WO2007097572A1

WO2007097572A1 - Sensing structure of biochip and method thereof

Info

Publication number: WO2007097572A1
Application number: PCT/KR2007/000909
Authority: WO
Inventors: Yong Hoon Cho
Original assignee: Chungbuk National University Industry-Academic Cooperation Foundation
Priority date: 2006-02-22
Filing date: 2007-02-21
Publication date: 2007-08-30

Abstract

A method of fabricating an integrated biochip sensing structure that enables easy measurement and mobility is provided by forming a linker layer comprising linkers separated from one another at regular intervals in a predetermined shape in an emitting surface of a light emitting diode having an active layer, and forming a bio-material layer including a luminous body or a fluorescent material thereon. Another method of fabricating a biochip sensing structure which has high emission efficiency and a simple detection system is also provided by using quantum dots instead of a conventional fluorescent material in labeling of a biochip.

Description

SENSING STRUCTURE OF BIOCHIP AND METHOD

THEREOF

Technical Field

[1] The present invention relates to a biochip, and more particularly, to a biochip sensing structure and a method of fabricating the same in which a biochip is integrated with a light emitting diode to enable easy measurement and mobility, and a method of fabricating a biochip sensing structure with heightened emission efficiency and a simplified measurement system by using quantum dots instead of a conventional fluorescent material for labeling the biochip. Background Art

[2] Biochips are hybrid devices made into the form of a conventional semiconductor chip by combining bio-organic materials derived from living organisms, for example, enzymes, proteins, antibodies, DNA, microorganisms, animal/plant cells and organs and neuronal cells and organs, with inorganic materials such as semiconductors and glass. The biochip acts to diagnose infectious diseases or analyze genes by using inherent functions of biomolecules and mimicking functions of organisms. The biochip is a device with a new function for processing new information.

[3] According to its biomaterials and systemization, the biochip can be classified as one of a DNA chip, an RNA chip, a protein chip, a cell chip, and a neuron chip. Also, by broad definition, the biochip includes a biosensor, which has detection and analysis functions for various biochemical materials, such as a lab-on-a-chip, which has automatic analysis functions including pretreatment of samples, biochemical reaction, detection, and data analysis.

[4] Meanwhile, in a method of analyzing the biochip and diagnosing a disease, a reactive material in the biochip is coated, and then light emitted by exciting the fluorescent material with light of a specific wavelength is detected. That is, in the method, when the light with a specific wavelength is applied to the fluorescent material, its internal energy goes up to a high level and then transitions to a low level, thereby emitting light with a longer wavelength than that of the excitation light.

[5] A laser is commonly used as an external light source to excite the fluorescent material. The laser is radiated onto the biochip and a fluorescence signal emitted from the biochip is detected by scanning.

[6] The biochip using excitation light requires several steps from fabrication to measurement. The fabricated biochip is operated by measuring emission wavelength and intensity when external excitation light is applied. Usually, the biochip is fabricated using a glass substrate such as a slide glass, and then reactivity and degree thereof are observed by a scanner, etc. [7] A conventional analyzing system for a biochip requires a system for exciting and detecting light in addition to the biochip itself. This usually means that an expensive laser has to be used, which boosts production costs. [8] Meanwhile, a matrix biochip sensing system using a light source formed from a light emitting diode (LED) matrix is disclosed in U.S. Patent Publication No.

2002/668865. FIG. 1 is a block diagram schematically illustrating a conventional matrix biochip system. [9] The system of FIG. 1 includes an LED matrix light source 1, a biochip clamping member 2, a light filter module 3, a lens array 4, a light sensor 5, and a signal processing and control module 6. [10] In the system, a biochip is disposed beneath the biochip clamping member 2, and each fragment of each sample matrix reacts with each light spot of the LED matrix light source 1. [11] A method of analyzing a biochip using the system of FIG. 1 includes depositing the reacted sample matrix biochip on a specific region of the biochip clamping member 2, and generating light using the LED matrix light source 1. [12] The biochip is excited by light generated from the LED matrix light source 1, thereby generating fluorescent light which is exclusively transmitted through the light filter module 3. [13] The fluorescent light transmitted through the light filter module 3 is collected by the light lens array 4, reaches the light sensor 5, and is sent to the signal processing and control module 6 to be analyzed. [14] This method requires separate equipment such as the LED matrix light source 1, and biochip analysis can only be performed at the location of the equipment including the LED matrix light source 1. [15] Also, the conventional art has problems of low fluorescence efficiency, a complicated detection method, high fabrication cost, and high power consumption, thereby making it impossible to carry a biochip. [16] Moreover, the conventional biochip has problems of low fluorescence efficiency, short lifespan, long detection time, complicated detection method, and high fabrication cost.

Disclosure of Invention

Technical Problem [17] The present invention is directed to providing a method of fabricating and detecting a low-cost biochip which has high fluorescence efficiency and a simplified detection method for a biochip/sensor.

[18] The present invention is also directed to proving a portable biochip/sensor of high practicality that can be driven by the low power of a mobile phone or the like.

[19] The present invention is also directed to providing a simplified method of detecting and imaging light by sensing change in fluorescent material color with the naked eye, a general microscope, etc., in addition to a detection method using a conventional scanning device.

[20] The present invention is also directed to providing a method of fabricating an effective and low-cost biochip sensing structure with heightened emission efficiency and a simple detection method for biochip/sensor by using quantum dots instead of a conventional fluorescent material.

[21] The present invention is also directed to providing a biochip/sensor that has excellent color sensitivity due to narrower wavelength and longer lifespan obtained by using quantum dots rather than a conventional fluorescent material.

[22] The present invention is also directed to providing a simplified method of detecting and imaging light by sensing change in fluorescent material color with the naked eye, a general microscope, etc., in addition to a detection method using a conventional scanning device. Technical Solution

[23] In one aspect, a biochip sensing structure is provided, which includes: a light emitting diode (LED) unit having an active layer; a surface-treated layer formed on the LED unit or under a substrate having the LED unit; a linker layer comprising several linker structures separated from one another at regular intervals on the surface-treated layer in a predetermined shape to connect bio-material; and a bio-material layer comprising a bio-material having a luminous body or a fluorescent material and in contact with the linker layer.

[24] The LED unit may have a light emitting diode (LED) structure or a laser diode (LD) structure. The LD may be a surface-emitting VCSEL. The LED unit may be a light source that can excite the luminous body.

[25] The LED unit may have a structure including a substrate and an active layer formed on the substrate, and the surface-treated layer may be formed on the substrate or on the structure. However, a top of the LED unit may be patterned in an array, or the LED units may be formed in an array.

[26] In another aspect, a method of fabricating a biochip sensing structure is provided, which includes: (a) forming a light emitting diode (LED) unit having an active layer; (b) treating a top surface of the LED unit or a bottom surface of a substrate having the LED unit; (c) forming a linker layer for connection with a bio-material in several linker structures which are separated from one another at regular intervals and formed in a predetermined shape on the surface-treated layer; and (d) forming a bio-material layer comprising a bio-material having a luminous body or a fluorescent material therein and in contact with the linker layer.

[27] In still another aspect, a method of fabricating a biochip sensing structure is provided, which includes: preparing quantum dots to which a target bio-material is attached; forming a capture bio-material layer in several arrays with a predetermined shape on a substrate; and interacting the quantum dots where the target bio-material is attached with the bio-material layer.

[28] The method may further include forming a linker layer between the capture bio- material and the substrate, and the quantum dots to which the target bio-material is attached may include a core nanocrystal, an inorganic shell, an organic coating layer, a functional group, an interlinker, and a target bio-material.

[29] Meanwhile, before forming the capture bio-material on the substrate, a surface of the substrate may be treated to easily attach the bio-material.

[30] In yet another aspect, a method of fabricating a biochip sensing structure is provided, which includes: preparing a target bio-material layer on which a fluorescent material is attached; forming a quantum dot layer, on which a capture bio-material is attached, on a substrate in several arrays having a predetermined shape; and interacting the target bio-material layer with the quantum dot layer.

[31] The method may further include forming a linker layer between the quantum dot layer and the substrate, and the quantum dots to which the capture bio-material is attached may include a core nanocrystal, an inorganic shell, an organic coating layer, a functional group, an interlinker, and a target bio-material.

Advantageous Effects

[32] The effects of the present invention are as follows.

[33] (1) The present invention provides a method of fabricating and detecting a simple, low-cost biochip having a high fluorescence efficiency and a simple detection method for a biochip/sensor, in which a biochip array and a sensor material are directly formed on a light emitting diode (LED) or a laser diode (LD).

[34] (2) The present invention provides a portable biochip/sensor of high practicality that can be driven by the low power of a mobile phone, etc., and a simplified method of detecting and imaging light by sensing change in fluorescent material color with the naked eye, a general microscope, etc., in addition to a conventional detection method using a scanning device.

[35] (3) In consideration of the numerous applications of multifunctional biochips and sensors in medicine and industry, the present invention is expected to contribute sig- nificantly to the development of the industry by providing a low-cost method of fabricating a light source integrated portable biochip and sensor.

[36] (4) The present invention provides a method of fabricating and detecting a simple, low-cost biochip having high fluorescence efficiency and a simple detection method for a biochip/sensor, in which a biochip array and a sensor material are formed using quantum dots.

[37] (5) Quantum dots have advantages over conventional fluorescent materials when used in biochips/sensors. Different sized quantum dots may have different emission wavelengths due to dependence of quantum effects on size. And, they may have a further smaller full width at half maximum of an emission peak than conventional fluorescent material. These properties enable more clear expression of an emission color and minimize spectrum overlap in multi-color labeling. Thus, various kinds of quantum dots with different wavelengths may be simultaneously used.

[38] (6) While a conventional fluorescent material absorbs only a limited range of wavelengths, quantum dots absorb a broad, continuous range of wavelengths shorter than the emission wavelength. Thus, in the case of excitation using the conventional fluorescent material, there is not a large difference in energy between the wavelength of the excitation light source and the emission wavelength of the fluorescent material. As a result, even using an appropriate filter, it is difficult to eliminate background noise caused by a signal of the excitation light source and detect a weak signal generated by the fluorescent material. However, using quantum dots, the wavelength of the excitation light source can be far removed from the emission wavelength, so that a higher S/N ratio can be achieved. In multi-color labeling, as many excitation light sources as kinds of fluorescent material are required to excite the fluorescent materials, each of which has a narrow absorption band. However, since the quantum dots have a continuous absorption band in a high energy range, even if several kinds of quantum dots with different wavelengths are used, all quantum dots may be simultaneously excited by only one excitation light source.

[39] (7) The quantum dots labeling biochip/sensor of the present invention can easily detect multi-color quantum dots with different quantum dots. In this case, different kinds of quantum dots may be excited using only one excitation light source. Thus, a simpler detection and imaging method can be implemented by using one excitation light source by sensing change in fluorescent material color with the naked eye, a general microscope, etc., in addition to a conventional detection method of using a scanning device.

[40] (8) The present invention provides a method of fabricating and detecting a simple, low-cost biochip having a high fluorescence efficiency and a simple detection method for a biochip/sensor, in which a biochip array and a sensor material are formed using quantum dots.

Brief Description of the Drawings

[41] FIG. 1 is a block diagram schematically illustrating a conventional biochip system.

[42] FIG. 2 is a schematic diagram of a biochip structure according to a first exemplary embodiment of the present invention. [43] FIG. 3 is a graph illustrating an emission wavelength band of a light emitting device and absorption and emission bands of a fluorescent material. [44] FIGS. 4 to 6 are flowcharts illustrating a method of fabricating the biochip structure according to a first exemplary embodiment of the present invention. [45] FIG. 7 is a schematic diagram of a biochip structure according to a second exemplary embodiment of the present invention. [46] FIG. 8 illustrates a method of fabricating a biochip sensing structure according to a third exemplary embodiment of the present invention.

[47] FIG. 9 is an enlarged view of quantum dots labeled with a target bio-material.

[48] FIG. 10 illustrates emission changes according to structure and size of the quantum dots for bonding with a bio-material. [49] FIGS. 11 and 12 are graphs illustrating absorption and emission spectrums of

EviDotTM quantum dots of CdSe/ZnS. [50] FIG. 13 is a graph showing intensity of photoluminescence (PL) obtained by forming quantum dots labeled with a bio-material. [51] FIG. 14 illustrates dip-pen lithography (DPN) technique used in the fabrication of nanoarrays. [52] FIG. 15 is a photograph of images obtained from a biochip sensing structure fabricated according to the present invention using a microchip scanner. [53] FIG. 16 illustrates a method of fabricating a biochip sensing structure according to a fourth exemplary embodiment of the present invention. [54] FIGS. 17 and 18 illustrate a method of fabricating a biochip sensing structure according to a fifth exemplary embodiment of the present invention.

Mode for the Invention [55] Hereinafter, a structure of a biochip and a method of fabricating the same according to exemplary embodiments of the present invention will be described with reference to the accompanying drawings. [56] (First Exemplary Embodiment)

[57] FIG. 2 is a schematic diagram of a biochip structure according to a first exemplary embodiment of the present invention. [58] Referring to FIG. 2, the biochip structure of the present invention includes a light emitting diode (LED) unit 10 including an active layer 14, a surface-treated layer 30 formed on the LED unit 10, a linker layer 40 having a plurality of linker structures spaced apart from one another at regular intervals in a predetermined shape on the surface-treated layer 30, and a bio-material layer 50 having a luminous body 60 or some other fluorescent material.

[59] The LED unit 10 serves as a light source to excite the luminous body in the bio- material layer 50. Here, the LED unit 10 may include an LED or laser diode (LD) structure. Although the active layer 14 and a clad layer 16 are illustrated as forming the LED unit 10, the present invention is not limited to such a structure, and if the LED unit 10 is formed in a LD structure, the LD may be a vertical-cavity surface-emitting laser (VCSEL) diode.

[60] Here, the active layer 14 may have various types of stack structures such as a bulk, quantum well, quantum dot, nanostick or super lattice structure, and the LED unit 10 may be formed of a compound semiconductor material consisting of Si series, SiGe series, II- VI series (ZnCdTeSe, ZnO, etc.), III-V series (InGaAlAs, InGaAlP, InGaAlN, etc.) or a combination thereof, and an organic semiconductor material. The upper layer 16 formed on the active layer 14 of the LED unit 10 may be a clad layer, and thereon an additional capping layer may be formed. The capping layer may be formed by depositing an appropriate material to perform surface treatment onto a top surface of the LED unit 10. For example, the capping layer may be formed to a proper thickness from the compound semiconductor, an organic semiconductor material, and at least one of Au, Ag, Al, Ti, Rh, Zn, Ni, Cu, Pd, Pt, Ru, Ir, Ta, Cr, Mo, W, Re, Fe and Sc.

[61] A top surface layer of the LED unit 10 may be formed of a semiconductor material, an oxide layer or a metal layer. Here, if it is formed of a semiconductor material, an amino group (-NH3) may be formed by silanization, thereby directly forming the bio- material layer 50 on the top surface of the LED unit 10.

[62] A substrate 12 may be formed of silicon, compound semiconductor, quartz or sapphire, depending on the LED unit 10, but is not limited to such materials. In the surface treatment performed on a bottom surface 12a of the substrate 12, an optimal combination of the surface treatment technique and the kind of substrate is selected. For example, a bottom surface of a glass substrate formed of quartz is treated by silanization, and then a bio-material layer may be formed using an immobilization technique to be described later.

[63] The surface-treated layer 30 serves to effectively form the bio-material layer 50 on the top surface of the LED unit 10 or the bottom surface 12a of the substrate. A linker layer 40 may be formed on the surface-treated layer 30 using the immobilization technique, and thereon the bio-material layer 50 may be formed. The linker layer 40 serves to stably fix materials (DNA, RNA, protein, etc.) of the bio-material layer 50 with a maximum activity onto the immobilized substrate, and the method of forming the same will be described later. It is understood that the surface-treated layer 30 and the linker layer 40 are compound layers having a functional group for immobilizing bio-materials on the surface of the biochip substrate. Also, for immobilization, a thin film having a regular array pattern may be added on the surface of the biochip substrate.

[64] The bio-material layer 50 includes a bio target material 60 such as a luminous body or some other luminescent material emitting light with a specific wavelength. The bio target material 60 reacts to external stimuli and emits light of a specific wavelength and intensity due to excitation by light applied from the LED unit 10.

[65] The "bio material" includes materials derived from living organisms or their analogs, or fabricated outside living organisms, for example, enzymes, proteins, antibodies, microorganisms, animal/plant cells and organs, neuronal cells, DNA, and RNA, and preferably, proteins, DNA and RNA. The DNA includes cDNA, genome DNA, oligonucleotides, the RNA includes genome RNA, mRNA and oligonucleotides, and the protein includes antibodies, antigens, enzymes and peptides. As fluorescent materials to target proteins, several fluorescent materials such as fluorescent proteins, quantum dots and nano particles, and luminous bodies, as well as chemical dyes such as Alexa546 (red), Cy5 (green) and Cy3 (yellow), may be used.

[66] In the biochip structure of FIG. 2, an electrode is formed to apply a voltage or current to the LED unit 10. The electrode 70 may be formed of at least one selected from the group consisting of Ni, Cu, Mg, Au, Pt, Ru, Rh, Ir, Ta, Cr, Mn, Mo, W, Re, Fe, Sc, Ti, Sn, Ge, Sb, Ag, Al, Zn and La, or an oxide thereof. Reference numeral 80 represents a power source. The electrode 70 may be formed in a random shape on the bottom surface of the substrate 12, or on the top layer 16 or the linker layer 40, so that the electrode 70 applies a voltage difference and a current to the LED unit 10, but is not limited thereto.

[67] An operating principle of the LED integrated biochip according to the first exemplary embodiment of the present invention will now be described.

[68] The bio-material is actuated by applying a specific stimulus to the bio material of the bio-material layer 50, and the LED unit 10 is driven to excite the luminous body 50 combined with the bio-material. The wavelength of light emitted from the LED unit 10 is filtered to block the light, and only fluorescence and a fluorescence image output from the luminous body 50 are detected. The light emitted from the LED unit 10 is Light 1, and the light emitted from the luminous body attached to the bio-material is Light2.

[69] FIG. 3 illustrates the fact that when the LED/LD emission wavelength band overlaps with the absorption band of the fluorescent material, the fluorescent material can emit light. Referring to FIG. 3, the LED/LD emission wavelength band overlaps with the fluorescent material absorption energy band, and thereby fluorescence is exhibited. According to the present invention, selection of the material and structure of the LED may facilitate change of the emission wavelength band of the LED/LD so as to accurately overlap with the fluorescent material, which leads to characteristic effects distinguished from conventional art.

[70] FIGS. 4 to 6 illustrate steps of fabricating the LED integrated biochip structure according to the first exemplary embodiment of the present invention.

[71] Referring to FIG. 4, an LED unit 10 including the active layer 14 and the clad layer

16 is formed on the substrate 12. The LED unit 10 may be formed by a known method in an LED or LD structure, which is described above.

[72] A surface-treated layer 30 is formed by silanization to form an amino group (-NH2,

-NH3, etc.) on a top surface of the topmost capping layer 16 of the LED unit 10. Also, for effective silanization, treatment for forming a hydroxy group may be performed in advance. The silanization treatment includes a pre-treatment step, for example, to form a bio-material layer.

[73] Subsequently, a plurality of linker layers 40 may be immobilized on the surface- treated layer 30 at regular intervals to form a bio-material layer 50. The linker layers 40 may be immobilized by surface chemical technology such as absorption technique, for example, hydrophobic interaction of carboxymethylcellulose, dextran, collagen and lipid mono-layers; technique using covalent bonding, for example, biotin-streptoavidin bonding technique; and technique using self-assembling linker molecules, for example, prolinker or alkanethiol-poly technique. The linker layers 40 may also be immobilized by coating treatment or formed in several films.

[74] Meanwhile, although FIG. 2 illustrates the fact that the surface-treated layer 30 is formed on the capping layer 16 of the LED unit 10, the surface-treated layer 30 may be formed on a bottom surface of the substrate 12, if necessary. That is, as emission direction of light generated from the active layer 14 of the LED unit 10 is changed upward or downward to the substrate, the surface-treated layer 30, the linker layers 40, and the bio-material layer 50 may be formed on the capping layer 16 or under the substrate 12.

[75] Particularly, when the surface-treated layer 30, etc. are formed under a substrate having a higher energy than the LED emission energy (e.g., InGaN LED/LD structure formed on a sapphire substrate), the light emitted from the LED may transmit through the substrate, so bio-material arrays may be directly formed on the bottom surface of the substrate (an opposite surface having no emission structure, for example, a bottom surface of the sapphire substrate) by surface treatment. Here, the substrate 12 may be used to grow the LED, or an additional substrate having the LED may be attached to the substrate 12 in a subsequent process.

[76] After the immobilization treatment, the bio-material layer 50 combined with the luminous body 60 is formed on the surface-treated layer 30 of the LED unit 10, and thus the LED integrated biochip can be completed. The formation of the bio-material layer 50 may be implemented by various conventional methods. Therefore, a process of fabricating a protein chip using a micro arrayer will now be described.

[77] A pin having a specific diameter, which can be spotted, is first equipped in a head of the micro arrayer, a pattern of a protein chip is designed using an appropriate program, and the designed pattern file is stored to drive the micro arrayer.

[78] Dehydration of protein may be prevented by maintaining proper humidity in a chamber of the micro arrayer at which a slide is disposed. A protein solution for immobilization is loaded in several wells to correspond to the pattern file, and protein is patterned on the slide chip by running the stored pattern file.

[79] The patterned protein spots are incubated in the chamber for a predetermined time, and fluorescent images are detected using a fluorescence scanner after washing and drying steps.

[80] A procedure for studying protein-protein interaction using a protein chip may include: 1) fabrication of an integrin protein micro array chip; 2) blocking using bovine serum albumin (BSA); 3) spotting of a ligand protein solution; 4) incubation; and 5) fluorescent image detection. Here, as labeling reagents for target proteins, chemical dyes such as Alexa546 (red), Cy5 (green) and Cy3 (yellow), all kinds of fluorescent material such as fluorescent proteins, quantum dots and nano particles, and luminous bodies may be used.

[81] (Second Exemplary Embodiment)

[82] FIG. 7 is a schematic diagram of a biochip structure according to a second exemplary embodiment of the present invention.

[83] Referring to FIG. 7, the biochip structure of the present invention includes an LED unit 10 having an active layer 14, a surface-treated layer 30 formed on the LED unit 10, a linker layers 40 formed of a plurality of linked structures in a predetermined shape, which are spaced apart from each other at regular intervals on the surface- treated layer 30, and a bio-material layer 50 having a luminous body 60 or a fluorescent material therein.

[84] The difference of the second embodiment from the first embodiment is that, in the second embodiment, patterns are formed in an array on the LED unit 10, or the LEDs themselves are aligned in an array.

[85] Referring to FIG. 7, after forming the active layer 14 and a clad layer 16, or the surface treatment layer 30, on the same substrate 12, patterns on the LED unit 10 may be formed in an array by lithography. In this case, in practical applications, various modifications may be possible. For example, the material of the active layer 14 may vary by array, or separation of each array may be performed down to the substrate 12 (e.g., in the lithography process, etching is performed down to the substrate), the active layer 14, or the clad layer 16.

[86] It should be clearly understood that, in the second embodiment, the surface-treated layer 30, the linker layer 40 and the bio-material layer 50 having a luminous body 60 or a fluorescent material may be formed on a bottom surface 12a of the substrate 12.

[87] (Third Exemplary Embodiment)

[88] FIG. 8 illustrates a method of fabricating a biochip sensing structure according to a third embodiment of the present invention.

[89] Referring to FIG. 8, a completed biochip sensing structure includes a substrate 110 whose surface is treated, a capture bio-material layer 112 (e.g., anti-high protein) formed in several arrays having a predetermined shape on the substrate 110, and a quantum dot target layer 114 having quantum dots labeled with a target bio-material.

[90] The method of fabricating a biochip sensing structure will be described in more detail.

[91] (1) Quantum dots labeled with a target bio-material are first prepared.

[92] FIG. 9 is an enlarged view of an example of quantum dots labeled with a target bio- material.

[93] Referring to FIG. 9, quantum dots of CdSe/ZnS are formed to a diameter of about

5nm, and surrounded by a IOnm diameter polymer. Around the polymer, NH2 amino groups are formed, each group being connected with a target bio-material by a cross linker.

[94] FIG. 10 illustrates a structure of quantum dots coupled with a bio-material on the left whose emission depends on the size of quantum dots on the right. Referring to FIG. 10, the quantum dots labeled with a bio-material may include a core nanocrystal (CdSe)/inorganic shell (ZnS), an organic coating layer (polymer), a functionality (NH2 amino group), a cross linker (SMCC), and a bio-material.

[95] In the method of fabricating the quantum dots labeled with a bio-material described above, using commercially available EviDotTM quantum dots, a solution with amino- polyethylene glycol (PEG) may be formed. However, the quantum dots labeled with a bio-material may be diluted with a mixed solution of PEG and phosphate buffered saline (PBS). FIGS. 11 and 12 are graphs illustrating emission and absorption spectrums of EviDotTM quantum dots of CdSe/ZnS.

[96] FIG. 13 is a photoluminescence (PL) spectrum graph of quantum dots labeled with a bio-material (e.g., MgG). Here, a wavelength of a light source is 488 nm and laser power is 0.5 mW. Referring to FIG. 10, even after quantum dots are combined with the bio-material, a clear PL peak occurs at about 525 nm. [97] (2) A substrate 110 is fabricated, and then a capture bio-material layer 112 having bio-materials arranged in several arrays in a predetermined shape is fabricated on the substrate 110.

[98] The substrate 110 may be formed of, for example, glass, silicon, compound semiconductor, quartz, or sapphire, irrespective of the kind of biochip, but the present invention is not limited to these substrate materials. A surface of the substrate 110 is specially treated. The surface treatment is performed to facilitate labeling of bio- materials and is useful for a glass or semiconductor substrate because amino groups (-NH3 or ?NH2) may be formed by silanization. Also, for effective silanization, a treatment for forming a hydroxyl (-OH) group may be performed in advance. The silanization treatment may include a pre-treatment step for forming a bio-material layer. The surface treatment may be performed to include a functional group to immobilize bio-materials on a substrate of the biochip.

[99] Selectively, a linker layer 111 may be formed between the surface treated substrate

110 and the capture biomaterial layer 112. The linker layer 111 facilitates immobilization between the capture bio-material layer 112 and the substrate 110. The linker layer 111 may be immobilized by surface chemical technology such as absorption technique, for example, hydrophobic interaction of carboxymethylcellulose, dextran, collagen and lipid mono-layers; technique using covalent bonding, for example, biotin- streptoavidin bonding technique; and technique using self-assembling linker molecules, for example, prolinker or alkanethiol-poly technique. The linker layers 111 may also be immobilized by coating treatment or formed in several films.

[100] The capture bio-material layer 112, which has bio-materials arranged in several arrays and formed in a predetermined shape, is formed on the surface-treated substrate 110 or the linker layer 111. It is clearly understood that the arrays may be micro arrays or nano arrays. FIG. 14 is a schematic view illustrating dip-pen lithography (DPN) technique used in fabrication of nano arrays. Referring to FIG. 14, molecules in an AFM tip are spotted on the substrate through a water meniscus formed in a pen shape.

[101] The term "bio-materials" refers to materials derived from living organisms or their analogs, or materials created from other organic substances. Bio-materials include, for example, enzymes, proteins, antibodies, microorganisms, animal/plant cells and organs, neuronal cells, DNA and RNA, etc., and preferably, proteins, DNA and RNA. Here, DNA may include cDNA, genome DNA and oligonucleotides, RNA may include genome RNA, mRNA and oligonucleotides, and protein may include antibodies, antigens, enzymes and peptides.

[102] The bio-material layer may be formed by various conventional methods, and therefore, a process of fabricating a protein chip using a micro arrayer, one of those methods, will be described below. [103] First, a predetermined diameter pin suitable for a spotting technique is equipped in a head of the micro arrayer, a pattern of a protein chip is designed using an appropriate program, and then the designed pattern file is stored to drive the micro arrayer.

[104] Dehydration of protein may be prevented by maintaining proper humidity in a chamber of the micro arrayer at which a slide is disposed. A protein solution for immobilization is loaded in several wells to correspond to the pattern file, and protein is patterned on the slide chip by running the stored pattern file.

[105] The patterned protein spots are incubated in the chamber for a predetermined time, and fluorescent images are detected using a fluorescence scanner after washing and drying steps.

[106] Micro arrays or nano arrays forming the capture bio-material layer 112, which has bio-materials arranged in several arrays and having a predetermined shape on the substrate 110, refer to a set of detectors that are addressable and spatially fixed on the surface in the form of spots, and several thousands probers (printed on the micro surface) probe the detectors to detect the quantum dots (in the solution) labeled with target bio-materials.

[107] (3) The quantum dots labeled with the target bio-materials are loaded on the substrate having the capture bio-materials to interact with each other.

[108] After that, a predetermined time later, the surface of the substrate is treated with a specific solution to remove non-interacted quantum dots from the substrate and leave interacted quantum dots.

[109] Fluorescent images are obtained using apparatuses such as a microchip scanner, confocal microscope, PL, and scanning near field optical microscopy (SNOM).

[110] FIG. 15 is a photograph of biochip sensing structures fabricated by the method of the present invention, which is obtained using a microchip scanner. The left side of FIG. 15 shows unit spots formed to a size of Φ= 300mm, and the right side thereof shows unit spots to a size of Φ= 100mm. Referring to FIG. 15, it can be noted that emission signals of the quantum dots may be detected using an excitation light source for exciting the quantum dots by the bio sensing structures fabricated by the method of the present invention.

[I l l] The excitation light source for the biochip may be a laser, a laser diode (LD), a light emitting diode (LED), or any light source having a lamp and a filter capable of exciting the quantum dots properly.

[112] One of the characteristic features of the present invention is that it uses quantum dots instead of a fluorescent material. Quantum dots have the following advantages over conventional fluorescent materials when used in biochips/sensors:

[113] (i) Different sized quantum dots may have different emission wavelengths due to dependence of quantum effects on size. And, they may have a further smaller full width at half maximum of an emission peak than conventional fluorescent material. These properties enable more clear expression of an emission color and minimize spectrum overlap in multi-color labeling. Thus, various kinds of quantum dots with different wavelengths may be simultaneously used.

[114] (ii) While a conventional fluorescent material absorbs only a limited range of wavelengths, quantum dots absorb a broad, continuous range of wavelengths shorter than the emission wavelength. Thus, in the case of excitation using the conventional fluorescent material, there is not a large difference in energy between the wavelength of the excitation light source and the emission wavelength of the fluorescent material. As a result, even using an appropriate filter, it is difficult to eliminate background noise caused by a signal of the excitation light source and detect a weak signal generated by the fluorescent material.

[115] However, using quantum dots, the wavelength of the excitation light source can be far removed from the emission wavelength, so that a higher S/N ratio can be achieved. In multi-color labeling, as many excitation light sources as kinds of fluorescent material are required to excite the fluorescent materials, each of which has a narrow absorption band. However, since the quantum dots have a continuous absorption band in a high energy range, even if several kinds of quantum dots with different wavelengths are used, all quantum dots may be simultaneously excited by only one excitation light source.

[116] (Fourth Exemplary Embodiment)

[117] FIG. 16 illustrates a method of fabricating a biochip sensing structure according to a fourth exemplary embodiment of the present invention.

[118] Referring to FIG. 16, a completed biochip sensing structure includes a substrate 120 whose surface is treated, a quantum dot layer 122 labeled with a capture bio-material on the substrate 120, and a target bio-material layer 124 labeled with a fluorescent material. As described above, a linker layer 121 may be added.

[119] A surface of the substrate 120 is treated to easily attach the bio-material thereto, and capture bio-materials are formed on the substrate 120. Here, surface-treated quantum dots QD are labeled with bio-materials (e.g., anti-high protein).

[120] Then, the target bio-material is also labeled with fluorescent materials to detect interaction between the target bio-material and the capture bio-material labeled with the quantum dots. Here, the fluorescent labeling reagents for proteins may include chemical dyes such as Alexa546 (red), Cy5 (green) and Cy3 (yellow), and fluorescent proteins.

[121] After that, using an excitation light source for exciting quantum dots (or appropriate single or several excitation light sources), an emission signal of the quantum dots or an emission signal generated from the fluorescent material attached to the target bio- material is detected.

[122] According to the fourth exemplary embodiment, when protein is labeled with conventional fluorescent materials, relatively little fluorescent material is attached to the bio-material. However, when protein is labeled with quantum dots, a large amount of bio-material is attached around the quantum dots because the quantum dots are relatively large in size. Thus, the bio-material attached to the quantum dots is bonded with the surface, and there (to which the fluorescent material may or may not be attached) the target bio-material is bonded with the capture bio-material around the quantum dots.

[123] In this case, since the bio-materials are attached around the three-dimensional surface of quantum dots, more bio-materials may take part in interaction with the capture bio-materials than in the conventional two-dimensional biochip/sensor structure, which leads to increase in reactivity and signal strength.

[124] In this case, the detected signal has various characteristics due to the emission of the quantum dots themselves and the fluorescent material. For example, when the quantum dots function as a donor and the fluorescent material as an acceptor by fluorescence resonance energy transfer (FRET) technique, a FRET signal is detected according to the interaction between the capture bio-material and the target bio-material. As a result, a conventional non-specific binding problem may be reduced, and the interaction between the capture bio-material and the target bio-material may be accurately detected.

[125] (Fifth Exemplary Embodiment)

[126] FIGS. 17 and 18 illustrate a method of fabricating a biochip sensing structure according to a fifth embodiment of the present invention.

[127] In the fifth exemplary embodiment, the concept of the LED integrated biochip and the concept of the quantum dots-attached biochip are combined. That is, a capture bio- material structure is formed using an LED as a substrate, and thereon a target bio- material with quantum dots is input to react with the capture bio-material, and then the reaction is detected.

[128] Referring to FIG. 17, the top drawing illustrates conventional art of multi-color emission using laser with several wavelengths and fluorescent materials, and the bottom drawing illustrates the fact that the multi-color emission is possible by one excitation light due to different absorption and emission bands of the quantum dots.

[129] Describing the operating principle in more detail, quantum dots (bonded with bio- materials) having a single or multiple colors by light of the LED spectrum are individually or simultaneously excited, and the interaction between the bio-materials may be detected by detecting light emitted by the quantum dots.

[130] Referring to FIG. 18, the left drawing illustrates a schematic diagram in which the bio structure connected with quantum dots is disposed on the LED structure, and the right drawing illustrates the exited spectrum of quantum dots (bonded with a bio- material) having multiple colors due to light from the LED.

[131] Meanwhile, in practical application of the embodiment, a color filter may be used to block unwanted wavelengths emitted from the LED and detect a specific wavelength emitted from the quantum dots.

[132] While the invention has been shown and described with reference to certain exemplary embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

[1] A sensing structure of a biochip, comprising: a light emitting diode (LED) unit having an active layer; a surface-treated layer formed on the LED unit or under a substrate having the

LED unit; a linker layer comprising several linker structures separated from one another at regular intervals on the surface-treated layer in a predetermined shape to connect bio-material; and a bio-material layer comprising a bio-material having a luminous body or a fluorescent material and in contact with the linker layer.

[2] The sensing structure of a biochip according to claim 1, wherein the LED unit is formed in an LED structure or a laser diode (LD) structure.

[3] The sensing structure of a biochip according to claim 1, wherein the LED unit comprises a structure having a substrate and an active layer formed on the substrate, and the surface-treated layer is formed under the substrate or on the structure.

[4] The sensing structure of a biochip according to claim 1, wherein a top of the

LED unit is patterned in an array or the LED units are formed in an array.

[5] A method of fabricating a biochip sensing structure, comprising:

(a) forming a light emitting diode (LED) unit having an active layer;

(b) treating a top surface of the LED unit or a bottom surface of a substrate having the LED unit;

(c) forming a linker layer for connection with a bio-material in several linker structures which are separated from one another at regular intervals and formed in a predetermined shape on the surface-treated layer; and

(d) forming a bio-material layer comprising a bio-material having a luminous body or a fluorescent material therein and in contact with the linker layer.

[6] The method according to claim 5, wherein, in step (b), the surface treatment is performed by silanization.

[7] The method according to claim 5, further comprising: patterning a top of the LED unit in an array or forming the LED unit in an array.

[8] A method of fabricating a biochip sensing structure, comprising: preparing quantum dots to which a target bio-material is attached; forming a capture bio-material layer in several arrays with a predetermined shape on a substrate; and interacting the quantum dots where the target bio-material is attached with the bio-material layer.

[9] The method according to claim 8, further comprising: forming a linker layer between the capture bio-material layer and the substrate.

[10] The method according to claim 8, wherein the quantum dots to which the target bio-material is attached comprise a core nanocrystal, an inorganic shell, an organic coating layer, a functional group, an interlinker, and a target bio- material.

[11] The method according to claim 8, further comprising: before forming a capture bio-material on the substrate, treating a surface of the substrate for easy attachment of bio-material.

[12] A method of fabricating a biochip sensing structure, comprising: preparing a target bio-material layer on which a fluorescent material is attached; forming a quantum dot layer, on which a capture bio-material is attached, on a substrate in several arrays having a predetermined shape; and interacting the target bio-material layer with the quantum dot layer.

[13] The method according to claim 12, further comprising: forming a linker layer between the quantum dot layer and the substrate.

[14] The method according to claim 12, wherein the quantum dots to which the capture bio-material is attached comprise a core nanocrystal, a inorganic shell, an organic coating layer, a functional group, an interlinker, and a capture bio- material.

[15] The method according to claim 12, further comprising: before forming the quantum dot layer on the substrate, treating a surface of the substrate for easy attachment of bio-material.