WO2008056837A1

WO2008056837A1 - Method for fabricating a biomaterial array using photoreaction

Info

Publication number: WO2008056837A1
Application number: PCT/KR2006/004646
Authority: WO
Inventors: Min-Gon Kim; Yong-Beom Shin; Bong Hyun Chung; Jun Hyoung Ahn; Hyou-Arm Joung
Original assignee: Korea Research Institute Of Bioscience And Biotechnology
Priority date: 2006-11-07
Filing date: 2006-11-07
Publication date: 2008-05-15

Abstract

The present invention relates to a method for fabricating a biomaterial array using photoreaction. The method comprises modifying a substrate surface with psoralen (PSO) to prepare a substrate having a PSO surface formed thereon, and immobilizing biomaterials (e.g., DNA, proteins, etc.) on the substrate having the PSO surface using photoreaction. According to the present invention, a biomaterial can be immobilized by photoreaction without separate modification of the biomaterial, thus making it possible to fabricate an array in a very simple and cost-effective manner. Also, when a biomaterial is immobilized using focused light, it is possible to fabricate an array having a very small spot size, which is impossible with the prior microarray.

Description

METHOD FOR FABRICATING A BIOMATERIAL ARRAY USING

PHOTOREACTION

TECHNICAL FIELD

The present invention relates to a method for fabricating a biomaterial array using photoreaction, and more particularly to a method for fabricating a biomaterial array, comprising modifying a substrate surface with psoralen (PSO) to prepare a substrate having a PSO surface formed thereon, and immobilizing biomaterials (e.g., DNA, proteins, etc.) on the substrate having PSO surface using photoreaction.

BACKGROUND ART

Biomaterial array technology has been applied to DNA microarrays, protein chips, etc. and is emerging as an important technology in genomics, proteomics and clinical diagnostic studies. To fabricate biomaterial arrays, a step of immobilizing biomaterials on surfaces is necessarily required.

Till now, various methods for immobilizing biomaterials have been developed to fabricate biomaterial arrays. In general immobilization methods for DNA microarrays, amine-terminated DNA is covalently attached to an aldehyde- modified surface. In the case of proteins, methods of covalently attaching proteins to an aldehyde- or carboxyl-modified surface are mainly used.

However, in the above immobilization methods, the following problems have been pointed out. First, DNA modification is required to immobilize DNA on a surface. Generally, a method of immobilizing amine-, thiol- or biotin-terminated DNA on a surface is used. Second, it is impossible to fabricate arrays having a spot size of less than 50 μm. When DNA microarrays are fabricated using a spotting method, pin-based dispensing or inkjet spray method is generally used, and thus it is difficult to control the spot size to 50 μm or less. A technology of controlling the spot size to 10 μm or less is required, because it makes it easy to measure a very small amount of a sample. Third, when the spotting method is used to fabricate arrays, the amount of spotting transferred to a surface is not constant, thus reducing the reproducibility of the arrays.

Thus, in the art, there has been a need to develop a method capable of fabricating a biomaterial array having small spot size in a simple manner without needing separate modification of the ends of a biomaterial for immobilization.

Accordingly, the present inventors have made extensive efforts to develop a method of fabricating a biomaterial array, in which a biomaterial is immobilized on a substrate without needing separate modification of the biomaterial. As a result, the present inventors have found that a biomaterial can be immobilized on a substrate modified with PSO by photoreaction, thereby completing the present invention.

SUMMARY OF INVENTION

Therefore, it is a main object of the present invention to provide a method for fabricating a biomaterial array, comprising modifying a substrate surface with psoralen (PSO) to prepare a substrate having a PSO surface, on which a biomaterial can be immobilized by photoreaction formed thereon, and immobilizing a biomaterial on the substrate having the PSO surface by photoreaction.

Another object of the present invention is to provide a biomaterial array fabricated by said method.

To achieve the above objects, the present invention provides a method for fabricating a biomaterial array, the method comprising the steps of: (a) modifying a substrate surface with psoralen (PSO), thereby preparing a substrate having a PSO surface formed thereon; and (b) bringing a biomaterial into contact with the substrate having the PSO surface, and then irradiating the substrate with light, thereby immobilizing the biomaterial on the substrate.

In the inventive method, the step (a) preferably comprises (i) modifying the substrate with an amine group and then treating the substrate with PSO-NHS, or (ii) modifying the substrate with streptavidin and then attaching PSO-biotin to the substrate. Also, the inventive method further comprises, before the step (a), a step of coating the substrate surface with a hydrophilic material selected from the group consisting of dextran, PEG, cellulose and levan.

Furthermore, a mask is preferably placed in a path of irradiating the light, and the light is preferably focused light having a wavelength of 300-600 nm.

Moreover, the substrate is preferably made of a material selected from the group consisting of glass, quartz, glass wafers, silicon wafers, fused silica, plastics and transparent polymers. Also, the biomaterial is at least one selected from the group consisting of proteins, peptides, amino acids, DNA, PNA, enzyme substrates, ligands, cofactors, carbohydrates, lipids, oligonucleotides and RNA.

In another aspect, the present invention provides a biomaterial array, which is fabricated according to said method and comprises a biomaterial bound to a PSO surface on a substrate.

The above and other objects, features and embodiments of the present invention will be more clearly understood from the following detailed description and accompanying claims. BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 schematically shows a method for fabricating a biomaterial according to the present invention. Reference numerals in FIG. 1 denote the following components:

1 : an initial surface with which a Psoralen (PSO) compound can react; 11: the PSO compound capable of reacting with the surface; 21: a surface modified with PSO 31 : a biomaterial; and 41 : a configuration in which the biomaterial is bound to the PSO surface.

FIG. 2 shows fluorescent images selectively hybridized with SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. In FIG. 2, a: SEQ ID NO: 1, b: SEQ ID NO: 2, and c: SEQ ID NO 3.

FIG. 3 is a fluorescent scanning image of a fabricated antibody microarray with which Alexa Fluor® 488-labelled goat anti-mouse IgGl(γl) reacted.

FIG. 4 is a fluorescence image of a fabricated antibody microarray with which IL-5 selectively reacted.

FIG. 5 is a fluorescence image of an oligonucleotide array having a spot size of less than 10 μm, fabricated using a shadow mask.

DETAILED DESCRIPTION OF THE INVENTION, AND

PREFERRED EMBODIMENTS

The present invention relates to a method for fabricating a biomaterial array, comprising bringing a biomaterial into contact with a substrate having a psoralen (PSO) surface formed thereon, and then irradiating the substrate with light to immobilize the biomaterial on the substrate. Also, the present invention relates to a biomaterial array, which is fabricated by said method and comprises a biomaterial immobilized on a PSO surface on a substrate.

FIG. 1 schematically shows a method for fabricating a biomaterial array. As shown in FIG. 1, a material, which is used to treat a substrate in order to bring a biomaterial into contact with the substrate surface, is a psoralen (PSO) having a structure of Formula 1. [Formula 1]

PSO is generally used in DNA tagging and is known to photoreact with DNA molecules while intercalating with DNA. Also, PSO does not react with water, but efficiently reacts with biomaterials. However, there has not been any attempt to make an array using such a material. The present invention provides a method capable of fabricating various biomaterial arrays by modifying a substrate surface with PSO on the basis of the characteristics of PSO.

The method of modifying a substrate surface with PSO can be approached in various ways. For example, PSO-NHS is allowed to react with an amine- modified surface, or PSO-biotin can be bound to a streptavidin-immobilized surface.

According to the method as described above, a substrate having a PSO surface formed thereon can be used to fabricate a biomaterial array. Specifically, a DNA microarray is fabricated by treating a PSO-modified surface with DNA solution using a microarrayer, irradiating the surface with UV light to allow DNA to react with PSO, and then washing the surface.

A general method of fabricating a DNA microarray requires a process of modifying DNA ends with an amine group or thiol group, but the inventive method as described above has an advantage in that the process of modifying DNA is not required.

PSO binds to not only DNA, but also proteins, and the use of this property of PSO enables a protein array to be fabricated in a very simple manner. According to the same method as the above-described method of fabricating the DNA microarray, an antibody microarray can be fabricated by treating a PSO-modified surface with an antibody using a microarrayer, irradiating the surface with UV light for a given time to allow the antibody to react with PSO, and then washing the surface.

A general method of fabricating a protein microarray has a problem in that the fabricated protein array has reduced protein stability, whereas the present invention has an advantage in that protein activity is not decreased because the irradiation wavelength of UV light and the time of exposure to light are controlled to 300-600 nm and 30 minutes, respectively.

Meanwhile, the method of fabricating the biomaterial array using photoreaction has an advantage in that the irradiation position of light can be easily controlled such that the biomaterial can be immobilized to a specific position. Study results showing that a biomaterial was immobilized to a specific position in a micro flui die channel using photoreaction were presented (Holden et al. Anal. Chem., 76(7): 1838, 2004), and the photoreaction method of the present invention can be applied to microfluidic chips.

In a particular case in which a microarray is fabricated using photoreaction, there is an advantage in that the size of spots can be reduced. Specifically, if UV light is focused by an optical lens, the size of spots can be reduced to less than 500 nm. A biomaterial array having a very small spot size can be fabricated by bringing a specific biomaterial solution into contact with a PSO-modified surface, irradiating light at a desired specific position to be immobilized with the biomaterial, bringing another biomaterial solution into contact with the PSO-modified surface, and then irradiating light to a desired specific position to be immobilized with the biomaterial.

According to the above-described method, it is possible to array various kinds of biomaterials in a very small area. The biomaterial array fabricated in a small area according to the above-described method is very useful for the measurement of a very small amount of a sample, such as the measurement of biomaterials in single cells.

Examples

Hereinafter, the present invention will be described in further detail with reference to examples. It is to be understood, however, that these examples are for illustrative purposes only and are not to be construed to limit the scope of the present invention.

Example 1: Modification with PSO

1-1 : Method using PSO-NHS

A glass slide was immersed in a mixed solution of 95% sulfuric acid and 30% hydrogen peroxide (3:1 v/v) at 60-65 ^°C for 20 minutes, and then washed sequentially with distilled water and ethanol. The washed glass slide was immersed in an ethanol solution of 1% (3~aminopropyl)trimethoxysilane (Aldrich, Milwaukee, WI, USA) at room temperature for about 30 minutes to form a self- assembled monomolecular film thereon, and then the glass slide surface was washed sequentially with ethanol and distilled water and dried with nitrogen gas. A solution of 1% glutaraldehyde (Sigma, St. Louis, MO, USA) in PBS (phosphate- buffered saline, pH 7.4) was allowed to react with the dried glass slide surface for 1 hour to modify the surface with an aldehyde group.

Meanwhile, a solution of 100 mM 2,2' -(ethyl enedioxy)bis(ethylaime) (Aldrich, Milwaukee, WI, USA) in PBS was allowed to react with the aldehyde-modified surface for 1 hour to modify the aldehyde-modified surface with an amine group. The amine-modified surface was allowed to react with a solution of lmg/mL PSO- NHS (succinimidyl-[4-(psoralen-8-yloxy)]-butyrate; Pierce, Rockford, IL, USA) in PBS to form a PSO-modified surface.

1-2: Method using PSO-biotin

A glass slide was immersed in a mixed solution of 95% sulfuric acid and 30% hydrogen peroxide (3: 1 v/v) at 60-65 ^°C for 20 minutes, and then washed clean sequentially with distilled water and ethanol. The washed glass slide was immersed in an ethanol solution of 1% (3-aminopropyl)trimethoxysilane at room temperature for about 30 minutes to form a self-assembled monomolecular film thereon, and then the glass slide surface was washed sequentially with ethanol and distilled water and dried with nitrogen gas. A solution of IM succinic anhydride (Aldrich, Milwaukee, WI, USA) in PBS (phosphate-buffered saline, pH 7.4) was allowed to react with the dried glass slide surface for 1 hour to modify the surface with a carboxyl group.

Meanwhile, a chip having the carboxyl-modified surface was immersed in a solution of 100 mM EDC (l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride) and 25 mM NHS (N-hydroxysuccinimide) in distilled water to activate the chip.

A solution (pH 7.5) of 0.1 mg/mL streptavidin (Pierce, Rockford, IL, USA) in PBS was allowed to react with the surface of the activated chip for 1 hour, and then the chip surface was treated with an aqueous solution (pH 8.5) of IM ethanolamine (Sigma, St. Louis, MO, USA) for 10 minutes to modify the surface with streptavidin. A solution of 0.01 mg/mL PSO-Biotin (EZ-Link® Psoralen-PEO₃- Biotin, Pierce, Rockford, IL, USA) in PBS was allowed to react with the streptavidin-modified surface to form a PSO-modified surface.

Example 2: Fabrication and analysis of DNA microarray

2-1 : Fabrication of DNA microarrays

The glass slide having the PSO surface formed thereon, fabricated in Example 1-1, was placed on a microarrayer (Proteogen, CM-1000, Korea). Meanwhile, each of oligonucleotides (Proligo Primers & Probes) having SEQ ID NOS: 1-3, respectively, was dissolved in TE buffer (10 mM Tris-HCl. 1 mM EDTA, pH 8.0) containing 10% polyethyleneglycol (MW: 200, Sigma, St. Louis, MO, USA), at a concentration of 50μg/m#, and each of the solutions was dispensed in a 384- microwell plate and then transferred into said microarrayer. SEQ ID NO: 1: 5¹-T15-ACTGCTGCCTCCCGTAG-3^l SEQ ID NO: 2: 5^l-T15-AAGGGTTGCGCTCGTTG-3¹ SEQ ID NO: 3: 5'-T15-CTCTCTGTACCAGCCATTGT-3^t

While the internal humidity of the microarrayer was maintained at 75%, the oligonucleotides of SEQ ID NOs: 1-3 were arrayed on the first, second and third lines of the glass slide, respectively, using a pin having a bore diameter of 335 μm.

The glass slide having the oligonucleotides arrayed thereon was irradiated with 365-nm UV light for 30 minutes and then washed with PBST (phosphate buffered saline: 0.01 M phosphate buffer with 0.138 M NaCl, 0.0027 M KCl, 0.05% Tween 20). Meanwhile, an oligonucleotide consisting of 15 T residues was dissolved in TE buffer containing 10% polyethyleneglycol (MW: 200), at a concentration of 50 βg/ml, and the solution was applied to cover the entire surface of the glass slide.

The glass slide was exposed again to 365-nm UV light for 30 minutes, washed sequentially with PBST and DW, and dried with nitrogen gas, thus fabricating DNA microarrays.

2-2: Hybridization analysis

Each of base sequences of SEQ ID NO: 4 (complementary to SEQ ID NO: 1), SEQ

ID NO: 5 (complementary to SEQ ID NO: 2) and SEQ ID NO: 6 (complementary to SEQ ID NO: 3), labeled with Cy5 fluorescent dyes, was dissolved in hybridization buffer (20 mM sodium phosphate, 300 mM sodium chloride and 1 mM EDTA, pH 7.7) at a concentration of 3μg/ml. Each of the solutions was hybridized to each of three DNA microarrays fabricated in Example 2-1 for 30 minutes. Then, the DNA microarrays were washed sequentially with PBST and distilled water at 4 ^°C and dried with nitrogen gas.

SEQ ID NO: 4: Cy5-5'-CTACGGGAGGCAGCAGT-3' SEQ ID NO: 5: Cy5-5'-CAACGAGCGCAACCCTT-3^f SEQ ID NO: 6: Cy5-5'-ACAATGGCTGGTACAGAGAG-3'

After completion of the hybridization, the three chips were analyzed with a fluorescent scanner (see FIG. 2). As a result, as shown in FIG. 2, it could be seen that SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3 were selectively hybridized.

Example 3: Fabrication and analysis of antibody microarrav 3-1 : Fabrication of antibody microarray

The glass slide having the PSO surface formed thereon, fabricated in Example 1-1, was placed on a microarrayer (Proteogen, CM-1000, Korea). Meanwhile, each of BSA, an anti-interleukin-5 antibody and an anti-interferon-gamma antibody was dissolved in TE buffer containing 10% polyethyleneglycol (MW: 200), at a concentration of 0.2 mg/mL. Each of the protein solutions was dispensed in a 384-microwell plate, and then the microwell plate was transferred into said microarrayer.

While the internal humidity of the microarrayer was maintained at 75%, the control BSA solution, the anti-interleukin-5 antibody solution and the anti-interferon- gamma antibody solution were arrayed on the first, second and third lines of the glass slide, respectively, using a pin having a bore diameter of 335 μm.

The glass slide having the proteins arrayed thereon was irradiated with 365-nm UV light for 30 minutes and then washed with PBST. BSA was dissolved in TEA buffer at a concentration of 1 mg/mL and applied to cover the entire surface of the glass slide.

The BSA-treated glass slide was exposed again to 365-nm UV light for 30 minutes, washed sequentially with PBST and DW, and then dried with nitrogen gas, thus fabricating a protein microarray.

In order to examine whether the antibodies were correctly immobilized on the microarray, the fabricated microarray was allowed to react with a solution of 10 μg/mL Alexa Fluor® 488-labelled goat anti-mouse IgGl(γl) (Molecular Probes, Eugene, Oregon, USA) in PBS for 30 minutes. Then, the microarray was washed sequentially with PBST and DW, dried with nitrogen gas and then analyzed with a fluorescent scanner (FIG. 3). As a result, as shown in FIG. 3, it could be seen that the antibodies were immobilized on the second and third lines.

3-2: Analysis of antibody chip

The antibody microarray fabricated in Example 3-1 was treated with a solution of 100 ng/mL IL-5 and 1 mg/mL BSA in PBS for 1 hour to induce antigen-antibody binding.

The chip was treated with an antibody for sensing the biotinylation of IL-5 and O.Olmg/mβ Cy5-labeled Streptavidin in PBS containing 1 mg/mL BSA, such that the Cy5 fluorescent dye was bound to spots in which antigen-antibody binding occurred. Then, the chip was washed sequentially with PBST and DW, dried with nitrogen gas and then analyzed with a fluorescent scanner (see FIG. 4). As a result, as shown in FUG. 4, it could be seen that strong spots appeared on the second line, suggesting that the antigen-antibody reaction selective to IL-5 occurred.

Example 4: Fabrication of DNA array using shadow mask and analysis thereof

The glass slide having the PSO surface formed thereon, fabricated in Example 1, was treated with a solution of 0.1 mg/mL biotinylated Tl 5 oligonucleotide (Proligo Primers & Probes) in TE buffer containing 10% PEG 200 (Polyethylene Glycol 200).

The slide was mounted on a microscope (Carl Zeiss International), and experimental instruments were arranged such that light would be focused on the upper surface of the slide from a mercury lamp through a shadow mask (20μm hole size, 20μmcenter to center) at the iris, a 330-380 nm bandpass filter and a 10x optical lens. In this state, the slide was irradiated with UV light for 30 minutes and then washed with PBST, thus fabricating a DNA array. In order to confirm that the fabricated DNA array was correctly formed, the DNA array was allowed to react with a solution of 10 μg/mL streptavidin-Cy5 in PBS for 10 minutes, and then washed sequentially with PBST and DW, and the fluorescent image thereof was measured (see FIG. 5). As a result, as shown in FIG. 5, it could be seen that an oligonucleotide pattern having small size was formed, and had a spot size of 9.7μm as measured with a fluorescent microscope.

From the above results, it can be found that, when UV light is focused with an optical lens, a biomaterial array having small spot size can be fabricated. Particularly, it can be found that, when confocal light is used, it is possible to fabricate even a nanoarray having a spot size of about 200 nm.

INDUSTRIAL APPLICABILITY

As described and proven in detail above, according to the present invention, a biomaterial array can be fabricated using photoreaction without separate modification of a biomaterial, and thus the fabrication cost of the array can be reduced. Furthermore, when a biomaterial is immobilized using focused light, an array having a very small spot size, which is impossible with the prior microarray, can also be fabricated, thus making it possible to measure a very small amount of a sample.

Although the present invention has been described in detail with reference to the specific features, it will be apparent to those skilled in the art that this description is only for a preferred embodiment and does not limit the scope of the present invention. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims

THE CLAIMS What is Claimed is:

1. A method for fabricating a biomaterial array, the method comprising the steps of: (a) modifying a substrate surface with psoralen (PSO), thereby preparing a substrate having a PSO surface formed thereon; and

(b) bringing a biomaterial into contact with the substrate having the PSO surface, and then irradiating the substrate with light, thereby immobilizing the biomaterial on the substrate.

2. The method for fabricating a biomaterial array according to claim 1, wherein the step (a) comprises:

(i) modifying the substrate with an amine group and then treating the substrate with PSO-NHS, or (ii) modifying the substrate with streptavidin and then attaching PSO-biotin to the substrate.

3. The method for fabricating a biomaterial array according to claim 1, which further comprises, before the step (a), a step of coating the substrate surface with a hydrophilic material selected from the group consisting of dextran, PEG, cellulose and levan.

4. The method for fabricating a biomaterial array according to claim 1, wherein a mask is placed in a path of irradiating the light.

5. The method for fabricating a biomaterial array according to claim 1, wherein said light is focused light having a wavelength of 300-600 nm.

6. The method for fabricating a biomaterial array according to claim 1, wherein the substrate is made of a material selected from the group consisting of glass, quartz, glass wafers, silicon wafers, fused silica, plastics and transparent polymers.

7. The method for fabricating a biomaterial array according to claim 1, wherein the biomaterial is at least one selected from the group consisting of proteins, peptides, amino acids, DNA, PNA, enzyme substrates, ligands, cofactors, carbohydrates, lipids, oligonucleotides and RNA.

8. A biomaterial array, which is fabricated according to the method of any one claim among claims 1-7, and comprises a biomaterial bound to a PSO surface on a substrate.