KR100641832B1 - A micro-well chip for bioreactor - Google Patents

Abstract

A micro-well chip for bioreactor is provided to simplify the operation, and reduce the time of analysis and amount of samples by performing the cultivation of transformed Escherichia coli, expression induction of recombinant proteins, cell crushing, and purification and elution of recombinant proteins with purification tags in one process on the chip. The micro-well chip for bioreactor comprises a substrate coated by a gold thin film and a perforated polymer plate, wherein the gold thin film-coating layer is covalently bound with the polymer plate by using self assembled monolayers, wherein the substrate is made of glass, silicon, silicon oxide, silicon nitride, polycarbonate, polystyrene or polypropylene; the polymer is poly-dimethylsiloxane, poly-methylmeta-acrylate, polycarbonate, polyethylene, polypropylene or polystyrene; the self assembled monolayer contains thiol group, silanol group or phosphine group; the thickness and diameter of polymer plate are 1-8 mm and 0.1-10 mm; and the thickness of gold thin film is 20-200 nm.

Description

A micro-well chip for bioreactor

1 is a schematic diagram showing a method of permanently bonding a gold thin film surface by forming a self-assembled monolayer on the perforated polymer plate.

FIG. 2A is a schematic diagram of a micro-well chip, and FIG. 2B is a diagram schematically illustrating a method of culturing cells to express and purify recombinant proteins, compared to using a micro-well chip and a conventional laboratory scale method.

3 is a micro-well chip prepared by binding a well-formed polymer plate to a gold thin film surface-treated with a substance that selectively binds to a recombinant protein purification tag, and E. coli cultured using the same, the supernatant SDS -PAGE picture.

Figure 4 is a graph showing the image and the relative image intensity measured by using a surface plasmon resonance image measuring the expression level of the recombinant protein expressed in the micro-well chip.

5 is a photograph confirmed by using protein electrophoresis after eluting the recombinant protein expressed in the micro-well chip using a purification tag.

6 is a graph showing the activity of the eluted protein using a fluorescence measurement device and a graph showing relative fluorescence intensity.

The present invention includes a substrate coated with a gold thin film and a perforated polymer plate, wherein one side of the gold thin film coating layer and the polymer plate is covalently bonded using self assembled monolayers. A micro-well chip and a method of manufacturing the chip.

Proteomics is the field of holistic study of the properties and functions of proteins produced by gene expression. Protein chips provide an extremely fast measurement technique that is very useful for proteomics and can rapidly analyze large quantities of protein-protein, protein-nucleic acid, protein-sugar, and protein-lipid binding. Protein chips can be used to predict new drug targets, make accurate assessments, and use high throughput screening (HTS) for new drug candidates. In addition, there is a great demand for disease diagnosis, environmental monitoring, hazard detection of environmental toxic substances.

The most important point in the development of new protein or peptide drugs using such protein chips is to efficiently reduce the time, resources, and costs in identifying and purifying the expression and degree of recombinant protein expression.

In order to efficiently shorten the time and resources required for a series of processes for expression and purification of recombinant proteins, the inventors have found that the culture of cells transformed with vectors expressing recombinant proteins, cell disruption, and elution of recombinant proteins are effective. In order to manufacture a micro-well chip for a bioreactor consisting of a single process.

Accordingly, a micro-well chip for a bioreactor including a substrate coated with a thin film of gold and a perforated polymer plate, wherein one side of the thin film coating layer and the polymer plate is covalently bonded using a self-assembled monomolecular film, and the chip The present invention was completed by confirming that the expression, purification and analysis of proteins can be performed in a single process in a short time.

One object of the present invention includes a substrate coated with a gold thin film and a perforated polymer plate, wherein one side of the gold thin film coating layer and the polymer plate is covalently bonded using self assembled monolayers. The present invention provides a micro-well chip for a bioreactor.

Still another object of the present invention is to (i) attach one side of the substrate with a thin gold film; (Ii) producing a perforated polymer plate having a self-assembled monolayer; (Iii) a method of manufacturing a micro-well chip for a bioreactor, comprising covalently bonding one side of a substrate coated with a thin film of gold to a polymer plate.

Still another object of the present invention is to (i) form and activate a self-assembled monolayer on the well gold thin film layer of the micro-well chip; (Ii) culturing the cells transformed with the vector expressing the tagged protein in wells of the chip; And (iii) crushing the cells and checking the amount of the protein adsorbed on the surface of the chip to provide a method for separating and purifying the protein using the micro-well chip.

One object of the present invention includes a substrate coated with a gold thin film and a perforated polymer plate, wherein one side of the gold thin film coating layer and the polymer plate is covalently bonded using self assembled monolayers. It relates to a micro-well chip for a bioreactor.

Chips formed using the self-assembled monolayer may be used in various ways as bioreactor chips.

As used herein, the term “micro-well chip for bioreactor” refers to a protein, nucleic acid (DNA or RNA), peptide nucleic acid (PNA), biological membrane, carbohydrates, lipids, small molecules, steroids, and variants and derivatives thereof derived from living organisms. It is a device that can reproduce biochemical reactions in vivo in artificial containers by integrating biomolecules such as microorganisms, animal and plant cells, organs and neurons on the chip surface and reacting with samples. It can be widely used for detecting environmental pollutants.

The chip of the invention comprises two different plates, in particular a substrate coated with a thin film of gold and a perforated polymer plate. The polymer plate of the chip has a micro-well having a thickness of 1 to 8 mm, a diameter of 0.1 to 10 mm, and the chip bottom has a gold thin film layer of 20 to 200 nm.

The substrate serves as a support for the micro-well chip, and may be glass, silicon, silicon oxide, silicon nitride, polycarbonate, polystyrene, polypropylene, or the like. However, the present invention is not limited thereto.

The polymer may include poly-dimethylsiloxane, poly-methylmethacrylate, polycarbonate, polyethylene, polypropylene, polystyrene, or the like. However, the present invention is not limited thereto. Among them, PDMS (Poly-dimethylsiloxane) is preferable. PDMS is known as a biological material that is nontoxic and has hydrophobic properties in liquid form.

The gold thin film coating layer and the polymer plate of the substrate are assembled using a self-assembled monomolecular film. As used herein, the term “self assembled monolayers (SAMs)” refers to regularly ordered organic molecular membranes spontaneously coated on the surface of a substrate so that metals and polymer plates form stable bonds. A self-assembled monomolecular film is formed on the surface to change the properties of the surface of the substrate or to change the reactivity of the surface The self-assembled monomolecular film may include, but is not limited to, for example, thiol groups, silanol groups or phosphine groups. The assembled monomolecular film may be formed on the polymer plate, the gold thin film coating layer of the substrate, or both.

Specifically, in the present invention, the polymer plate is oxidized by O ₂ plasma, and 3-mercapto-propyl trimethoxy silane (3-MPTS) is immersed to form a magnetic having a thiol group (-SH). Self-assembled monolayers were formed.

In still another aspect, the present invention provides a method for manufacturing a semiconductor device comprising: (i) attaching one side of a substrate with a thin film of gold; (Ii) preparing a perforated polymer plate having a self-assembled monolayer; (Iii) a method of manufacturing a micro-well chip for a bioreactor, comprising covalently bonding one side of a substrate coated with a thin film of gold to a polymer plate.

In the step (iii), since the bonding strength between gold and the substrate is generally weak, in order to improve the bonding strength between them, a metal such as chromium, titanium, titanium tungsten and nickel chromium, which is used as a binder, is first coated on the substrate, and then gold is attached. One side of the substrate has a thin gold layer.

In step (ii), the polymer plate is prepared by pouring the polymer solution into a mold in an appropriate shape and thickness, drying at an appropriate temperature of 40 to 80 ° C., and then punching a hole using a punch in the manufactured polymer plate.

In the step (iii), in order to covalently bond the polymer plate over the gold thin film layer of the substrate, it is oxidized using O ₂ plasma and immersed in 3-MPTS to form a self-assembled monomolecular film having a SH-functional group on the polymer plate. Lifting and pressing the polymer plate on the surface of the gold thin film of the substrate and baking in a 120 ℃ oven to form a strong covalent bond between the surface of the gold thin film and the polymer plate.

The micro-well chip of the present invention manufactured by the above method efficiently reduces the time and resources required for a series of processes from culturing cells transformed with the protein expressing the vector to expressing and purifying the recombinant protein. It can be used as a chip to allow cell culture directly on the gold thin film surface.

In still another aspect, the present invention provides a method for manufacturing a self-assembled monomolecular film on a gold thin film well of a micro-well chip; (Ii) culturing the cells transformed with the vector expressing the tagged protein in wells of the chip; And (iii) crushing the cells and confirming the amount of the protein adsorbed on the surface of the chip.

(Iii) is a step of activating a self-assembled monomolecular film by forming a self-assembled monomolecular film on the gold thin film layer of the well. In a specific embodiment of the present invention, the chip is treated with a mixture of sulfuric acid and hydrogen peroxide solution and immersed in a 11-mercapto-1-undecanoic acid (MUA) solution to form a self-assembled monomolecular film. The formed self-assembled monolayers were reacted in a mixture of NHS and EDC to enable the membranes to be activated with hydroxysuccinimidyl esters. The chip was then treated with reduced L-glutathione.

Step (ii) is a step of culturing cells and inducing protein expression on a chip.In a specific embodiment of the present invention, E. coli, which expresses a green fluorescent protein having a glutathione-S transferase (GST) tag, is cultured on a chip and the protein is expressed by IPTG. Expression was induced.

(Iii) is a step of checking the degree of protein expression, the identification method is not particularly limited. In a specific embodiment of the present invention, protein expression was confirmed by surface plasmon resonance (SPR). SPR image sensor is capable of ultra-fast analysis, there is no need to check the existing protein separation process, such as SDS-PAGE and Western blot, there is an advantage that can be confirmed in real time the expression of the recombinant protein of interest.

Cells that can be cultured using the chip of the present invention are not particularly limited, and are, for example, cells derived from animals, plants, algae, fungi, bacteria, viruses, and protozoa.

The tags for detecting proteins on chip are: glutathione-S transferase (GST), maltose binding protein (MBP), intain, thiorredoxine, dihydropol Dihydrofolate reductase (DHFR), cellulose binding domain (CBD), maltose binding protein (MBP), galactose-binding protein, calmodulin binding protein binding protein: CBP), hexa histidine (6 × His), T7gene10, lac repressor, hemagglutinin influenza protein (HIP), HSV-tag, staphylococcal protein A A), staphylococcal protein G, polycysteine, polyphenylalanine, hexa-arginine, flag (FLAG), streptoavidin tag II (Strep- tag II), c-myc, s-peptide, chitin-binding domain, and the like.

The micro-well chip of the present invention is a bioreactor chip in which a substance selectively binding to a purified tag of a recombinant protein is bound to the well surface of the chip. The micro-well chip has only a few microliters (μl) to several nanoliters (nL) of trait. Cultivation of the converted E. coli, induction of recombinant protein expression, cell disruption, purification using a purified tag of the recombinant protein, it can be made in a single process to elution.

Hereinafter, the present invention will be described in detail by examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.

Example 1 Micro-Well Chip Manufacturing

<1-1> Micro-Well Chip Manufacturing Process

PDMS was poured slowly into a flat Petri dish and bubbles were removed and dried at 60 ° C. for 3 hours to form a thin polymer plate of 2 mm. A punch having a diameter of 2 mm was punched into the polymer plate removed from the Petri dish to oxidize the surface with O ₂ plasma. Plasma treated polymer plate was immersed in 3-mercapto-propyl trimethoxy silane (3-MPTS) for 30 minutes to form self-assembled monolayers. By attaching the -SH functional group to the gold thin film, it was possible to bond firmly to the surface of the gold thin film. First, a 47 nm gold thin film with 2 nm chromium was deposited on a thin glass plate using a commercially available electron-beam evaporator. The PDMS plate with -SH functional group attached to the surface of the gold thin film was pressed and then baked in an oven at 120 ° C. for 1 hour to form a strong bond between the surface of the gold thin film and the polymer plate (FIG. 1).

<1-2> Glutathione Surface Preparation Process of Micro-Well Chip

The prepared micro-well chip was treated for 30 minutes in a mixed solution of 95% sulfuric acid and 30% hydrogen peroxide (volume ratio 3: 1) at 65 ° C., followed by 10 mM 11-mercapto-1-endeca dissolved in ethanol. It was immersed in a solution of 11-mercapto-1-undecanoic acid (MUA) for 16 hours to form a self-assembled monomolecular film, 0.1M N-hydroxysuccinimide (NHS) and 0.4M 1- The self-assembled monomolecular membrane was reacted with a mixture of ethyl-3-dimethylaminopropyl carbodiimide (EDC) for 10 minutes to activate the hydroxysuccinimidyl ester. The activated surface was reacted for 1 hour in an amino-dextran (M. W. 500,000) solution for 2 hours in a 0.1 mg / ml BMPS solution, and then the reaction solution was discarded and the chip was washed. Subsequently, the micro-well chip was immersed in reduced L-glutathione (GSH) 100 mM phosphate buffer (pH 7.0) dissolved at 44 mM concentration for 20 hours at 37 ° C, and then the reaction solution was discarded and distilled water. The chip was washed. In addition, in order to block the unreacted activator of the micro-well chip surface, the 1M ethanol amine solution was treated on the chip surface and reacted at 37 ° C. for 4 hours.

Example 2 Expression Analysis of Green Fluorescent Protein with Glutathione-S Transferase Tag

<2-1> Construction of Green Fluorescent Protein Gene with Glutathione-S Transferase Tag

For the preparation of the GFP gene having a GST tag at the N-terminus, two primers encoding the GFP gene were prepared. In order to insert into pGEX 4T-1, a GST tag expression vector, Bam HI was introduced into the N-terminal primer and Xba I restriction enzyme cleavage site was introduced into the C-terminal primer. The fragments obtained by PCR (polymerase chain reaction) were digested with restriction enzymes introduced into each primer, and then inserted into pGEX 4T-1 vectors cut with Bam HI and Xba I restriction enzymes to prepare pGST-GFP vectors.

<2-1> Green Fluorescent Protein Gene Expression with Glutathione-S Transferase Tag

E. coli BL21 transformed with the prepared pGST-GFP was shaken at 37 ° C. until the OD (Optical density, A600 nm) was 0.6, and protein expression was induced by adding IPTG with a total concentration of 1 mM. . After 4 hours, the recovered E. coli culture was crushed by ultrasound (Branson, Sonifier 450, 3㎑, 3W, 5min) to obtain a recombinant protein solution. The resulting protein solution was mixed with buffer (12 mM Tris-Cl, pH 6.8, 5% glycerol, 2.88 mM mercapto ethanol, 0.4% SDS, 0.02% phenol bromide blue), heated at 100 ° C. for 5 minutes and then loaded onto a polyacrylamide gel. By electrophoresis for 1 hour and stained with Coomassie stain to confirm the recombinant protein.

Example 3 Expression Analysis of Green Fluorescent Protein with Glutathione-S Transferase Tag Using Micro-Well Chip

<3-1> Expression of Green Fluorescent Protein with Glutathione-S Transferase Tag Using Micro-Well Chip

Several microliters (μL) to several nanoliters (nL) of transformed Escherichia coli (pGST-GFP / E. Coli BL21) cultures were placed in a micro-well chip until the OD ₆₀₀ was 0.6. It was. The external temperature was maintained at 37 ° C. and humidity to the extent that the medium did not dry. After 4 hours of incubation, recombinant protein expression was induced for 3 hours after adding IPTG to a final concentration of 1 mM. In stationary state, lysozyme solution was added to disrupt E. coli inside the micro-well chip. After crushing the supernatant was confirmed the presence of recombinant protein through protein electrophoresis as shown in FIG. In electrophoretic gels, little expression bands were expressed in IPTG-induced wells, indicating that the recombinant protein with the purified tag was completely adsorbed onto the surface of the gold thin film modified to specifically bind to the purified tag. The micro-well chip to which the recombinant protein (GST-GFP) was adsorbed was washed three times with distilled water, and then the degree of protein expression was confirmed by using a surface plasmon resonance image sensor (FIG. 4).

To the micro-well chip confirmed the recombinant protein expression, a few microliters (μl) to several nanoliters (nL) of 30 mM glutathione solution was added, and then left for about 10 minutes to try to elute the recombinant protein. The eluted protein was quantified and checked for activity using protein electrophoresis and fluorescence sensors (FIGS. 5 and 6).

The present invention includes a substrate coated with a gold thin film and a perforated polymer plate. Micro-well chips for bioreactors, in which a thin film coating layer and one side of the polymer plate are covalently bonded using self assembled monolayers, can be used for culturing very small amounts of transformed Escherichia coli, inducing recombinant protein expression, cell disruption, and recombination. Purification of proteins Using a tag, all the processes from elution to a single process on a chip are simple to operate, fast to analyze and can be analyzed with a small amount of sample.

Claims (11)

A micro-well for a bioreactor comprising a substrate coated with a gold thin film and a perforated polymer plate, wherein one side of the gold thin film coating layer and the polymer plate is covalently bonded using self assembled monolayers. chip. The micro-part according to claim 1, wherein the substrate is selected from the group consisting of glass, silicon, silicon oxide, silicon nitride, polycarbonate, polystyrene, and polypropylene. Well chip. The method of claim 1, wherein the polymer is polydimethylsiloxane, polymethylmethacrylate, polycarbonate, polyethylene, polypropylene, and polystyrene. Micro-well chips selected from the group consisting of. The micro-well chip of claim 1, wherein the self-assembled monomolecular film comprises a thiol group, a silanol group, or a phosphine group. The micro-well chip of claim 1, wherein the polymer plate has a thickness of 1 to 8 mm and a diameter of 0.1 to 10 mm. The micro-well chip of claim 1, wherein the gold thin film layer is 20 to 200 nm. The micro-well chip of claim 1, wherein cell culture, recombinant protein expression induction, and protein isolation purification are performed on a single well. (Iii) attaching one side of the substrate with a thin film of gold; (Ii) producing a perforated polymer plate having a self-assembled monolayer; (Iii) covalently bonding one side of the substrate and the polymer plate coated with a thin film of gold, a method of manufacturing a micro-well chip for a bioreactor. (Iii) forming and activating a self-assembled monolayer on the gold thin film well of the micro-well chip of claim 1; (Ii) culturing the cells transformed with the vector expressing the tagged protein in wells of the chip; And (iii) crushing the cells and confirming the amount of the protein adsorbed on the chip surface. The method of claim 9, wherein the tag is glutathione-S transferase (GST), maltose binding protein (MBP), intain, thiorredoxine, dihydrofolate Dihydrofolate reductase (DHFR), cellulose binding domain (CBD), maltose binding protein (MBP), galactose-binding protein, calmodulin binding protein protein: CBP), hexa histidine (6 × His), T7gene10, lac repressor, hemagglutinin influenza protein (HIP), HSV-tag, staphylococcal protein A ), Staphylococcal protein G, polycysteine, polyphenylalanine, hexa-arginine, flag (FLAG), streptoavidin tag II (Strep-tag II), C-myc, The method is selected from the group consisting of s-peptide and chitin-binding domain. The method of claim 9, wherein the protein expression is confirmed by surface plasmon resonance (SPR).

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