TECHNICAL FIELD The present invention relates to a method of fabricating a biochip using mi crospotting, and more particularly, to a method of immobilizing an integrated array of probes or probe-attached beads on a surface of a substrate of a biochip.

BACKGROUND ART Biochips are devices with various kinds of probes immobilized on the surface of a chip substrate. Using such biochips, diagnoses of diseases and experiments, such as high throughput screening (HTS), enzyme assay, etc., can be easily performed on a large scale using small amounts of samples.

Most methods of immobilizing probes on a substrate use a glass substrate havin g a surface pretreated with coating materials. For such surface-treated substrates, var ious surface chemical species have been suggested, and a technology using self-asse mbled monolayers has been widely used (Korean Patent Laid-open No. 2003-0038932)

In regard this, to immobilize more probes on the substrate and maintain the acti vity of the probes, a three-dimensional immobilization method has been developed (Gill. I. and Ballesteros, Trends in Biotechnology 18:282, 2000). For example, a Hydrogel ^{r M} coating slide from Packard Bioscience, which has been recently undertaken by Perkin Elmer, a polyethylene glycol hydrogel from Biocept. Inc, a Solgel from LG Chem, etc., a re used for a three-dimensional immobilization method. In particular, the Hydrogel™ coating slide from Packard Bioscience utilizes a tec hnology using a 3-dimensinoal polyacrylamide gel. Optically flat silanized Swiss glass used as a base substrate material is surface-modified with acrylamide polymer to enha nee protein binding and maintain the three-dimensional structure preserving the activity of protein. According to the above-described methods, probes are encapsulated into a 3-di mensional gel microstructure maintaining the activity of the probes in spots of samples.

However, such a 3-dimensional hydrogel structure has pores of tens of nanometers i n size, and thus requires a special mixing device, etc. for analysis. In addition, it takes

time to sufficiently transfer biomolecules into the gel of probes, such as protein or DNA. In particular, these limitations are more serious when supplying a target biomolecule i nto a microchannel of a lab-on-a-chip.

To overcome these drawbacks, a method of immobilizing probes on a substrate via beads having sizes from tens of nanometers to several micrometers has been sugg ested (K. Sato, Adv Drug Deliv Rev., 55, 379 (2003); H. Andersoon, Electrophoresis, 22 , 249 (2001 ); J.-W. Choi, Biomed. Microdevices, 3, 191 (2001 )). According to this met hod, target probes are immobilized on a solid bead support and then introduced into a microchannel of a lab-on-a-chip. The large surface area of 3-demensional beads can be used as an immobilizing surface area so that more biomolecules can be immobilized . Since solid beads, which are easy to handle, are used, chip processibility is improve d.

Representative methods of applying or immobilizing beads on a lab-on-a-chip in elude a method of trapping a bead in a microchannel and a method of using magnetic fi elds (K. Sato, Adv Drug Deliv Rev., 55, 379 (2003); H. Andersoon, Electrophoresis, 22,

249 (2001 ); J.-W. Choi, Biomed. Microdevices, 3, 191 (2001 )). In the method of trappi ng a bead to form a physical barrier in a microchannel of a lab-on-a-chip, the size of the bead is limited to tens of microns or larger to prevent loss of the bead. Therefore, thi s method is unsuitable for manufacturing a lab-on-a-chip on which a predetermined am ount of biological probes has to be immobilized. In the method of using magnetic field s, magnets are installed inside or outside a chip, which inhibits chip miniaturization. In addition, the use of opaque magnetic beads causes limitations in optical measurement.

Methods of introducing or fixing a bead in a microchannel of a lab-on-a-chip incl ude a method of using ultrasonic waves or a laser tweezer. However, this method doe s not facilitate the manufacturing of a low-cost, miniaturized lab-on-a-chip (A. Meng, Tra nsducers, Sendai, Japan, 876 (1999); K. Dorre, Bioimaging, 5, 139 (1997)).

Therefore, a method of directly immobilizing a bead inside or outside a biochip without using an additional external device is required. When immobilizing a bead on a substrate in a biochip without using an external device, electrical binding between the bead and the substrate surface, chemical binding between the bead and the substrate surface, or biochemical binding between the subs trate and the bead to which biochemical materials, such as biotin-streptavidin, are resp

ectively bound, have to be considered (H. Andersson, Electrophoresis, 22, 3876 (2001 ); I. Place, Langmuir, 16, 9042 (2000)). However, in most cases, the binding is not suffi ciently strong, and the bead separates from the substrate surface. In addition, to ensu re strong binding, the substrate must be processed using plasma, UV light, etc., or a ch emical treatment has to be performed to activate the surface of the substrate, which are complicated processes.

DETAILED DESCRIPTION OF THE INVENTION

TECHNICAL PROBLEM The present invention provides a method of fabricating a biochip using microspo tting, the method including fixing probe-attached beads to a surface of a substrate witho ut chemical activation of the substrate.

The present invention also provides a method of immobilizing probes not attach ed to beads on a surface of a substrate.

TECHNICAL SOLUTION

According to an aspect of the present invention, there is provided a method of fa bricating a biochip using microspotting, the method comprising immobilizing probes or p robe-attached beads on a surface of a substrate using an adhesive. The immobilization of the probes or probe-attached beads on the substrate may comprise: preparing an aqueous probe or probe-attached bead suspension containing probes or probe-attached beads in an aqueous medium; preparing a water dispersing a dhesive containing an aqueous medium, an aqueous adhesive, and an emulsifier; mixin g the aqueous probe or probe-attached bead suspension and the water dispersing adh esive to obtain a mixture; and spotting the mixture of the aqueous probe or probe-attac hed bead suspension and the water dispersing adhesive onto the substrate to immobiliz e the probes or probe-attached beads on the surface of the substrate.

A method used to spotting the mixture of the aqueous probe or probe-attached bead suspension and the water dispersing adhesive may be, but is not limited to, ink j etting. Any commonly used spotting method can be used.

In the preparing of the water dispersing adhesive, a main monomer selected fro m the group consisting of butadiene, ethyl acrylate, butyl acrylate, ethylhexyl acrylate, o ctyl acrylate, and a mixture thereof, a comonomer selected from the group consisting of

vinyl acetate, acrylonitrile, acrylamide, styrene, methacrylate, methylacrylate, and a mi xture thereof, and a hydrophilic monomer may be added as the aqueous adhesive into t he aqueous medium together with the emulsifier.

The hydrophilic monomer may be selected from the group consisting of methacr ylic acid, acrylic acid, itaconic acid, hydroxyethylmethacrylate, hydroxypropylmethacrylat e, acrylamide, glycidylmethacrylate, polyethyleneglycol acrylate, polyethyleneglycol met hacrylate, and a mixture of thereof.

In the mixing of the aqueous probe or probe-attached bead suspension and the water dispersing adhesive, a dispersant may be further added. The dispersant can be a water soluble polymer.

Any arbitrary water soluble polymer commonly known to those of ordinary skill in the art can be used as the dispersant. For example, the water soluble polymer may b e selected from, but is not limited to, the group consisting of polyacrylic acid, polymetha crylic acid, polyvinyl alcohol, polyvinyl acetate, polyvinyl pyrrolidone, methylcellulose, ca rboxymethylcellulose, and a mixture thereof.

The substrate on which the probe-attached beads are immobilized may be one of a micro-well, a slide, and a micro-channel of a lab-on-a-chip.

The substrate can be, but is not limited to, a plastic substrate made of a material selected from the group consisting of polymethylmethacrylate (PMMA), polycarbonate ( PC), polystyrene (PS), a cyclic olefin copolymer, polynorbonene, a styrene-butadiene c opolymer (SBC), and acrylonitrile butadiene styrene.

Alternatively, the substrate can be a substrate made of a material selected from the group consisting of glass, silicon, hydrogel, metal, ceramic, and a porous membran e.

ADVANTAGEOUS EFFECTS

As described above, in a method of fabricating a biochip according to the presen t invention, probe-attached beads are directly immobilized on a substrate using an adhe sive, not a physical barrier or an electric field. In other words, a small biochip can be manufactured even using beads to immobilize probes on the substrate and economicall y due to the use of cheap adhesive.

In addition, the method according to the present invention using beads is especi ally useful to a lab-on-a-chip and can be used to detect various kinds of materials in a s

hort time using small amounts of samples in a single chip and diagnose various disease s in a short time.

DESCRIPTION OF THE DRAWINGS FIG. 1 is a diagram illustrating a process of spotting a mixture of an aqueous bea d suspension and a water dispersing adhesive onto a substrate using ink jetting when f abricating a biochip according to the present invention and the state of spots fixed to th e substrate after being dried;

FIG. 2A is a scanning electron microscopic (SEM) photograph showing the mor phology of a spot of a dispersion of beads and an adhesive fixed onto a substrate using ink jetting;

FIG. 2B is a SEM photograph showing particles of the adhesive having a size of 100 nm dispersed between 600 nm beads in the spot of FIG. 2A;

FIG. 3A is a graph of signal-to-noise ratio (SNR) resulting from the measuremen t of autofluorescence of spots jetted onto Corning glasses using the method according t o the present invention in Example 2;

FIG. 3B is a graph of SNR resulting from the measurement of autofluorescence of spots jetted onto polymethylmetacrylate (PMMA) slides using the method according t o the present invention in Example 2; FIG. 4 is a graph of SNR resulting from the measurement of fluorescence indica ting the occurrence of non-specific protein binding in spots jetted using the method ace ording to the present invention in Example 3;

FIG. 5A is a photograph of the results of scanning fluorescent spots after specifi c protein binding to the spots formed using adhesives having different glass transition te mperatures in Example 4 using the method according to the present invention;

FIG. 5B is a graph of quantitated fluorescence of spots measured after specific protein binding to the spots formed using the adhesives having different glass transition temperatures in Example 4 using the method according to the present invention;

FIG. 6A is a photograph of the results of scanning fluorescent spots after specifi c protein binding to the spots formed using different concentrations of adhesive in Exa mple 5 using the method according to the present invention;

FIG. 6B is a graph of quantitated fluorescence of spots measured after specific protein binding to the spots formed using different concentrations of adhesive in Examp

Ie 5 using the method according to the present invention;

FIG. 7 A is a graph of autofluorescence of spots formed using water dispersing a dhesives in Example 6;

FIG. 7B is a comparative graph of autofluorescence of spots formed using water dispersing adhesives #4 and #5 and a blank (PMMA slide) in Example 6;

FIG. 8 is a graph of protein immobilization efficiency of spots of mixtures of vario us water dispersing adhesives and anti-mouse lgG-Cy3 measured in Example 7; and

FIG. 9 is a graph of SNR of spots of the mixtures of various water dispersing adh esives and a protein after immunological reaction to the protein in Example 8.

MODE OF INVENTION

Hereinafter, the present invention will be described in detail. The present invention relates to a method of immobilizing probes on a substrate , which is a main process in the fabricating of a biochip, and more particularly, to a meth od of immobilizing probe-attached beads on a substrate using an adhesive.

Beads having a diameter ranging from tens of nanometers to several micromete rs can be used in the method according to the present invention. In addition, materials of beads include, but are not limited to, polystyrene, polymethylmethacrylate, cellulose

, polyglycidyl methacrylate, etc., which can be appropriately selected according to the ty pes of probes to be immobilized, the character of a substrate material, and the type of t he adhesive used to bind beads. .

The inventors of the present invention provide a method of immobilizing probe-a ttached beads on a surface of a substrate using an adhesive, the method including disp ersing probe-attached beads in an aqueous medium to prepare an aqueous bead susp ension, adding a water dispersing adhesive containing an aqueous adhesive dispersed in an aqueous medium into the aqueous bead suspension and mixing the mixture to obt ain a mixture of the probes or probe-attached beads and the aqueous adhesive, and mi crospotting the mixture onto a surface of a substrate to immobilize the probe-attached b eads on the surface of the substrate in three dimensions. In addition, when preparing the mixture of the probe-attached beads and the aqueous adhesive by mixing the aque ous bead suspension and the water dispersing adhesive, a dispersant may be added to effectively maintain the dispersion of the beads and allow uniform and stable jetting of the mixture using microspotting.

The method of immobilizing probe-attached beads on a substrate using an adhe sive may include: preparing an aqueous bead suspension containing probes or probe-a ttached beads in an aqueous medium; preparing a water dispersing adhesive containin g an aqueous medium, an aqueous adhesive, and an emulsifier; mixing the aqueous be ad suspension and the water dispersing adhesive to obtain a mixture; and spotting the mixture of the aqueous bead suspension and the water dispersing adhesive onto the su bstrate to immobilize the probes or probe-attached beads on the substrate.

The aqueous bead suspension may be prepared by adding and dispersing prob e-attached beads in an aqueous medium. Examples of the aqueous medium include, but are not limited to, any aqueous solvent, for example, water, ethanol, methanol, dime thylformamide (DMF), dimethyl sulfoxide (DMSO), acetone, N-methylpyrrolidone (NMP), etc. However, water is preferred.

To control the drying speed to prevent formation of doughnut-shaped spots and to increase the activity of biological molecules in the spots, a hydrophilic polymer, such as polyethylene glycol, methylcellulose, hydroxylpropyl cellulose, polyvinyl alcohol, poly acrylic acid, etc., may be added into the aqueous bead suspension.

In the preparing of the water dispersing adhesive, the water dispersing adhesive may be obtained by mixing an aqueous adhesive and an emulsifier in an aqueous me dium. Examples of the aqueous medium include, but are not limited to, any aqueous s olvent, for example, water, ethanol, methanol, DMF, DMSO, acetone, NMP, etc. Howe ver, water is preferred.

The aqueous adhesive refers to an adhesive having adhesive properties in an a queous medium. In the preparing of the water dispersing adhesive, a main monomer, a comonomer, and a hydrophilic monomer are added into the aqueous medium as the adhesive material. Any arbitrary main monomer and comonomer widely known to thos e of ordinary skill in the art can be used as the main monomer and comonomer, which a re base materials, of the aqueous adhesive. Examples of the main monomer include, but are not limited to, butadiene, ethyl acrylate, butyl acrylate, ethylhexyl acrylate, octyl acrylate, and a mixture thereof. Examples of the comonomer include, but are not limit ed to, vinyl acetate, acrylonitrile, acrylamide, styrene, methyl methacrylate, methylacryla te, and a mixture thereof. The main monomer provides a target material to be adhere d with soft and adhesive properties, and the comonomer provides the adhesive with tra nsparency, processibility, etc.

The hydrophilic monomer used to prepare the water dispersing adhesive improv es the dispersibility in an aqueous medium and adhesion of the water dispersing adhesi ve. Any arbitrary hydrophilic monomer widely known to those of ordinary skill in the art can be used. Examples of the hydrophilic monomer include, but are not limited to, m ethacrylic acid, acrylic acid, itaconic acid, hydroxyethylmethacrylate, hydroxypropylmeth acrylate, acrylamide, glycidylmethacrylate, polyethyleneglycol acrylate, polyethyleneglyc ol methacrylate, and a mixture of thereof.

The water dispersing adhesive prepared using the materials described above is mixed with the aqueous bead suspension prepared prior to the preparation of the water dispersing adhesive. The mixture of the water dispersing adhesive and the aqueous b ead suspension is spotted onto a surface of a substrate to immobilize the beads on the substrate. To uniformly immobilize the beads on the surface of the substrate, the bea ds must be uniformly and stably dispersed in the mixture. In particular, when using be ads larger than few hundreds of nanometers, the gravity of the beads is larger than the dispersing ability of the beads, making it difficult to form a uniform dispersion, which aff ects the uniformity of bead spots. To uniformly disperse the beads in the mixture, a di spersant may be further added when mixing the aqueous bead suspension and the wat er dispersing adhesive.

Any dispersant known to those of ordinary skill in the art can be used. Exampl es of the dispersant include, but are not limited to, polyacrylic acid, polymethacrylic acid , polyvinyl alcohol, polyvinyl acetate, polyvinyl pyrrolidone, methylcellulose, carboxymet hylcellulose, and a mixture thereof.

After the mixture of the aqueous bead suspension and the water dispersing adh esive is prepared, the mixture is spotted onto a substrate to immobilize the probe-attach ed beads on the surface of the substrate. The spotting process may be performed usi ng any arbitrary spotting method known to those of ordinary skill in the art, for example, using ink jetting.

Ink jetting makes it easier to accurately jet a desired quantity of the mixture of th e aqueous bead suspension and the water dispersing adhesive according to the presen t invention onto the substrate.

After the spotting, spots of the mixture are dried. The drying may be performed using a method commonly used to fabricate a biochip using microspotting. For exam pies, the spots of the mixture may be left at room temperature. The optimal drying tern

perature varies according to materials to be dried. The optimal drying temperature can be 15-35°C for protein, and 15-90 ⁰ C for DNA.

The process of spotting the mixture of the aqueous bead suspension and the w ater dispersing adhesive onto a substrate using ink jetting and the state of spots fixed to the substrate through the drying process are illustrated in FIG. 1. Referring to FIG. 1 , beads 1 with probes 2 attached thereto are immobilized on the substrate via an aqueo us adhesive 3. The beads 1 are continuously connected one to another in three dime nsions via an aqueous adhesive 3.

In the method of immobilizing probe-attached beads on the substrate according to the present invention, various substrates widely used in the biochip field can be used . Examples of substrates include, but are not limited to, a micro-well, a slide substrate, or a micro-channel of a lab-on-a-chip.

Although substrates made of various materials can be used, a substrate made o f a material with a high affinity to the used aqueous adhesive may be used favorably. A substrate made of a material with a high affinity to the used aqueous adhesive can be selected by those of ordinary skill in the art. Examples of the substrate that can be us ed include, but are not limited to, polymethylmethacrylate (PMMA), polycarbonate (PC), polystyrene (PS), cyclic olefin copolymer, polynorbonene, styrene-butadiene copolyme r (SBC), and acrylonitrile butadiene styrene. In addition, a substrate made of a material selected from the group consisting of glass, silicon, hydrogel, metal, ceramic, and a porous membrane can be used.

In the method according to the present invention, probes are initially attached to beads, and then the beads with the probes are immobilized on the substrate. Due to the large surface area of the beads and the large space between the beads, the metho d according to the present invention is suitable to immobilize probes, especially in a mic ro-channel of a lab-on-a-chip. In addition, an array of the bead spots can be effectivel y used as a platform of a protein chip or a gene chip.

The method according to the present invention provides a technology of spottin g tens of spots containing different biological materials onto a single substrate. A bioc hip fabricated using this method can be used to diagnose various kinds of diseases in a short time using small amounts of samples in the single chip as a substitute for a conv entional diagnostic method, such as enzyme immunoassay (EIA). The biochip can be u sed as a new, high-sensitivity diagnostic tool with thousands to tens of thousands times

the sensitivity of conventional devices.

In addition, materials used in the present invention, such as beads, an aqueous adhesive, a dispersant, a plastic substrate, etc., are economical, and the manufacturing costs are low. The method according to the present invention is suitable for mass pro duction of biochips.

The aqueous adhesive used in the present invention may be used to immobilize probes without using beads. In this case, an aqueous solution of probes is mixed wit h the water dispersing adhesive and then spotted onto a substrate to fabricate a biochip . In this case, rather than being fixed to the surfaces of the beads, probes are immobili zed by being three-dimensionally encapsulated in the adhesive.

In a method of fabricating a biochip by spotting a mixture of probes not attached to beads and the water dispersing adhesive onto a substrate, solvents, dispersants, aq ueous adhesive, emulsifiers, reaction conditions, etc., described above in connection wi th the method of fabricating a biochip using the mixture of probe-attached beads and th e water dispersing adhesive, can be used.

Hereinafter, the present invention will be described in detail with reference to the following examples. The following examples are only for illustrative purposes and are not intended to limit the scope of the invention.

Example 1 : Preparation of Water Dispersing Adhesive

167.4 g of deionized water was put into a reactor, and the temperature of the re actor was raised to 75 ⁰ C . When the temperature of the deionized water reached 75 ⁰ C , 10.4 g of butylacrylate, 7.7 g of styrene, 0.18 g of sodium lauryl sulfate (SLS) were add ed to the deionized water. A solution of 0.14 g of potassium sulfate dissolved in 9.0 g of deionized water was added to form polymerized seeds while the temperature of the r eactor was maintained at 75 ^° C .

A suspension of 54.0 g of styrene, 108.2 g of butylacrylate, 1.4 g of allyl methac rylate, 9.7 g of itaconic acid, and 0.27 g of SLS in 167.4 g of deionized water and a solu tion of 0.38 g of potassium sulfate in 18.0 g of deionized water were simultaneously add ed to the above reactor containing the seeds through several times over 3 hours to obta in a water dispersing adhesive.

Example 2: Measurement of Autofluorescence of Spots To examine whether a water dispersing adhesive to be added to an aqueous be ad suspension is autofluorescence, the water dispersing adhesive and the aqueous bea d suspension were spotted onto each of a Corning glass and a polymethyl methacrylate (PMMS) slide for tests.

Water dispersing adhesives #1 through #3 were prepared in the same manner a s in Example 1 using different kinds and amounts of copolymers and hydrophilic mono mers.

Water dispersing adhesive #1 was a mixture containing a copolymer of styrene and butadiene and 10% by weight of itaconic acid and having a glass transition temper ature of 0 ⁰ C. Water dispersing adhesive #2 was a mixture containing a copolymer of s tyrene and butadiene and 12% by weight of itaconic acid and having a glass transition t emperature of 32 ^° C. Water dispersing adhesive #3 was a mixture containing a copoly mer of methyl methacrylate and butadiene and 5.6% by weight of itaconic acid and havi ng a glass transition temperature of 30 ⁰ C . Water dispersing adhesives #4 and #5 were prepared by adding 5.6% by weight of polyethylene glycol and acrylic acid, respectivel y, instead of itaconic acid and had glass transition temperatures of -20 ^° C and 10 ^° C , res pectively.

Bovine serum albumin (BSA)-coated polystyrene beads having a diameter of 60 0 nm were used as beads. The bovine serum albumin-coated polystyrene beads were dispersed in a 1X PBS buffer to prepare an aqueous bead suspension.

Mixed solutions 1 through 3 were prepared by mixing the aqueous bead suspen sion and each of the water dispersing adhesives #1 through #3to be containing 0.2% by weight of the beads and 0.2% by weight of the adhesive to prepare mixed solutions 1 t hrough 3.

50 nl_ of each of the mixed solutions 1 through 3 was spotted onto a Corning gla ss and a PMMA slide, dried at a 70% humidity and 20 ⁰ C for one day or longer, and sea nned at PMT(Photo mutiplier tube) 600 and 430. The intensity of autofluorescence of spots of each of the mixed solutions 1 through 3 was measured. The scanned results are shown in FIGS. 3A and 3B. FIG. 3A is the results for t he Corning glasses, and FIG. 3B is the results for the PMMA slides. Referring to FIGS . 3A and 3B, the signal-to-noise (SNR) values of the spots on the Corning glasses and t he PMMA slides are not greater than 3 on average at both PMT 600 and 430, indicating

that the intensity of autofluorescence of the spots of each of the mixed solutions 1 thro ugh 3 is very low.

This result indicates that the method of immobilization probe-attached beads on a substrate using an adhesive according to the present invention can be used to fabrica te a biochip because the mixtures of the beads and adhesives do not interfere with fluor escence detection after reaction with a target analyte.

Example 3: Non-specific protein binding in spots

To investigate the degree of occurrence of non-specific protein binding, not cau sed by an immunological reaction with an antigen or antibody protein, in the bead spots immobilized on the substrate using adhesives, the following experiment was performe d.

The bead-spotted PMMA slides in Example 2 were used. A blocking reaction a nd an incubation with an antibody were performed on each of the PMMA slides in a hyb ridization chamber, and washing was performed by dipping the PMMA slides in a washi ng solution in a bath.

In particular, the immunological reaction was performed as follows. Blocking reaction was performed with PBS containing 1 % by weight BSA and 0. 05% by weight Tween 20 for 10 minutes. 10OμL of Cy3-labeled (0.01 mg/mL) anti-mou se IgG antibody was added to each of the PMMA slides and reacted for 30 minutes. T he PMMA slides were washed using PBS containing 0.05% by weight Tween 20 and th en PBS. Since the used Cy3-labeled protein does not cause an immunological reactio n with the BSA protein coated on the beads, fluorescent signals detected in the experim ent are derived from non-specific protein binding. After the reaction, the PMMA slides were scanned using an Axon scanner to qua ntitate the non-specific protein binding. The results are shown in FIG. 4. Referring to

FIG. 4, the non-specific protein binding is almost zero when the water dispersing adhe sives #2 and #3 are used. In addition, the SNR value is not greater than 3 when the w ater dispersing adhesive #1 is used, indicating that the non-specific protein binding is n egligible.

From the results described above, it is apparent that only a target analyte, not a ny other materials, adhere to spots on the substrate in a biochip fabricated using an aq ueous adhesive to fix beads to a substrate according to the present invention. That indi

cates that the water dispersing adhesive does not affect the function of the biochip, and thus the present invention can be used to fabricate a biochip.

Example 4: Adhesion Test Using Adhesives having Different Glass Transition Temperatures

The adhesion of beads to the substrate when adhesives having different glass tr ansition temperatures were used was measured.

As in Example 2, anti-CRP monoclonal antibody-coated bead-spotted PMMA sli des were used. An immunological reaction was performed in the same manner as in Example 3, except that Cy3-labeled anti-mouse IgG antibody, which detects the BSA co ated on the beads as an antigen, was added.

After the immnological reaction, the PMMA slides were scanned using an Axon scanner. A photograph of the scanned results is shown in FIG. 5A, and the quantitate d intensities of fluorescence are shown in FIG. 5B. The results in FIGS. 5A and 5B indicate that the adhesion of beads to the substr ate is strong when an adhesive having a glass transition temperature lower than room t emperature is used.

Example 5: Adhesion Test at Different Adhesive Concentrations The adhesion of beads to a substrate according to the concentration of a used a dhesive was tested as follows.

The mixture of water dispersing adhesive 1 and the aqueous bead suspension i n Example 2 was spotted onto a PMMS slide using the same method as used in Examp Ie 2 to obtain the bead-spotted substrate. Here, the concentration of the aqueous adh esive was adjusted to be 0.1 % by weight, 0.05% by weight, 0.01 % by weight, 0.005% b y weight, 0.001% by weight, and 0.0005% by weight in the final mixtures to be spotted. The mixtures containing the different concentrations of aqueous adhesive were spotte d onto a single substrate. Here, the concentration of beads in each of the mixture was constant at 0.1 % by weight. A bead spot containing 0.001 % by weight of the adhesive was observed using a scanning electron microscope. The results are shown in FIGS. 2A and 2B. FIG. 2A is a scanning electron microscopic (SEM) photograph of a spot of the dispersion of the b eads and the adhesive jetted onto a substrate using ink jetting. FIG. 2B is a SEM phot

ograph of the adhesive having a particle size of 100 nm, dispersed among beads havin g a size of 600 nm. A 100-nm particle of the adhesive is indicated by an arrow in FIG. 2B.

After the microscopic observation, an immnological reaction was performed usin g the bead-spotted PMMA slide in the same manner as in Example 4.

The PMMA slide after the immnological reaction was analyzed using an Axon sc anner. A photograph of the scanned results is shown in FIG. 6A, and the quantitated i ntensities of fluorescence are shown in FIG. 6B.

Referring to FIGS. 6A and 6B, when the ratio of the adhesive to the beads on a % by weight basis is equal to or greater than 1/20, spots of the mixture can be maintain ed on the substrate. Preferably, when the ratio of the adhesive to the beads is equal t o or greater than 1/10, the adhesion of spots to the substrate is maintained, and sufficie ntly high immnological reaction signals are detected.

Example 6: Measurement of Autofluorescence of Adhesive

To investigate whether the adhesive can immobilize a biomolecule without bead s, the autofluorescence of the adhesive was measured. Since adhesives form spots w ith various surface morphologies with respect to polymethylmetacrylate (PMMA), even when mixtures containing different adhesives are spotted at the same volume and the s ame concentration, the spots have different sizes and shapes. Therefore, to minimize distortion in the measurement of signals due to spot widths, the total intensity of autoflu orescence of all of the spots of each of the adhesives was measured.

In particular, after measuring the total intensity of spots of each of the water disp ersing adhesives, the total intensity of a blank region having the same size as the area of the spots was measured. The total intensity of the blank region was subtracted fro m the total intensity of spots of each of the water dispersing adhesives. The results of the subtraction are shown in FIG. 7A.

Referring to FIG. 7A, the lowest autofluorescence is exhibited by the water disp ersing adhesive 4. The autofluorescence of the water dispersing adhesive 4 is 1.6 tim es higher than the autofluorescence of the blank region (PMMA) (refer to FIG. 7B).

Example 7: Protein Immobilization Efficiency Test To compare the protein immobilization efficiency between adhesives, 10ug/mL

of anti-mouse lgG-Cy3 was mixed with each of the water dispersing adhesives having a final solid content of 6.4% by weight. Each of the mixtures containing the different wa ter dispersing adhesives was spotted to form an array of 10 spots each having a volum e of 10 nL The arrays were dried at room temperature and a humidity of 50% or great er and washed using 1X phophate buffered saline (PBS) at room temperature for 30 mi nutes while stirring.

The fluorescence of Cyanine 3 in each of the arrays was measured at 532 nm, and the immobilization efficiency was calculated as follows. The results are shown in FIG. 8.

(Fluorescence after washing - Fluorescence of Blank Re gion) x 1 OU

(Fluorescence before washing - Fluorescence of Blank Re gion)

Referring to FIG. 8, most of the water dispersing adhesives, except for water dis persing adhesive 3, exhibits an immobilization efficiency of about 80% at the 6.4% adhe sive concentration.

Example 8: lmmonoassay using Adhesives

An immunoassay was performed using adhesives. In particular, 100 ug/ml_ of anti-CRP polyclonal IgG was mixed with each of the adhesives having a concentration of 6.4% by weight, spotted as an array of spots, and dried at room temperature and a h umidity of 50% or greater in the same manner as in Example 7.

After blocking PMMA slides spotted with the mixtures of each of the water dispe rsing adhesives 1 through 5 and the protein (anti-CRP polyclonal IgG) using 0.5% by w eight BSA in 1X PBS for 5-10 hours, fluorophore Cy-3-conjugated anti-goat polyclonal I gG was spread thereon, reacted at room temperature for 30 minutes, and then washed three times using 1X PBS for 2 minutes each time.

The results of the immunoassay are shown as a signal-to-noise ratio (SNR) in Fl G. 9. In particular, the signal level is a value obtained by subtracting the intensity of flu orescence of a control group from the total intensity of fluorescence of the combination of the adhesive and the protein resulting from the immunoassay. The noise level is a v alue obtained by subtracting the total intensity of a blank from the total intensity of fluor escence of the water dispersing adhesive.

Referring to FIG. 9, the water dispersing adhesive 4 containing polyethylene gly col exhibits the highest SNR. This can be attributed to the increased stability of the fix ed protein resulting from the high biocompatibility of the hydrophilic polyethylene glycol polymer.

While the present invention has been particularly shown and described with refer ence to exemplary embodiments thereof, it will be understood by those of ordinary skill i n the art that various changes in form and details may be made therein without departin g from the spirit and scope of the present invention as defined by the following claims.