WO2007123313A1

WO2007123313A1 - Biochip and method for manufacturing the same

Info

Publication number: WO2007123313A1
Application number: PCT/KR2007/001727
Authority: WO
Inventors: Mi-Kyung Kim; Dong-Kyu Park; Ju-Young Kim; Chang-Woo Park
Original assignee: Advanced Nano Products Co., Ltd.
Priority date: 2006-04-20
Filing date: 2007-04-10
Publication date: 2007-11-01
Also published as: KR100720155B1

Abstract

Disclosed are a biochip and a method for manufacturing the same. The biochip comprises a substrate; a thin metal pattern formed on the substrate by patterning a metal solution with an inkjet printer; and a biomaterial immobilized on the thin metal pattern via a linker. The biochip, in which a thin metal pattern is freely formed through an inkjet printer, allows biomaterials to be formed in various patterns. The thin metal pattern, which can be easily formed in an inkjet printing ejection manner, can be produced in a mass scale. Also, the apparatus for manufacturing the biochip of the present invention is small and inexpensive. Further, thin metal pattern can be formed to a size as small as micrometers so that biomaterials can be integrated at high density. In addition, the method is environmentally friendly because there is no need to carry out a washing process for an etching process. No limitations are imposed on the kinds of the metal that can be used in the present invention, and silver can be used, which results in an improvement in the adhesiveness of the substrate to the thin metal pattern as well as in the immobilization efficiency of biomolecules.

Description

BIOCHIP AND METHOD FOR MANUFACTURING THE SAME

Technical Field

[1] The present invention relates to a biochip and a method of manufacturing the same.

More particularly, the present invention relates to a biochip in which metal thin layers can be patterned on the substrate using an inkjet printer, thereby allowing a variety of fine patterns of biomaterials to be integrated thereon at a high degree, and a method for manufacturing the same, by which the biochip can be produced on a mass scale at low cost. Background Art

[2] The development of biochips is a major thrust of the rapidly growing biotechnology industry, which encompasses a very diverse range of research effort covering nucleic acids (DNA, RNA, & PNA), proteins, enzymes, antigens, antibodies, microorganisms, cells, etc.

[3] Depending on the biomaterials immobilized thereon, there are various biochips. For example, biochips are defined as DNA chips when DNA probes are immobilized thereon, protein chips when enzymes, antigens, or antibodies are immobilized thereon, cell chips when microorganisms or various kinds of cells are immobilized thereon, and neuron chips when neurons are immobilized thereon. In addition, biochips have also evolved into lab chips, on which a collection of pretreatment, biochemical reaction, and data interpretation functions are arranged so as to permit many tests to be automatically conducted, and biosensors which allow a variety of biochemical materials to be detected and analyzed.

[4] Further, a biochip can undergo many reactions with genes in order to obtain genetic information within a short period of time. Moreover, biochips enable researchers to screen large numbers of biological analytes for the diagnosis of various diseases. When subject DNA is applied thereto, for example, a biochip on which cancer-inducing genes are integrated can tell whether the subject retains cancer-associated DNA or not.

[5]

[6] It is very important to immobilize biomaterials on substrates when manufacturing biochips. Various methods are currently used to immobilize biomaterials. For example, silane compounds or aminosilane oligomers, which have amine functional groups, are used as linkers to immobilize probe DNA.

[7] U. S. Pat. No. 5,760,130 discloses a method of immobilizing carboxylated DNA on an aminated glass substrate. Further, aminated DNA is found to be immobilized on isothiocyanated substrates, epoxylated substrates or aldehyded substrates (hereinafter referred to as "aldehyde substrates").

[8] Because they undergo multi-stage reaction pathways, these immobilization methods require a long processing time and show very slow reaction rates, thereby having low yield. Further, the general use of the conventional methods is poor. Recently, a substrate having a metal thin layer formed one surface thereof (hereinafter referred to as a "thin metal pattern substrate") has been preferred. Simpler immobilization methods are applicable to metal thin layer substrates in comparison with polymer- treated substrates, such as aldehyde substrates. In addition, the metal thin layer substrate can be applied to the biosensors and the like, and so the metal thin layer substrate has high general-purpose property.

[9] In general, in a process for manufacturing the metal thin layer substrate, an entire surface of the substrate is coated with metal through a vapor deposition method or a sputtering method, or the metal layer is patterned on the substrate surface by a photolithography method. Biomaterials are immobilized on metal thin layer using a deposition device called a spotter. Biomaterials may be immobilized on the metal thin layer as organic compounds which serve as linkers for self-assembly.

[10] U. S . Pat. Nos. 4,964,972 and 6, 127, 127 disclose the immobilization of biomolecules on metal using an organic compound having a hydrogen sulfide (-SH) or disulfide (-S-S-) group which can form a covalent bond with metal.

[11] Generally, biomolecules are required to be immobilized on substrates in a desired pattern (preferably a micropattern) as well as at high efficiency. Above all, it is important to immobilize biomaterials at a high integration rate within specialized spots on the micrometer scale in the case of DNA chips or protein chips. Biochips are improved in the capacity of decoding genetic information as biomaterials are highly integrated thereon.

[12]

[13] Typically, etching processes have been used to immobilize biomaterials in predetermined patterns. For example, U. S. Pat. No. 5,143,854 discloses the use of photolithography in immobilizing polypeptides on a substrate. However, this photolithographic method requires the construction of masks having respective suitable patterns whenever chips are manufactured, and a washing and masking step for each process. Accordingly, the photolithographic method is complicated and requires expensive apparatus, thus incurring increased costs. In addition, the lithographic method suffers from the disadvantage of limiting pattern designs and not being environmentally friendly.

[14] Korean Pat. Publication No. 2001-0004339 discloses a biochip consisting of a substrate, a metal reflection layer (gold or aluminum) formed on the substrate, and an active layer (silicon oxide), formed on the metal reflection layer, comprising a functional group capable of reacting with biomaterials, and a technique in which the biochip is rotated with a pulse of laser light radiated thereon so as to activate a predetermined region of the active layer, and a biomaterial is immobilized on the activated region to form a pattern. However, this method needs complicated apparatus and processes because the biochip rotating process and the laser irradiating process are conducted in addition to the metal etching process. Also, the biochip rotating process limits the pattern of biomaterials as well as the substrate itself to circular patterns.

[15] Conventional techniques including the aforementioned patents have the problem of being low in the immobilization rate of biomaterials. Particularly, the immobilization of biomaterials on substrates must be followed by washing and drying processes. These post-processes cause biomaterials to fall off of the substrate, lowering the immobilization rate.

[16] In addition, conventional techniques impose a limitation on the kinds of metal constituting the metal thin layer. For example, available metals are limited to gold (Au), platinum (Pt) and aluminum (Al).

[17] According to the study report, it is expected that silver(Ag) has a covalent strength which is higher than those of the above listed metals so that the immobilization rate of silver is high. However, the application of silver (Ag) to products is avoided as its stability is poor due to strong oxidation power. Disclosure of Invention

Technical Problem

[18] Accordingly, the present invention has been made keeping in mind the above problems occurring in the prior art, and an object of the present invention is to provide a biochip which allows a variety of fine patterns of biomaterials to be integrated thereon at a high degree, and a method for manufacturing the same, by which the biochip can be produced on a mass scale at low cost.

[19] It is another object of the present invention to provide a biochip which imposes no limitation on the kinds of the metal to be used and which shows excellent immobilization rates of biomaterials. Technical Solution

[20] In accordance with an aspect of the present invention, there is provided a biochip, comprising: a substrate; a thin metal pattern formed on the substrate; and a biomaterial immobilized to the thin metal pattern via a linker, wherein the thin metal pattern is formed by patterning a metal solution through an inkjet printer, the metal solution containing metal nanoparticles independently dispersed therein.

[21] In the biochip, the thin metal pattern consist of one kind of metal, or an alloy or a mixture of two or more different kinds of metal. No limitations are imposed on the metal used in the present invention. The metal useful in the present invention include gold (Au), platinum (Pt), aluminum (Al), copper (Cu), palladium (Pd) and nickel (Ni) as well as silver (Ag).

[22] In accordance with another aspect of the present invention, there is provided a method for manufacturing a biochip, comprising the steps of: preparing a metal solution in which metal nanoparticles are independently dispersed; patterning the metal solution on a substrate through an inkjet printer to form a thin metal pattern; thermally heating the thin metal pattern to improve adhesiveness between the thin metal pattern and the substrate; bonding a biomaterial to a linker; and immobilizing the linker- coupled biomaterial onto the thin metal pattern.

[23] In accordance with a further aspect of the present invention, there is provided a method for manufacturing a biochip, comprising the steps of: preparing a metal solution in which metal nanoparticles are independently dispersed; patterning the metal solution on a substrate through an inkjet printer to form a thin metal pattern; thermally heating the thin metal pattern to improve adhesiveness between the thin metal pattern and the substrate; bonding a linker to thin metal pattern; and immobilizing a biomaterial to the linker.

[24] In the methods, the thin metal pattern are preferably thermally treated at a temperature of 450⁰C or higher. Upon the heat treatment at such a high temperature, the metal solution is a solution of metal nanoparticles made from an alloy or mixture of a first metal that is able to form a strong covalent bond with the linker and a second metal that provides thermal stability, with the nanoparticles being independently dispersed in the metal solution.

[25] In accordance with the present invention, the biochip in which the thin metal pattern is freely formed using an inkjet patterning method allows biomaterials to be formed in various patterns. The thin metal pattern can be easily formed in a simple inkjet printing manner so that the chip can be produced on a mass scale at low cost. Further, thin metal pattern can be patterned to a size as small as micrometers so that biomaterials can be integrated at high density. No limitations are imposed on the kinds of the metal useful in the present invention, and silver can be used, which results in an improvement in the adhesiveness of substrate to the thin metal pattern as well as in the immobilization efficiency of biomolecules. Advantageous Effects

[26] As described hitherto, a biochip in which a thin metal pattern is freely formed using an inkjet patterning method allows biomaterials to be formed in various patterns. The thin metal pattern which can be easily formed in an inkjet printing manner can be produced on a mass scale. Also, the apparatus for manufacturing the biochip of the present invention is small and inexpensive. Further, the thin metal pattern can be formed to a size as small as micrometers so that biomaterials can be integrated at high density. In addition, the method is environmentally friendly because there is no need to carry out a washing process for an etching process. [27] No limitations are imposed on the kinds of the metal useful in the present invention, and silver can be used, which results in an improvement in the adhesiveness of substrate to the thin metal pattern as well as in the immobilization efficiency of biomolecules.

Brief Description of the Drawings [28] The above and other objects, features and other advantages of the present invention will be more clearly understood from the following detailed description taken in conjunction with the accompanying drawings, in which: [29] FIG. 1 a graph showing the particle size distribution of Ag/Pd nanoparticles according to an embodiment of the present invention; [30] FIG. 2 is a TEM photograph of Ag/Pd nanoparticle dispersion according to an embodiment of the present invention; [31] FIG. 3 is a SEM photograph of an Ag/Pd-patterned substrate according to an embodiment of the present invention; [32] FIG. 4 is a scanned image of DNA immobilized on the Ag/Pd-patterned substrate according to an embodiment of the present invention; [33] FIG. 5 is a scanned image of DNA hybridized on the Ag/Pd-patterned substrate according to an embodiment of the present invention; [34] FIG. 6 is a scanned image of DNA hybridized on a conventional substrate

(Au-coated substrate); [35] FIG. 7 is a scanned image of DNA hybridized on a conventional substrate (aldehyde substrate); and [36] FIG. 8 is a graph showing the performance of DNA chips according to the kind of linker.

Best Mode for Carrying Out the Invention

[37] A detailed description will be given of the present invention, below.

[38] In accordance with an aspect thereof, the present invention pertains to a biochip comprising a substrate as a support, a thin metal pattern formed on the substrate, and a biomaterial immobilized to the thin metal pattern via a linker. [39] Any solid substrate that can be printed with an inkjet printer can be used in the present invention. Examples of the substrate useful in the present invention include plastic substrates made of polypropylene, polyacryl amide, polycarbonate, polytetraflu- oroethylene, or polystyrene; silicon or silicon oxide substrates, glass substrates (modified surface); and paper substrates. The paper substrates may be printing paper.

[40] The metal solution is patterned through an inkjet printer to form a thin metal pattern.

No limitations are imposed on the pattern of the thin metal pattern. For example, the thin metal pattern may be formed into various shapes including lines, circles, triangles, rectangles, etc. depending on the use purpose and the degree of integration of the bio- materials. The circular, triangular or rectangular shaped metal patterns may be arranged in the form of a grid array. A diameter of the metal pattern(in a case where the thin metal pattern has a circular shape) or a length of one side of the thin metal pattern (in a case where the thin metal pattern has a triangular or rectangular shape) may be at least 5 nm. For example, the length may range from 20 to 1,000 μm upon the fabrication of DNA chips. In addition, the thin metal pattern may have a thickness of at least 5 nm to several millimeters.

[41] As long as it is able to be patterned through an inkjet printer, any metal solution may be used in the present invention. The metal solution ranges in viscosity from 1 to 100 mPaDs and preferably from 1 to 50 mPaDs and in surface tension from 25 to 80 mN/m and preferably from 30 to 60 mN/m, which is the range within which patterning can be freely conducted using an inkjet printer.

[42]

[43] The metal solution used in the present invention comprises metal nanoparticles (A)

100 nm in size or smaller and a dispersion medium (B). The metal nanoparticles (A) are not particularly limited, but are made of a material selected from a group consisting of gold (Au), platinum (Pt), aluminum (Al), copper (Cu), palladium (Pd), nickel (Ni), silver (Ag), combinations thereof, and alloys of at least two thereof.

[44] The metal nanoparticles (A) are 100 nm or less in size, for example, ranging from 1 nm to 100 nm. Metal nanoparticles (A) exceeding 100 nm are likely to plug up the nozzle of an inkjet printer. Preferably, the metal nanoparticles (A) are 50 nm or less in size so that inkjetting can be smoothly performed.

[45] Adapted to independently disperse the metal nanoparticles (A) without aggregation, the dispersion medium comprises a dispersant (B-I) and a solvent (B-2). The dispersant (B-I) is an organic compound capable of forming a complex on the surface of the thin metal pattern. Examples of the dispersant (B-I) useful in the present invention include alkyl amine, carboxylic acid amide, aminocarboxylic acid salts, and sodium citrate. In order to sufficiently disperse the metal nanoparticles (A) in the solvent (B-2), the alkyl group of the alkyl amine has 4 to 20 carbon atoms, and preferably 4 to 12 carbon atoms. Alternatively, the dispersant (B-I) may be polyvinylpyrrolidone (PVP), which ranges in molecular weight from 1,000 to 40,000 and preferably from 10,000 to 20,000, or polyvinyl alcohol (PVA), which ranges in molecular weight from 1,000 to 40,000 and preferably from 10,000 to 20,000. On the other hand, the dispersant is a commercially available one, such as that identified as BYK-108, BYK-1000, or BYK-antiterra-U, from BYK, Germany, or mixtures thereof. The solvent (B-2) of the dispersion medium (B) may be at least one selected from among non-polar hydrocarbons having 6-20 carbon atoms, water, cellosolves, and polar alcohols.

[46] The metal solution, in which the metal nanoparticles (A) having a size of 100 nm or less are independently dispersed by the dispersant (B-I), can be applied to an inkjet printer for patterning. Depending on inkjet patterning, the thin metal pattern may be formed into various shapes irrespective of size and thickness. The metal nanoparticles (A) useful in the present invention are not limited to particular kinds. For example, silver (A), which has been not used owing to the high oxidative power thereof, can be used in the present invention. This is, in our opinion, attributable to the fact that the dispersant (B-I) functions to prevent the oxidation by forming a coat on the silver surface.

[47] In the metal solution, optionally, metal oxide nanoparticles (C) and/or partially poly- condensed metal oxide nanoparticles (D) may be independently dispersed together with the metal nanoparticles (A). In greater detail, the metal solution preferably comprises the metal nanoparticles (A), and metal oxide nanoparticles (C) and/or partially polycondensed metal oxide nanoparticles (D) in a dispersion medium (B).

[48] A prerequisite for the high immobilization efficiency of biomaterials is attachment between the substrate and the thin metal pattern. The metal oxide nanoparticles (C) or the partially polycondensed metal oxide nanoparticles (D) are adapted to provide high adhesiveness between the substrate and the thin metal pattern.

[49] Like the metal nanoparticles, both the metal oxide nanoparticles (C) and the partially polycondensed metal oxide nanoparticles (D) have a size of 100 nm or less, for example, from 1 to 100 nm. Preferably, their size is not more than 50 nm, in order that inkjetting can be efficiently conducted.

[50] The metal oxide nanoparticles (C) are selected from a group consisting of oxides of silicon (Si), magnesium (Mg), yttrium (Y), cerium (Ce), titanium (Ti), zirconium (Zr), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), neodymium (Nd), copper (Cu), silver (Ag), zinc (Zn), aluminum (Al), gallium (Ga), indium (In), tin (Sn), antimony (Sb), and combinations thereof. Examples of the metal oxide nanoparticles include silica (SiO2), tin oxide (SnO2), indium oxide (In2O3), titanium oxide (TiO2), zinc oxide (ZnO2), antimony oxide (Sb2O3), magnesium oxide (MgO), calcium oxide (CaO) and iron oxide (FeO2).

[51] The partially polycondensed metal oxide nanoparticles (D) may be at least one metal alkoxide represented by the following General Formula 1 or may be a polycondensate of one or more of the metal alkoxides. [52]

[53] [General Formula 1]

[54] M(OR)n

[55] (wherein M is one selected from among Si, Sn, In, Ti, Zn, Mg, Ca, and Sb, R is hydrogen or a hydrocarbon having at least one functional group (alkyl, aryl, etc.), and n is an integer of 1 ~ 10.)

[56]

[57] Also, the partially polycondensed metal oxide nanoparticles (D) may be a poly- condensed polymer represented by the following general formula 2.

[58]

[59] [General Formula 2]

[60] MxOy(OR)Z

[61] (wherein, M is one selected from among Si, Mg, Y, Ce, Ti, Zr, V, Cr, Mn, Fe, Co,

Ni, Nd, Cu, Ag, Zn, Al, Ga, In, Sn, and Sb, R is hydrogen or a hydrocarbon having at least one functional group (alkyl, aryl, etc.), and x, y and z are integers or decimals larger than zero.)

[62] In a preferred embodiment of the present invention, the metal solution comprises the metal nanoparticles independently dispersed therein, wherein the metal nanoparticles consist of at least two different kinds of metal nanoparticles. In this regard, the metal solution comprises metal nanoparticles (A) and a dispersion medium (B), the metal nanoparticles (A) being made of an alloy or a mixture of a first metal that is able to form a strong covalent bond with the linker and a second metal providing thermal stability. More preferably, the second metal does not vaporize, even at 450⁰C or higher. The first metal may be selected from among silver (Ag), gold (Au), platinum (Pt) and copper (Cu), and the second metal may be selected from among palladium (Pd) and nickel (Ni).

[63] In the case where the metal nanoparticles (A) consist of an alloy or a mixture of the first metal and the second metal, the metal solution is patterned and then thermally treated at a temperature of 450⁰C or higher in accordance with a preferred embodiment of the present invention. When the metal nanoparticles (A) do not exist as an alloy but are in the form of a mixture of two different kinds of metal nanoparticles dispersed independently, the mixture changes into an alloy during the post-patterning heat treatment.

[64] In accordance with a preferred embodiment, the metal nanoparticles (A) are composed of silver (Ag) alone or Ag in combination with at least one other metal as an alloy or a mixture thereof, because silver has a high covalent bonding strengh so as to increase the immobilization rate of biomaterials. More preferably, the metal nanoparticles are made of an alloy of silver (Ag) and palladium (Pd) (Ag/Pd alloy) or a mixture of Ag and Pd.

[65] In the case wherein the metal nanoparticles (C) are composed of an alloy of the first metal, which is able to form a strong covalent bond with the linker, and the second metal, which provides thermal stability, the post-patterning heat treatment can be conducted at a temperature of 450⁰C or higher, preferably at a temperature of 650⁰C. Typically, evaporation takes place during heat treatment at such a high temperature, but the second metal functions to prevent the evaporation, thereby making heat treatment possible. The heat treatment at such a high temperature remarkably increases the adhesiveness to the substrate.

[66] The metal solution can be prepared by mixing a dispersion of the metal nanoparticles

(A) with a mixture or a dispersion of the metal oxide nanoparticles (C) and/or the partially polycondensed metal oxide nanoparticles (D). For this preparation, a liquid phase reduction method may be useful. In this regard, the solid content (A, A+C, A+D, or A+C+D) is allowed to amount to 1 to 70 % by weight based on the total weight of the solution, and preferably to 10 to 55 % by weight, so that the solution ranges in viscosity from 1 to 100 mPaDs and in surface tension from 25 to 80mN/m. Accordingly, the metal solution exhibits features of an ink capable of being patterned by an inkjet printer.

[67] In the case where the metal nanoparticles (A) are Ag/Pd alloy or mixture nanoparticles, the second metal (for example, Pd) is preferably contained in an amount of 0.05 ~ 50 % by weight based on the total weight of the entire metal (for example, Ag+Pd), and more preferably in an amount of 0.1 ~ 50 % by weight. When the content of Pd is less than 0.05 % by weight, evaporation takes place upon heat treatment at a temperature of 450⁰C or higher, degrading the adhesiveness. If Pd content exceeds 50% by weight, a bonding strength between the linker and silver(Ag) can be decreased due to a low bonding strength of Pd. When the Pd content increases to 0.1 % by weight or higher, the evaporation phenomenon disappears, with a significant improvement in thermal stability.

[68] The metal oxide nanoparticles (C) and/or the partially polycondensed metal oxide particles (D) are preferably used in an amount of 0.01 ~ 30 % by weight on the basis of the total content of the solid. For example, the content of C and/or D (C, D or C+D) preferably amounts to 0.01 ~ 30 % by weight in the total content (A+C, A+D or A+C+D) and more preferably to 0.1 ~ 10 % by weight. If the weight ratio of the oxide nanoparticles (C and/D) to the metal nanoparticles (A) is too high, the adhesiveness to the substrate is increased, whereas the bonding strength to the linker can be decreased.

[69] After the metal solution as described above is patterned on a substrate, the heat treatment process is performed to form various shaped metal pattern. The heat treatment process may be conducted at a temperature of 60⁰C or higher, and preferably at a temperature of 120⁰C. When the metal solution comprises an alloy or a mixture of the first metal and the second metal, the heat treatment is conducted at a temperature of 450 ⁰C or higher, for example, up to 650⁰C. For the heat treatment, hot-air blowing or oven heating may be used.

[70] Onto the thin metal pattern, biomaterials are immobilized via a linker. As long as it is able to form covalent bonds with both metal and biomaterial, any linker may be used in the present invention. The linker may be selected from organic compounds which are capable of self-assembling. For example, an organic compound having a sulfhydryl group (-SH) or a disulfide group (-S-S-), such as alkane thiol, may be used as a linker. Preferable is an organic compound having 6 - 24 carbon atoms.

[71] The immobilization of a biomaterial to the thin metal pattern with a linker interposed between biomaterial and the thin metal pattern may be achieved by first bonding the biomaterial to the linker and then reacting the linker with the thin metal pattern, or vice versa.

[72] The immobilization may be achieved by spray coating, spotting with a spotter, or depositing. For the reaction of a linker-bonded biomaterial with the thin metal pattern, in detail, the linker-bonded biomaterial is applied over the thin metal pattern by spraying or spotting, or the thin metal pattern is immersed in a solution of the linker- bonded biomaterial. The reaction is preferably conducted for a period ranging from 1 ~ 20 hrs. In addition, when the immobilization is conducted by bonding a linker onto the thin metal pattern and then reacting the linker, bound to thin metal pattern, with a biomaterial, a spin-coating method, a spray coating method, a spotting method using a spotter or a depositing method is useful.

[73] The pattern of the biomaterial is dependent on thin metal pattern. When immobilized by spotting, spin-coating, spray coating, or depositing, the biomaterial can have the same pattern as the thin metal pattern. The thin metal pattern can be formed to have the size of several micrometers to several nanometers (in thickness or diameter) using an inkjet printer. Accordingly, the biomaterial can be formed in a micro pattern as small as the micrometer to nanometer scale with a high degree of integration.

[74] As used herein, the term "biomaterials" is intended to include materials originating from organisms or extracellularly prepared equivalents thereof, such as DNA, RNA, PNA, proteins, enzymes, antigens, antibodies, cells (neurons), and microorganisms. Mode for the Invention

[75] Having generally described this invention, a further understanding can be obtained by reference to certain specific examples which are provided herein for purposes of illustration only and are not intended to be limiting unless otherwise specified.

[76] First, when metal nanoparticles are dispersed by the dispersant, the metal solution was analyzed for adhesiveness according to the composition so as to examine whether, even if devoid of cohesion, it could be patterned using an inkjet printer. In this regard, a silver (Ag) solution was used to determine the applicability of silver to biochips. This examination was carried in the following Examples 1 to 7.

[77] Next, the silver (Ag) solution was finely patterned on substrates to fabricate biochips, which were examined for commercial applicability and immobilization efficiency with reference to conventional biochips in Examples 8 and 9.

[78] Biochips were also analyzed for performance according to linker in Example 10.

[79]

[80] [EXAMPLE 1]

[81] <Preparation of a Dispersion of Ag Nanoparticles>

[82]

[83] 236.247 g of silver nitrate (AgNCβ) was dissolved in tertiarily distilled water to prepare an aqueous silver nitrate solution comprising Ag in an amount of 30 % by weight. To the silver nitrate solution was added polyvinylpyrrolidone (PVP, Mw=40,000) in an amount of 10 % by weight of the metal (Ag) (Ag:PVP=0.1:l) in a nitrogen atmosphere. Stirring was conducted until the polymer was completely dissolved and the solution was maintained at a temperature of 60⁰C. To the resulting solution was added 4 liters of a mixture of 2 parts of ethanol and 8 parts of decane, followed by the reduction of the metal in the presence of the reducing agent potassium borohydride in an amount twice as much as the number of moles of the metal to produce a dispersion of Ag nanoparticles. To this dispersion was further added BYK- 108 (BYK, Germany) and PVP. Following the completion of the reduction, the dispersion of 6 liter, was found to have Ag nanoparticles uniformly dispersed therein.

[84] Then, the dispersion was changed in polarity through the addition of ethanol thereto so as to separate the Ag nanoparticles therefrom. The Ag nanoparticles were washed several times with distilled water and acetone to remove impurities therefrom. The nanoparticles thus purified were dispersed in a mixture of hexane, decane and toluene. Ag nanoparticles were found to have a diameter of about 3-7 nm and to be uniformly dispersed. The resulting dispersion was measured to comprise the Ag nanoparticles in an amount of 53.4 % by weight based on the total weight thereof, and to have a viscosity of 8.7mPaDS at 25°C, which permits inkjet patterning. Even after being allowed to stand for 30 days at room temperature, the dispersion remained un- precipitated.

[85]

[86] <Specimen precipitation>

[87] The Ag nanoparticle dispersion prepared as mentioned above was patterned through a InkJet Model 70 system available from Litrex, U.S.A. on which Spectra SE need available from spectra, U.S.A. was mounted. InkJet printing was conducted twice to form patterns which had a total length of 1,160 mm and a thickness of 70 ~ 90 μm. The dispersion was observed to be effectively ejected without plugging the nozzle and then patterned.

[88] Next, the pattern metal wire was thermally treated at 250⁰C for 30 min or 560⁰C for

20 min.

[89]

[90] <Test for adhesiveness and thermal stability>

[91] The specimen thus prepared was tested for adhesiveness and thermal stability. An adhesiveness test was conducted in such a manner that the metal wires formed by the patterning/thermal treatment were observed with the naked eye to determine the degree of damage when a pressure sensitive tape (commercially available from 3M, U.S.A.) was applied to the metal wires and peeled off. In a test for thermal stability, the light transmittance of the specimen was measured before and after the patterning/heat treatment to examine whether evaporation took place at high temperatures.

[92] The results are shown in Tables 1 and 2, below.

[93]

[94] [EXAMPLE 2]

[95] <Ag/Pd Nanoparticle Dispersion 1>

[96]

[97] First, a dispersion of Pd nanoparticles was prepared in a manner similar to that of

Example 1, with the exception of using Pd(NCβ). In the dispersion, Pd nanoparticles were found to have a diameter of about 5-10 nm and to be uniformly dispersed while remaining completely isolated from each other. The resulting dispersion was measured to comprise the Pd nanoparticles in an amount of 45 % by weight, based on the total weight thereof, and to have a viscosity of 13.4 mPaDS at a temperature of 25°C. Even after being allowed to stand for 30 days at room temperature, the dispersion remained stable without precipitation.

[98] Next, the Pd nanoparticle dispersion was mixed with the Ag nanoparticle dispersion of Example 1 in such a manner that Pd was contained in an amount of 0.3 % by weight based on the total weight of the metal (Ag+Pd). FIG. 1 is a graph showing the particle size distribution of the Ag/Pd nanoparticles, obtained using a particle size analyzer (Model UPA- 150, Mirotrek Japan). HG. 2 is a TEM photograph of the Ag/Pd nanoparticle dispersion.

[99] As in Example 1, the resulting dispersion was patterned and thermally treated, followed by testing for adhesiveness and thermal stability. The dispersion was freely ejected without plugging up the nozzle. No evaporation was detected upon heat treatment at a temperature of 250⁰C and 560⁰C. The results are given in Tables 1 and 2, below.

[100]

[101] [EXAMPLE 3]

[ 102] < Ag/Pd Nanoparticle Dispersion 2>

[103]

[104] A mixture of the Pd nanoparticle dispersion and the Ag nanoparticle dispersion was prepared in a manner similar to that of Example 2, with the exception that Pd was contained in an amount of 30 % by weight based on the total weight of the metal (Ag+Pd).

[105]

[106] As in Example 1, the resulting dispersion was patterned and thermally treated, followed by testing for adhesiveness and thermal stability. The dispersion was freely ejected without plugging up the nozzle. No evaporation was detected upon heat treatment at a temperature of 250⁰C and 560⁰C. The results are summarized in Tables 1 and 2, below.

[107]

[108] [EXAMPLE 4]

[ 109] < Ag/Pd/SiO2 Nanoparticle Dispersion 1 >

[HO]

[111] To the Ag/Pd nanoparticle dispersion prepared in Example 2, silica sol comprising silica particles with a diameter of 50 nm or less (available from Snowtex, Nissan Chemical, Japan) was added in an amount of 3% by weight based on the total weight of the metal (Ag+Pd) to produce an Ag/Pd/SiO2 nanoparticle dispersion in which silver (Ag), palladium (Pd), and silica (SiO2) were independently dispersed.

[112] As in Example 1, the resulting dispersion was patterned and thermally treated, followed by testing for adhesiveness and thermal stability. The dispersion was freely ejected without plugging up the nozzle. No evaporation was detected upon heat treatment at a temperature of 250⁰C and 560⁰C. The results are summarized in Tables 1 and 2, below.

[113]

[114] [EXAMPLE 5]

[115] <Ag/Pd/SiO2 Nanoparticle Dispersion 2>

[116]

[117] To the Ag/Pd nanoparticle dispersion prepared in Example 3, silica sol comprising silica particles with a diameter of 50 nm or less (available from Snowtex, Nissan Chemical, Japan) was added in an amount of 3% by weight based on the total weight of the metal (Ag+Pd) to produce an Ag/Pd/SiO2 nanoparticle dispersion in which silver (Ag), palladium (Pd) and silica (SiO2) were independently dispersed. [118] As in Example 1, the resulting dispersion was patterned and thermally treated, followed by testing for adhesiveness and thermal stability. The dispersion was freely ejected without plugging up the nozzle. No evaporation was detected upon heat treatment at a temperature of 250⁰C and 560⁰C. The results are summarized in Tables 1 and 2, below.

[119]

[120] [EXAMPLE 6]

[121] <Ag/SiO Nanoparticle Dispersion>

[122]

[123] To the Ag nanoparticle dispersion prepared in Example 1, silica sol comprising silica particles with a diameter of 50 nm or less (available from Snowtex, Nissan Chemical, Japan) was added in an amount of 3% by weight based on the total solid content to produce an Ag/SiO nanoparticle dispersion in which silver (Ag) and silica (SiO2) were independently dispersed.

[124] As in Example 1, the resulting dispersion was patterned and thermally treated, followed by testing for adhesiveness and thermal stability. The dispersion was freely ejected without plugging up the nozzle. No evaporation was detected upon heat treatment at a temperature of 560⁰C. The results are summarized in Tables 1 and 2, below.

[125]

[126] [EXAMPLE 7]

[ 127] <Partially Poly condensed Dispersion of Ag/Pd/SiO2 Nanoparticles>

[128]

[ 129] 20 g of tetraethoxysilane (TEOS) (TSL8124, Toshiba Chemical, Japan) was added 20 g of hexyltrimethoxysilane (TSL8241, Toshiba Chemical, Japan) with stirring. 20 g of dodecane and 10 g of a 20 wt% sodium hydroxide solution were then added, followed by ball milling to prepare partially polycondensed silica. After the removal of the aqueous layer, the partially polycondensed silica, contained in the organic layer, was concentrated to 30 % by dry weight. The partially polycondensed silica thus prepared was observed to be uniformly dispersed in a solvent.

[130] The partially polycondensed silica was added to the Ag/Pd nanoparticle dispersion in an amount of 3 % by weight on the basis of the weight of the solid content. This was sufficiently stirred in tetradecane, which served as a dispersion medium. The resulting dispersion was measured to comprise Ag/Pd metal in an amount of 51.8 % by weight and to have a viscosity of 13.4 mPa.S at a temperature of 25°C with Pd measuring 0.5 % by weight of the total metal.

[131] As in Example 1, the resulting dispersion was patterned and thermally treated, followed by testing for adhesiveness and thermal stability. The dispersion was freely ejected without plugging up the nozzle. No evaporation was detected upon heat treatment at a temperature of 250⁰C and 560⁰C. The results are summarized in Tables 1 and 2, below.

[132] Table 1 [Table 1] [Table ] <Test for Adhesiveness of Metal Wires>

[133] In Table 1, X stands for falling off at 80 % or greater, Δ for falling off at 10 ~ 20 %,

O for falling off at less than 10 %, and © for not falling off. [134] Table 2

[Table 2]

[Table ]

[135] In Tables 1 and 2, "S" represents silica and "PcS" represents silica poly condensed compound.

[136] [137] Some of Examples 1 to 7 are not good in adhesiveness or thermal stability, as assayed in the taping test at a temperature of 560⁰C, but the results are sufficient enough to apply to biochips.

[138] As understood from data of Tables 1 and 2, when metal nanoparticles (Ag, Pd) are dispersed with a dispersant, the dispersion, even if devoid of cohesion, can be freely patterned by an inkjet printer. Also, metal oxide nanoparticles (silica) or partially poly- condensed metal oxide nanoparticles (silica polycondensate) can improve the adhesiveness of the metal wires to substrates (e.g. glass).

[139] In addition, the metal nanoparticles, when mixed with metal having high thermal stability, showed highly improved adhesiveness to substrates (glass).

[140] [141] [EXAMPLE 8] [142] <Preparation of Ag/Pd-Patterned Substrate> [143]

[144] A glass substrate was cleaned with an aqueous ethanol solution and treated with a

2wt% surface treatment solution (XC95-C1658) before incubation at 50⁰C for 20 min. In order to examine the surface properties of the glass substrate (hydrophobicity), the substrate was measured for contact angle using a contact angle meter.

[145] Next, the Ag/Pd nanoparticle dispersion, prepared in Example 2, was used to fabricate an Ag/Pd-patterned substrate as follows.

[146] A desired pattern was designed using CAD and stored in a file (*.dwg). This file was opened in Adobe Illustrator and stored as a file (*.bmp). this file was again converted into a *.tif file in Photoshop. Thereafter, the rate of dropping from a nozzle provided for a head of a patterning instrument (an inkjet printer, Model 70 System, commercially available from Litrex, U.S.A. in which the spectra SE head, Spectra, U.S.A., was mounted) was controlled by modulating voltage and time. After inspecting the nozzle state, the pattern design file (*.tif) was opened and sent to the printer to print the pattern on photopaper so as to identify the starting point. Thereafter, the pattern design file was sent to the printer to print the pattern on one side of a glass substrate (the drop size of the ink and patterns were monitored with a microscope, and the image was adjusted by modulating the resolution). After completion of the patterning, heat treatment was carried out at a temperature of 560⁰C for 20 minutes.

[147] An SEM image of the Ag/Pd-patterned substrate was shown in FIG. 3.

[148]

[149] <Self-assembly Immobilization of Linker DNA>

[150] The Ag/Pd-patterned substrate was analyzed for DNA immobilization with well known HPV (Human Papilloma Virus)- 16 DNA. In order to read the degree of immobilization, the DNA was modified to prepare a 30-mer probe DNA, in which a thiol group was added to the 5 '-end and a fluorescent material (TAMRA) was labeled at the 3'-end. For use in the immobilization, the probe DNA was dissolved in 3x SSC (0.45 M sodium chloride/0.05 M sodium citrate, pH 7.0). HPV-16 DNA was used at a concentration of 10 μM.

[151] Afterwards, the thiol-probe DNA-TAMRA thus prepared was immobilized on the Ag/Pd-patterned substrate by deposition. After the deposition, the substrate was incubated for 14 hrs at 70% humidity in a incubator under the condition of a temperature of 25°C. During the incubation, the probe DNA was self-assembled to be immobilized on the substrate.

[152] After completion of the immobilization, the substrate was washed with 0.2% (w/v) sodium dodecyl sulfate (SDS) solution for 1 min and with de-ionized water for 1 min, and this washing process was conducted twice with stirring at 50 rpm.

[153] In order to analyze the immobilization rate of the probe DNA, the probe DNA- immobilized substrate was scanned with a Genepix 4000B scanner from Exon. Fluorescence intensity was analyzed (excitation 650nm, emission 668nm). The scanned images are shown in FIG. 4 and the analyzed fluorescence intensity is given in Table 3, below.

[154] [155] [COMPARATIVE EXAMPLE 1] [156] On a glass (available from Erie Scientific Co.) substrate which was entirely coated with gold, the same probe DNA as in Example 8 (thiol-probe DNA-TAMRA) was immobilized using a process of spotting with a microarray (Genetix Qarray mini). The substrate was washed, dried and measured for fluorescence intensity in the same manner as in Example 8. The results are summarized in Table 3, below.

[157] [158] [COMPARATIVE EXAMPLE 2] [159] [160] HPV-16 DNA (probe DNA), to which an amine group was attached at the 5'-end and fluorescent material (TAMRA) was attached at the 3 '-end for labeling, was immobilized on an aldehyde substrate (available from CeI Associate) using a spotting method as in Comparative Example 1. The substrate was washed, dried and measured for fluorescence intensity in the same manner as in Example 8. The results are summarized in Table 3, below.

[161] [162] Table 3 [Table 3] [Table ] <Test Results of Immobilization of Probe DNA>

[163] [164] Data of Table 3 indicate that the immobilization efficiency on the Ag/Pd-patterned substrate prepared through inkjet patterning (Example 8) was found to be 10 times greater than that on that of the gold-coated substrate (Comparative Example 1) and twice as large as that on the aldehyde substrate (Comparative Example 2), determined by measuring fluorescence intensity.

[165] [166] EXAMPLE 9

[167] <Test for Degree of Hybridization with Complementary Target DNA and Sensitivity >

[168]

[169] In order to examine the performance (hybridization) and sensitivity (S/N ratio) of a DNA chip based on the Ag/Pd-patterned substrate, 100 μM of the HPV- 16 DNA (probe DNA) was immobilized on the Ag/Pd-patterned substrate prepared in Example 8 through a self-assembly process, followed by incubation at 70% humidity for 14 hrs in a 25°C incubator. Thereafter, the substrate was washed with a 0.2% SDS buffer for 1 min and with sterile distilled water for 1 min., and this washing was repeated again before drying the substrate. Various kinds of chambers (cover slip, Grace Bio-Labs) for hybridization according to pattern designs were applied to the dried substrate.

[170] In the chambers, complementary target DNA labeled with 300 μl of CY5-dUTP was allowed to undergo hybridization. The target DNA was amplified by PCR from the DNA, isolated, and purified from a SiHa cell line (HPV-16, KCLB 30035, Human squamous carcinoma, cervix) and a HeLa cell line, both purchased from the Korean Cell Line Bank (KCLB), located at 28 Yeonkun-Dong, Jongno-Gu, Seoul, Korea. For use in the detection of hybridization, the fluorescent material Cy5 was labeled to the target DNA at the 5' end thereof. For the hybridization, a 6x saline-sodium phosphate- EDTA buffer (SSPE, Sigma Chemical CO., St. Louis, Mo, U.S.A.) and a 0.2% sodium dodecylsulfate (SDS) buffer were used. Following deposition, incubation was conducted at 70% humidity for 15 hrs in a 40⁰C incubator. Thereafter, the chip was washed with 3x SSPE for 2 min and with Ix SSPE for 2 min., and then dried in air.

[171] Next, the chip sample was analyzed for fluorescence intensity using a confocal laser scanner (Genepix 4000B) (excitation 650 nm, emission 668 nm). Scanned images after hybridization are shown in FIG. 5. The fluorescence intensity was represented as an S/ N ratio (sensitivity), and calculated according to the following mathematical formula. In the formula, intensities measured in a DNA patterned region and in the background around the region were indicated as spot intensity and background intensity, respectively. The results are summarized in Table 4, along with the spot diameters measured in the patterned region after the hybridization.

[172]

[173] Sensitivity (S/N Ratio) = Spot-Intensity / Background-Intensity

[174]

[175] [COMPARATIVE EXAMPLE 3]

[176]

[177] The same probe DNA as in Example 9 was immobilized on a glass substrate which was entirely coated with gold (Erie Scientific Co.) using a spotting method. Hy- bridization with target DNA and analysis for hybridization and sensitivity (S/N Ratio) were conducted in the same manner as in Example 9. The results are summarized in Table 4. The scanned images after hybridization on the substrate are shown in FIG. 6.

[178] [179] [COMPARATIVE EXAMPLE 4] [180] [181] 100 μM of HPV- 16 DNA, to which an amine group was added at the 5 '-end, was immobilized as a probe DNA on an aldehyde substrate (CeI Associate) using a spotting method. Hybridization with the complementary target DNA and analysis of hybridization and sensitivity (S/N ratio) were conducted in the same manner as in Example 9. The results are summarized in Table 4. The scanned images after hybridization on the substrate are shown in FIG. 7.

[182] [183] Table 4 [Table 4] [Table ]

[184] [185] As is apparent from the data of Table 4, the Ag/Pd-patterned chip substrate prepared according to the present invention (Example 9) is far superior to the Au-coated substrate (Comparative Example 3) and the aldehyde substrate (Comparative Example 4) with respect to spot intensity and sensitivity (S/N ratio). A high S/N ratio means a high degree of DNA immobilization and hybridization, implying that the chip has excellent ability to analyze DNA. As shown in Table 4, high spot intensity was detected from the chip of the present invention (Example 8), even though it has a small spot diameter, which indicates that the chip has excellent DNA analysis performance with a high degree of DNA immobilization and hybridization thereof.

[186] In addition, as observed with the naked eye in FIGS. 5 to 7, the Ag/Pd-patterned chip substrate according to the present invention (FIG. 5) is far superior to the Au-coated substrate (FIG. 6) and the aldehyde (FIG. 7) with respect to fluorescence intensity.

[187] [188] [EXAMPLE 10] [189] <Test for Performance of DNA Chips According to Linker> [190] [191] In order to examine the degree of hybridization and sensitivity of DNA chips according to kinds (lengths) of linkers, alkane thiols (GenoTech, Korea) having 6, 18 and 24 carbon atoms, respectively, were linked to the 5'-end of HPV-16 DNA (probe DNA). The HPV-16 DNA was used at concentrations of 10 nM and 100 nM to examine the effect of the linkers according to the concentration of the probe DNA.

[192] The linker-probe DNAs were immobilized on the Ag/Pd-patterned substrate prepared in Example 8 using a deposition method through a self-assembly process. Hybridization with the complementary target DNA and analysis of hybridization and sensitivity (S/N ratio) were conducted in the same manner as in Example 9. The results are summarized in Table 5. The scanned images after hybridization on the substrate are shown in FIG. 8.

[193] Table 5 [Table 5] [Table ] <Test Results of DNA Chip Performance According to Linker>

[194] [195] As understood from the data of Table 5 and FIG. 8, the kinds and length of linker played a critical role in determining the performance of the DNA chip. Favorable S/N ratios were obtained with the linkers having 6 to 24 carbon atoms, rather than less or more carbon atoms, with the best result detected at C18. Industrial Applicability

[196] As described hitherto, a biochip in which a thin metal pattern is freely formed through an inkjet patterning method allows biomaterials to be formed in various patterns. The thin metal pattern which can be easily formed an inkjet printing manner can be produced on a mass scale. Also, the apparatus for manufacturing the biochip of the present invention is small and inexpensive. Further, thin metal pattern can be formed to a size as small as micrometers so that biomaterials can be integrated at high density. In addition, the method is environmentally friendly because there is no need to carry out a washing process for an etching process.

[197] No limitations are imposed on the kinds of the metal useful in the present invention, and silver can be used, which results in an improvement in the adhesiveness of substrate to the thin metal pattern as well as in the immobilization efficiency of biomolecules.

[198] No limitations are imposed on the kinds of the metal useful in the present invention, and silver can be used, which results in an improvement in the adhesiveness of substrate to the thin metal pattern as well as in the immobilization efficiency of biomolecules.

[199] The present invention has been described in an illustrative manner, and it is to be understood that the terminology used is intended to be in the nature of description rather than of limitation. Many modifications and variations of the present invention are possible in the light of the present inventions. Therefore, it is to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

[200]

[201]

Claims

[ 1 ] A biochip, comprising ; a substrate; a thin metal pattern formed on the substrate; and a biomaterial immobilized to the thin metal pattern via a linker, wherein the thin metal pattern is formed by patterning a metal solution with an inkjet printer, said metal solution containing metal nanoparticles independently dispersed therein.

[2] The biochip according to claim 1, wherein the metal solution contains the metal nanoparticles in combination with oxide nanoparticles selected from a group consisting of metal oxide nanoparticles, partially polycondensed metal oxide nanoparticles and combinations thereof, said nanoparticles being independently dispersed in the metal solution.

[3] The biochip according to claim 1 or 2, wherein the metal nanoparticles are made from an alloy of a first metal that is able to form a strong covalent bond with the linker and a second metal that provides thermal stability, or a mixture of the first metal and the second metal.

[4] The biochip according to claim 1 or 2, wherein the metal nanoparticles are made from silver (Ag) alone or from an alloy or mixture based on silver.

[5] The biochip according to claim 2, wherein the metal oxide nanoparticles are made from at least one of oxides of a metal selected from a group consisting of silicon (Si), magnesium (Mg), yttrium (Y), cerium (Ce), titanium (Ti), zirconium (Zr), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), neodymium (Nd), copper (Cu), silver (Ag), zinc (Zn), aluminum (Al), gallium (Ga), indium (In), tin (Sn), antimony (Sb), and combinations thereof.

[6] The biochip according to claim 2, wherein the partially polycondensed metal oxide nanoparticles are made from an inorganic polycondensed polymer represented by the following General Formula 2. [General Formula 2] MxOy(OR)Z

(wherein, M is one selected from among Si, Mg, Y, Ce, Ti, Zr, V, Cr, Mn, Fe, Co, Ni, Nd, Cu, Ag, Zn, Al, Ga, In, Sn, and Sb, R is hydrogen or a hydrocarbon, and x, y and z are integers or decimals larger than zero.)

[7] The biochip according to claim 1, wherein the linker is an organic compound having 6 - 24 carbon atoms and a sulfhydryl group (-SH) or a disulfide group (-S-S-).

[8] A method for manufacturing a biochip, comprising the steps of; preparing a metal solution in which metal nanoparticles are independently dispersed; patterning the metal solution on a substrate through an inkjet printer to form thin metal patterns; thermally heating the thin metal pattern to improve adhesiveness between the thin metal pattern and the substrate; bonding a biomaterial to a linker; and immobilizing the linker-coupled biomaterial onto the thin metal pattern.

[9] A method for manufacturing a biochip, comprising the steps of: preparing a metal solution in which metal nanoparticles are independently dispersed; patterning the metal solution on a substrate through an inkjet printer to form a thin metal pattern; thermally heating the thin metal pattern to improve adhesiveness between the thin metal pattern and the substrate; bonding a linker to the thin metal pattern; and immobilizing a biomaterial to the linker.

[10] The method according to claim 8 or 9, wherein the metal solution contains the metal nanoparticles in combination with oxide nanoparticles selected from a group consisting of metal oxide nanoparticles, partially polycondensed metal oxide nanoparticles, and combinations thereof, said nanoparticles being independently dispersed in the metal solution.

[11] The method according to claim 8 or 9, wherein the thin metal patterns are thermally treated at a temperature of 450⁰C ~ 650⁰C and the metal solution is a solution of metal nanoparticles made from an alloy or mixture of a first metal that is able to form a strong covalent bond with the linker and a second metal that provides thermal stability, said nanoparticles being independently dispersed in the metal solution.

[12] The method according to claim 8 or 9, wherein the metal solution is a silver (Ag) solution or a solution of a silver (Ag)-based alloy or solution of mixture containing silver.

[13] The method according to claim 8, wherein the linker-coupled biomaterial is immobilized by spin-coating, spray-coating, spotting or depositing the linker- coupled biomaterial on the thin metal pattern.

[14] The method according to claim 9, wherein the biomaterial is immobilized to the linker by spin-coating, spray-coating, spotting or depositing the biomaterial on th e linker-bound thin metal pattern.

[15] The method according to claim 10, wherein the metal oxide nanoparticles are made from at least one of oxides of a metal selected from a group consisting of silicon (Si), magnesium (Mg), yttrium (Y), cerium (Ce), titanium (Ti), zirconium (Zr), vanadium (V), chromium (Cr), manganese (Mn), iron (Fe), cobalt (Co), nickel (Ni), neodymium (Nd), copper (Cu), silver (Ag), zinc (Zn), aluminum (Al), gallium (Ga), indium (In), tin (Sn), antimony (Sb), and combinations thereof.

[16] The method according to claim 10, wherein the partially poly condensed metal oxide nanoparticles are made from an inorganic polycondensed polymer represented by the following general formula 2. [General Formula 2] MxOy(OR)Z

(wherein M is one selected from among Si, Mg, Y, Ce, Ti, Zr, V, Cr, Mn, Fe, Co, Ni, Nd, Cu, Ag, Zn, Al, Ga, In, Sn, and Sb, R is hydrogen or a hydrocarbon, and x, y and z are integers or decimals greater than zero.)

[17] The method according to claim 8 or 9, wherein the linker is an organic compound having 6 - 24 carbon atoms and a sulfhydryl group (-SH) or a disulfide group (-S-S-).