flow path 12 at the upper part and connected to the second chamber 31 at the lower part. Although the first chamber 21 is illustrated as a large-diameter circular hole in which the first chamber 21 is located at the central portion of the flat plate and penetrates the flat plate in the vertical direction, the first chamber 21 may have a different shape such as a polygonal shape, Do not. For example, the diameter of the first chamber 21 may be 50 to 90% of the width of the second layer 20.

The second suction port 22 is formed for vacuum suction as in the case of the first suction port 13 and is vertically aligned with the first suction port 13 or partially connected to at least the first suction port 13 . Although the second suction port 22 is illustrated as a hole through which the flat plate is vertically penetrated, the second suction port 22 may be inclined or may extend only to a certain depth without penetrating the flat plate.

The second flow path 23 has one end connected to the second suction port 22 and the other end connected to the third flow path 24 to thereby connect the second suction port 22 and the third flow path 24 do. Although the second flow path 23 is illustrated as a groove formed in the bottom of the flat plate in the lower horizontal direction, the second flow path 23 may be formed in the shape of a hole and the direction thereof may be set differently.

The third flow path 24 is connected to the third flow path 24 and may be selectively connected to the fourth flow path 41 and the fifth flow path 43. A part of the fourth flow path 41, a part of the separation part 42, and a part of the fifth flow path 43 may be positioned below the third flow path 24. In the drawing, the third flow path 24 is illustrated as a groove formed in a lower horizontal direction on a lower surface of a flat plate. Alternatively, the third flow path 24 may be formed in a hole shape and its direction may be set differently.

The second outlet 25 is located in the vertical direction with respect to the first outlet 14 as shown in the figure or partially connected to at least the first outlet 14 as the first outlet 14, . Although the second outlet 25 is illustrated as a hole through which the flat plate passes in the vertical direction, the second outlet 25 may be inclined in a different manner.

The third layer 30 is a layer in the form of a thin film disposed on the lower side of the second layer 20 and includes a second chamber 31 connected to the first chamber 21, 3 outlet 32. In this embodiment,

The second chamber 31 provides a space in which the nanofiber sheet 50 is inserted, like the first chamber 21. The second chamber 31 is connected to the first chamber 21 at the top and to a part of the fourth flow path 41 at the bottom. The second chamber 31 is formed at a position vertically aligned with the first chamber 21 or partially connected to at least the first chamber 21 as shown in the figure. The shape and size of the second chamber 31 may be the same as or different from that of the first chamber 21. Although the second chamber 31 is illustrated as a large-diameter circular hole in which the second chamber 31 is located at the center of the thin film and penetrates the thin film in the vertical direction, the second chamber 31 may have a different shape such as a polygonal shape, Do not. For example, the diameter of the second chamber 31 may be 50 to 90% of the width of the third layer 30.

The third outlet 32 is formed at a position where the fluid is discharged, aligned vertically with the second outlet 25 as shown in the drawing, or at least partially connected to the second outlet 25. Although the third outlet 32 is illustrated as a hole through which the thin film passes in the vertical direction, the third outlet 32 may be inclined.

The fourth layer 40 is a layer in the form of a flat plate (third flat plate) disposed at the lower part of the third layer 30 and has a fourth flow path 41 connected to the second chamber 31, A fifth flow path 43 and a fifth flow path 43 which are spaced apart from the fourth flow path 41 through the separation part 42. The fifth flow path 43, And a fourth outlet 44 connected to the third outlet 32, respectively.

One end of the fourth flow path 41 is connected to the second chamber 31 and the other end of the fourth flow path 41 is blocked by the separating part 42 and can be selectively connected to the third flow path 24. Although the fourth flow path 41 is illustrated as a groove in which the fourth flow path 41 is formed on the upper surface of the flat plate in the horizontal direction on the right side, it may be formed differently.

The separating portion 42 separates the fourth flow path 41 and the fifth flow path 43 from the fourth flow path 41 and the fifth flow path 43, 3 layer 30 to block the flow path. The separating portion 42 is located below the third flow path 24 and is smaller than the third flow path 24.

One end of the fifth flow path 43 is blocked by the separating part 42 and the other end is connected to the fourth discharge port 44 and selectively connected to the third flow path 24. Although the fifth flow path 43 is illustrated as a groove in which the fifth flow path 43 is formed on the upper surface of the flat plate in the horizontal direction on the right side, it may be formed differently.

The fourth outlet (44) is connected to the fifth flow path (43) and the third outlet (32), respectively, where the fluid is discharged. The fourth outlet 44 is formed at a position vertically aligned with the third outlet 32 as shown in the figure, or at least partially connected to the third outlet 32. Although the fourth exhaust port 44 is illustrated as a groove formed on the upper surface of the flat plate, the fourth exhaust port 44 may be formed differently.

The fluid flows into the first chamber 21 and the second chamber 31 through the inlet 11 and the first flow path 12 and flows into the second chamber 31 through the fourth flow path 41 and the third flow path 24, 43, and out through the outlets 44, 32, 25, 14.

3, the third layer 30 is in close contact with the separating portion 42 to block the flow of the fluid from the fourth flow path 41 to the fifth flow path 43 during normal operation (in the closed state). That is, the flow paths 41 and 43 of the flow path 24 and the fourth layer 40 of the second layer 30 are blocked by the third layer 30 and are not connected to each other.

4, when a tube is inserted into the first suction port 13 and the vacuum pump is operated to perform vacuum suction, the first suction port 13, the second suction port 22, The suction force acts on the upper surface of the third layer 30 through the first flow path 23 and the third flow path 24 so that the third layer 30 is lifted up in the third flow path 24, Since the width of the separating portion 42 is smaller than that of the third flow path 24, both sides of the separating portion 42 are opened, Through the third flow path (24) to the fifth flow path (43). Thus, the second suction port 22, the third flow path 24, the third layer 30, the fourth flow path 41, the separation part 42, the fifth flow path 43, It is possible to open and close the valve.

The size and shape of the first layer (10) to the fourth layer (40) are not particularly limited and may be suitably set as necessary. For example, the thickness of the first layer 10, the second layer 20 and the fourth layer 40 may be 0.1 to 5 cm, and the thickness of the third layer 30 may be 1 to 100 m have. The size and shape of chambers, holes, grooves, flow paths, etc. formed in each layer are not particularly limited, and can be appropriately set as necessary.

The first layer 10 to the fourth layer 40 may be formed of a siloxane-based polymer. As the siloxane-based polymer, for example, polydimethylsiloxane (PDMS) or the like can be used. When PDMS is used, there is an advantage that each layer can be easily manufactured by using photolithography or the like. Polymethylmethacrylate (PMMA) or the like may be used. However, since PMMA has a high hardness, mechanical work is required to form holes and grooves.

The nanofiber sheet 50 is installed in the first chamber 21 and the second chamber 31, and is patterned with a hydrogel, and the enzyme-detecting peptide can be immobilized.

The nanofibers may be selected from the group consisting of polystyrene / poly (styrene-co-maleic anhydride) (PS / PSMA), chitosan, elastin, hyaluronic acid, alginate, gelatin, collagen, cellulose, polyethylene glycol (PEG), polyethylene oxide (PLGA), poly [(3-hydroxybutyrate) -co- (3-hydroxycyclohexyl) -L-lactone (PCL), polylactic acid (PLA), polyglycolic acid (L-lactide) -co- (caprolactone), poly (ester urethane) (PEUU), poly [(L-lactide) (PVA), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), polystyrene (PS), polyvinyl alcohol (PVA) ) And polyaniline (PAN), and PS / PSMA can be preferably used. When PS / PSMA is used as the nanofiber, there is an advantage that since the peptide is directly bonded to the MA group, a separate pretreatment step for bonding the peptide is not necessary.

Hydrogels include polyethylene glycol-diacrylate (PEG-DA), polyethylene glycol (PEG), polyethylene oxide (PEO), polyhydroxyethyl methacrylate (PHEMA), polyacrylic acid (PAA), polyvinyl alcohol , Poly (N-isopropylacrylamide) (PNIPAM), polyvinylpyrrolidone (PVP), polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), gelatin, alginate, May be formed of at least one member selected from the group consisting of hydroxyalkylcellulose, alkylcellulose, silicone, rubber, agar, carboxyvinyl copolymer, polydioxolane, polyacrylate, polyvinyl chloride and maleic anhydride / vinyl ether PEG-DA can be used. When PEG-DA is used as the hydrogel, there is an advantage that it is easy to produce a nanofiber sheet patterned with hydrogel.

Hydrogel patterning of nanofibers can be used to improve the mechanical strength of nanofibers and to fabricate nanofiber sheets that are easy to manipulate.

The substance to be detected may be an enzyme or the like, the enzyme may be a protease, and the protease may be, for example, a matrix metalloprotease (MMP). In the present invention, any protease that can be used for detection by immobilizing a peptide or a derivative thereof can be detected, and is not limited to MMP.

The peptide may be a peptide that is specifically degraded to MMP, and specifically, a peptide having the amino acid sequence of Arg-Ser-Trp-Met-Gly-Leu-Pro-Gly.

A fluorescent dye may be attached to the end of the peptide. As the fluorescent dye, for example, fluorescein isothiocyanate (FITC) and the like can be used.

The peptides can be immobilized in hydrogel free areas, i.e. exposed nanofiber areas.

The present invention also provides a method of manufacturing a microchip. A method of fabricating a microchip according to the present invention includes: forming a nanofiber scaffold; Patterning a nanofiber scaffold with a hydrogel to form a nanofiber sheet; Immobilizing the peptide in the exposed region of the nanofiber sheet; Forming the first to fourth layers as described above; And inserting a peptide-immobilized nanofiber sheet into the first chamber and the second chamber.

The nanofiber scaffold can be formed by electrospinning a polymer solution. As the polymer, for example, PS / PSMA and the like may be used, and the content of the polymer in the solution may be, for example, 10 to 30% by weight. As the solvent for dissolving the polymer, for example, dimethylformamide (DMF), chloroform and the like can be used. The nanofiber scaffold can be formed and then subjected to oxygen plasma treatment. The oxygen plasma treatment of the nanofiber scaffold can increase the hydrophilicity of the nanofiber scaffold, and the increase in hydrophilicity can increase the permeability of the hydrogel precursor solution.

Hydrogel patterning may be performed using photolithography or soft lithography. Specifically, a nanofiber scaffold is sufficiently immersed in a photopolymerizable precursor solution containing a precursor and a photopolymerization initiator, and then a light is irradiated by placing a photomask on the scaffold, the photomask including a portion through which light passes and a portion through which light is not passed. The portion irradiated with light is crosslinked by photopolymerization and is not dissolved in a solvent such as water, and the portion not irradiated with light is washed out by a solvent. That is, the hydrogel is not formed at the portion not irradiated with light, so that the nanofiber is exposed to the outside. As the precursor, for example, PEG-DA and the like can be used, and as the photopolymerization initiator, for example, 2-hydroxy-2-methylpropiophenone (HOMPP) can be used. The content of the precursor in the solution may be, for example, 30 to 70% by weight, the content of the photopolymerization initiator may be, for example, 1 to 10% by weight, and the balance may be composed of a solvent such as water. The light can be irradiated with ultraviolet rays (UV).

To immobilize the peptide on the exposed nanofibers, the nanofiber sheet is immersed in a solution containing a peptide in which the fluorescent dye is immobilized on the end. The concentration of the peptide in the solution may be, for example, 10 to 100 占 퐂 / mL.

Each layer of the microchip may be fabricated using photolithography or the like. For example, PDMS or the like may be used to fabricate a plurality of layers, and then the layers may be combined. More specifically, a plurality of layers including a flow path pattern and the like can be manufactured using a patterned substrate after coating a photosensitive agent on the substrate, covering the mask, exposing and developing the patterned substrate, The layers may be bonded by corona treatment or the like.

After the nanofiber sheet is inserted into the microchip, the analyte is flowed, the valve is closed and reacted, and the reacted solution is discharged and analyzed. After the reaction is completed, the nanofiber sheet is removed, Reusable.

The present invention also relates to the above-mentioned microchip; And an enzyme detection system including a fluorescence measurement device. The enzyme detection system according to the present invention may further include a vacuum pump or the like.

The present invention also provides an enzyme detection method characterized by using the enzyme detection system described above. In the enzyme detection method according to the present invention, the concentration of the enzyme can be analyzed by measuring the fluorescence intensity of the sample discharged from the enzyme detection system.

5, a peptide having a FITC dye attached to Arg-Ser-Trp-Met-Gly-Leu-Pro-Gly which is a peptide capable of specifically degrading to MMP-9 was attached to the nanofiber, The MMP-9 concentration can be measured by measuring the fluorescence intensity of the broken FITC using the property of MMP-9 which breaks the Met-Gly loop of the FITC.

Thus, in order to effectively manipulate the nanofibers having low mechanical strength, a hydrogel is patterned to form a support, an enzyme-specific peptide is immobilized on the exposed nanofiber portion, and then inserted into the microchip, A microchip that can be detected can be produced.

The nanofibers used in the present invention can effectively fix a peptide as an analyte by using a very large surface area and can detect the enzyme with high sensitivity because a large amount of peptides are effectively fixed.

Hereinafter, the present invention will be described in more detail by way of examples. However, the following examples are illustrative of the invention, and the scope of the present invention is not limited by the following examples.

[Example]

1. Formation of Nanofiber Scaffolds by Electrospinning and Oxygen Plasma Treatment

10 g of PS / PSMA was mixed with 40 mL of DMF, and the mixture was left at 80 ° C. for 12 hours to completely dissolve PS / PSMA. The PS / PSMA solution prepared for electrospinning was injected into a 10 mL syringe and spun at a constant rate of 0.5 mL / hr, and a voltage of 8 kV was applied using a high voltage device. Due to the voltage difference, the solution was pulled into a grounded stainless steel floor to form nanofibers. The resulting nanofibers were left at 50 < 0 > C and under vacuum for 24 hours to remove residual solvent. To increase the hydrophilicity of the nanofiber scaffold, the nanofiber scaffold was subjected to oxygen plasma treatment. The oxygen plasma treatment was carried out for 5 minutes at a radio frequency output of 40 W and a pressure of 1 x 10 ^-1 mm Hg.

2. Fabrication of nanofiber sheet using PEG hydrogel

The hydrogel structure was prepared using PEG-DA, and the precursor solution was prepared by mixing 20 μl of HOMPP as a photopolymerization initiator and 1 mL of a PEG-DA aqueous solution. In order to prepare a hydrogel sheet of a desired shape, a nanofiber was sufficiently immersed in a hydrogel precursor solution. A photomask consisting of a light passing portion and a non-passing portion was placed on the scaffold, and a UV light having a wavelength of 365 nm The light was irradiated for 1.2 seconds at an output of 18 W / cm < 2 >. The part of the precursor solution irradiated with UV became crosslinked and became insoluble in water, and the part not irradiated with UV was washed out with water. The portion not irradiated with UV was not formed with a hydrogel, and the sheet was produced in a form in which the nanofibers were outside (Fig. 6).

3. Peptide immobilization in the exposed nanofiber region

For peptide immobilization on the exposed nanofibers, a peptide solution with FITC end fixed at 50 μg / mL was made using HCL buffer and 300 μl of peptide solution was injected into a well-plate Then, one nanofiber sheet was immersed in each well and reacted for 24 hours. After 24 hours of reaction, immersed in HCL buffer for 24 hours in order to remove the peptides in the hydrogel. The thus-formed peptide-immobilized nanofiber sheet was immersed in 4 ° C HCl buffer and taken out when necessary (FIG. 7).

4. Formation of PDMS plate (1st layer, 2nd layer, 4th layer)

Three silicon substrates for patterning with a photosensitizer were reacted at 100 ° C for 15 minutes using a pyran solution (9 mL of sulfuric acid + 3 mL of hydrogen peroxide). The silicon substrate after the reaction was coated with a photosensitizer by spin coating an SU-8 50 photosensitive agent at 1500 rpm. Pre-baking was carried out using a hot plate at 65 DEG C for 10 minutes and at 95 DEG C for 30 minutes. Three pre-baked silicon substrates were covered with three different masks and exposed for about 14 seconds using UV. The exposed substrate was placed on a hot plate and post-baked at 65 ° C for 1 minute and at 95 ° C for 10 minutes. The post-baked substrate was developed using a developer solution to remove all of the photosensitizer in the uncured portions to prepare a patterned substrate. A pre-aqueous solution of PDMS (Sylgard 184A and Sylgard 184B) in a ratio of 10: 1 was made and poured onto the patterned substrate. After removing the bubbles, the cured PDMS plate was prepared by reacting in an oven at 80 ° C. for 5 hours or more.

5. Fabrication of PDMS thin film (3rd layer) into microchip

The silicon substrate washed with the pyranase solution was placed in a suitable container, and 1 mL of 1H, 1H, 2H, 2H-perfluorooctyl trichlorosilane solution was dropped around the substrate. The container was placed in a vacuum oven, and a vacuum of -500 mmHg was applied, and then the surface of the silicon substrate was subjected to silane treatment for about 1 hour. The PDMS pre-aqueous solution mixed in the above ratio was poured onto the surface-treated silicon substrate, and the PDMS was thinly coated by spin coating at 1000 rpm. The coated substrate was reacted in an oven at 80 ° C for at least 5 hours to prepare a cured PDMS thin film.

6. Combination of PDMS plate and PDMS thin film

Holes with 2 cm diameter were drilled vertically through the PDMS plate to be disposed as the second layer, and the PDMS thin film was corona treated for 1 minute each, and then the PDMS plate was bonded. After removing the silicon substrate from the thin-film coated substrate, the PDMS plate to be placed as the fourth layer on the other side of the PDMS thin film was bonded in the same manner and the PDMS plate for the first layer was bonded to the PDMS plate for the second layer To complete the microchip.

7. Insert nanofiber sheet into microchip

The peptide-immobilized nanofiber sheet was inserted through the hole in the upper part of the fabricated PDMS microchip, and covered with a cover glass having a size of 18 mm × 18 mm to block the inflow of the solution through the hole (FIG. 8 ).

8. Measurement of fluorescence intensity by MMP-9 concentration

MMP-9 solution was diluted to make 1, 5, 10, 100, and 1000 ng / mL solutions, respectively. Vacuum the pump to open the valve, inject five different solutions of each concentration into each microchip, close the valve and wait for about 30 minutes. After the reaction was completed, the valve was opened and a low pressure was applied to the outlet to allow the solutions to flow out. The concentration of MMP was analyzed through the fluorescence intensity of the flowing solution, or the fluorescence intensity was measured using a separate fluorescence measurement device of the flowing solution, and the concentration of MMP was analyzed using the fluorescence intensity (FIG. 9).

10: First layer
11: Inlet
12: First Euro
13: First inlet
14: First outlet
20: Second layer
21: First chamber
22: Second intake port
23: 2nd Euro
24: Third Euro
25: Second outlet
30: Third floor
31: Second chamber
32: Third outlet
40: fourth floor
41: Fourth Euro
42:
43: the fifth euro
44: Fourth outlet
50: Nanofiber sheet

Claims

A flat plate-shaped layer having an inlet through which the fluid flows, a first flow path in the form of a groove connected to the inlet and formed on the lower surface of the flat plate in the right horizontal direction, a first suction port for vacuum suction, A first layer;
A layer in the form of a flat plate disposed at the lower part of the first layer and having a large-diameter circular hole shape connected to the first flow path and penetrating the flat plate in the vertical direction, wherein the hole diameter is 50 to 90% A second channel connected to the first suction port and connected to the second suction port and formed in a lower horizontal direction on the lower surface of the flat plate, a second channel connected to the second channel, a lower horizontal direction A second layer having a third flow path in the form of a groove formed therein and a second discharge port connected to the first discharge port;
Layer in the form of a large-diameter circular hole connected to the first chamber and penetrating the flat plate in the vertical direction, wherein the hole diameter is 50 to 90% of the width of the third layer A third chamber having a second chamber and a third outlet connected to the second outlet;
A fourth channel in the form of a groove connected to the second chamber and selectively connected to the third channel and formed on the upper surface of the flat plate in the right horizontal direction, A fifth flow path in the form of a groove, which is located on the upper surface of the flat plate and is horizontally disposed on the right side of the flat plate, A fourth layer having a groove and a fourth outlet connected to the flow path and the third outlet and formed on the upper surface of the plate; And
A nanofiber sheet installed in the first chamber and the second chamber, patterned with a hydrogel and fixed with an enzyme detection peptide,
The nanofibers are formed of polystyrene / poly (styrene-co-maleic anhydride) (PS / PSMA)
The hydrogel is formed of polyethylene glycol-diacrylate (PEG-DA)
The enzyme is a protease,
Wherein the peptide has an amino acid sequence of Arg-Ser-Trp-Met-Gly-Leu-Pro-Gly.

The method according to claim 1,
Wherein the fluid flows into the first chamber and the second chamber through the inlet and the first flow path, and is discharged through the fourth outlet and the first outlet through the fourth flow path, the third flow path and the fifth flow path. .

3. The method of claim 2,
The third layer is brought into close contact with the separating portion to block the flow of the fluid from the fourth flow path to the fifth flow path and the third layer is moved upward in the third flow path by the vacuum suction through the first suction port and the second suction port And the third layer is separated from the separating portion so that the fluid passes from the fourth flow path to the fifth flow path through the third flow path.

The method according to claim 1,
Wherein the microchip is formed of a siloxane-based polymer.

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The method according to claim 1,
Wherein the proteolytic enzyme is a matrix metalloprotease (MMP).

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The method according to claim 1,
A microchip characterized in that a fluorescent dye is attached to the end of the peptide.

A manufacturing method of a microchip according to claim 1,
Forming a nanofiber scaffold;
Patterning a nanofiber scaffold with a hydrogel to form a nanofiber sheet;
Immobilizing the peptide in the exposed region of the nanofiber sheet;
Forming a first layer to a fourth layer according to claim 1; And
And inserting a nanofiber sheet on which a peptide is immobilized in the first chamber and the second chamber.

12. The method of claim 11,
Wherein the nanofiber scaffold is formed through electrospinning.

12. The method of claim 11,
Forming a nanofiber scaffold and then performing an oxygen plasma treatment.

12. The method of claim 11,
Wherein the hydrogel patterning and the first to fourth layer formation are performed using photolithography or soft lithography.

A microchip according to claim 1; And
An enzyme detection system comprising a fluorescence measurement device.

16. The method of claim 15,
An enzyme detection system further comprising a vacuum pump.

A method for detecting an enzyme, characterized by using the enzyme detection system according to claim 15.

18. The method of claim 17,
Wherein the fluorescence intensity of the sample discharged from the enzyme detection system is measured to analyze the concentration of the enzyme.