KR100539318B1 - Method for Manufacturing a Biosensor Using the High Density Carbon Nanotube Pattern - Google Patents

Abstract

The present invention relates to a CNT-biosensor in which a bioreceptor coupled to a target biomaterial is chemically or physicochemically bonded to a high-density carbon nanotube (CNT) pattern exposed to a carboxyl group, and a method of manufacturing the same.

According to the present invention, it is possible to manufacture a CNT-biosensor in which various bioreceptors are chemically or physicochemically coupled to a CNT pattern in which a carboxyl group is exposed or a CNT pattern in which the carboxyl group is modified with various chemical functional groups.

Description

Method for manufacturing a biosensor using the high density carbon nanotube pattern

The present invention relates to a biosensor in which a bioreceptor coupled to a target biomaterial is chemically or physicochemically bonded to a high density carbon nanotube (CNT) pattern exposed to a carboxyl group, and a method of manufacturing the same.

CNT is a carbon allotrope composed of carbon present on the earth in large quantity. It is a material in which one carbon is combined with other carbon atoms in hexagonal honeycomb pattern to form a tube, and the diameter of the tube is nanometer (nm = 1 billionth of a meter). ) Is an extremely small area of matter. CNT is known as a perfect new material that has excellent mechanical properties, electrical selectivity, excellent field emission characteristics, high efficiency hydrogen storage medium properties and few defects among existing materials.

CNTs have excellent mechanical robustness and chemical stability, can exhibit both semiconductor and conductor properties, and because of their small diameter and relatively long length, CNTs are excellent materials for flat panel display devices, transistors and energy storage materials. , Nanosized, various electronic devices are very useful.

In order to apply the above-described characteristics more diversely, single-wall CNTs were started to be chopped using strong acids. The CNTs thus cut have mainly -COOH chemical groups on the cut ends and part of the sidewalls. These chemical functional groups have been used to chemically attach various materials to modify the properties of CNTs. Furthermore, by substituting the -SH group for the functional group of CNT by chemical mechanism, the report was patterned on the surface by micro contact printing technique (Nan, X. et al. , J. Colloid). Interface Sci., 245: 311-8, 2002) and a report on the multi-layer adhesion of CNTs to surfaces using electrostatic methods (Rouse, JH et al. , Nano Lett., 3:59). -62, 2003). However, the former has the disadvantage that the surface density of CNTs is low and the bonding strength is weak, and the latter has the fatal disadvantage that the patterning method of selectively fixing to the surface cannot be applied. Therefore, a new type of surface fixation method is urgently needed.

        Of the 100,000 human genes currently estimated, about 10,000 functions have been identified, and most of these genes are known to be directly related to disease. In addition, since most diseases are caused at the protein level, not at the genetic level, more than 95% of the drugs developed or under development target proteins. Therefore, the study of biomolecules interacting with specific proteins and ligands, and based on the data obtained through protein function analysis and network analysis, developed a method for treating and preventing diseases that were not possible with classical methods. Essential is an efficient protein-protein and protein-ligand reaction detection technique.

Recently, studies on reaction detection through electrochemical changes of CNTs immobilized with biomaterials have been made using the electrical, semiconductor properties, or structurally stable properties of CNTs (Dai, H. et al. , ACC. Chem). Res., 35: 1035-44, 2002; Sotiropoulou, S. et al. , Anal.Bioanal.Chem., 375: 103-5, 2003; Erlanger, BF et al. , Nano Lett., 1: 465-. 7, 2001; Azamian, BR et al. , JACS, 124: 12664-5, 2002).

A representative example of the protein-ligand reaction is an avidin-biotin reaction, in which a channel is formed on a substrate treated with polymer using CNTs, followed by electrochemical method for streptoavidin. Binding was measured (Star, A. et al. , Nano Lett., 3: 459-63, 2003).

CNTs are attracting attention as biosensors because, first, they do not require labeling, second, they are very sensitive to signal changes, and third, they can proceed in aqueous solution without modification of proteins. The combination of new nanomaterials and biological systems will create important applications in disease diagnosis, genetics, and nanobiotechnology.

In order to develop faster and cheaper biosensors, much research has recently been conducted on DNA hybridization detection technology. Various labeling techniques have been developed to detect hybridization of DNA. Fluorescent materials are most commonly used for labeling. By fixing a single DNA chain that can detect complementary DNA, the complementary DNA in aqueous solution is recognized, and the signal converter converts the DNA hybridization signal into a signal that can be analyzed.

      In order to efficiently detect DNA hybridization with a DNA chip, an effective surface treatment is required to increase the hybridization efficiency and to remove the background caused by nonspecific binding. Many studies have been conducted to make such surface-treated DNA chip platforms (Anal. Biochem., 266: 23-30, 1999; Nuc. Acid. Res., 29 (21): 107, 2001).

In addition, various methods for detecting DNA hybridization have been sought, such as the scanmetric method, the calorimetric method, the method using nanoparticles, and the method using electrochemistry (Science, 289: 1757). -60, 2000; Anal. Biochem., 295: 1-8, 2001; Analyst., 127: 803-8, 2002; Anal. Bioanal. Chem., 375: 287-93, 2003).

On the other hand, the application of CNTs in the field of biotechnology has recently appeared. Glucose sensor, detection of proteins, detection of specific DNA sequences (Anal. Bioanal. Chem., 375: 103-5, 2003; Proc. Natl. Acad. Sci. USA, 100 (9): 4984-9, 2003; Anal. Bioanal. Chem., 375: 287-93, 2003). Biomolecular screening in CNT-based multilayers can increase the amount of biomolecules such as DNA immobilized due to its large surface area and excellent electrical conductivity, and increase detection sensitivity to biomolecules.

Currently, the method of detecting reaction results in biochips is the most common method using conventional fluorescent materials and isotopes (Toriba, A. et al. , Biomed. Chromatography: BMC., 17: 126-32, 2003; Raj, SU et al. , Anal. Chim. Acta, 484: 1-14, 2003; Peggy, AT et al. , J. Microbio.Meth., 53: 221-33, 2003), more easily and accurately As new methods of measurement are attempted, the need for a new material called CNT is growing.

Creating a high-density CNT multilayer, attaching DNA to it, and detecting complementary binding DNA can be done by genotyping, mutation detection, pathogen identification, etc. useful. PNA (peptide nucleic acid: DNA analogue) has been reported to be site-specifically fixed to a single-walled CNT and detected complementary binding to the target DNA (Williams, KA et al. , Nature, 420 : 761, 2001). In addition, an oligonucleotide is immobilized to a CNT array by an electrochemical method and a DNA is detected by a guanidine oxidation method (Li, J. et al. , Nano Lett., 3: 597-602). , 2003). However, they do not apply CNTs to the manufacture and development of biosensors.

Recently, a high capacity biomolecular detection sensor (WO 03/016901 A1) using CNTs is known. The present invention relates to a multi-channel biochip obtained by arranging a plurality of CNTs by attaching a plurality of CNTs on a substrate and attaching various kinds of receptors.

Accordingly, the inventors of the present invention devised a method of manufacturing a CNT-biosensor by chemically bonding a bioreceptor to the CNT pattern after forming a high-density CNT pattern exposed to the carboxyl group by stacking the CNTs through chemical bonding to the desired site. The present invention was completed.

It is an object of the present invention to provide a CNT-biosensor having a bioreceptor attached to a high-density CNT pattern laminated by chemical bonding at a desired position, and a method of manufacturing the same.

Another object of the present invention is to provide a method for detecting various target biomaterials that bind to or react with various types of bioreceptors using the CNT-biosensor.

In order to achieve the above object, the present invention comprises the steps of (a) reacting a substrate having an amino group exposed on the surface and CNT exposed carboxyl group to form an amide bond between the amino group and the carboxyl group to form a CNT monolayer on the substrate surface; (b) reacting the CNT monolayer with a diamine-based organic compound to form an organic amine layer on the CNT monolayer, and stacking CNTs by reacting the organic amine with CNT exposed carboxyl groups; And (c) repeating step (b) n times to form a high-density CNT pattern exposing the carboxyl group by n- alternating lamination of the CNT layer and the organic amine layer n times, and fixed to the substrate with high density. The present invention provides a CNT-biosensor having a bioreceptor having a chemical functional group bonded to the carboxyl group to a pattern of carbon nanotubes (CNT) exposed to carboxyl groups by chemical or physicochemical bonding.

The present invention also comprises the steps of (a) reacting a substrate having an amino group exposed on the surface and a CNT exposed carboxyl group to form an amide bond between the amino group and the carboxyl group to form a CNT monolayer on the substrate surface; (b) reacting the CNT monolayer with a diamine-based organic compound to form an organic amine layer on the CNT monolayer, and stacking CNTs by reacting the organic amine with CNT exposed carboxyl groups; And (c) repeating step (b) n times to stack the CNT layer and the organic amine layer n times to form a high-density CNT pattern exposed to the carboxyl group, and fix the carboxyl group on the substrate. The modified CNT pattern is modified with a chemical compound having a functional group which combines the carboxyl group with a chemical functional group selected from the group consisting of an amino group, an aldehyde group, a hydroxyl group, a thiol group, and a halogen, and then an amino group and an aldehyde group exposed by the modification. It provides a CNT-biosensor, which is obtained by chemically or physicochemically bonding a bioreceptor having a functional group which binds the chemical functional group to a chemical functional group selected from the group consisting of a hydroxyl group, a thiol group and a halogen.

delete

The present invention also comprises the steps of (a) reacting a substrate having an amino group exposed on the surface and a CNT exposed carboxyl group to form an amide bond between the amino group and the carboxyl group to form a CNT monolayer on the substrate surface; (b) reacting the CNT monolayer with a diamine-based organic compound to form an organic amine layer on the CNT monolayer, and stacking CNTs by reacting the organic amine with CNT exposed carboxyl groups; (c) repeating step (b) n times to alternately stack the CNT layer and the organic amine layer n times to form a high-density CNT pattern exposed to the carboxyl group; And (d) chemically or physically coupling a bioreceptor having a chemical functional group that binds the carboxyl group to the high-density CNT pattern exposed to the carboxyl group, in which the bioreceptor is coupled to the CNT pattern. Provides a method of manufacturing a biosensor.

The present invention also comprises the steps of (a) reacting a substrate having an amino group exposed on the surface and a CNT exposed carboxyl group to form an amide bond between the amino group and the carboxyl group to form a CNT monolayer on the substrate surface; (b) reacting the CNT monolayer with a diamine-based organic compound to form an organic amine layer on the CNT monolayer, and stacking CNTs by reacting the organic amine with CNT exposed carboxyl groups; (c) repeating step (b) n times to alternately stack the CNT layer and the organic amine layer n times to form a high-density CNT pattern exposed to the carboxyl group; (d) modifying the high-density CNT pattern with the carboxyl group exposed to a chemical having a functional group which binds the carboxyl group and a chemical functional group selected from the group consisting of an amino group, an aldehyde group, a hydroxyl group, a thiol group, and a halogen; And (e) chemically or physicochemically binding a bioreceptor having a functional group that binds the chemical functional group to a chemical functional group selected from the group consisting of an amino group, an aldehyde group, a hydroxyl group, a thiol group and a halogen exposed by the modification. It provides a method for producing a CNT-biosensor is a bioreceptor coupled to the CNT pattern characterized in that.

In the present invention, the substrate exposed to the amino functional group on the surface can be obtained by treating the substrate with aminoalkyloxysilane, the amino group exposed on the surface of the substrate is exposed in the form of a pattern to bind the CNT to the desired position It can be characterized.

In addition, in the present invention, the substrate in which the amino group is exposed in a pattern form may be obtained by forming a photoresist or an organic supramolecular pattern on the substrate in which the amino group is exposed. The CNTs may be stacked or fixed in the vertical or horizontal direction. In the case of the nanopattern of organic supermolecules, the CNTs are preferably fixed in the vertical direction.

The substrate in which the amino group is exposed in the form of a pattern is obtained by forming a photoresist pattern on the substrate to which the amino group is exposed using a photolithography method commonly used in semiconductor processes, or forming a photoresist or organic supramolecular pattern on the substrate. It may then be obtained by treatment with aminoalkyloxysilane.

The present invention also provides a method for detecting a target biomaterial that binds to or reacts with a bioreceptor, wherein the CNT-biosensor is used.

The present invention also provides a CNT-DNA chip, wherein the bioreceptor is DNA, and a method for detecting a DNA hybridization reaction, wherein the CNT-DNA chip is used.

In this invention, it is preferable that the chemical functional group couple | bonded with a carboxyl group is an amino group or a hydroxyl group.

In the present invention, the bioreceptor may be an enzyme substrate having a carboxyl group, an amino group, a hydroxyl group, an aldehyde group, or a thiol group, a ligand, an amino acid, a peptide, a protein, a nucleic acid (DNA, RNA), a lipid, a cofactor or a carbohydrate. have.

In the present invention, the target biomaterial is a substance capable of serving as a target to be detected by reacting with or binding to a bioreceptor, preferably a protein, nucleic acid, antibody, enzyme, carbohydrate, lipid or other bio-derived biomolecule, More preferably nucleic acid (DNA, RNA) or protein.

The term 'CNT-biosensor' used in the present invention encompasses the concept that the bioreceptor is chemically or physicochemically coupled to the CNT pattern. It is defined to include a biochip attached to).

In the present invention, the substrate may be characterized in that the material selected from the group consisting of silicon, glass, molten silica, plastic and PDMS.

In the present invention, CNT is repeatedly laminated on a solid substrate coated with a chemical functional group (amino group) through chemical bonding to prepare a CNT pattern exposed to a carboxyl group having a high surface density, and then chemically reacts with the carboxyl group. , Bioreceptors with hydroxyl groups, etc.) were attached to fabricate CNT-biosensors that can directly detect various types of target biomaterials or detect them using electrochemical signals.

According to the present invention, it is possible to form a pattern of a desired shape at a desired position, deviating from the limitations of the prior art, which is completed by growing CNTs from a catalyst placed at a predetermined position. The present invention improves the disadvantages of the prior art by forming a pattern of the substrate using a polymer or organic supramolecules so as to take full advantage of the chemical method.

In the present invention, a biosensor is manufactured by chemically or physicochemically bonding a bioreceptor having an amino group or a hydroxyl group reacting with the carboxyl group to a high-density CNT pattern exposed to the carboxyl group.

In addition, a biosensor is manufactured by chemically or physically combining a bioreceptor having a carboxyl group or an aldehyde group reacting with the amino group on a high-density CNT pattern in which an amino group is exposed.

On the other hand, in order to attach a bioreceptor having no functional group which binds the carboxyl group or an amino group, at the same time, a chemical functional group which binds the exposed CNT pattern to the carboxyl group and a chemical functional group which binds to the functional group possessed by the target bioreceptor Eggplants can be modified using chemicals. Thus, almost all bioreceptors can be chemically or physicochemically bound to the high density CNT pattern.

For example, in order to attach a bioreceptor having a thiol group, the thiol functional group is modified on the surface of the CNT pattern by modifying it by using a chemical functional group which combines with a carboxyl group and a chemical compound having a thiol functional group, and then having the thiol group via an SS bond. Bioreceptors may be attached.

In the present invention, the CNTs may be connected to a power source through at least one conductive nanowires so that each electric charge can be applied, wherein the conductive nanowires may be formed as a single atom using conventional techniques (Science, 275: 1896-97, 1997), and a conductive pattern may be formed to form a conductive pattern, and then ion implantation or sputtering may be used to deposit a conductive wire.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1 Preparation of CNT Exposed to Carboxyl Group

The CNTs used in the present invention are not particularly limited, and commercially available products may be purchased and used, or may be manufactured and used by conventional methods. Pure CNTs must be carboxylated on the surface and / or both ends of the CNTs for use in the present invention.

The carboxyl-exposed CNTs were refluxed for 24 hours in a sonicator containing a strong acid (mixture of nitric acid and sulfuric acid) solution, filtered through a 0.2 μm filter, and the filtrate was immersed in jinsan for 45 hours. It was refluxed and then centrifuged. The supernatant was recovered, filtered through a 0.1 μm filter and dried. CNTs exposed to the dried carboxyl group were dispersed in distilled water or an organic solvent, and then filtered through a 0.1 μm filter to select CNTs having a constant size.

Example 2 Preparation of Substrate Exposed Amino Group in Pattern

In the present invention, two methods can be used to expose the patterned amino groups on the substrate. First, a polymer, photoresist, or organic supramolecular pattern is formed on a substrate such as silicon, glass, molten silica, plastic, or polydimethylsiloxane (PDMS), and then the aminoalkyloxysilane is fixed to the surface by using the pattern as a mask. This is a method of exposing the surface of the substrate in a pattern form. The second method is to treat the surface of the substrate with an aminoalkyloxysilane, and then form a polymer, photoresist, or organic supramolecular pattern to expose the amino group in a pattern form on the surface of the substrate. It is preferable to use aminopropyl triethoxysilane as said aminoalkyloxysilane.

The polymer or photoresist pattern can be easily formed using a photolithography method commonly used in semiconductor processes, and the pattern of organic supramolecules is disclosed in Korean Patent Application Nos. 10-2003-0032514 and 10- It can form by the method of 2003-0037752.

Example 3: Formation of High Density CNT Patterns

The CNT exposed carboxyl group prepared in Example 1 is reacted with the substrate in which the amino group prepared in Example 2 is exposed in a pattern form to form a CNT monolayer on the substrate through an amide bond between the carboxyl group and the amino group.

In the present invention, in order to promote the amide bond, DCC (1,3-dicyclohexyl carbodiimide), HATU ( O- (7-azabenzotriazol-1-yl) -1,1: 3,3-tetramethyl urofluor hexafluorphosphate) as a coupling agent , HBTU ( O- (benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate), HAPyU ( O- (7-azabenzotriazol-1-yl) -1,1: 3,3-bis (tetramethylene ) uronium hexafluorphosphate), HAMDU ( O- (7-azabenzotriazol-1-yl) -1,3-dimethyl-1,3-dimethyleneuronium hexafluorphosphate), HBMDU ( O- (benzotriazol-1-yl) -1,3-dimethyl It is preferable to use -1,3-dimethyleneuronium hexafluorphosphate (DI) (diisopropylethylamine), TMP (2,4,6-trimethylpyridine), NMI ( N -methylimidazole) or the like as a base. In addition, using EDC (1-ethyl-3- ( 3-dimethylamini-propyl) arbodiimide hydrochloride) as a coupling agent when using water as a solvent and, NHS N -hydroxysuccinimide) as an adjuvant of a coupling agent, NHSS (N-hydroxysulfosuccinimide), etc. Preference is given to using. The coupling agent serves to form an amide bond (-CONH-) with a -COOH functional group and a -NH ₂ functional group, and the base and the auxiliary agent help to increase efficiency when the coupling agent forms an amide bond. do.

Next, the CNT attached to the substrate by the amide bond is reacted with the diamine organic compound having a double amino functional group, and the CNT exposed carboxyl group is reacted with the other amino group of the diamine organic compound.

In the present invention, a substance having a chemical formula of H ₂ NR ₁ -NH ₂ may be used as the diamine organic compound. Here, R ₁ is C _1-20 saturated hydrocarbons, unsaturated hydrocarbons or aromatic organic groups.

Subsequently, the chemical reaction between the CNT exposed to the carboxyl group and the diamine-based organic compound is repeatedly performed to alternately stack the CNT layer and the organic amine layer n times to form a high density CNT pattern exposed to the carboxyl group.

The CNT pattern in which the carboxyl group is exposed has a chemical functional group (amino group, hydroxyl group, etc.) that reacts with the carboxyl group and a chemical functional group (amino group, hydroxyl group, thiol group, aldehyde group, etc.) that combines with the functional group possessed by the target bioreceptor. It can be modified using chemicals. Chemicals that can be used for the modification include H ₂ NR ₁ -NH ₂ , H ₂ NR ₂ -SH, H ₂ NR ₃ -OH, H ₂ NR ₄ -CHO Etc. can be used. Where R ₁ , R ₂ , R ₃ and R ₄ are independently C _1-20 saturated hydrocarbons, unsaturated hydrocarbons or aromatic organic groups.

Example 4 Fabrication of CNT-DNA Chip

In this example, a DNA chip was prepared by chemically binding DNA to a CNT pattern exposed to a carboxyl group. A DNA chip was prepared by attaching an amino group of DNA to the CNT pattern to which the carboxyl group prepared in Example 2 was exposed (FIG. 1). Alternatively, the carboxyl group prepared in Example 2 was exposed to a CNT pattern exposed with a diamine-based organic compound having amino functional groups on both sides to expose amino functional groups, and then a DNA chip was prepared by attaching a carboxyl group of DNA to the amino group. (FIG. 2). In this example, a CNT-DNA chip was prepared using the oligonucleotide of SEQ ID NO: 1 having an amino group or a carboxyl group at the terminal.

SEQ ID NO: 5'-TGT GCC ACC TAC AAG CTG TG (C3) -3 '

The presence of DNA on the CNT pattern was confirmed by X-ray photoelectron spectroscope (XPS) spectra for phosphorus atoms using the fact that all DNA contained phosphate groups (FIG. 3). . As shown in FIG. 3, phosphorus was detected in the XPS surface analysis, and it was confirmed that DNA was bound to the CNT surface.

Example 5 Analysis of Hybridization Reaction Using CNT-DNA Chips

The DNA chip prepared in Example 3 was placed in a hybridization chamber, and the hybridization solution was pipetted to the site where the CNT was fixed and covered with a cover slide. At this time, the hybridization solution is filled with a solution containing 32 μl of complementary nucleotide oligonucleotides, and the final concentration is 3XSSC (0.45M NaCl, 0.045M sodium citrate), 0.3% sodium dodecyl sulfate (SDS), and the total volume. To 40 μl. Here, the complementary oligonucleotide sequence of the hybridization solution was used in the following SEQ ID NO: 2 having a fluorescein isothiocyanate (FITC) attached to the terminal.

SEQ ID NO: 5'-CAC AGC TTG TAG GTG GCA CA-3 '

To remove nonspecific binding of the two oligonucleotide strands, the solution was left at 100 ° C. for 2 minutes and then centrifuged at 12000 rpm for 2 minutes. In order to prevent the hybridization solution from drying in the hybridization chamber, 30 μl of 3 × SSC (0.45 M NaCl, 0.045 M sodium citrate) was added to the depressions on both sides of the chamber. The chamber lid was closed and left in a 55 ° C. thermostat for 10 hours.

After 10 hours, the hybridization chamber was removed from the thermostat and soaked in 2XSSC solution for 2 minutes, then soaked in 0.1XSSC (0.015M NaCl, 0.0015M sodium citrate) and 0.1% SDS solution for 5 minutes, and finally 5 minutes in 0.1XSSC. Soak for a while. In order to remove the remaining solution on the DNA chip, the chip was placed in a centrifuge and centrifuged at 600 rpm for 5 minutes.

Hybridization reactions detected fluorescence using FITC labeled at the oligonucleotide terminus of SEQ ID NO: 2. Fluorescence images were obtained using ScanArray 5000 Packard BioScience, BioChip Tecnologies LLC confocal microscopy and QuantArray Microarray Analysis Software (FIG. 4). Figure 4 (a) is a CNT-exposed substrate of the carboxyl group exposed to the high density before binding the DNA (b) is a complementary DNA after binding the DNA having an amino group terminal group on the CNT pattern (C) shows the result of fluorescence detection in the case of hybridization of non-complementary DNA. When the hybridization reaction of the oligonucleotide having a nucleotide sequence complementary to the oligonucleotide bound to the CNT pattern was confirmed that the fluorescence was clear and even (b). However, it was found that fluorescence did not appear at all when hybridizing oligonucleotides having non-complementary nucleotide sequences to oligonucleotides bound to the CNT pattern and to the CNT pattern in which the oligonucleotide was not fixed ((a) and (c)). From this result, it was confirmed that almost no specific reaction occurred.

Example 6

As shown in FIG. 2, a DNA chip was prepared by chemically binding a DNA chain (SEQ ID NO: 1) having a carboxyl terminal functional group to an amino group-modified CNT, and the same hybridization reaction as in Example 4 was performed (FIG. 5). In FIG. 5, (a) shows fluorescence detection results when binding complementary DNA, and (b) shows fluorescence detection results when binding non-complementary DNA. As a result of hybridizing oligonucleotides with non-complementary nucleotide sequences and oligonucleotides with complementary nucleotide sequences, it was possible to clearly distinguish between hybridized and non-combined oligonucleotides.

      As described in detail above, the present invention has the effect of providing a biosensor chemically bonded to the bioreceptor to a high-density CNT pattern obtained by stacking the CNT exposed to the carboxyl group on a desired position on the substrate.

According to the present invention, it is possible to manufacture a CNT-biosensor in which various bioreceptors are chemically or physicochemically coupled to a CNT pattern in which a carboxyl group is exposed or a CNT pattern in which the carboxyl group is modified with various chemical functional groups.

FIG. 1 shows that complementary DNA binds to a CNT-DNA chip prepared by binding a DNA having an amino group to a CNT pattern in which a carboxyl group is exposed.

Figure 2 shows that the complementary DNA is bonded to the CNT-DNA chip prepared by modifying the CNT pattern exposed to the carboxyl group to an amino group, and then combine the DNA having a carboxyl end group.

Figure 3 is an XPS spectrum of phosphorus on the surface of a CNT pattern to which DNA is chemically bound.

Figure 4 (a) is a CNT-exposed substrate of the carboxyl group exposed to the high density before binding the DNA (b) is a complementary DNA after binding the DNA having an amino group terminal group on the CNT pattern (C) shows the result of fluorescence detection in the case of hybridization of non-complementary DNA.

       FIG. 5 (a) shows fluorescence detection results when the DNA having the carboxyl end group is coupled to the CNT pattern modified with an amino group, followed by hybridization of complementary DNA, and (b) shows non-complementary DNA. The fluorescence detection result of the hybridization reaction is shown.

<110> KAIST <120> Method for Manufacturing a Biosensor Using the High Density Carbon Nanotube Pattern <160> 2 <170> KopatentIn 1.71 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide for DNA-chip <400> 1 tgtgccacct acaagctgtg 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> oligonucleotide for DNA-chip <400> 2 cacagcttgt aggtggcaca 20

Claims (19)

A bioreceptor having a chemical functional group that bonds the carboxyl group to a pattern of carboxyl group exposed carbon nanotubes (CNT), which is prepared through the following steps and is fixed at a high density on a substrate, is attached by chemical or physicochemical bonding. CNT-biosensor characterized by: (a) reacting a substrate having an amino group exposed to the surface with a CNT having a carboxyl group exposed thereto to form an amide bond between the amino group and the carboxyl group to form a CNT monolayer on the substrate surface; (b) reacting the CNT monolayer with a diamine-based organic compound to form an organic amine layer on the CNT monolayer, and stacking CNTs by reacting the organic amine with CNT exposed carboxyl groups; And (c) repeating step (b) n times to alternately stack the CNT layer and the organic amine layer n times to form a high-density CNT pattern exposed to the carboxyl group. In the group consisting of a functional group, an amino group, an aldehyde group, a hydroxyl group, a thiol group and a halogen, which combines the carboxyl group-exposed pattern of carbon nanotubes (CNTs), which are prepared by the following steps and fixed at a high density on a substrate, with the carboxyl group A bioreceptor having a functional group that binds the chemical functional group to a chemical functional group selected from the group consisting of an amino group, an aldehyde group, a hydroxyl group, a thiol group, and a halogen exposed by the modification CNT-biosensors obtained by chemical or physicochemical bonding: (a) reacting a substrate having an amino group exposed to the surface with a CNT having a carboxyl group exposed thereto to form an amide bond between the amino group and the carboxyl group to form a CNT monolayer on the substrate surface; (b) reacting the CNT monolayer with a diamine-based organic compound to form an organic amine layer on the CNT monolayer, and stacking CNTs by reacting the organic amine with CNT exposed carboxyl groups; And (c) repeating step (b) n times to alternately stack the CNT layer and the organic amine layer n times to form a high-density CNT pattern exposed to the carboxyl group. The CNT-biosensor of claim 1 or 2, wherein the bioreceptor is an enzyme substrate, ligand, amino acid, peptide, protein, nucleic acid (DNA, RNA), lipid, cofactor or carbohydrate. The CNT-biosensor of claim 3, wherein the bioreceptor is a protein or nucleic acid (DNA, RNA). The CNT-biosensor according to claim 1 or 2, wherein the chemical functional group which binds to the carboxyl group is an amino group or a hydroxyl group. delete The CNT-biosensor according to claim 1 or 2, wherein the substrate having an amino functional group exposed to the surface is obtained by treating the substrate with an aminoalkyloxysilane. The CNT-biosensor of claim 1 or 2, wherein the amino group exposed on the surface of the substrate is exposed in a pattern to bind the CNT to a desired position. Method of manufacturing a CNT-biosensor is combined with a bioreceptor in a CNT pattern, characterized in that it comprises the following steps: (a) reacting a substrate having an amino group exposed to the surface with a CNT having a carboxyl group exposed thereto to form an amide bond between the amino group and the carboxyl group to form a CNT monolayer on the substrate surface; (b) reacting the CNT monolayer with a diamine-based organic compound to form an organic amine layer on the CNT monolayer, and stacking CNTs by reacting the organic amine with CNT exposed carboxyl groups; (c) repeating step (b) n times to alternately stack the CNT layer and the organic amine layer n times to form a high-density CNT pattern exposed to the carboxyl group; And (d) chemically or physicochemically binding a bioreceptor having a chemical functional group that binds the carboxyl group to the high density CNT pattern to which the carboxyl group is exposed. Method of manufacturing a CNT-biosensor is combined with a bioreceptor in a CNT pattern, characterized in that it comprises the following steps: (a) reacting a substrate having an amino group exposed to the surface with a CNT having a carboxyl group exposed thereto to form an amide bond between the amino group and the carboxyl group to form a CNT monolayer on the substrate surface; (b) reacting the CNT monolayer with a diamine-based organic compound to form an organic amine layer on the CNT monolayer, and stacking CNTs by reacting the organic amine with CNT exposed carboxyl groups; (c) repeating step (b) n times to alternately stack the CNT layer and the organic amine layer n times to form a high-density CNT pattern exposed to the carboxyl group; (d) modifying the high-density CNT pattern with the carboxyl group exposed to a chemical having a functional group which binds the carboxyl group and a chemical functional group selected from the group consisting of an amino group, an aldehyde group, a hydroxyl group, a thiol group, and a halogen; And (e) chemically or physicochemically binding a bioreceptor having a functional group that binds the chemical functional group to a chemical functional group selected from the group consisting of an amino group, an aldehyde group, a hydroxyl group, a thiol group and a halogen exposed by the modification. The method according to claim 9 or 10, wherein the substrate having an amino functional group exposed to the surface is obtained by treating the substrate with aminoalkyloxysilane. The method of claim 9 or 10, wherein the amino group exposed on the surface of the substrate is exposed in a pattern to bind the CNT at a desired position. The method of claim 12, wherein the pattern of the amino group is obtained by forming a photoresist or organic supramolecular pattern on a substrate to which the amino group is exposed. The method of claim 12, wherein the pattern of the amino group is obtained by forming a photoresist or organic supramolecular pattern on a substrate and then treating with an aminoalkyloxysilane. The method according to claim 9 or 10, wherein the chemical functional group which binds to the carboxyl group is an amino group or a hydroxyl group. The method of claim 9 or 10, wherein the bioreceptor is an enzyme substrate, ligand, amino acid, peptide, protein, nucleic acid (DNA, RNA), lipid, cofactor or carbohydrate. A method for detecting a target biomaterial that binds to or reacts with a bioreceptor, using the CNT-biosensor of claim 1. The CNT-DNA chip according to claim 1 or 2, wherein the bioreceptor is DNA. A method for detecting a DNA hybridization reaction, wherein the CNT-DNA chip of claim 18 is used.