KR20120038352A

KR20120038352A - Biochip and manufacturing method for the same

Info

Publication number: KR20120038352A
Application number: KR1020110015705A
Authority: KR
Inventors: 이내림
Original assignee: 한국전자통신연구원
Priority date: 2010-10-13
Filing date: 2011-02-22
Publication date: 2012-04-23

Abstract

The technical problem to be achieved by the present invention is to increase the surface area of the biochip to which the biomaterial can be fixed and to improve the efficiency of the reaction. The biochip according to the present invention includes a substrate, a coating layer formed on the substrate, and nanoparticles fixed to the substrate by the coating layer and attached to a surface of a material bonded to the target material.

Description

Biochip and its manufacturing method {BIOCHIP AND MANUFACTURING METHOD FOR THE SAME}

The present invention relates to a biochip used in various fields such as gene expression patterns, gene defects, protein distribution analysis, disease diagnosis, new drug development, etc. using a biochemical reaction between a target material and a biomaterial. .

Silicon, quartz glass, and the like have been generally used to fabricate biochip substrates, but metals, inorganic materials, or polymer materials have gradually been used. The manufacturing process of plastic chip, which has been studied a lot recently, is slightly different from the manufacturing process of glass chip and PDMS chip. The manufacturing process of a plastic chip requires a mold similarly to the manufacturing process of a PDMS chip. Unlike the SU-8 mold, the plastic chip mold is made of hard materials such as Si, glass, and metal. The mold of the plastic chip is manufactured through a photolithography process and an etching process. Then, a hot embossing process of imprinting a pattern on a thermoplastic substrate such as PMMA using a mold of the manufactured plastic chip is performed. The plastic chip is finally completed by bonding the plastic plate with the pattern imprinted thermoplastic substrate. The advantage of the hot embossing process is that the pattern can be imprinted repeatedly using a mold, thus simplifying the manufacturing process and reducing the cost. However, plastic chips manufactured by such methods have problems in surface modification, which is essential for fixing biomaterials on the chip surface. That is, the conventional plastic chip has a small amount of biomaterial fixed on the surface of the chip, and thus is unsuitable for use in a low concentration sample.

The present invention is to solve the above problems, the technical problem to be achieved by the present invention is to increase the surface area of the biochip to which the biomaterial can be fixed and to improve the efficiency of the reaction.

Technical problems of the present invention are not limited to the above-mentioned technical problems, and other technical problems that are not mentioned will be clearly understood by those skilled in the art from the following description.

Biochip according to the present invention is a substrate; A coating layer formed on the substrate; And nanoparticles fixed to the substrate by the coating layer and to which a material that binds a target material is attached to a surface.

In addition, the substrate may be formed of any one of silicon, glass, metal, inorganic material, polymer, and polymer material.

In addition, the substrate may be made of polydimethylsiloxane (PDMS), polymethyl methacrylate (poly (methyl methacrylate), PMMA), cyclic olefin copolymer (cyclic olefin copolymer, COC), polycarbonate , PC), polyamide (PA), polyethylene (PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene , POM), polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), polybutylene Selected from the group consisting of terephthalate (polybutylene terephthalate, PBT), fluorinated ethylene propylene (FEP), and perfluoro alkoxyalkane (PFA) It may be formed of either one or a mixture thereof.

In addition, the coating layer may be a silica coating layer.

In addition, the silica coating layer may be formed by applying a silica sol containing silica (silica) having a diameter of 5 to 50 nm to the substrate.

In addition, the nanoparticles may be silica nanoparticles.

In addition, the diameter of the nanoparticles (nanoparticle) may be 100 ~ 800 nm.

In addition, the biomaterial further comprises a biological material attached to the surface of the nanoparticles and binds to the target material, wherein the biomaterial is an antibody, enzyme, protein, DNA, RNA, PNA, nucleic acid, At least one of the oligopeptides.

Method for producing a biochip according to the present invention comprises the steps of (a) forming a coating layer on the surface of the substrate; And (b) attaching nanoparticles to the coating layer to attach a material that binds to the target material.

In addition, the step (a) is at least any one of a drop-casting method, a dip-coating method or a method of injecting a sol solution into a channel formed in a predetermined region by a syringe pump. It may include one.

Also, the method may include drying the substrate on which the coating layer is formed between the steps (a) and (b).

In addition, the step (b) may be a step of positioning the nanoparticles using a pipette in the region to which the biomaterial is to be immobilized, or using the ink-jet printing method.

In addition, the coating layer is a silica coating layer, the step (a) may comprise the step of applying a silica sol containing silica (silica) having a diameter of 5 to 50 nm to the substrate.

In addition, the nanoparticles may be silica nanoparticles having a diameter of 100 to 800 nm.

In addition, after the step (b) may further comprise the step of placing a biomaterial on the nanoparticles.

In addition, after the step (b), the amine group, the carboxyl group, the aldehyde group, the maleimide group, the epoxy group 3-aminopropyltriethoxysilane (APTES), glutaraldehyde, in order to introduce functional groups such as thiol group, NHS-ester group (N-hydroxysuccinimide ester group) (glutaraldehyde) and sulfo-SMCC (Sulfosuccinimidyl-4- [N-maleimidomethyl] cyclohexane-1-carboxylate) can be treated.

According to the present invention has an effect that the surface area to which the biomaterial can be fixed is increased. In addition, the hydrophilicity of the hydrophobic surface can be increased by the silica sol coating, thereby facilitating the control of the fluid on the substrate. In addition, the silica nanoparticles enhance the mixing effect of the fluid on the substrate.

The technical effects of the present invention are not limited to the above-mentioned effects, and other technical effects not mentioned will be clearly understood by those skilled in the art from the following description.

FIG. 1A shows a substrate, and FIG. 1B shows a state where silica sol coating is performed on the surface of the substrate. FIG. 1C illustrates a state in which silica nanoparticles are placed on a surface of a substrate coated with silica sol.
FIG. 2A illustrates a state in which an antibody is immobilized on a surface of a substrate on which silica sol coating is not performed, and FIG. 2B illustrates a state in which an antibody is immobilized on a substrate surface on which a silica sol coating is performed, and FIG. 2C illustrates silica nanoparticles. The state in which the antibody is fixed after being placed on the surface of the silica sol coated substrate is shown.
3 is a photograph of a portion (1) subjected to silica sol coating and a portion (2) not subjected to silica sol coating on the substrate surface.
FIG. 4A is a photograph of a surface on which silica particles having a size of about 10 nm are formed by performing a silica sol coating by using a scanning electron microscope (SEM) apparatus, and FIG. 4B is a surface on which a silica sol coating is performed. It is a picture taken about 300 nm size silica nanoparticles are located.
FIG. 5A is a fluorescence image obtained from an experiment using a substrate without silica sol coating and fixation of silica nanoparticles. FIG. 5B is a fluorescence image obtained from an experiment using a substrate which is subjected to silica sol coating and which is not fixed with silica nanoparticles. 5C is a fluorescence image obtained from an experiment using a substrate on which silica sol coating and silica nanoparticles were fixed.
Figure 6 is a graph showing the fluorescence intensity of the fluorescence image obtained from the experiments associated with Figures 5a-5c.
7 is a flowchart illustrating a method of manufacturing a biochip according to an embodiment of the present invention.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, it should be understood that the present invention is not limited to the disclosed embodiments, but may be implemented in various forms, and the present embodiments are not intended to be exhaustive or to limit the scope of the invention to those skilled in the art. It is provided to let you know completely. Shapes of the elements in the drawings may be exaggerated parts for a more clear description, elements denoted by the same reference numerals in the drawings means the same element.

(1) substrate material

Inexpensive plastic polymers can be used to make substrates. Plastic polymers include, for example, poly (dimethylsiloxane), PDMS, polymethyl methacrylate (PMMA), cyclic olefin copolymers (COC), polycarbonates, PC), polyamide (PA), polyethylene (PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (polyoxymethylene, POM), polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), polybutylene tere In the group consisting of phthalate (polybutylene terephthalate (PBT), fluorinated ethylene propylene (FEP), and perfluoro alkoxyalkane (PFA) It may be any one selected.

(2) Preparation of Silica Sol Coating Liquid

Reagents used include tetraethoxysilane (TEOS), dodecyltriethoxysilane (C12TES), anhydrous ethanol and hydrochloric acid solution, using reagents purchased from Sigma Aldrich Co. without pretreatment. It was.

In order to prepare a silica colloid (particularly, silica sol) coating liquid, TEOS (0.036 mol) and C12TES (0.004 mol) were first added to 10 ml of anhydrous ethanol and stirred at 60 ° C. for 2 hours.

Next, in order to hydrolyze the ethoxy group of TEOS with a hydroxy group, hydrochloric acid solution (˜38%; 0.008 mol) is stirred slowly adding dropwise for 10 minutes. Finally, the molar ratio of TEOS: C12TES: H ₂ O: ethanol: HCl was 0.9: 0.1: 4 (x): 9: 0.2. Here, the ratio (x) of water can be changed in the range of 1-10 as the case may be.

Next, the flask is sealed and then reacted at 80 ° C. for 2 hours to finally complete the silica sol solution. The silica included in the silica sol may have a diameter of about 5 to 50 nm, and in this embodiment, a silica colloid including silica having a size of about 10 to 20 nm was used.

(3) silica nanoparticles ( silica nanoparticle Manufacturing

Silica nanoparticles were synthesized using a water-in-oil method. First, 1.77 g of Triton x-100, 1.6 ml of 1-hexanol, and 400 μl of water are added to 7.6 ml of cyclohexane and stirred.

Next, add 100 μl of tetraethyl orthosilicate and 60 μl of ammonia water and stir for 24 hours.

Next, after washing several times with ethanol, acetone, water, the nanoparticles are separated using a centrifuge.

Next, the silica nanoparticles are completed by drying the separated nanoparticles at 80 ° C. for 24 hours using a vacuum oven. The diameter of the silica nanoparticles may be approximately 100 ~ 800 nm, in this embodiment silica nanoparticles having a diameter of about 300 nm was used.

(4) surface treatment of substrate

First, the substrate is immersed in acetone, methanol, distilled water and the like and cleaned with an ultrasonic generator for 5 to 15 minutes. Next, the substrate is subjected to oxygen plasma treatment under a power condition of 100 W for 60 seconds using a plasma surface modification apparatus (S01 in FIG. 7).

(5) silica sol coating

After surface treatment of the substrate is performed, silica sol coating is performed on the surface of the substrate by a drop-casting method at about 1 wt% using the prepared silica sol solution. Alternatively, the coating may be performed by performing a silica sol coating by a dip-coating method, or by forming a channel in a desired portion and then injecting a silica sol solution using a syringe pump. (S02 in Fig. 7)

In more detail, first, the silica sol coating liquid prepared by the aforementioned method is diluted to 1 wt% (weight percentage). Next, 10 μl of the silica sol coating solution is dropped in an area of 5 nm in width and 5 cm in length of a substrate composed of 70% polystyrene and 30% PMMA, followed by drying. The substrate thus prepared was immersed in ethanol or water for several hours, and it was confirmed that the silica sol coating was well fixed. On the other hand, the amount to be treated may vary depending on the sol coating area or area.

FIG. 1A shows a substrate, and FIG. 1B shows a state where silica sol coating is performed on the surface of the substrate.

3 is a photograph of a portion (1) subjected to silica sol coating and a portion (2) not subjected to silica sol coating on the substrate surface.

FIG. 4A is a photograph of a surface on which silica particles having a size of about 10 nm are formed by performing a silica sol coating by using a scanning electron microscope (SEM) apparatus, and FIG. 4B is a surface on which a silica sol coating is performed. It is a picture taken about 300 nm size silica nanoparticles are located.

Coating with a silica sol is preferred, but the coating may also be carried out using other equivalent materials that increase the hydrophilicity of the hydrophobic surface to inhibit non-specific reactions and facilitate the flow of fluid over the surface. have.

(6) surface treatment of substrate

After the silica sol coating is completed, the coated substrate is dried for 10 to 15 minutes at 65 ℃ using a vacuum oven. Next, the substrate is subjected to plasma treatment again. (S03 in Fig. 7)

(7) Fixation of Silica Nanoparticles and Biomaterials

After the surface treatment of the substrate is completed, 5-10 wt% (weight percentage) of the silica nanoparticles are placed by 0.5-1 μl using a pipette in the spot portion where the biomaterial is immobilized (S04 in FIG. 7).

As another example, silica nanoparticles may be positioned using an ink-jet printing method.

Next, the substrate is dried for 10 to 15 minutes at 65 ℃ using a vacuum oven.

FIG. 1C illustrates a state in which silica nanoparticles are placed on a surface of a substrate coated with silica sol.

FIG. 2A illustrates a state in which an antibody is immobilized on a surface of a substrate on which silica sol coating is not performed, and FIG. 2B illustrates a state in which an antibody is immobilized on a substrate surface on which a silica sol coating is performed, and FIG. 2C illustrates silica nanoparticles. The state in which the antibody is fixed after being placed on the surface of the silica sol coated substrate is shown.

2 shows that as the surface area of the substrate increases, the number of immobilized biomaterials (antibodies, enzymes, proteins, DNA, RNA, PNA, nucleic acids, proteins, oligopeptides, etc.) increases.

In order to fix the biomaterial to the spot where the silica nanoparticles are located, a step of introducing a functional group may be performed (S05 of FIG. 7). That is, for example, 3-aminopropyltriethoxysilane (APTES), sulfo-SMCC (Sulfosuccinimidyl-4- [N-maleimidomethyl] cyclohexane-1-carboxylate), glutaraldehyde, etc. Treatment of the reagent with the substrate may be performed.

Next, the step of placing the biomaterial in the spot where the silica nanoparticles are located may be performed (S06 of Figure 7). The biomaterial that can be immobilized on the biochip is a material that can specifically bind to a target material, and may be, for example, an antibody, an enzyme, a protein, DNA, RNA, PNA, nucleic acid, a protein, an oligopeptide, or the like.

While it is preferred to use silica nanoparticles, it is also possible to use nanoparticles made of other materials that serve to increase the surface area of the biochip or increase the mixing of the fluid.

(8) Detection of target substance

When the target material is administered after the biomaterial is fixed to the spot where the silica nanoparticles are located, a biochemical reaction occurs between the biomaterial and the target material. Fluorescent or luminescent materials can be used to confirm the presence or absence of such biochemical reactions. As an embodiment using the fluorescent material, a method of measuring the fluorescent material remaining after the biochemical reaction caused by administering the target material to which the fluorescent material is bound to the biological material using an external light source may be used. As an embodiment using the light emitting material, a method of measuring light emitted from the light emitting material remaining after the biochemical reaction caused by administering the target material to which the light emitting material is bound may be used.

FIG. 5A is a fluorescence image obtained from an experiment using a substrate without silica sol coating and fixation of silica nanoparticles. FIG. 5B is a fluorescence image obtained from an experiment using a substrate which is subjected to silica sol coating and which is not fixed with silica nanoparticles. 5C is a fluorescence image obtained from an experiment using a substrate on which silica sol coating and silica nanoparticles were fixed.

Figure 6 is a graph showing the fluorescence intensity of the fluorescence image obtained from the experiments associated with Figures 5a-5c.

In this example, a reagent such as 3-aminopropyltriethoxysilane (APTES) was treated on a substrate made of a copolymer of 70% polystyrene and 30% PMMA. Next, glutaraldehyde was administered to the surface treated with APTES to introduce a functional group (for example, an aldehyde group) capable of immobilizing a biomaterial. Next, on the surface to which glutaraldehyde was administered, concentrations of 0 ng / ml (zone 1), 10 ng / ml (zone 2), 10 ² ng / ml (zone 3), 10 ³ ng / ml (zone 4), 10 ⁴ ng / ml (region 5) and 10 ⁵ ng / ml (region 6) of mouse IgG were spotted, respectively. Next, a high concentration of BSA was reacted to block the activity of the aldehyde group. 5A-5C show images of biochips obtained by introducing anti-mouse IgG labeled with fluorescent material alexa 555 into a biochip and then using a fluorescent scanner. Figure 6 is a graph recording the average value and the error value of the fluorescence intensity obtained by performing the same experiment three times. Fluorescence intensity was measured using a program called ImageJ, and the relative value of fluorescence intensity was plotted on the vertical axis of the graph.

As can be seen from these experimental results, the fluorescence intensity of the fluorescence image obtained in the experiment using the substrate on which the silica sol coating and the silica nanoparticles were fixed on the substrate surface was the highest. That is, it was confirmed that the reaction efficiency was improved by performing silica sol coating and fixing of the silica nanoparticles.

The target material detectable by the biochip according to the present invention is a material capable of specific binding with a biomaterial. For example, the target material may be an antigen, a protein, a peptide, an oligopeptide, a nucleic acid, a small molecule, a microorganism, a fungus, a DNA, a carbohydrate, a herbal medicine, or the like.

The biochip according to the present invention can be applied to various devices using specific binding between a biomaterial and a target material. For example, it can be used as a protein chip, DNA chip, drug discovery chip, environmental analysis chip, toxicity analysis chip, or food bacteria analysis chip.

An embodiment of the present invention described above and illustrated in the drawings should not be construed as limiting the technical idea of the present invention. The scope of protection of the present invention is limited only by the matters described in the claims, and those skilled in the art will be able to modify the technical idea of the present invention in various forms. Accordingly, such improvements and modifications will fall within the scope of the present invention as long as they are obvious to those skilled in the art.

Claims

Board;
A coating layer formed on the substrate; And
And a nanoparticle fixed to the substrate by the coating layer and having a material attached to a surface thereof attached to a surface thereof.

The method of claim 1,
The substrate is a biochip formed of at least one of silicon, glass, metal, inorganic material, polymer, polymer material.

The method of claim 1,
The substrate is polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), cyclic olefin copolymer (cyclic olefin copolymer, COC), polycarbonate, PC ), Polyamide (PA), polyethylene (PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM) ), Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (polybutylene terephthalate (PBT), fluorinated ethylene propylene (FEP), perfluoro alkoxyalkane (PFA) Or or a bio chip formed from a mixture thereof.

The method of claim 1,
The coating layer is a biochip silica coating layer.

The method of claim 4, wherein
The silica coating layer is formed by applying a silica sol containing silica (silica) having a diameter of 5 to 50 nm to the substrate.

The method of claim 1,
The nanoparticles are silica nanoparticles.

The method of claim 1,
The diameter of the nanoparticles (nanoparticle) is a biochip of 100 ~ 800 nm.

The method of claim 1,
It further comprises a biomaterial attached to the surface of the nanoparticles and binds to the target material,
The biomaterial is at least one of an antibody, an enzyme, a protein, DNA, RNA, PNA, nucleic acid, and an oligopeptide capable of specific binding with a target material.

(a) forming a coating layer on the surface of the substrate; And
(b) attaching nanoparticles to the coating layer to attach a material that binds to a target material.

10. The method of claim 9,
In step (a), at least one of a drop-casting method, a dip-coating method, or a method of injecting a sol solution with a syringe pump into a channel formed in a predetermined region may be used. Method for producing a biochip containing.

10. The method of claim 9,
Method of manufacturing a biochip comprising the step of drying the substrate having a coating layer formed between the step (a) and the step (b).

10. The method of claim 9,
Step (b) is a method of manufacturing a biochip to position the nanoparticles using a pipette in the area to be immobilized biomaterial, or using the ink-jet printing (ink-jet printing) method.

10. The method of claim 9,
The substrate is polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), cyclic olefin copolymer (cyclic olefin copolymer, COC), polycarbonate, PC ), Polyamide (PA), polyethylene (PE), polypropylene (PP), polyphenylene ether (PPE), polystyrene (PS), polyoxymethylene (POM) ), Polyetheretherketone (PEEK), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), polybutylene terephthalate (polybutylene terephthalate (PBT), fluorinated ethylene propylene (FEP), perfluoro alkoxyalkane (PFA) Method for manufacturing a biochip consisting of or or a mixture thereof.

10. The method of claim 9,
The coating layer is a silica coating layer,
The step (a) is a method of manufacturing a biochip comprising the step of applying a silica sol comprising a silica (silica) having a diameter of 5 to 50 nm to the substrate.

10. The method of claim 9,
The nanoparticle (nanoparticle) is a silica nanoparticles (silica nanoparticles) of 100 ~ 800 nm in diameter manufacturing method of the biochip.

10. The method of claim 9,
The method of manufacturing a biochip further comprising the step of placing a biomaterial on the nanoparticles after the step (b).

The method of claim 16,
The biomaterial is at least any one of an antibody, enzyme, protein, DNA, RNA, PNA, nucleic acid, protein, oligopeptide which can achieve specific binding with the target material.