KR20090083784A - Microfluidic devices incorporating non-spherical hydrogel microparticles for bioassay - Google Patents

Abstract

A microfluidic system is provided to synthesize hydrogel microparticles containing various biomaterials to various shapes, and to fix the hydrogel microparticles to a signal analysis chamber. A microfluidic system comprises one or more microfluidic channels. The microfluidic channel includes a polymerization chamber which synthesizes hydrogel particle by using fluid flowed from the outside; a detection chamber which analyzes signal by using the hydrogel particles and is connected to the hydrogel synthesis chamber; a fluid inlet for supplying the fluid to synthetic chamber; and a fluid outlet for discharging the fluid flown from the analysis chamber to the outside.

Description

Microfluidic devices incorporating non-spherical hydrogel microparticles for bioassay based on non-spherical hydrogel microparticles

The present invention relates to a bioanalytical microfluidic system based on non-spherical hydrogel microparticles, which is synthesized due to the hydrogel synthesis portion and hydrophobic surface treatment for producing non-spherical hydrogel microparticles using photolithography. The present invention relates to a microanalysis system for bioanalysis, which is manufactured to include a signal analysis portion capable of easily separating a hydrogel from a surface of a chip and fixing the hydrogel at a desired position.

Biofluidic microfluidic systems using microfluidics mainly use technology to detect biological and chemical changes before and after the reaction by attaching biomaterials to the inner surface of channels and injecting the sample to be analyzed into them. ( Anal . Chem . 2001, 73, 8-12; Anal Chem . 2002, 74, 379-385). Since then, research has been conducted on the expansion of reaction surface area using microparticles to improve the reaction system that depends only on diffusion along the flow of the fluid ( Anal . Chem . 2003, 75, 3161-3167; J. Am . Chem . Soc . 2002, 124, 13360-13361). In addition, studies have been conducted using magnetic particles or hydrogels as part of performing complex reactions or controlling the reaction rate ( Anal . Chem . 2002, 74, 3372-3377; Lap). Chip , 2006, 6, 555-560).

In addition, the conventional optical signal identification method used in bio analysis mainly used the color difference of fluorescence, but recently, studies using morphological characteristics of non-spherical microparticles have been conducted ( Chem . Mater . 2004, 16, 5574-). 5580; Nature Materials . 2006, 5, 365-369; Science, 2007, 315, 1393-1396; Langmuir , 2007, 23, 4669-4674; Lap Chip , 2007, 7, 818-828).

Hydrogel is a material having a water-insoluble hydrophilic three-dimensional polymer network structure and has the characteristic of swelling by containing a large amount of water in the aqueous solution inside. Hydrogels are mainly manufactured from synthetic polymers such as polyethylene glycol, polyvinyl alcohol, polyacrylamide, and natural polymers such as alginate, agarose, chitosan, and the like, and may also be prepared using self-assembled peptides or proteins. . In addition, the hydrogel shows various functions according to the types of functional groups present in the polymer network structure. The introduction of such functional groups into the hydrogel can be easily performed by changing the type of monomer used in the hydrogel synthesis. have. Certain hydrogels react significantly and rapidly to external chemical and physical stimuli such as temperature, pH, and chemicals, depending on the type of functional group being introduced. These hydrogels are called intelligent polymer hydrogels or smart polymer hydrogels. do. Due to its excellent biocompatibility, environmental sensitivity, and specific molecular cognition, intelligent polymer hydrogels have long been used in biomedical fields such as artificial organs, drug delivery systems (DDS), biosensors, and tissue engineering. .

On the other hand, in the case of a biological sample, especially a protein, a qualitative or quantitative analysis is performed by using a biochip form using a microarray. A common problem with these plate arrays is that the protein is bound to a solid two-dimensional solid surface, thereby altering the protein structure. It is easy to lose weight and dry quickly in the air. Therefore, this problem can be solved when a protein chip is prepared by immobilizing a protein such as an enzyme inside a hydrogel microarray which may contain a large amount of water. Currently, most hydrogel microarrays are manufactured by combining the conventional hydrogel synthesis techniques such as photolithography, micromolding, microspotting, and the like. The hydrogel structure constituting the microarray may be manufactured in nano or micrometer size having a three-dimensional structure, and recently, in the form of a high density integrated microarray by mounting a bioactive material such as a protein inside the hydrogel structure. Efforts are being made to manufacture biochips.

An object of the present invention is a bioanalytical device using the morphological characteristics of non-spherical microparticles, the synthesis of the hydrogel microparticles containing a biomaterial in an integrated system and a microchannel consisting of a signal analysis at a fixed position It is to provide a fluid system and a method of manufacturing the same.

It is another object of the present invention to provide a method for producing non-spherical hydrogel microparticles of various forms including a biomaterial.

Still another object of the present invention is to provide a method for analyzing biomaterials using the microfluidic system that combines the advantages of a suspension array and a flat plate array.

In order to achieve the above object, the present invention

Synthesis chamber for synthesizing hydrogel particles using a fluid introduced from the outside; And

It is connected to the hydrogel synthesis chamber, and provides a microfluidic system comprising at least one microfluidic channel including an analysis chamber for analyzing a signal using the introduced hydrogel particles.

The invention also

Forming a pattern corresponding to the chamber and the microchannel on the substrate;

Forming a high molecular compound layer or a glass layer on the entire structure of the substrate including the pattern, curing the separation, and separating the high molecular compound layer or the glass layer to form an upper plate formed of the high molecular compound layer or the glass layer;

Forming a lower plate by forming a polymer compound layer or a glass layer on the substrate;

Activating corresponding surfaces of the upper plate and the lower plate; And

It provides a method of manufacturing a microfluidic system of the present invention comprising the step of joining the active corresponding surface of the upper plate and the lower plate to form one or more microfluidic channels.

The invention also

Introducing a water-soluble polymer precursor solution into the synthesis chamber; And

It provides a method for producing hydrogel microparticles using the microfluidic system of the present invention, characterized in that it comprises the step of producing hydrogel microparticles by photolithography in the synthesis chamber.

The invention also

Synthesizing hydrogel microparticles containing a probe capable of reacting with a target material in the sample in a hydrogel synthesis chamber;

Fixing the hydrogel microparticles into a signal analysis chamber;

Reacting the target material in the sample with a probe contained in the hydrogel microparticles fixed in the signal analysis chamber; And

It provides a method for analyzing a sample using the microfluidic system of the present invention, characterized in that it comprises the step of analyzing the reaction result.

The microfluidic system of the present invention is composed of a channel including a hydrogel synthesis chamber and a signal analysis chamber integrally, and thus the amount and morphological characteristics of the hydrogel microparticles are manufactured by using photolithography in the synthesis chamber. Can be easily adjusted.

In addition, the microfluidic system of the present invention can synthesize hydrogel microparticles containing various biomaterials in various shapes, and can simultaneously fix hydrogel microparticles of various shapes in a signal analysis chamber to analyze biomaterials. It has a quick and simple effect.

In addition, the microfluidic system of the present invention is easy to separate the synthesized hydrogel from the channel substrate surface due to the hydrophobic surface treatment and to fix it in the desired position.

Hereinafter, the configuration of the present invention will be described in detail.

The present invention

Synthesis chamber for synthesizing hydrogel particles using a fluid introduced from the outside; And

It is connected to the hydrogel synthesis chamber, and relates to a microfluidic system comprising at least one microfluidic channel including an analysis chamber for analyzing a signal using the introduced hydrogel particles.

The microfluidic system

A fluid inlet port for supplying fluid to the synthesis chamber; And

The apparatus may further include a fluid discharge port for discharging the fluid introduced from the fluid inflow port to the outside from the analysis chamber.

Each chamber of the microfluidic channel has a width and a height of 10 μm to 900 μm because the size of the microfluidic channel is too small, which causes difficulty in producing the hydrogel particles, and the size of the microfluidic channel is too low. More preferably, 10 micrometers-500 micrometers are good. It is preferable that the length is 10 micrometers-900 mm.

The signal analysis chamber includes a filter mounted therein to fix the hydrogel particles.

The filter is preferably maintained at 10% to 90% of the hydrogel microparticle size in order to efficiently fix the hydrogel microparticles. If the spacing of the filter is more than 90% of the size of the hydrogel microparticles, due to the elasticity of the gel itself, the synthesized hydrogel microparticles may be lost through the filters. This may work so large that fluid flow may not be smooth.

The fluid inlet port and a hydrogel synthesis chamber; Hydrogel synthesis chamber and signal analysis chamber; And the signal analysis chamber and the fluid outlet port are each connected by a fine flow path.

Each of the fine flow paths is difficult to operate when the size is too small, and when the size is too large, the consumption of the sample increases and the analysis time increases. 10 micrometers-500 micrometers are preferable. It is preferable that the length is 10 micrometers-900 mm.

In addition, the microfluidic channel of the present invention is formed by an upper plate and a lower plate made of a polymer compound or a glass material. At least one of the upper plate and the lower plate preferably has a rate, more specifically, a UV transmittance of 80% or more.

The polymer compound is not particularly limited, but may be a polyalkylsiloxane, poly (meth) acrylate, polyalkyl (meth) acrylates, poly It is preferable to use carbonates, polycyclic olefins, polyimides, or polyurethanes alone or in combination of two or more.

The invention also

Forming a pattern corresponding to the chamber and the microchannel on the substrate;

Forming a high molecular compound layer or a glass layer on the entire structure of the substrate including the pattern, curing the separation, and separating the high molecular compound layer or the glass layer to form an upper plate formed of the high molecular compound layer or the glass layer;

Forming a lower plate by forming a polymer compound layer or a glass layer on the substrate;

Activating corresponding surfaces of the upper plate and the lower plate; And

A method of manufacturing a microfluidic system of the present invention comprising the step of forming at least one microfluidic channel by bonding an activated corresponding surface of an upper plate and a lower plate.

The method of manufacturing the pattern is not particularly limited, but a photoresist for intaglio or embossing, preferably SU-8, is spin-coated to a desired height on a substrate, exposed to ultraviolet light through a photomask, and the photoresist of the exposed area. It is preferable to prepare a mold by cross-polymerizing and washing away the unreacted photoresist with a developer.

Preferably, the substrate is either a silicon wafer or a glass slide.

The polymer compound layer or glass layer has a hydrophobic surface so that hydrogel particles are well separated from the surface during hydrogel synthesis. That is, thermodynamically hydrophilic hydrogel particles are due to the interfacial energy increases when it meets the hydrophobic surface, it is easy to separate the two surfaces.

Corresponding surfaces of the upper plate and the lower plate are preferably activated by exposure to oxygen plasma for 1 to 5 minutes.

In addition, since the number of the microfluidic channels is determined when the mold is manufactured, the microfluidic system of the present invention preferably includes one or more channels. The spacing between the microfluidic channels is not particularly limited.

The invention also

Introducing a water-soluble polymer precursor solution into the synthesis chamber; And

It relates to a method for producing hydrogel microparticles using the microfluidic system of the present invention, comprising the step of preparing hydrogel microparticles by photolithography in a synthesis chamber.

The water-soluble polymer for the hydrogel synthesis is not particularly limited, gelatin, alginate, carrageenan, chitosan, polyhydroxyalkyl (meth) acrylate, hydroxyalkyl cellulose, alkyl cellulose, silicone rubber, agar, carboxyvinyl copolymer , Polyvinyl alcohol, polyvinylpyrrolidone, polyethylene oxide, polyethylene glycol, polyacrylamide, polydioxolane, poly (meth) acrylic acid, polyacrylic acetate, polyvinyl chloride or maleic anhydride / vinyl ether copolymer, or the like Two or more kinds can be used.

The water-soluble polymer is preferably included in 20 to 80 parts by weight based on 100 parts by weight of the hydrogel precursor solution. When the content is in the above range, the water content of the hydrogel of the present invention may be 50 to 97% by weight, so that the probe capable of reacting with the target material in the sample may be fixed while maintaining the structure and activity inside the hydrogel. Can be.

Further, the non-spherical hydrogel microparticles are not particularly limited as long as they are polymerized by conventional photolithography, but are preferably prepared by radical polymerization of a polymer using a photomask under ultraviolet exposure.

The ultraviolet exposure is preferably carried out at 50 to 200 W for 0.5 to 30 seconds. This is because crosslinking between polymers may not occur sufficiently when the UV exposure is not sufficient. On the contrary, when the UV exposure is excessive, the shape of the mask may not be accurately reflected on the appearance of the hydrogel by radical diffusion.

In addition, the hydrogel microparticles can control the amount of light and the shape of the exposed surface when using a photomask under ultraviolet light exposure. The form of the photomask is not particularly limited because any form except for a sphere can be used.

In addition, the physical / chemical properties of the hydrogel microparticles can be adjusted while changing the molecular weight of the water-soluble polymer. For example, a hydrogel made of PEG 575 has a water content of about 60% while a hydrogel made of PEG 20000 has a water content of 95%. Due to this high water content, various biomaterials including enzymes can be immobilized while maintaining structure and activity inside the hydrogel.

Therefore, it is preferable that the molecular weight of the said water-soluble polymer is 500-30000.

In addition, the microfluidic system of the present invention can be prepared by moving the prepared hydrogel along the flow path to be fixed to the filter in the signal analysis chamber and injecting a new precursor solution to produce a new hydrogel. This process can be repeated several times.

In addition, the precursor solution may physically, chemically or physically bind to the hydrogel microparticles, and may further include a probe (or biomaterial) capable of reacting with a target material in a sample.

The probe is not particularly limited as long as it is a substance capable of reacting (including physical binding) with any target substance in the sample to be analyzed, and for example, proteins, lipids, polysaccharides, etc., such as nucleic acids, amino acids, peptides, or polypeptides. Carbohydrates, nucleotides, polynucleotides, or hybrid molecules thereof may be used alone or in combination of two or more thereof.

The protein includes a residue capable of binding to a fluorescent marker, for example, lysine, cysteine, and the like, and is not particularly limited as long as the protein has a sufficient molecular weight to be fixed to a filter in the signal analysis chamber of the present invention. Do not. Preferably, enzymes such as glucose oxidase, urease, or alkaline phosphatase, or antibodies thereof, or the like may be used alone or in combination.

The lipid is not particularly limited, but lysophosphatidylcholine, phosphatidylcholine, sphingomyelin, cholesterol, or a mixture thereof is preferable.

The carbohydrate is not particularly limited, but cellulose, glucose starch, alginic acid, chitosan, hyaluronan, dextran, aminodextran, heparin, or lectin is preferable.

Meanwhile, the method of preparing the hydrogel microparticles to which the probe is bound is not particularly limited, but the hydrogel including the enzyme as a probe is photo-crosslinked through a photomask with a precursor solution in which an enzyme solution is added to PEG-DA and a photoinitiator solution. It is preferable to make it. At this time, the enzyme is physically fixed inside the hydrogel as crosslinking occurs, which is called physical encapsulation. In addition, physical encapsulation can be chemically immobilized using a bifunctional linker such as acryloyl-PEG-Nhydroxysuccinimide (acryloyl-PEG-NHS). In this case, a protein such as an enzyme binds to one NHS group, and the other acrylic group participates in radical polymerization to chemically bind to PEG hydrogel so that the enzyme is chemically bound to the hydrogel chain and immobilized inside the hydrogel. .

In addition, the probe is preferably included in an amount of 0.001 to 1 parts by weight based on 100 parts by weight of the precursor solution for hydrogel microparticle synthesis. When the content is within the above range, the probe may be fixed while maintaining its structure and activity in the hydrogel of the present invention.

In addition, the transparent properties of the hydrogels enable a variety of optical analytical methods, in which additional fluorescent markers can be added to the hydrogels coupled with the probes to detect biochemical reactions occurring within the hydrogels.

The fluorescent marker is not particularly limited, but SNAFL, SNARF, SNAFL, calcium green, Amplex Red, Texas Red, BIODIPY, Oregon Green, Alexa Fluor, Cascade Blue, Dapoxyl, coumarin, rhodamine, N-methyl-4-hydrazine- Organic dye fluorophores including 7-nitrobenzofurazane, monoethyl ethylenediamine, monoethyl cadaverine, monoyl hydrazine, or a mixture thereof may be used alone or in combination of two or more thereof.

In particular, SNAFL is a pH-sensitive fluorescent marker, which has dual absorption and dual excitation properties because it has different absorption and emission peaks under acidic and basic conditions. In other words, at pH 8 and below, optimal absorption and emission spectra are shown at 510 nm and 545 nm, but at pH 8 and above, optimal absorption and emission are shifted to 545 nm and 645 nm, respectively. In this case, when the ratio of two emission intensities (intensity at 545 nm / intensity at 645 nm) is measured according to pH, the emission ratio changes linearly with pH. In addition, when reacting with a buffer solution of pH 5, SNAFL shows a strong light green and weak red color, but at pH 12, the emission peak shifts to a strong red and light green color.

In addition, the enzyme reacts with the substrate to cause a pH change, such as glucose oxidase, urease, or alkaline phosphatase. Glucose oxidase and alkaline phosphatase react with glucose and p-nitrophenylphosphate (pNPP) to produce gluconic acid and phosphoric acid to lower the surrounding pH, and urease to react with NH ³ By generating ⁺ to increase the surrounding pH, the resulting change in the ratio of the emission intensity of the pH-sensitive fluorescent substance SNAFL can be measured to detect the target material.

In addition, according to one embodiment of the present invention, it is possible to detect the glucose concentration by using a hydrogel prepared inside the microfluidic system using Amplex Red. In the presence of glucose oxidase, Amplex Red reacts with H ₂ O ₂ in 1: 1 to produce a red fluorescent oxide called resorufin. When glucose and glucose oxidase react, H ₂ O ₂ is produced. Therefore, the higher the glucose concentration, the more H ₂ O ₂ is produced and the stronger the release intensity of resorufin. Glucose can be measured.

The fluorescent marker is also conjugated to a probe in a hydrogel or generated inside the gel by diffusion of the substrate. E.g,

Nucleic Acids: Nucleic acid binding dyes change fluorescent properties in the presence of DNA or RNA.

Proteins: Especially for enzymes, the fluorescence signal can be labeled with fluorescent dyes and fluorescent quenching molecules generated in the presence of certain enzymes such as proteases.

Lipids: Lipophilic dyes can change the fluorescence properties in the presence of cell membranes or other lipid rich analytes.

Polysaccharides: Certain lectins, such as ConA, bind glucose, and suitably labeled lectins can be prepared as probes for glucose.

In addition, the fluorescent marker is preferably included in 0.001 to 1 parts by weight based on 100 parts by weight of the precursor solution for the synthesis of hydrogel microparticles. If the content is within the above range, it is suitable for measuring the change in the biochemical environment of the biomaterial inside the hydrogel of the present invention.

In addition, the microfluidic system of the present invention can fix various types of hydrogels containing various types of biomaterials simultaneously to the signal analysis chamber by using photomasks having different shapes.

The invention also

i) synthesizing hydrogel microparticles containing a probe capable of reacting with a target material in the sample in a hydrogel synthesis chamber;

ii) fixing the hydrogel microparticles in a signal analysis chamber;

iii) reacting the target material in the sample with a probe contained in the hydrogel microparticles fixed in the signal analysis chamber; And

iv) a method for analyzing a sample using the microfluidic system of the present invention, characterized in that it comprises the step of analyzing the reaction result.

Step i) is a step of synthesizing hydrogel microparticles containing a probe that can react with the target material in the sample in the hydrogel synthesis chamber.

Hydrogel microparticles containing the probe are preferably prepared from a precursor solution comprising a water-soluble polymer for hydrogel synthesis, a probe and a fluorescent marker for measuring changes in the biochemical environment of the probe. In addition, the polymerization initiator may further include a polymerization initiator for initiating the polymerization of the water-soluble polymer.

The precursor solution preferably contains 20 to 80 parts by weight of the water-soluble polymer, 0.001 to 1 parts by weight of the probe, and 0.001 to 1 parts by weight of the fluorescent marker. When the content is in the above range, the water content of the hydrogel of the present invention may be 50 to 97% so that the probe may be fixed while maintaining its structure and activity inside the hydrogel.

The hydrogel microparticles are preferably prepared by a radical polymerization method using a photomask under ultraviolet exposure.

In addition, the hydrogel microparticles can control the amount of light and the shape of the exposed surface when using a photomask under ultraviolet light exposure.

Therefore, according to the microfluidic system of the present invention, after fixing the hydrogel microparticles prepared through the precursor solution to the signal analysis chamber, the process of synthesizing the new hydrogel microparticles through the new precursor solution may be repeated one to several times. Various types of photomasks may be used to synthesize hydrogel microparticles of various shapes containing various kinds of biomaterials, and may be simultaneously fixed to the signal analysis chamber. Therefore, the speed of the signal analysis of the biological material is remarkably fast compared to the conventional analysis device.

According to an embodiment of the present invention, a polymer including glucose oxidase, 10-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), and polyethylene glycol (PEG) to synthesize hydrogel microparticles containing enzymes as a biomaterial And a precursor solution composed of a photoinitiator. The precursor solution is injected into the liquid inlet of the microfluidic system of the present invention and placed in a hydrogel synthesis chamber. When the ultraviolet light is irradiated through the photomask, the initiator generates free radicals, and the generated radicals attack the acrylate of the polymer to start the polymerization reaction. After completion of the reaction, hydrogel particles having a size of 10 to 500 µm containing 1 mg / g of enzyme are produced.

In step ii), the hydrogel microparticles are moved through the microchannels and fixed to the filters in the signal analysis chamber.

Hydrogel microparticles synthesized in the hydrogel synthesis chamber flow distilled water from the inlet and fixed to the filter in the signal analysis chamber along the flow path.

In step iii), the target material in the sample is reacted with a probe contained in the hydrogel microparticles fixed in the signal analysis chamber.

According to one embodiment of the present invention, an enzyme, for example, glucose oxidase is used as a probe, and glucose is added as a target to induce a reaction between substrate and enzyme.

Step iv) is to analyze the reaction result. The reaction results include optical characteristics such as color development or fluorescence intensity of the fluorescent marker, electrochemical characteristics, or physical characteristics, etc., depending on the probe and target material used, and the like, and are not particularly limited. In the case of using the fluorescent marker according to an embodiment of the present invention using a filter corresponding to the excitation wavelength 571nm, emission wavelength 585nm using a 100W mercury lamp as a light source after adjusting the wavelength of light to the light exposure time of the fluorescent marker to 1 second You can use the average of the ten measurements that are fixed.

According to one embodiment of the invention, glucose generates H ₂ O ₂ upon oxidation. Amplex Red, a fluorescent marker included in the synthesis of hydrogel microparticles, has resorufins produced by reaction with H ₂ O ₂ and have absorption and emission wavelengths at 563 nm and 585 nm, respectively. Therefore, the fluorescence spectrum is investigated to measure the activity of the biomaterial, ie, glucose oxidase.

Hereinafter, preferred examples are provided to help understanding of the present invention, but the following examples are merely to illustrate the present invention, and the scope of the present invention is not limited to the following examples.

Example 1 Preparation of Microfluidic System of the Present Invention

(Production of pattern)

The photoresist SU-8 for the intaglio pattern was poured on a silicon wafer or glass slide on a 1 mL basis per 3 cm ² , and spin-coated for 10 seconds at 1,000 rpm and 30 seconds at 3,000 rpm.

The coated photoresist was cured by heating at 65 ° C. for 10 minutes and 95 ° C. for 30 minutes, and then exposed to 100W ultraviolet rays for 7 seconds through a photomask as shown in FIG. 1.

After UV exposure for 1 minute at 65 ℃, 10 minutes at 95 ℃ to cross-polymerize the photoresist of the exposed area and then to wash the unreacted photoresist with a developer to prepare a pattern.

(Microfluidic channel manufacturing)

3cm ^{2 of a} mixed solution of poly (dimethylsiloxane, PDMS) and a curing agent in a weight ratio of 10: 1 on the fabricated pattern Poured based on 1mL per sugar and reacted at 60 ℃ for 2 hours.

Next, 3 cm ² of the mixed solution for the pretreatment of the glass or silicon wafer constituting the bottom plate of the system. Poured based on 1mL per sugar and spin-coated for 30 seconds at 3,000rpm and then heated at the same 60 ℃ for 2 hours.

Next, after 2 hours, the PDMS cured in the step was removed from the mold and then exposed to oxygen plasma for 2 minutes with the lower plate prepared in the step to activate the joint surface of the upper plate and the lower plate.

Considering the diameter of the tube to inject fluid, the entrance was made and the activated both sides were joined to complete the microfluidic channel system including one or more microfluidic channels.

Example 2 Synthesis of Aspheric Hydrogel Microparticles

In order to synthesize non-spherical hydrogel microparticles in the hydrogel synthesis chamber in the microfluidic system prepared in Example 1, a polymer containing 0.005 parts by weight of glucose oxidase, 50 parts by weight of polyethylene glycol (PEG) based on 100 parts by weight of the precursor solution And 0.1 part by weight of a photoinitiator to prepare a precursor solution. The precursor solution was injected into the channel and passed through a photomask to the hydrogel synthesis region as shown in FIG.

After completion of the reaction, hydrogel particles having a size of 400 μm containing 1 mg / g of enzyme were produced.

Experimental Example 1 Analysis of Biomaterials

Distilled water is flowed to move the hydrogel prepared in Example 2 to a signal analysis chamber, and when the particles are fixed by a filter, a bio-analysis is performed by injecting reactants, that is, 0.5 to 11 mM glucose solution by concentration. It was. In this step, after adjusting the wavelength of light using a filter corresponding to excitation wavelength 571 nm and emission wavelength 585 nm using a 100 W mercury lamp as a light source, the light exposure time of the fluorescent marker is fixed to 1 second for 10 times. The measured values were averaged.

As shown in Figure 5, as the amount of H ₂ O ₂ was increased it was confirmed that the fluorescence by Amplex Red counted.

In addition, the change in absorbance intensity of resorufin in the particles according to the concentration change of various H ₂ O ₂ was observed and shown in FIG. 6.

As shown in FIG. 6, the strength increased as the concentration of glucose increased.

From the above results, it can be seen that the microfluidic system of the present invention is effective for biological material analysis.

1 shows a schematic diagram of a microfluidic channel of the present invention.

2 is a schematic diagram schematically showing the synthesis of non-spherical hydrogel microparticles using the microfluidic system of the present invention.

3 briefly illustrates a process of manufacturing the microfluidic channel of the present invention.

Figure 4 shows the microfluidic channel pattern and the hydrogel microparticles of the present invention.

Figure 5 shows the results of measuring the activity of the enzyme through the change in fluorescence intensity according to the substrate concentration using Amplex Red.

Figure 6 shows the results of measuring the activity of the enzyme using a change in the ratio of the emission intensity (Emission intensity) of Resorufin.

Claims (19)

Synthesis chamber for synthesizing hydrogel particles using a fluid introduced from the outside; And And at least one microfluidic channel connected to the hydrogel synthesis chamber and including an analysis chamber for analyzing a signal using the introduced hydrogel particles. The method of claim 1, A fluid inlet port for supplying fluid to the synthesis chamber; And And a fluid discharge port for discharging the fluid introduced from the fluid inlet port to the outside from the analysis chamber. The method of claim 1, Each chamber has a width and a height of 10 μm to 900 μm, and a length of 10 μm to 900 mm. The method of claim 1, The signal analysis chamber is mounted therein, the microfluidic system comprising a filter capable of fixing the hydrogel particles. The method of claim 2, Fluid inlet port and hydrogel synthesis chamber; Hydrogel synthesis chamber and signal analysis chamber; And the signal analysis chamber and the fluid outlet port are connected by microchannels, respectively. The method of claim 5, Each microchannel has a width and a height of 10 μm to 900 μm and a length of 10 μm to 900 mm. The method of claim 1, Microfluidic channel is a microfluidic system, characterized in that formed by the upper plate and the lower plate made of a polymeric compound or glass material. The method of claim 7, wherein Polymer compounds include poly (alkylsiloxane), poly (meth) acrylate, polyalkyl (meth) acrylates, polycarbonates, Microfluidic system, characterized in that at least one member selected from the group consisting of polycyclic olefins, polyimides (polyimides) and polyurethanes (polyurethanes). The method of claim 7, wherein At least one of the upper plate and the lower plate has a light (UV) transmittance of 80% or more microfluidic system, characterized in that. In the method of manufacturing a microfluidic system according to any one of claims 1 to 9, Forming a pattern corresponding to the chamber and the microchannel on the substrate; Forming a high molecular compound layer or a glass layer on the entire structure of the substrate including the pattern and curing, and then separating the high molecular compound layer or the glass layer to form an upper plate formed of the high molecular compound layer or the glass layer; Forming a lower plate by forming a polymer compound layer or a glass layer on the substrate; Activating corresponding surfaces of the upper plate and the lower plate; And A method of manufacturing a microfluidic system comprising the step of joining an activated corresponding surface of an upper plate and a lower plate to form one or more microfluidic channels. The method of claim 10, The substrate is a method of manufacturing a microfluidic system, characterized in that any one of a silicon wafer or a glass slide. The method of claim 10, The corresponding surface of the upper plate or the lower plate is activated by exposure to oxygen plasma. In the method for producing hydrogel microparticles using the microfluidic system according to any one of claims 1 to 9, Introducing a water-soluble polymer precursor solution into the synthesis chamber; And A method of producing hydrogel microparticles, comprising the step of preparing hydrogel microparticles by photolithography in a synthesis chamber. The method of claim 13, The water-soluble polymer is gelatin, alginate, carrageenan, chitosan, polyhydroxyalkyl (meth) acrylate, hydroxyalkyl cellulose, alkyl cellulose, silicone rubber, agar, carboxyvinyl copolymer, polyvinyl alcohol, polyvinylpyrrolidone, Hydrogel fine, characterized in that it is at least one selected from the group consisting of polyethylene oxide, polyethylene glycol, polyacrylamide, polydioxolane, poly (meth) acrylic acid, polyacrylacetate, polyvinylchloride and maleic anhydride / vinylether copolymer Method for Producing Particles. The method of claim 13, The precursor solution further comprises a probe capable of reacting with the target material in the sample. The method of claim 15, A method for producing hydrogel microparticles, characterized in that the probe is at least one selected from the group consisting of nucleic acids, proteins, lipids, carbohydrates, nucleotides, polynucleotides and hybrid molecules thereof. The method of claim 13, Method for producing a hydrogel microparticles, characterized in that the precursor solution further comprises a fluorescent marker. The method of claim 17, Fluorescent markers are SNAFL, SNARF, SNAFL, Calcium Green, Amplex Red, Texas Red, BIODIPY, Oregon Green, Alexa Fluor, Cascade Blue, Dapoxyl, Coumarin, Rhodamine, N-methyl-4-hydrazine-7-nitrobenzofurazane The method for producing hydrogel microparticles, characterized in that at least one selected from the group consisting of an organic dye fluorophore, including single ethylene diamine, single thread cadaverine, single hydrazine and mixtures thereof. In the analysis method of the sample using the microfluidic system according to any one of claims 1 to 9, Synthesizing hydrogel microparticles containing a probe capable of reacting with a target material in the sample in a hydrogel synthesis chamber; Fixing the hydrogel microparticles into a signal analysis chamber; Reacting the target material in the sample with a probe contained in the hydrogel microparticles fixed in the signal analysis chamber; And Analysis method of a sample using a microfluidic system comprising the step of analyzing the reaction result.

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