KR101440542B1 - Biosensor Using the Conductive Graphene and Manufacturing Method Thereof - Google Patents
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Abstract
The present invention relates to a biosensor using a conductive graphene and a method of manufacturing the same, and more particularly, to a biosensor using a conductive graphene and a method of manufacturing the same. The present invention relates to a biosensor using a conductive graphene having a biosensor that selectively binds to a target biomolecule, and a method for manufacturing the biosensor.
The conductive graphene biosensor according to the present invention has a wide surface area and is excellent in electric conductivity, so that the amount of immobilized biomolecules such as DNA can be increased and the detection sensitivity to biomolecules can be increased. In addition, by directly detecting various target biomolecules or measuring electrochemical signals, it is possible not only to detect a reaction between a biomolecule and a bioreceptor in a large amount at a time, but also to obtain an accurate measurement value with only a small amount of a source It is possible to introduce a detection method.
Graphene, graphite, biosensor, detection method, conductive, electrochemical
Description
The present invention relates to a biosensor using a conductive graphene and a method of manufacturing the same, and more particularly, to a biosensor using a conductive graphene and a method of manufacturing the same. The present invention relates to a biosensor using a conductive graphene having a biosensor that selectively binds to a target biomolecule, and a method for manufacturing the biosensor.
A graphene is a two-dimensional thin film of a honeycomb structure made of one layer of carbon atoms. The carbon atoms are sp 2 When chemically bonded by a hybrid orbit, a hexagonal carbon hexagonal surface is formed which is spread in two dimensions. The aggregate of carbon atoms with this planar structure is graphene, first found by Novoselov and Geim of Manchester University in England, 2004, with a thickness of only 0.3 nm, just one carbon atom (Novoselor K et al ., Science , 306: 666, 2004). In particular, graphene is similar to carbon nanotubes in most of its properties, including strength and conductivity, but can be etched on a silicon wafer as a sheet in a sheet form, It is known that it can be formed more easily than forming a circuit from pieces (Berger et al ., Science , 312: 1191, 2006).
Conventional silicon-based semiconductor processing technology can not manufacture semiconductor devices with a high degree of integration of 30 nm or less. This is because, when the thickness of the metal atom layer such as gold or aluminum deposited on the substrate is less than 30 nm, the metal atoms become unstable due to thermodynamic instability and a uniform thin film can not be obtained. Also, This is because the size becomes uneven. However, graphene has the potential to overcome the integration limitations of these silicon-based semiconductor device technologies.
In addition, graphene is characterized in that its characteristic is metallic and its thickness is very thin, which is several nm or less, corresponding to the screening length, and the electric resistance changes due to the change of the charge density depending on the gate voltage. The metal transistor can be realized by this, and the mobility of the charge carrier can be increased to realize a high-speed electronic device. Further, since the charge of the charge carrier can be changed from electron to hole according to the polarity of the gate voltage, It is expected to be.
For example, graphene can be used as an electron emitter for various devices, a vacuum fluorescent display (VFD), a white light source, a field emission display (FED), a lithium ion secondary battery electrode, a hydrogen storage fuel cell, Capsules, nano-tweezers, AFM / STM tips, single electron devices, gas sensors, biomedical components, and high-performance complexes. Since graphene is excellent in mechanical durability and chemical stability, and has both semiconductor and conductor properties, and has a small diameter and a long length, it exhibits excellent properties as a material for a flat panel display device, a transistor, and an energy storage material. Is very useful as various electronic devices.
In order to apply various of the above-mentioned characteristics, we began to cut the graphenes. These cut-out grapins have mainly -COOH chemical groups on the cut ends and sidewalls. These chemical functional groups have also been used to chemically bond various materials and modify the properties of the graphene (Hashimoto et al ., Nature , 430: 870, 2004). Further, it has been reported that the functional groups of graphene are replaced with -SH groups by a chemical mechanism and then patterned on the surface using a micro contact printing technique (Nan, X. et al ., J. Colloid Interface Sci . , 245: 311, 2002) and an electrostatic method to fix the graphene to the surface with multiple membranes (Rouse, JH et al ., Nano Lett . , 3: 59-62, 2003). However, the electron has a disadvantage that the surface density of the graphene is low and the bonding force is weak, and the latter has a fatal disadvantage that the patterning method of selectively fixing to the surface can not be applied. Therefore, it is urgently required to develop a new type of surface fixing method.
On the other hand, since most diseases are caused at the protein level rather than at the genetic level, more than 95% of the drugs that have been developed or under development are targeting proteins. In this study, we have developed biomolecules that interact with specific proteins and ligands, and have developed methods for the treatment and prevention of diseases, which were impossible with classical methods based on data obtained through protein function analysis and network analysis What is essential is an efficient protein-protein and protein-ligand-reaction detection technique. In addition, detection techniques using DNA-DNA hybridization have been performed in parallel.
The technology for detecting the reaction between proteins and proteins has been developed so far, and it is a protein chip technology. The orientation of biomolecules is controlled at the molecular level by using an affinity tag to a target protein, and a single layer of uniform and stable protein is specifically And then analyzing protein-protein interactions (Hergenrother, PJ et < RTI ID = 0.0 > al ., JACS , 122: 7849-7850, 2000; Vijayendran, RJ, A. et al ., Anal . Chem . , ≪ / RTI > 73: 471-480, 2001; Benjamin, T. et al ., Tibtech . , ≪ / RTI > 20: 279-281, 2002). Recently, studies have been carried out to detect the reaction between protein-protein and protein-ligand by using electrochemical changes of carbon nanotubes after fixing biomaterials on carbon nanotubes (Dai, H. et al ., ACC . Chem . Res . , ≪ / RTI > 35: 1035-44, 2002; Sotiropoulou, S. et al ., Anal . Bioanal . Chem . , 375: 103-5, 2003; Erlanger, BF et al ., Nano Lett . , ≪ / RTI > 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. Star et al. Form a polymer channel on a substrate treated with a polymer and then form a polymer channel on the substrate by an electrochemical method. Avidin was determined (Star, A. et al ., Nano Lett . , 3: 459-63, 2003).
The DNA-DNA detection technology is a technology of DNA chip, which detects the presence or absence of reaction of a target substance by detecting a signal that appears when a target DNA is hydrogen bonded to a receptor DNA bound to a chip surface. Methods for detecting DNA complementarily bound to DNA by making a dense carbon nanotube multilayer include genotyping, mutation detection, pathogen identification (pathogen identification) ). There is a report that PNA (peptide nucleic acid) is locally and specifically bound to a single walled carbon nanotube and complementarily bound to a target DNA (Williams, KA et al., Nature , 420: 761, 2001). In addition, an oligonucleotide is immobilized on a carbon nanotube array through an electrochemical method and DNA is detected by a guanidine oxidation method (Li, J. et al., Nano Lett., 3: 597 -602, 2003). However, these have a disadvantage that they are relatively difficult to analyze accurately because of their relatively low electrical conductivity.
As a result, the present inventors have made extensive efforts to improve the problems of the prior art. As a result, the present inventors have found that the conductive graphene is produced using a chemical functional group, and the conductive graphene is repeatedly laminated on the substrate to have a high surface density, When a graphene film is produced and then a biosensor that selectively binds to the target biomaterial is attached to the conductive graphene or conductive graphene film, a variety of target biomaterials can be directly or electrochemically And the present invention has been completed.
An object of the present invention is to provide a conductive graphene having excellent electrical conductivity and a method for producing the same.
Another object of the present invention is to provide a method for forming a conductive graphene pattern by laminating graphene on a substrate.
It is still another object of the present invention to provide a graphene film having a high surface density and an excellent electrical conductivity and a method of manufacturing the same.
It is still another object of the present invention to provide a conductive graphene biosensor having various types of biosensors attached to conductive graphene or a film and a method of manufacturing the same.
In order to achieve the above object, the present invention provides a method for producing a graphene comprising: (a) preparing graphene having a carboxyl group; And (b) combining the carboxyl group of the graphene with an amino group of a chemical substance having an amino group and a thiol group at the same time to prepare a graphene modified with a thiol group, and a method for producing the conductive graphene , And a graphene- (CONH-R 1 -S) r wherein r is a natural number of 1 or more and R 1 is a saturated hydrocarbon, an unsaturated hydrocarbon or an aromatic organic group.
The present invention also provides a method for preparing a substrate, comprising: (a) preparing a substrate having a thiol group exposed on its surface; (b) laminating the conductive graphene by bonding the conductive graphene to a thiol group on the surface of the substrate; And (c) bonding the conductive graphene to the conductive graphene attached to the substrate to form a conductive graphene pattern.
The present invention also provides a method for preparing a substrate, comprising: (a) providing a substrate having a thiol group exposed on its surface; (b) laminating the conductive graphene by bonding the conductive graphene to a thiol group on the surface of the substrate; (c) bonding the conductive graphene to the conductive graphene attached to the substrate, thereby laminating the conductive graphene; And (d) repeating the above step (c) to increase the density of the conductive graphene, and a method for producing a conductive GRF film comprising the steps of: (1) preparing a substrate - [(CONH-R 2 -S- (SR 3 -S-graphene) p] q wherein p and q are natural numbers of 1 or more, and R 2 and R 3 are C 1 -20 saturated hydrocarbon, unsaturated hydrocarbons or aromatic organic groups. ). ≪ / RTI >
The present invention also provides a conductive graphene biosensor characterized in that a conductive graphene or conductive graphene film produced by the above method is attached to a biosensor that is bound to or reacts with a target biomaterial.
The present invention also provides a method for detecting a target biomaterial which is in combination with or reacts with a bio-receptor, which uses the conductive graphene biosensor.
The present invention also relates to a process for the preparation of conductive graphene having the form of graphene- (CONH-R 1 -S) r, wherein r is one or more natural numbers and R 1 is a saturated hydrocarbon, an unsaturated hydrocarbon or an aromatic organic group And a nucleic acid is attached to the nucleic acid molecule.
The present invention also provides a method for producing a nucleic acid chip, wherein the conductive grapina nucleic acid complex is immobilized on a substrate having an amine and / or a lysine group attached thereto.
The present invention also provides a method for detecting a DNA hybridization reaction, wherein the conductive graphene-DNA complex is immobilized on a substrate to which an amine and / or a lysine group is attached, .
The present invention also relates to a process for the preparation of conductive graphene having the form of graphene- (CONH-R 1 -S) r, wherein r is one or more natural numbers and R 1 is a saturated hydrocarbon, an unsaturated hydrocarbon or an aromatic organic group And an enzyme substrate is attached to the substrate.
The conductive graphene biosensor according to the present invention has a wide surface area and is excellent in electric conductivity, so that the amount of immobilized biomolecules such as DNA can be increased and the detection sensitivity to biomolecules can be increased. In addition, by directly detecting various target biomolecules or measuring electrochemical signals, it is possible not only to detect a large amount of reaction between a biomaterial and a bioreceptor at a time, but also to overcome a special situation to be measured in a liquid phase , It is possible to introduce a detection method capable of obtaining accurate measurement values even with a small amount of a raw material.
According to one aspect of the present invention, there is provided a method for producing a graphene comprising: (a) preparing graphene having a carboxyl group; And (b) combining the carboxyl group of the graphene with an amino group of a chemical substance having both an amino group and a thiol group to produce graphene modified with a thiol group.
Specifically, the graphene is cleaved by an acid to have a carboxyl group (-COOH), and the carboxyl group (-COOH) of the graphene is combined with an amino group of a chemical substance having an amino group and a thiol group at the same time to prepare a graphene modified with a thiol group can do.
In the present invention, it is preferable that the chemical substance having both the amino group and the thiol group is NH 2 -R 1 -SH, wherein R 1 is a saturated hydrocarbon, an unsaturated hydrocarbon, or an aromatic organic group of C 1 -20 . In the step (a), graphene may be treated with a strong acid such as hydrochloric acid, sulfuric acid, or the like.
The present invention relates, in another aspect, to conductive graphenes having the form of graphene- (CONH-R 1 -S) r. Here, r is a natural number of 1 or more, and R 1 is a saturated hydrocarbon, an unsaturated hydrocarbon, or an aromatic organic group having C 1 - 20 .
In the present invention, the conductive graphene is not required to be labeled, and the reaction can be proceeded in an aqueous solution without any modification of the protein, and the production process is easy, so that introduction into a mass production system is sufficiently possible, Which can be used as a basic constituent material of a biosensor by simultaneously taking advantage of the characteristics of the semiconductor and the electrochemical application scalability while reducing the production cost.
According to another aspect of the present invention, there is provided a process for preparing a substrate, comprising the steps of: (a) preparing a substrate on which a thiol group is exposed; (b) bonding the conductive graphene to the thiol group on the surface of the substrate to attach the conductive graphene to the substrate; And (c) bonding the conductive graphene to the conductive graphene attached to the substrate to form a conductive graphene pattern.
According to another aspect of the present invention, there is provided a process for preparing a substrate, comprising the steps of: (a) preparing a substrate on which a thiol group is exposed; (b) bonding the conductive graphene to the thiol group on the surface of the substrate to attach the conductive graphene to the substrate; (c) laminating the conductive graphene by bonding another conductive graphene to the conductive graphene attached to the substrate; And (d) repeating the step (c) to increase the density of the conductive graphene.
The conductive graphene pattern or film according to the present invention may be produced by bonding conductive graphene to a substrate by bonding a thiol group of a substrate having a thiol group exposed on its surface to conductive graphene and then attaching conductive graphene to the conductive graphene stacked on the substrate The conductive graphene can be formed by laminating the conductive graphene to form a conductive graphene pattern, and the conductive graphene having the structure of the substrate - [CONH-R 2 -S-graphene- (SR 3 -S-graphene) p] A pin pattern can be formed. Here, p and q represent a natural number of 1 or more, and R 2 and R 3 represent saturated hydrocarbons, unsaturated hydrocarbons or aromatic organic groups of C 1 -20 .
On the other hand, by repeating the above-described step of laminating the conductive graphene, a high-density conductive graphene film can be produced. Wherein the conductive graphene film may have the structure of a substrate - [CONH-R 2 -S-] - (SR 3 -S-graphene) p] q wherein p and q are natural numbers of 1 or more, and R 2 And R 3 means a saturated hydrocarbon, an unsaturated hydrocarbon or an aromatic organic group.
In the present invention, a substrate having a thiol group exposed on its surface is treated with a chemical substance having an amino functional group exposed on the surface thereof with a chemical substance having both a carboxyl group and a thiol group, thereby forming an amide bond between the amino group on the substrate and the carboxyl group of the chemical substance, A substrate having a thiol group exposed on its surface can be produced. The chemical substance having both the carboxyl group and the thiol group is preferably a substance of HOOC-R 2 -SH, wherein R 2 represents a saturated hydrocarbon, an unsaturated hydrocarbon or an aromatic organic group of C 1 -20 .
The substrate having the amino functional group exposed on the surface can be prepared by treating the substrate with an aminoalkyloxysilane. A coupling agent and a base can be used when the amino group and the carboxyl group are bonded to each other. When the conductive graphene is attached to the substrate by attaching the conductive graphene to the substrate, a linker having a double thiol functional group may be used. The linker having the double thiol functional group is preferably HS-R 3 -SH, wherein R 3 is a saturated hydrocarbon, an unsaturated hydrocarbon, or an aromatic organic group having C 1 -20 .
In the present invention, the substrate is formed with a photoresist or a polymer pattern for attaching the conductive graphene to a desired position, and is selected from the group consisting of glass, silicon, fused silica, plastic, and polydimethylsiloxane (PDMS) .
The invention, in another aspect graphene - (CONH-R 1 -S) r ( where, r is a natural number of 1 or more, R 1 is C 1 -20, saturated hydrocarbons, unsaturated hydrocarbons or aromatic organic group being ) conductivity of graphene or a substrate having the form of a - [CONH-R 2 -S- graphene - (SR 3 -S- graphene) p] q (where, p and q is a natural number of 1 or more, R 2 and R 3 is a saturated hydrocarbon having 1 to 20 carbon atoms, an unsaturated hydrocarbon or an aromatic organic group), or a biosensor that reacts with or reacts with a target biomaterial is adhered to the conductive graphene film. Pin biosensor.
In the present invention, a target biomolecule is a substance capable of acting as a target to be detected or reacted with a bioreceptor, preferably a protein, a nucleic acid, an antibody, an enzyme, a carbohydrate, a lipid, More preferably, the protein is a disease-related protein.
In the present invention, the bioreceptor may be characterized by being an enzyme substrate, a ligand, an amino acid, a peptide, a protein, a nucleic acid, a lipid, a cofactor or a carbohydrate, and the bioreceptor also has a thiol group.
'Conductive graphene' according to the present invention is a concept encompassing that a chemical functional group is attached to graphene. 'Conductive graphene-biosensor' refers to a conductive graphene having a receptor that reacts with a biomaterial, , It can be defined to include a biochip bonded to a conductive graphene. In addition, 'enzyme substrate' can be defined as a generic term for reaction materials involved in enzyme reaction.
The present invention relates to a method for detecting a target biomaterial to be combined with or reacting with a bio-receptor, characterized by using the conductive graphene-biosensor from another viewpoint.
The present invention is, in a further aspect, characterized in that a nucleic acid is attached to a conductive graphene having the form of graphene- (CONH-R 1 -S) r (wherein R 1 and r are as defined above) The present invention relates to a method for producing a nucleic acid chip characterized by binding a conductive graphene-nucleic acid complex and the nucleic acid complex to a substrate to which an amine and / or a lysine group is attached to a surface.
In the present invention, the binding of the graphene-nucleic acid on the substrate may be characterized by using crosslinking by ultraviolet (UV) irradiation, and the nucleic acid may be DNA.
In another aspect, the present invention relates to a DNA chip characterized in that a conductive graphene-DNA complex is attached to a solid substrate, and a method for detecting DNA hybridization using the DNA chip, And an electric signal is used.
In another aspect, the present invention is characterized in that an enzyme substrate is attached to a conductive graphene having the form of graphene (CONH-R 1 -S) r (wherein R 1 and r are as described above) , Wherein the enzyme substrate is a substrate peptide (S P ) of carnauba.
According to another aspect of the present invention, there is provided a method for detecting an enzyme reaction involving carnauba using the conductive graphene- SP complex, wherein the detection is performed using an electrical signal.
In the present invention, a conductive graphene pattern (or film) having high surface density was produced by repeatedly laminating graphene on a solid substrate coated with a chemical functional group through chemical bonding. In addition, various bioreceptors having functional groups present in a high-density graphene pattern were attached to the graphene pattern or film to directly detect various kinds of target biomaterials or to produce biosensors that can be detected using electrochemical signals .
According to the present invention, a desired pattern can be formed at a desired position at a room temperature at a desired position, without departing from the limitations of the prior art in which graphene is grown from a catalyst disposed at a predetermined position. That is, there are two methods of attaching graphene to a substrate: electrical and chemical. While the electrical method can adjust the position of the grapins relatively freely, the chemical method is to immerse the graphene in the suspended solution for a certain time after modifying the substrate to a specific functional group, It is very difficult to attach.
Further, in order to form various patterns by bonding graphene to a desired position of the graphene, it is necessary to expose only a specific portion of the substrate and to withstand a long time in the solution containing the graphene. After the graphene is deposited, It should be easy to attach to substrates such as PDMS.
Accordingly, the present invention improves disadvantages of the prior art by forming a pattern of a substrate using a polymer so that the advantages of a chemical method can be utilized to the utmost. In addition, it is possible to solve the problems of the prior art such as the difficulty of polymer patterning due to the high-temperature mechanism such as the plasma chemical vapor deposition method, the thermal chemical vapor deposition method and the like and the absence of the chemical functional group such as -COOH obtained in the cutting process in strong acid .
In addition, when the biosensor of the present invention is used, an accurate value can be measured using only a small amount of reactant, and the concentration of the ionic substance deposited on the surface can be electrically measured in a liquid phase.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention will now be described in more detail with reference to the accompanying drawings.
One. Graphene - ( CONH - R One -S) r < / RTI > Graphene Produce
FIG. 1 schematically shows a process for producing graphene having a carboxylic functional group (-COOH) as a defect by using an oxidation-reduction method on graphene cut at a strong acid. The carboxyl functional group of the graphene was bound to the amino functional group of the linker having amino (-NH 2 ) functional group and thiol (-SH) functional group at the same time. In this case, DCC (1,3-dicyclohexyl carbodiimide), HATU (O- (7-azabenzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate), HBTU (benzotriazol-1-yl) -1,1,3,3-tetramethyluronium hexafluorophosphate), HAPyU (O- (7-azabenzotriazol-1-yl) -1,1,3,3- bis (tetramethylene) uronium hexafluorophosphate) HAMDU (O- (7-azabenzotriazol-1-yl) -1,3-dimethyl-1,3-dimethylenuronium hexafluorophosphate), HBMDU (O- (benzotriazol- dimethylethyleneuronium hexafluorophosphate and the like and diisopropylethylamine (DIEA), 2,4,6-trimethylpyridine (TMP) and N-methylimidazole (NMI) are preferably used as a base.
When water is used as a solvent, EDC (1-ethyl-3- (3-dimethylamini-propyl) arbodiimide hydrochloride) is used as a coupling agent and N-hydroxysuccinimide (NHS) ) Or the like is preferably used. The coupling agent serves to form an amide bond (-CONH-) with the -COOH functional group and the -NH 2 functional group, and the base and the auxiliary agent serve to enhance the efficiency when the coupling agent forms the amide bond do.
The linker having both the amino functional group and the thiol functional group is preferably a chemical substance represented by NH 2 -R 1 -SH. Here, R 1 means a saturated hydrocarbon, an unsaturated hydrocarbon or an aromatic organic group having C 1 - 20 . Finally, a conductive graphene having the form of graphene (CONH-R 1 -S) r is obtained, wherein r is a natural number of 1 or more.
2. On the substrate Graphene Layer Laminated Graphene Method of forming film
In the present invention, a method is used in which a polymer or a photoresist pattern is formed on a substrate such as glass, silicon wafer or plastic, and then the aminoalkyloxy silane is fixed on the surface of the substrate using the pattern as a mask to expose the amino group to the substrate surface . As the aminoalkyloxysilane, it is preferable to use aminopropyltriethoxysilane.
To expose the thiol functional groups on the surface of said fixed group, HOOC-R 2 the amino group -SH (wherein, R 2 is C 1 -20 saturated hydrocarbons being unsaturated hydrocarbons or aromatic organic group), a thiol such as The amide bond is linked to the carboxyl group of the chemical having both the functional group and the carboxyl functional group at the same time. As a result, a structure in the form of a substrate-CONH-R 2 -SH 'in which a thiol group is exposed on the substrate surface is formed.
At this time, it is preferable to use DIEA, TMP, NMI or the like as a base with DCC, HATU, HBTU, HAPyU, HAMDU, HBMDU and the like as the coupling agent for the amide bond. When water is used as a solvent, it is preferable to use EDC as a coupling agent and NHS or NHSS as a coupling aid.
As shown in FIG. 2, the conductive graphene dotted with gold particles binds to the substrate, 'substrate -CONH-X-SH', in which the thiol functional group is exposed. At this time, an Au-S link is formed between the thiol functional group on the surface of the substrate and the gold crystal dotted on the graphene, so that graphene binds to the substrate, and the structure of 'substrate -CONH-XS-Au- (Fig. 2A).
Next, a chemical represented by HS-R 3 -SH, which is a linker having a double thiol functional group, and gold that is dotted on graphene selectively attached to the substrate is reacted, and the gold-dotted conductive graphene is reacted with the linker React with the other thiol functional group. This reaction forms a structure of the form 'substrate - [CONH-XS-Au-Graphin-Au-SR 3 -S-Au-Graphin-Au]' (FIG.
Next, a chemical reaction between the gold-dotted conductive graphene and the chemical having the double thiol functional group is repeatedly carried out to increase the surface density of the conductive graphene on the surface. A conductive graphene pattern or a conductive graphene film having a structure of 'substrate - [CONH-XS-Au-Graphin-Au- (S-R3-S-Au-Graphin-Au) p] q' (Figs. 2C and 2D). Where p and q are natural numbers greater than or equal to one.
In the case of a biochip using existing graphene, the graphene is grown in a certain region to measure electrical and optical results. However, the present invention has an advantage that graphene can be adhered or deposited at a desired position.
As described above, in order to electrically detect biomaterials attached to a substrate having graphene arranged therein, it is necessary to maintain a liquid phase. In this case, a space for containing a fluid having a thickness of several millimeters to several micrometers should be secured. A variety of polymeric materials such as polydimethylsiloxane (PDMS), polymethylmethacrylate (PMMA), polycarbonate (PC), polyethylene (PE), polypropylene (PP) .
In the present invention, the graphene can be connected to a power source via at least one conductive nanowire (nanowires) to be applied to each of the charge, in which the conductive nanowires can form a single reactor, using conventional techniques, and (Science , 275: 1896-97, 1997), a conductive pattern can be formed with a conductive metal, and then a conductor capable of flowing current can be deposited using implantation or sputtering.
3. On the substrate Thiol The functional group (- SH ) Produce
In the present invention, a method is used in which a polymer or a photoresist pattern is formed on a substrate such as glass, silicon wafer or plastic, and then the aminoalkyloxy silane is fixed on the surface of the substrate using the pattern as a mask to expose the amino group to the substrate surface . As the aminoalkyloxysilane, it is preferable to use aminopropyltriethoxysilane.
To expose the thiol functional groups on the surface of which the amino group is fixed, the unexposed O groups such as HOOC-R 2 -SH (wherein, R 2 is C 1 -20, saturated hydrocarbons, unsaturated hydrocarbons or aromatic organic group) The amide bond is linked to the carboxyl group of the chemical that has the thiol and carboxyl groups at the same time. As a result, a structure in the form of a substrate-CONH-R 2 -SH 'in which a thiol group is exposed on the substrate surface is formed.
At this time, it is preferable to use DIEA, TMP, NMI or the like as a base with DCC, HATU, HBTU, HAPyU, HAMDU, HBMDU and the like as the coupling agent of the amide bond. When water is used as a solvent, it is preferable to use EDC as a coupling agent and NHS or NHSS as a coupling aid.
4. Conductivity On graphene Receptor How to Combine
In the present invention, a bioreceptor is a substance that binds or reacts with a target biomaterial, and is preferably a substance that serves as a probe capable of detecting the binding or reaction. Such bio-receptors include nucleic acids, proteins, peptides, amino acids, ligands, enzyme substrates, cofactors, and the like. In the present invention, a target biomolecule is a substance capable of acting as a target to be detected by binding to or reacting with a receptor, and includes proteins, nucleic acids, enzymes or other biomolecules.
Figure 3 is a schematic diagram showing selective interaction of various types of target biomaterials after attachment of various receptors with functional groups that bind or react with gold on the surface of the conductive graphene. The functional group which reacts with the gold nanocrystals preferably contains a thiol group. 3, 1 and 2 represent a bioreceptor capable of reacting with a target biomaterial, and 4 represent a target biomaterial capable of reacting with the bioreceptor. 3 denotes an oligonucleotide in the bio-receptor, 5 denotes a complementary nucleic acid capable of hybridizing with the oligonucleotide fixed to the metal of the conductive graphene, and 6 denotes a non-reactive biomaterial.
Fig. 4 shows a graphene-Au-substrate peptide complex in which a substrate peptide ( SP ) having a thiol functional group on the conductive graphene is immobilized for kinase enzyme reaction. The electrochemical change of the graphene can be measured by applying it to the phosphorylation reaction by the various carinase enzymes.
As a method of detecting the reaction between the bio-receptor and the bio-material, an electric detection method, a resonance method or a method using a fluorescent material, which are well known in the art, can be used as a built-in detection system. It is preferable to use a method of detecting by electric signals. In this case, a change in the minute potential difference generated in the graphene when the bioreceptor is reacted with the target biomaterial can be monitored and detected through a suitable circuit
5. Coupling Detection System
The reaction result can be measured using a probe station for measuring the electrical characteristics of the biosensor and a fluorescence microscope for detecting the fluorescent substance generated from the biosensor. It is also possible to use an existing method of attaching a radioactive isotope to a reactant and measuring the radiation using a measuring device after the reaction.
In the present invention, a method using electrical properties of the above-mentioned method is embodied in that it utilizes the sensitive electrical properties of graphene. Since it is often necessary to measure in liquid phase due to the nature of the bio material, the present invention focuses on measuring the electrical value of the graphene in the liquid phase. In order to measure the ion concentration of the biomaterial attached to the surface of the graphene, three methods were used in the present invention.
The first one is the measurement using a device such as a potentiostat after inducing a redox reaction using a special solute. The second is to use the concept of a capacitor to measure the amount of ions inside the capacitor plate by an electrical control And the third is to measure the degree to which the thin film of the charging plate is spread according to the intensity of the surrounding ions by using the principle of the large whole.
The first oxidation-reduction reaction is currently a common electrochemical detection method. It uses cyclic voltammetry, potentiometry and amperometry (Potentiostat / Galbanostat, Ametech co ), As shown in Fig. 4, electrodes are immersed in a liquid containing a lead wire connected to the graphene and a specific solute surrounding the bio-material to measure the result before and after the reaction.
Specifically, the graphene-Au-substrate peptide complex according to FIG. 5, in which the substrate peptide of the kinase enzyme is bound to the surface of the graphene, is applied to the kinase enzyme reaction, It is possible to measure by branch method.
FIG. 6 is a schematic diagram showing the detection of an inhibitory action of an agricultural chemical using a conductive GRP-enzyme complex in which AChE is fused with a thiol functional group or a gold binding protein to conductive GRP according to the present invention. The enzyme reaction can be induced by inducing the transfer of electrons generated by the enzyme reaction that converts the substrate to the reactant using the enzyme immobilized on the graphene and AChE inducing the hydrolysis reaction of acetylcholine can be detected by the organic phosphorus Or carbamate-based pesticides. Since the movement of ions and electrons is also inhibited, it can be used as a residual pesticide sensor by a method of measuring the degree of inhibition.
FIG. 7 is a schematic view showing a biosensor using a conductive GRF-enzyme complex in which a GOx fused with a thiol functional group or a gold binding protein is immobilized on the conductive GRF according to the present invention, It is possible to apply the present invention to an enzyme reaction involved in all oxidation and reduction by measuring the redox reaction by inducing the movement of ions / electrons generated by the reaction of oxidizing the substrate using an enzyme, The movement of ions and electrons can be utilized as a sensor by electrochemically changing signals.
1 is a schematic diagram showing a process for functionalizing graphene with an amine or thiol group.
FIG. 2 is a process diagram showing the process of integrating a conductive graphene pattern according to the present invention. FIG. 2 (a) is a cross-sectional view showing a step of exposing a thiol group (-SH) to a surface of a substrate on which a pattern is formed, (B) is a schematic view of fixing a graphen dotted with another gold particle by using a chemical substance having two thiol groups in the graphene monolayer formed in (a), (c) (D) is a method in which the method of (c) above is repeated to laminate graphenes having gold particles dotted thereon at a high density FIG.
Figure 3 is a schematic diagram showing selective interaction with various types of target biomaterials after attachment of various receptors having functional groups on the surface of the conductive graphene. 1 and 2 represent a bioreceptor capable of reacting with a target biomaterial, and 4 represent a target biomaterial capable of reacting with the bioreceptor. 3 denotes an oligonucleotide in the bio-receptor, 5 denotes a complementary nucleic acid capable of hybridizing with the oligonucleotide fixed to the metal of the conductive graphene, and 6 denotes a non-reactive biomaterial.
FIG. 4 is a schematic diagram for utilizing graphene as a fusion protein in which streptavidin is bound so that DNA can be bound to the conductive graphene and using the biotin-DNA complex to bind the DNA locally on the graphene wall surface to be.
FIG. 5 is a schematic view showing a biosensor using a conductive graphene-peptide substrate complex in which a thiourea or a gold binding protein and a fused substrate peptide of the fused form are immobilized on the conductive graphene according to the present invention.
FIG. 6 is a schematic view showing the detection of an inhibitory action of an agrochemical using a conductive carbon nanotube-enzyme complex in which AChE fused with a thiol functional group or a gold binding protein is immobilized on the conductive carbon nanotube according to the present invention.
7 is a schematic view showing a biosensor using a conductive carbon nanotube-enzyme complex in which a GOx fused with a thiol functional group or a gold binding protein is immobilized on the conductive carbon nanotube according to the present invention.
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