KR20070031759A - Method for Fabricating a Biochip Using Polymer?gel - Google Patents

Abstract

The present invention relates to a method for manufacturing a biochip using a polymer gel, and more particularly, to prepare a bead suspension by fixing a biomolecule to the bead, and adding a mixture of a monomer and a crosslinking agent capable of forming a polymer gel to the bead suspension. And then mix, spotting on the substrate, and then polymerizing and fixing the spotted spot on the substrate.

The biochip manufactured by the manufacturing method of the present invention encapsulates the beads fixed with the biomolecule inside the porous structure of the polymer gel, thereby creating a bio-friendly environment and forming pores having a micro-level size, thereby facilitating mass transfer of the biomolecule. can do.

Polymer gel, biochip, biomolecule

Description

Manufacturing method of biochip using polymer gel {Method for Fabricating a Biochip Using Polymer-gel}

1 is a schematic diagram showing a process of forming a spot made of a polymer gel on a substrate by gelling a monomer mixture including beads in which a biomolecule is immobilized in the method of manufacturing a biochip according to the present invention.

Figure 2a is a photograph of the observation of the polymer gel spot fixed on the substrate with a scanning electron microscope, Figure 2b is a micrograph observing that the bead fixed to the probe is supported on the polymer gel having a micropore.

Figure 3 is a photograph observing each spot having a monomer content of 1 to 20% by volume.

4 is a photograph observing the size of the spot formed on the substrate according to the volume of the droplet to be spotted.

FIG. 5A is a graph showing specific peaks (N1s) of amine groups detected by an Electron Spectroscopy for Chemical Analysis (ESCA) analysis of an amine-modified polymethylmethacrylate substrate, and FIG. 5B is a fluorescence analysis of an amine-modified substrate surface amine group And surface roughness values measured by atomic force microscopy.

Figure 6a is a graph comparing the fixing force according to the substrate material of the spot produced according to the method of the present invention, Figure 6b is a photograph comparing the fixing force according to the substrate material.

7 is a graph showing the results of measuring the bead loss rate generated during the washing process for the spot formed by mixing the additive in the method of manufacturing a biochip according to the present invention.

Figure 8a is a photograph of the pore channel structure of the polymer gel spot by electron microscope, Figure 8b is a photograph of the micropore structure of the polymer gel spot by electron microscope.

Figure 9a is a graph quantifying the self-fluorescence of the spot in which the protein-coated beads are fixed on the chip substrate by forming a polymer gel, Figure 9b is a graph quantifying the degree of nonspecific binding of the protein.

FIG. 10A is a fluorescence scanner picture of detecting various concentrations of SAH (S-adenosyl-L-homocysteine) using a competitive immunodetection method using a biochip manufactured according to the method of the present invention, and FIG. 10B is a method of the present invention. Competitive immunodetection against the target material SAH was performed on the manufactured biochip and MaxiSorp chip, and the results are compared with each other.

The present invention relates to a method for manufacturing a biochip using a polymer gel, and more particularly, to prepare a bead suspension by fixing a biomolecule to the bead, and adding a mixture of a monomer and a crosslinking agent capable of forming a polymer gel to the bead suspension. And then mix, spotting on the substrate, and then polymerizing and fixing the spotted spot on the substrate.

Biochip is immobilized various types of biological probes on the surface of solid support of unit area.By using biochip, it is possible to diagnose disease, high throughput screening (HTS) and enzyme activity measurement even with a small amount of sample. Experiments can be easily performed.

The technique of fixing biomolecules to a substrate has been developed for a long time, and a method of directly fixing biomolecules onto a substrate on which a self-assembled monolayer has been traditionally applied has been applied (Korean Patent Publication 2003-0038932; Korean Patent Published 2005-0023753). However, the two-dimensional fixation method as described above is difficult to fix high-density biomolecules in a small area, and it is difficult to preserve the bioactive function of the bound biomolecules.

In order to solve the above problems of the two-dimensional fixing method, a three-dimensional immobilization technology using a polymer matrix or a porous gel has been developed (Korean Patent Publication 2003-0095645; Lofas, SJ, Colloid Interface Sci . 65: 423, 1993; Haab, BB, Genome Biology 2: 0004.1-0004.13, 2001). For example, Xantec's coating slide used a three-dimensional polymer matrix composed of biocompatible polymers such as glucose, polylysine, chitosan, dextran, polyallylamine, and polyvinyl alcohol on a glass substrate as a fixed screen. The Hydrogel ^TM slide of Packard Biosciences, by placing a porous polyacrylamide gel on a silane-treated glass substrate, widens the surface area to which the biomolecules are fixed and improves steric hindrance of the biomolecules.

On the other hand, as a method for increasing the fixation rate of the biomolecule to the porous gel, by mixing the biomolecule having a reactive derivative from the gelation process to fix the biomolecule inside the polymer or inorganic gel to improve the binding force of the biomolecule, Methods to improve loss of bioactivity by maintaining conformation have been developed (US Patent 2004/0241713; US Patent 5,034,428; US Patent 5,482,996; Yakovleva, J., Biosens . Bioelectron ., 19:21, 2003; Gill, I., Trends in Biotechnology 18: 282, 2000).

However, the above methods still have technical limitations. The polymer matrix method is limited to fixation of biomolecules around the surface of the matrix, and also, when covalently bound to increase the anchoring force of biomolecules, There are significant limitations in maintaining the bioactivity of other proteins.

On the other hand, the three-dimensional porous gel structure is very effective in maintaining the bioactivity of the biomolecule, but in general, the pore size is within several tens of nanometers, it is troublesome to use special mixing equipment for the analysis of the experimental results In addition, there is a disadvantage that sufficient time is required for mass transfer of biomolecules such as proteins or DNA into the gel. In addition, such disadvantages are more limited when the target biomolecules are introduced into the microfluidic channel such as lab-on-a-chip to proceed with an immune response under limited conditions.

In order to solve the above problems, a new three-dimensional immobilization technology has been developed for immobilizing bead-borne biomolecules ranging from tens of nanometers to several microns (Sato, K., Adv Drug Deliv Rev. , 55). : 379, 2003; Andersoon, H., Electrophoresis , 22: 249, 2001; Choi, JW, Biomed . Microdevices , 3: 191, 2001). The above method is to fix a biomolecule of a desired purpose to a solid bead support, and then to introduce it into the micro-channel of the lab-on-a-chip, the large surface area of the three-dimensional bead can be utilized as a fixed screen fixed amount of biomolecule It is a method that can improve the processability of the chip by using a solid bead that can improve the, and easy to handle.

However, in order to introduce or fix the beads on a chip, a method of confining beads in a microchannel using a physical barrier, a method of fixing using a magnetic field, and a method using an ultrasonic wave or a laser tweezer are typically used. However, the methods described above are limited in the selection of beads, complicated processing process for manufacturing chips, and high in noise during optical measurement, and require a separate device inside or outside the chip. is not economical (Sato, K., Adv Drug Deliv Rev, 55: 379, 2003; Andersoon, H., Electrophoresis, 22:. 249, 2001; Choi, JW, Biomed Microdevices, 3:. 191, 2001; Meng, A., Transducers, Sendai , Japan, 876, 1999; Dorre, K., Bioimaging , 5: 139, 1997).

On the other hand, the present applicant filed a patent for a method of manufacturing a biochip, characterized in that the bead fixed to the surface of the substrate using a pressure-sensitive adhesive as a probe or a probe is fixed as part of the method for introducing the beads on the chip (Korean patent Application No. 10-2004-104944). According to the above patent, it is possible to fix the bead to which the probe is fixed to the substrate by means of an adhesive without a physical barrier or an electric field, but this still hass to add a separate adhesive to fix the beads to the substrate. do.

In order to solve the problems of the present invention, the applicant has the characteristics as a pressure-sensitive adhesive to the surface of the support and the chip substrate fixing the biomolecules at the same time to be directly fixed on the biochip without a separate device and treatment process and using the same He developed a biochip and filed a patent application (Korean Patent Application No. 10-2005-0086284). However, the present applicant's invention relates to economical and convenient adhesive beads attached to a substrate, and does not suggest a method for introducing a conventional bead into or out of a chip other than the adhesive beads.

Therefore, in the art, the beads fixed with biomolecules can be directly immobilized on the biochips through a simple process without a separate device and processing process inside or outside the chip, and biochips suitable for maintaining the biomolecule bioactivity There is an urgent need for the development of a process for the preparation of methacrylates.

Accordingly, the present inventors can directly fix the beads fixed on the biomolecule on the biochip through a simple process without a separate device and processing process inside or outside the chip, and suitable for maintaining the biological activity of the biomolecule Efforts have been made to develop the present invention.

Eventually, the main object of the present invention is to prepare a bead suspension by immobilizing the biomolecules to the beads, add a mixture of monomer and crosslinking agent capable of forming a polymer gel to the bead suspension, then mix and spot it on the substrate. The present invention provides a method of manufacturing a biochip, which comprises polymerizing the spotted spot and then fixing the spotted spot on a substrate, and a biochip manufactured by the method.

Still another object of the present invention is to provide a method for detecting a target material in a sample, comprising applying a sample containing the target material to the biochip and detecting a target material that specifically binds to the biomolecule on the biochip. .

In order to achieve the above object, the present invention comprises the steps of (a) fixing the biomolecules to the beads to prepare a bead suspension; (b) adding a mixture of a monomer capable of forming a polymer gel and a crosslinking agent to the bead suspension, followed by mixing; (c) spotting the mixture on a substrate; And (d) polymerizing the spotted spots to fix the spotted spots onto a substrate.

In the present invention, (e) it may be characterized in that it further comprises the step of drying the polymerized spot, the drying temperature is 15 ~ 33 ℃ for protein, 15 ~ 90 ℃ for DNA It may be characterized in that, the bead suspension of step (a) may be characterized in that the aqueous suspension, the biomolecule is a nucleic acid, amino acids, proteins, peptides, lipids, carbohydrates, enzyme substrates, ligands and cofactors It may be characterized in that any one selected from the group consisting of.

In the present invention, the bead of step (a) from the group consisting of polystyrene, polymethyl methacrylate, cellulose, polyglycidyl methacrylate, polypyrrole, polythiophene, glass, quartz, silica and metal-based beads It may be characterized in that any one selected, the method of immobilizing the biomolecule of the (a) to the bead may be characterized in that any one selected from the group consisting of hydrophobic adsorption, covalent bonds and electrostatic attraction.

In the present invention, the monomer mixture of step (b) is prepared by mixing the monomer and the crosslinking agent in any one or more aqueous medium selected from the group consisting of water, ethanol, methanol, DM, DMS, acetone and NMP. The monomer of step (b) may be acrylamide, isopropylacrylamide, polyethylene glycol methacrylate, polyethylene glycol acrylate, acrylic acid, methacrylic acid, paritoleic acid, oleic acid, linoleic acid, arachidonic acid, It may be characterized in that any one or more selected from the group consisting of linolenic acid, allyl alcohol and vinyl alcohol, wherein the crosslinking agent of step (b) is made of bisacrylamide, polyethylene glycol diacrylate and polyethylene glycol dimethacrylate It may be characterized in that any one or more selected from the group have.

In the present invention, it may be characterized in that the addition of the polymerization initiator in step (b), the polymerization initiator is ammonium persulfate, tetramethylethylenediamine (TEMED), riboflavin, riboflavin-5'-phosphate, It may be characterized in that any one or more selected from the group consisting of 2-hydroxy-2-methylpropanone and 2,2-diethoxyacetophenone.

In the present invention, in addition to the addition of any one or more additives selected from the group consisting of glycidyl methacrylate, N-hydroxysuccinimide polyethylene glycol acrylate and branched polyethylene glycol in the step (b) It may be characterized in that, in step (b) sodium chloride, magnesium chloride, calcium chloride, ammonium chloride, sodium sulfate, magnesium sulfate, calcium sulfate, ammonium sulfate, sodium carbonate, magnesium carbonate, calcium carbonate, ammonium carbonate, sodium nitrate, calcium nitrate, It may be characterized by further adding any one or more salts selected from the group consisting of ammonium nitrate.

In the present invention, the spotting of the step (d) may be performed by inkjet, the polymerization of the step (e) is from the group consisting of chemical polymerization, UV polymerization and photochemical polymerization It may be characterized in that it is any one method selected.

In the present invention, the substrate may be any one selected from the group consisting of microchannels of a microwell, a slide substrate, and a lab-on-a-chip, and the material of the substrate is polymethyl methacrylate (PMMA), poly Carbonate (PC), polystyrene (PS), cyclic olefin copolymer, polynorbonene, styrene-butadiene copolymer (SBC), acrylonitrile butadiene styrene, glass, It may be characterized in that any one or more selected from the group consisting of silicon, hydrogel, silicon, metal, ceramic and porous membrane, the surface of the substrate may be characterized in that the functional group modified, the functional group is an amine group, Carboxyl group, epoxy group, isocyanyl group, imide group, azide group, halogen group, phosphate group, sulfate group, ammonium group, benzo Not less than one selected from the group consisting of rice and anthraquinone can be characterized.

The present invention also provides a biochip manufactured by the above method.

The present invention also comprises the steps of: (a) applying a sample containing a target material to the biochip of claim 21; And (b) detecting a target material that specifically binds to the biomolecule on the biochip, wherein the detection of the target material comprises radioisotopes or luminescence or From the group consisting of a detection method using a biolabel such as a coloring dye, an enzyme-linked immunoassay (ELISA), an electrochemical immunoassay, a particle turbidimetric immunoassay (particle turbidimetric immunoassay) It may be characterized by using any one or more selected.

Hereinafter, the present invention will be described in more detail.

(1) Preparation of Bead-Aqueous Suspensions Fixing Biomolecules

The pore size of the polymer gel can be controlled using the content of monomers and crosslinkers and the additives in the total mixture. The content of the monomer is preferably in the range of 10 to 60% by volume, more preferably 10 to 40% by volume, the content of the crosslinking agent is preferably in the range of 1 to 20% by volume, and more preferably in the range of 1 to 10% by volume. Do.

Mixtures of one or more of the monomers described above and one or more of crosslinking agents may be used as the base material, and additives may be additionally mixed to improve the fixing force of the polymer gel to the chip surface. The content of the additive is preferably in the range of 1 to 20% by volume, more preferably in the range of 1 to 10% by volume.

In addition, salts may be added to the monomer mixture to form macroporous polymer gel spots so as to promote diffusion of biomolecules.

The present invention involves a gelation process for polymerizing monomers to form polymer gels for bead fixation. In this case, in order to increase the efficiency of the polymerization, an appropriate polymerization initiator may be added according to the polymerization method, and the content of the polymerization initiator is preferably 0.1 to 10% by volume.

As the aqueous medium used to prepare the monomer mixture, any solvent having aqueous properties may be used, and examples thereof include water, ethanol, methanol, DM, DMS, acetone, and NMP, but are not limited thereto. However, water may be used.

The above-mentioned bead aqueous suspension and monomer mixture are combined and spotted, wherein each compounding ratio is determined according to the concentration range of the detection source to be detected in the biochip to be formed and the pore size of the desired porous structure. Suitable aqueous suspensions range from 0.1 to 5% by volume and monomer mixtures from 5 to 30% by volume. If the monomer mixture exceeds 30% by volume in total, the compatibility is poor and spot formation on the chip substrate is poor.

(2) Immobilization of Biomolecule-immobilized Beads on a Chip Substrate by Polymer Gelation

Once the mixed solution of the bead aqueous suspension and monomer mixture is prepared, the beads fixed with the biomolecules are fixed to the substrate surface by spotting the mixed solution on a substrate. The spotting may use any spotting method conventionally used in the art, and typically, spotting may be performed by inkjet. When using an inkjet it is advantageous because it is easy to quantitatively spray the monomer mixture solution developed in the present invention on a substrate.

The gelation reaction of the spots is basically a polymer polymerization process of the monomer mixture, such as a chemical polymerization process that proceeds through chemical decomposition of an initiator and a photochemical polymerization process that proceeds by photoinitiation such as UV or plasma. It is possible.

1 is a schematic diagram showing a process of forming a spot made of a polymer gel on a substrate by gelling a monomer mixture including beads in which a biomolecule is immobilized in the method of manufacturing a biochip according to the present invention.

After performing the spotting step and the gelling step, a step of drying the aqueous medium of the spot may be added as necessary. Drying may be used a conventional drying method used when manufacturing a biochip by spotting, for example, room temperature drying. There is an appropriate drying temperature depending on the material, 15-33 ℃ for proteins and 15-90 ℃ for DNA.

Figure 2a is a photograph taken by observing the polymer gel spot fixed on the substrate with a scanning electron microscope, Figure 2b is a micrograph observing that the bead is fixed to the biomolecule on the polymer gel having a macropore.

Example  One: On a chip Gelation reaction  through Spot  formation

The fluorescent dye Cyanine 3 labeled immunoglobulin was immobilized on polystyrene beads (600 nm in diameter) to prepare an aqueous suspension of 1% by weight of beads. In addition, polyethylene glycol methacrylate (average molecular weight 360) as a monomer, polyethylene glycol damethacrylate (average molecular weight 550) as a crosslinking agent, and polymerization initiator 2-hydroxy-2-methylpropanone [Shiba Gaiji] are used in distilled water as an aqueous medium. DAROCUR ^™ 1173] (Ciba Geigy) was mixed at a volume ratio of 90: 9: 1, respectively, to prepare a monomer mixture having a monomer content of 40% by volume.

The prepared bead aqueous suspension and monomer mixture were mixed in a 1: 1 volume ratio to prepare a mixture having a monomer content of 20% by volume and a bead content of 0.5% by weight. In addition, a mixture of 10% by volume, 5% by volume and 1% by volume of the monomer was prepared in the same manner as described above.

20% by volume, 10% by volume, 5% by volume and 1% by volume of the prepared monomers were spotted onto the PMMA slide with a volume of 0.5 μl, and gelled by irradiation of UV (365 nm, 100 mW / cm ² ) for 1 minute. (FIG. 3). As a result, as shown in Figure 3, it was confirmed that the beads of the spot having a polymer gel content of 10 to 20% by volume has excellent uniformity.

On the other hand, the size of the spot formed on the substrate was adjusted by adjusting the volume of the droplet to be spotted. In the same manner as described above, 20% by volume and 15% by volume of the monomer mixture were prepared, and 50 and 20 nL of droplets were respectively dispensed onto the amine-modified PMMA (AmPMMA) substrate using an inkjet arrayer (FIG. 4). As a result, as shown in FIG. 4, the size of the polymer gel spot fixed to the substrate was reduced by reducing the volume of the droplet to be spotted, and the distribution of the fluorescent beads was uniform even when 20 nL of the polymer gel was dispensed. .

Example  2: surface of PMMA substrate Amination  Equation

The substrate surface was aminated to modify the covalent bond between the polymer gel spot and the substrate. Ethylenediamine and 2.5 M butyllithium were prepared in a volume ratio of 67:33, and reacted for 5 hours under argon atmosphere to prepare an amine activation solution.

The polymethyl methacrylate (PMMA) substrate was ultrasonically washed with isopropyl alcohol and distilled water, immersed in the amine activation solution for 30 seconds, and then ultrasonically washed with ethanol and distilled water, dried and amine-modified polymethylmethacryl. Rate substrates were prepared. The prepared substrate was analyzed by Electron Spectroscopy for Chemical Analysis (ESCA) (FIG. 5A). As a result, as shown in FIG. 5A, the specific peak (N1s) of the modified amine group was confirmed. In addition, the prepared substrate was measured by AFM (Atomic Force Microscopy) analysis to measure the roughness of the substrate surface (FIG. 5B). As a result, as shown in FIG. 5B, the substrate has an even surface roughness of 4 nm (RMS) level. And it was found.

Fluorescence quantitation was used to evaluate the relative substitution rates of the modified amine groups. 5- (6-) carboxytetramethylhodamine succinic ester (TAMRA) was used as a reactive fluorescent dye, and an fluorescence scanner of Axon Corporation was used as a device for quantification. As shown in Figure 5b, the prepared substrate was found to be prepared at a level close to the amine substitution rate of Corning (Corning), a representative commercial amine silanated glass slide.

Comparative example  1: according to the substrate material Polymer gel Spot  Shape comparison and Holding force  exam

The fixing force of the polymer gel spots according to the substrate material was compared. In the same manner as in Example 1, 18% by volume monomer mixture containing 0.5 wt% of polystyrene beads coated with Cyanine 3 fluorescently labeled immunoglobulin was prepared.

Comparative glass substrates (Corning), aminosilane-treated glass (Xenopore), PMMA (EG920, IF870 from LG Chem), amine-modified PMMA, cyclic olefin copolymer (Euray slide, Exiqon) slides were prepared, and 20 μl of a monomer mixture solution was spotted on each substrate, followed by gelation.

The substrate was washed by shaking 2 0.1% by volume of Tween (Tween 20) containing a phosphate buffer solution for 10 min (pH 7.4) times, using a stronger washing conditions to note rack (Memorec)'s aHyb ^TM device 150㎕ / Strong washing was performed twice for 1 minute at a flow rate of minutes. The number of spots before and after each washing step was counted to relatively evaluate the fixing force of the polymer gel spots on the substrate (FIG. 6A). As a result, as shown in Figure 6a, it was found that the spot fixing force is excellent for the PMMA series substrate. In addition, it was found that the shape of the spot formed on the substrate of the plastic material is more uniform than the spot formed on the glass material (FIG. 6B).

Example  3: with additive Of polymer gel Holding force  Improving

An additive covalently bonded to the surface reactor of the amine-modified PMMA substrate was added to the monomer mixture to improve the fixing force of the spot to the substrate.

To 100 μl of a mixture of polyethylene glycol methacrylate (average molecular weight 360) and polyethyleneglycol dimethacrylate (average molecular weight 550) as a crosslinking agent in 310 μl of aqueous carbonate buffer solution (pH 11) as an aqueous medium. 40 μl of 2-hydroxy-2-methylpropanone (DAROCUR ^™ 1173, manufactured by Ciba Geigy), a polymerization initiator, and 50 μl of glycidylmethacrylic acid as an additive were added and mixed.

500 µl of an aqueous suspension of 1% by weight of polystyrene beads coated with Cyanine3 fluorescently labeled immunoglobulin was mixed with the mixture, and the monomers contained 5% by volume and 0.5% by volume, respectively, and the total monomer content was 15% by volume. A mixture was prepared.

In the above-described method, a monomer mixture containing 1% by volume of an additive (glycidyl methacrylate) and a total monomer content of 15% by volume and a 15% by volume monomer mixture containing no additives were prepared, respectively.

0.5 μl of each of the prepared mixture containing 5% by volume and 1% by volume of the additive and the mixture without the additive was spotted on an amine-modified PMMA substrate, and gelled in the same manner as in Example 1. Washing was vigorously twice for 1 minute at 150 μl / min flow rate using a-Hyb ^™ instrument from Memorec. In the strong washing process, the beads present in the spot are lost, and the loss rate of the beads was measured by measuring the change in fluorescence intensity of the spot before and after washing (FIG. 7). As a result, as shown in Figure 7, it can be seen that as the additive content increases the loss rate of the beads decreases.

Example  4: Macroporus ( macroporous ) Of polymer gel  formation

The pores of the polymer gel were largely formed at the micro level. When salt is added to the monomer mixture, the aqueous medium and the phase separation are promoted in the process of gelling the spotting liquid, so that the pore of the formed polymer gel can be made to a micro level.

First, the monomer acrylamide and bisacrylamide are mixed in a volume ratio of 37.5: 1, and 5 to 10 wt% of the monomer is added to a 3.3 M aqueous sodium chloride solution, and then 2 μl of a 5 wt% aqueous solution of ammonium persulfate is added to 40 μl of the aqueous solution. After mixing 1.2 μl of tetramethylethylenediamine, the mixture was spotted on a PMMA substrate, and then gelled for 40 minutes as in Example 1 (FIGS. 8A and 8B). As a result, as shown in FIGS. 8A and 8B, micropores of 2 to 3 μm were formed simultaneously with deep pore channels of 10 to 20 μm.

In addition, when the monomer mixture including the aqueous bead suspension is carried out in the same manner as described above, as shown in Figure 2b it is possible to prepare a spot on which beads are supported in the micropores.

Example  5: Spot  Self-fluorescence measurement and nonspecific protein binding

As a preliminary experiment for applying the bead fixation method using the polymer gel of the present invention to a biochip, the monomer mixture containing the beads is nanospotted on a chip substrate, and the degree of autofluorescence and non-specific binding is different. Was measured. To fix the biomolecules into beads, 200 μl of an aqueous solution of bovine serum albumin phosphate at a concentration of 3.1 mg / μl and 200 μl of an aqueous suspension of 2 wt% polystyrene beads (600 nm in diameter) were mixed and reacted by shaking at room temperature for 15 hours.

The suspension was centrifuged and washed repeatedly to prepare 1.0 or 2.5 wt% aqueous beads suspension, respectively. 15 μl of the aqueous bead suspension, 15 μl of a 10% by weight monomer mixture (acrylamide: bisacrylamide 37.5: 1 volume ratio, 5M aqueous sodium chloride solution) and 2-hydroxy-2-methylpropanone as a polymerization initiator [Ciba Gaiji (Ciba) 0.4 μl of DAROCUR ^™ 1173 Co., Ltd. was mixed to prepare a monomer mixture having a final bead content of 0.5% and 1.25% by weight.

The monomer mixture was spotted on the PMMA substrate using an inkjet array, and spots were formed by irradiating UV (365 nm, 100 mW / cm ² ) for 30 minutes. In this case, 50 nL of the monomer mixture containing 0.5 wt% beads and the monomer mixture containing 1.25 wt% beads were spotted in a volume of 20 μl. After drying the substrate for 10 hours at room temperature, the fluorescence intensity of the spot adhered by using a fluorescence image scanner (Exxon) was measured (Fig. 9a). As a result, as shown in Fig. 9a, the self-fluorescence intensity of the polymer gel spot was less than 3 compared to the signal-to-noise ratio (SNR) of the background, indicating that the self-fluorescence was insignificant. .

On the other hand, the bovine serum albumin-coated beads immobilized on the substrate by the polymer gel as described above was treated with an anti-SAH (S-adenosyl-L-homocysteine) aqueous solution of the protein to determine the degree of nonspecific binding (Fig. 9b). As a result, as shown in Figure 9b, the fluorescence intensity of the spot measured after the non-specific binding reaction of the anti-SAH antibody did not exceed 3 compared to the base fluorescence intensity, it can be seen that there is almost no non-specific binding of the protein.

The results of the experiment that the self-fluorescence intensity and non-specific binding as described above are insignificant means that the performance of the biochip manufactured by fixing the adhesive beads prepared in the present invention to the substrate is excellent.

Example  6: SAH (S- Adenosyl -L-homocysteine) competitive immunodetection test

The bead support was nanospotted on a plastic substrate to fabricate a simple biochip capable of detecting SAH targets and to test for immune detection.

In the same manner as in Example 5, spotting a monomer mixture comprising bovine serum albumin and a bovine serum albumin coated with SAH (0.5 wt% and 1.25 wt%) onto a PMMA substrate with 50 nL and 20 nL volume After gelation, gelled and dried at room temperature for 20 hours. The chip was blocked for 30 minutes with phosphate buffer (pH 7.4) containing 3 wt% bovine serum albumin and 0.05 vol% Tween and washed. After detecting the Cy3-labeled secondary antibody (secondary antibody) with anti-SAH antibody for 30 minutes in advance for fluorescence detection, SAH is mixed by concentration as a target substance to be detected therein, and a competitive immune reaction is performed with the spot of the chip. It was. Fluorescence scanner photographs and detection results of spots according to SAH concentration are shown in FIGS. 10A and 10B (-●-,-▼-), respectively. The fluorescence signal was expressed as a relative value with a signal of 100 when the target material SAH was not added. In the SAH detection by competitive immunodetection using the chip fabricated in this embodiment, the fluorescence signal was reduced by more than 80% compared to the absence of the target material SAH, and the upper limit of detection was 0.5 ~ 1 μM.

Comparative example  2: Comparison with 2D Biomolecule Immobilization

In order to verify the superiority of the three-dimensional immobilization method using the polymer gel of the present invention, compared with the conventional two-dimensional immobilization method and the immunodetection performance commercialized as another biomolecule immobilization method.

As a representative chip capable of two-dimensional immobilization of biomolecules, Nunc's MaxiSorp chip was selected to test competitive immunodetection against SAH target material and the results were compared with Example 6.

Bovine serum albumin and SAH (S-adenosyl-L-homocysteine) labeled bovine serum albumin were prepared in a solution of 0.5 mg / mL in a phosphate buffer containing 20% by volume of glycerol, respectively, and spotted on maxi chip. And dried for 20 hours in a humidity chamber. After washing the spotted chip, a competitive immune response was performed in the same manner as in Example 6. As a result, as shown in Figure 10b (-▲-), the maximum reduction value of the fluorescent signal by the competition reaction is 50% level, the standard quantification range is 0.01 ~ 0.5μM immobilization method using the polymer gel of Example 6 It did not reach the level of detection significantly.

The above results are due to the fact that the conventional two-dimensional immobilization method using the maxi chip is low in the degree of integration of biomolecules and large steric hindrance to the chip surface. From this result, it was confirmed that the biochip using the polymer gel according to the present invention is relatively superior to the conventional biochip.

As described above in detail specific parts of the present invention, for those of ordinary skill in the art, these specific descriptions are only preferred embodiments, which are not intended to limit the scope of the present invention. Will be obvious. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

According to the present invention, since a carrier having a large surface area such as a bead is used, high density biomolecules can be efficiently integrated on the surface of the biochip substrate, and beads fixed with the biomolecules are separately disposed in or outside the chip. The beads can be fixed directly on the biochip through a simple process without a process, which is economically effective.

The biochip according to the present invention encapsulates the beads in which the biomolecules are immobilized inside the porous structure of the polymer gel, thereby creating a bio-friendly environment and forming pores having a micro level size to maintain the biomolecule's physiological activity as well as the immunity. It is possible to facilitate the delivery of the biomaterial in the detection reaction.

Claims (23)

Biochip manufacturing method comprising the following steps: (a) immobilizing the biomolecules to the beads to prepare a bead suspension; (b) adding a mixture of a monomer capable of forming a polymer gel and a crosslinking agent to the bead suspension, followed by mixing; (c) spotting the mixture on a substrate; And (d) polymerizing and fixing the spotted spots on a substrate. The method of claim 1 further comprising (e) drying the polymerized spot. The method of claim 2, wherein the drying temperature is 15 to 33 ° C. for proteins and 15 to 90 ° C. for DNA. The method of claim 1 wherein the bead suspension of step (a) is an aqueous suspension. The method of claim 1, wherein the biomolecule is any one selected from the group consisting of nucleic acids, amino acids, proteins, peptides, lipids, carbohydrates, enzyme substrates, ligands and cofactors. The group of claim 1, wherein the bead of step (a) comprises polystyrene, polymethylmethacrylate, cellulose, polyglycidyl methacrylate, polypyrrole, polythiophene, glass, quartz, silica, and metal-based beads. And any one selected from. The method of claim 1, wherein the method of immobilizing the biomolecule of the step (a) is any one selected from the group consisting of hydrophobic adsorption, covalent bonding and electrostatic attraction. The method of claim 1, wherein the monomer mixture of step (b) is prepared by mixing the monomer and the crosslinking agent in any one or more aqueous medium selected from the group consisting of water, ethanol, methanol, DM, DMSO, acetone and NMP. Characterized in that the method. The method of claim 1, wherein the monomer of step (b) is acrylamide, isopropylacrylamide, polyethylene glycol methacrylate, polyethylene glycol acrylate, acrylic acid, methacrylic acid, paritoleic acid, oleic acid, linoleic acid, arachidonic acid, At least one selected from the group consisting of linolenic acid, allyl alcohol and vinyl alcohol. The method of claim 1, wherein the crosslinking agent of step (b) is at least one selected from the group consisting of bisacrylamide, polyethylene glycol diacrylate, and polyethylene glycol dimethacrylate. The method of claim 1, further comprising adding a polymerization initiator in the step (b). 12. The method of claim 11, wherein the polymerization initiator is ammonium persulfate, tetramethylethylenediamine (TEMED), riboflavin, riboflavin-5'-phosphate, 2-hydroxy-2-methylpropanone and 2,2-diethoxyacetophenone At least one selected from the group consisting of a method. The method of claim 1, wherein in step (b) further adding at least one additive selected from the group consisting of glycidyl methacrylate, N-hydroxysuccinimide-ized polyethylene glycol acrylate and branched polyethylene glycol Characterized in that the method. The method of claim 1, wherein in step (b), sodium chloride, magnesium chloride, calcium chloride, ammonium chloride, sodium sulfate, magnesium sulfate, calcium sulfate, ammonium sulfate, sodium carbonate, magnesium carbonate, calcium carbonate, ammonium carbonate, sodium nitrate, calcium nitrate, And further adding at least one salt selected from the group consisting of ammonium nitrate. The method of claim 1, wherein the spotting in step (d) is performed by inkjet. The method of claim 1, wherein the polymerization in the step (e) is any one selected from the group consisting of chemical polymerization, UV polymerization and photochemical polymerization. The method of claim 1, wherein the substrate is any one selected from the group consisting of microchannels of a microwell, a slide substrate, and a lab-on-a-chip. The method of claim 17, wherein the material of the substrate is polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), cyclic olefin copolymer (Cyclic olefin copolymer), polynorbonene, styrene Butadiene copolymer (SBC), acrylonitrile butadiene styrene, glass, silicone, hydrogel, silicone, metal, ceramic and porous membrane. The method of claim 1, wherein the surface of the substrate is modified with a functional group. The method according to claim 19, wherein the functional group is any selected from the group consisting of amine group, carboxyl group, epoxy group, isocyanyl group, imide group, azide group, halogen group, phosphate group, sulfate group, ammonium group, benzoquinone and anthraquinone At least one method. A biochip manufactured by the method of any one of claims 1 to 20. A method for detecting a target substance in a sample comprising the following steps: (a) applying a sample containing a target substance to the biochip of claim 21; And (b) detecting a target material that specifically binds to the biomolecule on the biochip. The method of claim 22, wherein the detection of the target material is performed using a biolabel such as a radioisotope, a luminescence or a coloring dye, an immunoassay using an enzyme, or an enzyme-linked immunoassay (ELISA). Electrochemical immunoassay, particle turbidimetric immunoassay and fluorophore characterized in that any one or more selected from the group consisting of.

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