KR20220031523A - Graphene Channel Member for Detecting Anti-stress Hormone and Sensor Comprising the Same - Google Patents

Abstract

The present invention relates to: a graphene channel member comprising a graphene film and a bioprobe specifically binding to the anti-stress hormone immobilized on the graphene film; a graphene transistor including a substrate, the graphene channel member, and a pair of electrodes; and a biosensor including the same.

Description

Graphene Channel Member for Detecting Anti-stress Hormone and Sensor Comprising the Same

The present invention relates to a graphene channel member used for detection of an anti-stress hormone and a sensor comprising the same.

In modern society, increasing mental stress due to unlimited competition, employment, and lifestyle is gradually emerging as a serious social problem, and it is known that it causes diseases directly related to life such as depression and heart attack. is the trend

Cortisol is a glucocorticoid hormone and is produced in the adrenal cortex. When you are in a stressful situation, the adrenal cortex secretes cortisol, which is one of the hormones that help you overcome stress and adapt. In addition, serotonin (serotonin) is one of the chemicals that function as a neurotransmitter present in the hypothalamus of the brain, and is involved in food, sleep, mood, gastrointestinal motility, pain, and is generally deeply involved in depression. .

The secretion of anti-stress hormones such as cortisol and serotonin as described above is known to be directly related to the induction of related diseases such as depression, and the importance of rapid, accurate and quantitative measurement and testing thereof is increasing.

However, most of the conventional research is limited to instantaneous detection in saliva, sweat, and blood of a subject. In the past, sensors for detecting neurotransmitters using optical and electrical signals through antigen-antibody immune response have been mainly studied. Since the noise intensity of these conventional sensors is often higher than that of an effective signal, it is difficult to distinguish a signal generated by an actual detection target material from a signal generated by other materials.

Therefore, by using a bio-probe that specifically or selectively binds only the detection target material, the problem of various biomaterials reacting directly with the conductive material to decrease the sensitivity is solved, and the signal is greatly amplified even in the presence of a small amount of the detection target material Research is underway to enable multiple detection of various anti-stress hormones.

The present invention has been devised to overcome the limitations of the conventional anti-stress hormone sensor as described above, and a graphene channel member in which a bioprobe that specifically or selectively binds only an anti-stress hormone is immobilized on a graphene film. It is intended to provide a graphene transistor sensor with high sensitivity.

The present invention provides a graphene film and an anti-stress hormone immobilized on the graphene film, which includes a bio-probe specifically or selectively binding to the graphene channel member.

In addition, the present invention is a substrate; the graphene channel member; And a pair of electrodes; provides a graphene transistor comprising a.

The present invention also provides a biosensor including the graphene transistor.

Since the graphene channel member of the present invention includes bioprobes that are each selective for various anti-stress hormones, multiple detection through each bioprobe is possible, so there is a limitation in diagnosing depression only with a change of a single anti-stress hormone can overcome Ultimately, there is an effect of rapidly and accurately detecting various anti-stress hormones that are directly related to human mental diseases (depression, etc.) at the same time.

1 and 2 are exemplary views illustrating miniaturization of a graphene transistor according to an embodiment of the present invention in a form that can be connected to a SIM chip.
3 shows ¹ H-NMR spectrum of carbene compound 1 according to Preparation Example 1.
4 shows ¹ H-NMR spectrum of carbene compound 2 according to Preparation Example 2.
5 shows a ¹ H-NMR spectrum of the carbene compound 3 according to Preparation Example 3.
6 shows a ¹ H-NMR spectrum of the carbene compound 4 according to Preparation Example 4.
7 shows a ¹ H-NMR spectrum of the carbene compound 5 according to Preparation Example 5.
8 is a view showing a schematic diagram showing the process of functionalizing the graphene film in one embodiment of the present invention.
9 shows Keithley equipment used in specific experimental examples of the present invention.
10 shows the detection result of cortisol measured using Keithley equipment according to Experimental Example 1 of the present invention.
11 shows the detection result of serotonin measured using Keithley equipment according to Experimental Example 1 of the present invention.
12 shows the detection result of cortisol in human saliva measured using the graphene transistor of Example 3 according to Experimental Example 2 of the present invention.
13 shows the detection result of serotonin in human saliva measured using the graphene transistor of Example 3 according to Experimental Example 2 of the present invention.
14 shows the detection results for each concentration of cortisol measured using the graphene transistor of Example 3 according to Experimental Example 3 of the present invention.
15 shows the detection results for each concentration of serotonin measured using the graphene transistor of Example 3 according to Experimental Example 3 of the present invention.
16 shows cortisol (A) and serotonin (B) in serum samples derived from patients suffering from depression and patients without depression using the graphene transistor of Example 3 according to Experimental Example 4 of the present invention. , and the dotted lines in (A) and (B) of FIG. 16 are quantitative curves for each concentration of cortisol and serotonin, respectively.
17 shows the results of analyzing cortisol (A) and serotonin (B) in a serum sample derived from patient A suffering from depression using the graphene transistor of Example 3 according to Experimental Example 4 of the present invention. will be.

Hereinafter, the present invention will be described in detail.

1. Graphene Channel Absence

One aspect of the present invention provides a graphene channel member.

The graphene channel member of the present invention includes a graphene film, and a bio-probe.

The graphene film is for supporting the bio-probe, and may be configured in a form in which a graphene layer is laminated as a single layer or a multi-layer. When the graphene layer is formed as a multilayer, the surface resistance of the graphene film itself is reduced, and thus the sensitivity of a device such as a transistor to which a graphene channel member is applied may be reduced. Therefore, the graphene film is more preferably composed of a single graphene layer. In particular, the thickness of the graphene film may be 0.1 to 1 nm, which may mean the thickness of a single-layer graphene film. When the thickness of the graphene film satisfies the above range, it has properties of high conductivity and high charge mobility, and it is possible to implement a device such as a transistor with high sensitivity by using the graphene channel member using the same.

On the other hand, the graphene film may be patterned, specifically, may be finely patterned. For example, the graphene film may be variously patterned in a polygonal shape such as a circle, a triangle, a square, a pentagon, or a hexagon (honeycomb). Since the shape of the graphene film can be diversified by patterning the graphene film as described above, the graphene channel member using the same can be applied to devices having various shapes without design restrictions.

The bioprobe is a bioprobe that specifically or selectively binds to an anti-stress hormone, in particular an antibody, an aptamer, a receptor, etc. there is. For example, the antibodies may be commercially available as anti-cortisol antibodies and/or anti-serotonin antibodies. In addition, the aptamer may be obtained commercially as specifically or selectively binding to cortisol and serotonin, or may be prepared by a method known in the art. For example, the aptamer for cortisol is 5'-ATG GGC AAT GCG GGG TGG AGA ATG GTT GCC GCA CTT CGG C/3AmMO/-3' may have a nucleotide sequence, and the aptamer for serotonin is 5'-/5AmMC6/CGA CTG GTA GGC AGA TAG GGG AAG CTG ATT CGA GCG TGG GTC It may be one having the nucleotide sequence of G-3'. In addition, the receptor may be a 2Q1V receptor, a 5-HT _1E receptor, or the like. The 2Q1V receptor specifically or selectively binds to cortisol, and may have an amino acid sequence reported as Genbank Accession 2Q1V_A. In addition, the 5-HT _1E receptor binds specifically or selectively to serotonin, and has an amino acid sequence reported as Genbank Accession NP_000856, for example, encoded by a gene having a nucleotide sequence reported as Genbank Accession NM_000856 it may be The 2Q1V receptor and/or 5-HT _1E receptor may be obtained commercially or directly synthesized and expressed in a host cell such as E. coli.

The bioprobe is fixed on the graphene film. The immobilization may be accomplished through physical or chemical bonding, and in particular, the bioprobe may be chemically immobilized through a linker. That is, the linker may serve as a medium for immobilizing the bioprobe to the graphene film.

In this case, the linker is a carbene compound represented by Formula 1 or Formula 2; And 1,5-diaminonaphthalene (1,5-diaminonaphthalene, DAN); may include at least one selected from the group consisting of.

[Formula 1]

[Formula 2]

In Formulas 1 and 2,

R1, R2, R5 and R6 are the same as or different from each other and are each independently hydrogen, a substituted or unsubstituted alkyl group having 1 to 20 carbon atoms, a substituted or unsubstituted cycloalkyl group having 3 to 20 carbon atoms, a substituted or unsubstituted cycloalkyl group having 6 to 30 carbon atoms, or an unsubstituted aryl group or a substituted or unsubstituted heteroaryl group having 2 to 30 carbon atoms;

R3, R4, R7, R8, R9 and R10 are the same as or different from each other and each independently represent hydrogen, a substituted or unsubstituted alkyl group having 1 to 20 carbon atoms, a substituted or unsubstituted cycloalkyl group having 3 to 20 carbon atoms, and 6 to carbon atoms A substituted or unsubstituted aryl group of 30, a substituted or unsubstituted heteroaryl group having 2 to 30 carbon atoms, or a structure represented by the following Chemical Formula 3, or two or more adjacent substituents of R7 to R10 combine to form a hydrocarbon ring and,

At least one of R3 and R4 is a structure represented by the following formula (3),

At least one of R7 to R10 is a structure represented by the following formula (3), or when two or more substituents adjacent to each other of R7 to R10 combine to form a hydrocarbon ring, at least one of the hydrogens bonded to the carbon forming the hydrocarbon ring is substituted with a structure represented by the following formula (3),

[Formula 3]

In Formula 3,

n is an integer from 1 to 30 as the number of repeating units in parentheses,

A is an alkyl group having 1 to 20 carbon atoms including a nitrogen (N) atom or a substituted or unsubstituted heteroaryl group having 2 to 30 carbon atoms including a nitrogen (N) atom.

R1, R2, R5 and R6 may be the same as or different from each other and each independently represent hydrogen, an alkyl group having 1 to 20 carbon atoms, or an aryl group having 6 to 30 carbon atoms.

R1, R2, R5 and R6 may be the same as or different from each other and each independently represent hydrogen, an alkyl group having 1 to 10 carbon atoms, or an aryl group having 6 to 10 carbon atoms.

R1, R2, R5 and R6 may be the same as or different from each other and each independently hydrogen or an alkyl group having 1 to 10 carbon atoms.

R1, R2, R5 and R6 may be the same as or different from each other and each independently hydrogen, isopropyl or benzyl.

At least one of R1 and R2 and at least one of R5 and R6 may be the same as or different from each other and may each independently be an alkyl group having 1 to 20 carbon atoms or an aryl group having 6 to 30 carbon atoms.

At least one of R1 and R2 and at least one of R5 and R6 may be the same as or different from each other, and each independently may be isopropyl or benzyl.

R3, R4, R7, R8, R9 and R10 are the same as or different from each other and each independently represent hydrogen, an alkyl group having 1 to 20 carbon atoms, a cycloalkyl group having 3 to 20 carbon atoms, an aryl group having 6 to 30 carbon atoms, and 2 to 30 carbon atoms. of a heteroaryl group or a structure represented by Formula 3, or two or more substituents adjacent to each other among R7 to R10 may combine to form a hydrocarbon ring.

Wherein R3, R4, R7, R8, R9 and R10 are the same as or different from each other and each independently represent hydrogen or a structure represented by Formula 3, or two or more adjacent substituents of R7 to R10 combine to form a hydrocarbon ring can

When two or more adjacent substituents of R7 to R10 combine to form a hydrocarbon ring, at least one of carbons forming the hydrocarbon ring may be substituted with a structure represented by Formula 3 above.

At least one of R3 and R4 may have a structure represented by Formula 3 above. In addition, at least one of R7 to R10 may have a structure represented by Formula 3 above.

The n may be an integer of 1 or more to 30 or less as the number of repetitions of units in parentheses. In particular, n may be 1 or more, 2 or more, or 3 or more, and 30 or less, 27 or less, 25 or less, 23 or less, 20 or less, 17 or less, 15 or less, 13 or less, 10 or less, 7 or less, or 5 or less.

A is an alkyl group having 1 to 20 carbon atoms including a nitrogen (N) atom or a heteroaryl group having 2 to 30 carbon atoms including a nitrogen (N) atom. Specifically, A may be azide, phthalimide, or amine.

In the present invention, "adjacent" group means a substituent substituted on an atom directly connected to the atom in which the substituent is substituted, a substituent sterically closest to the substituent, or another substituent substituted on the atom in which the substituent is substituted. can For example, two substituents substituted at an ortho position in a benzene ring and two substituents substituted at the same carbon in an aliphatic ring may be interpreted as "adjacent" groups.

The alkyl group may be linear or branched, and may have 1 to 20 carbon atoms, and preferably 1 to 10 carbon atoms. More preferably, it may have 1 to 6 carbon atoms. Specific examples of the alkyl group include methyl group, ethyl group, propyl group, n-propyl group, isopropyl group, butyl group, n-butyl group, isobutyl group, tert-butyl group, sec-butyl group, 1-methylbutyl group, 1-ethylbutyl group, pentyl group, n-pentyl group, isopentyl group, neopentyl group, tert-pentyl group, hexyl group, n-hexyl group, 1-methylpentyl group, 2-methylpentyl group, 4-methyl group -2-pentyl group, 3,3-dimethyl butyl group, 2-ethylbutyl group, heptyl group, n-heptyl group, 1-methylhexyl group, cyclopentylmethyl group, cyclohexylmethyl group, octyl group, n-octyl group, tert-octyl group, 1-methylheptyl group, 2-ethylhexyl group, 2-propylpentyl group, n-nonyl group, 2,2-dimethylheptyl group, 1-ethylpropyl group, 1,1-dimethylpropyl group, an isohexyl group, a 4-methylhexyl group, a 5-methylhexyl group, or a benzyl group, but is not limited thereto.

The cycloalkyl group may have 3 to 20 carbon atoms, preferably 3 to 10 carbon atoms. Specific examples of the cycloalkyl group include a cyclopropyl group, a cyclobutyl group, a cyclopentyl group, a 3-methylcyclopentyl group, a 2,3-dimethylcyclopentyl group, a cyclohexyl group, a 3-methylcyclohexyl group, and a 4-methylcyclo a hexyl group, a 2,3-dimethylcyclohexyl group, a 3,4,5-trimethylcyclohexyl group, a 4-tert-butylcyclohexyl group, a cycloheptyl group, or a cyclooctyl group, but is not limited thereto.

The aryl group may have 6 to 30 carbon atoms, preferably 6 to 10 carbon atoms. The aryl group may be a monocyclic aryl group or a polycyclic aryl group. Specific examples of the monocyclic aryl group include a phenyl group, a biphenyl group, or a terphenyl group, and specific examples of the polycyclic aryl group include a naphthyl group, an anthracenyl group, a phenanthryl group, a pyrenyl group, a perylenyl group, and cry There is a cenyl group, a fluorenyl group, or a triphenylene group, but is not limited thereto.

The heteroaryl group is an aromatic ring group including at least one selected from N, O, P, S, Si and Se as a heteroatom, and may have 2 to 30 carbon atoms, preferably 2 to 20 carbon atoms. . Specific examples of the heteroaryl group include a thiophene group, a furan group, a pyrrole group, an imidazole group, a thiazole group, an oxazole group, an oxadiazole group, a triazole group, a pyridyl group, a pyrimidyl group, a triazine group, a triazole group, acri Diyl group, quinolinyl group, quinazoline group, quinoxalinyl group, phthalazinyl group, isoquinoline group, indole group, carbazole group, benzoxazole group, benzimidazole group, benzothiazole group, benzocarbazole group, benzothiophene group , a dibenzothiophene group, or a benzofuranyl group, but is not limited thereto.

In addition, the alkyl group, cycloalkyl group, aryl group, heteroaryl group or hydrocarbon ring may be unsubstituted or substituted with an alkyl group, a cycloalkyl group, an aryl group or a heteroaryl group.

When the carbene compound represented by Formula 1 or Formula 2 is used as a linker, the lone pair of electrons of the carbene compound may be chemically bonded (covalently bonded) to the graphene film, for this purpose, for example, the carbene compound is attached to the graphene film. After the compound is treated, it can be reacted by applying a temperature of 100° C. or higher.

Meanwhile, the 1,5-diaminonaphthalene is a compound represented by the following Chemical Formula 4, and may be commercially obtained or directly synthesized.

[Formula 4]

When the 1,5-diaminonaphthalene is used as a linker, the naphthalene functional group of the 1,5-diaminonaphthalene may be bonded to the graphene film through a pi-pi interaction, and this For example, a solution of 1,5-diaminonaphthalene and glutaraldehyde in distilled water at a concentration of 1 to 10 μM is added to the surface of the graphene film by 5 to 10 μl, and the result is at room temperature for about 10 to 25 minutes. A method of washing the surface of the graphene film with distilled water after the reaction can be used. In this case, the glutaraldehyde may serve as a coupling agent for chemically bonding 1,5-diaminonaphthalene and the bio-probe that specifically or selectively binds to the anti-stress hormone.

In addition, the bioprobe that specifically or selectively binds to the anti-stress hormone may physically or chemically bind to the linker, and when the linker is a carbene compound or 1,5-diaminonaphthalene, chemical and, for example, the functional group of the carbene compound or 1,5-diaminonaphthalene compound and the functional group of the bioprobe may be combined with a coupling agent (eg, DMTMM (4-(4,6-Dimethoxy-1, 3,5-triazin-2-yl)-4-methylmorpholinium tetrafluoroborate), EDC-NHS (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide), glutaraldehyde (GA), etc. ) can be chemically combined.

When the bioprobe is immobilized on the graphene film through the linker as described above, there is an advantage in that the bioprobe becomes more stable from changes in the external environment, and stability against external environmental changes is improved.

On the other hand, when the graphene film is patterned, the bioprobe may be coupled to the surface of the patterned graphene film. At this time, as described above, the bioprobe may be immobilized on the patterned graphene film by a chemical bond using the carbene compound or 1,5-diaminonaphthalene represented by Formula 1 or Formula 2 as a linker.

As described above, the graphene channel member of the present invention in which the bioprobe is immobilized using the carbene compound represented by Formula 1 or Formula 2 and/or 1,5-diaminonaphthalene as a linker on the graphene film, graphene Because the layer is used, high current flows even in the off state when no voltage is applied to the gate, and the on/off ratio of the operating current is very low. Since it shows high stability, it has the advantage of being able to implement a device with high usability and usability. In addition, as a bioprobe for the anti-stress hormone on the graphene film, a bioprobe such as a 2Q1V receptor that specifically or selectively binds to cortisol and a 5-HT _1E receptor that specifically or selectively binds to serotonin When all of the bio-probes are immobilized, there is also an advantage that real-time multiplex detection of various anti-stress hormones is possible.

2. Graphene Transistor

The present invention also provides a graphene transistor including the graphene channel member.

The graphene transistor of the present invention includes: a substrate; the aforementioned graphene channel member; and a pair of electrodes.

The substrate serves as a support on which the components of the graphene transistor of the present invention are supported, and an insulating inorganic substrate such as a Si substrate, a glass substrate, a GaN substrate, a silica (SiO ₂ ) substrate, and a metal such as Ni, Cu, W A substrate or a plastic substrate (eg, polyethylene naphthalate (PEN), etc.) may be used, and when an insulating substrate is used, in terms of excellent affinity with the graphene channel member, silica (SiO ₂ ) It is preferable that it is a board|substrate or a silicon wafer. In addition, the substrate may be selected from various materials capable of depositing graphene, for example, may be made of a material such as silicon-germanium or silicon carbide (SiC), and an epitaxial layer, silicon-on. - may include a silicon-on-insulator layer, a semiconductor-on-insulator layer, and the like.

The graphene channel member may be formed on the substrate.

Specifically, the graphene film may be formed by growing graphene on the substrate by a chemical vapor deposition method using a hydrocarbon gas as a carbon source.

The graphene film may be formed using, for example, chemical vapor deposition, and by using this, single to several layers of graphene having excellent crystallinity can be obtained over a large area. The chemical vapor deposition method is a method of growing graphene by adsorbing, decomposing, or reacting a carbon precursor in the form of a gas or vapor having high kinetic energy on the substrate surface to separate it into carbon atoms, and making the carbon atoms bond with each other. .

The chemical vapor deposition method may be at least one selected from the group consisting of Plasma Enhanced Chemical Vapor Deposition (PECVD), Atmospheric Pressure Chemical Vapor Deposition (APCVD), and Low Pressure Chemical Vapor Deposition (LPCVD), and minimize defects in a large area It is preferable that the chemical vapor deposition method is LPCVD in that deposition is possible.

As a specific method of the chemical vapor deposition, for example, a metal catalyst such as nickel, copper, aluminum, iron is deposited on a wafer having a silicon oxide layer using a sputtering device and an electron beam evaporation device to form a metal catalyst layer, CH ₄ , C ₂ H ₂ It is put into a reactor together with a gas containing carbon, such as, heated, so that carbon is absorbed in the metal catalyst layer, cooled to separate carbon from the metal catalyst layer and crystallized, and finally the metal By removing the catalyst layer, a graphene film can be formed.

However, the method for forming the graphene film is not limited to the chemical vapor deposition method, and various methods may be used to form the graphene film.

For example, in a graphite crystal composed of multiple layers, a physical exfoliation method to make graphene by peeling off one layer by mechanical force, a chemical exfoliation method utilizing oxidation-reduction properties, or SiC, where carbon is adsorbed or contained in the crystal A graphene film can be formed by using an epitaxial synthesis method in which a material is heat-treated at a high temperature of 1,500 °C.

The pair of electrodes may be a source electrode and a drain electrode formed to be spaced apart from each other on the graphene film in order to apply a voltage to the graphene channel member.

The source electrode and the drain electrode may be electrically connected through the graphene film, may include a conductive material, and may be formed of, for example, a metal, a metal alloy, a conductive metal oxide, or a conductive metal nitride. .

The source electrode and the drain electrode are each independently Cu, Co, Bi, Be, Ag, Al, Au, Hf, Cr, In, Mn, Mo, Mg, Ni, Nb, Pb, Pd, Pt, Re, Rh, Sb, Ta, Te, Ti, W, V, Zr, Zn, and may include at least one selected from the group consisting of Zn and combinations thereof, but is not limited thereto, in terms of contact with graphene and ease of etching. , Au, or a Cr/Au alloy is preferred.

The pair of electrodes may be formed by a method known in the art, but for example, photolithography, thermal deposition, E-beam deposition, plasma enhanced (PECVD) It may be formed by a deposition method such as Chemical Vapor Deposition), LPCVD (Low Pressure Chemical Vapor Deposition), PVD (Physical Vapor Deposition), sputtering, or ALD (Atomic Layer Deposition).

The graphene channel member may be a bio-probe that specifically or selectively binds to an anti-stress hormone on the graphene film is directly bound (immobilized) by physical adsorption, or to an anti-stress hormone. The bio-probe that specifically or selectively binds may be bound (immobilized) via the carbene compound or 1,5-diaminonaphthalene compound. The graphene channel member, the bioprobe that specifically or selectively binds to the anti-stress hormone, the carbene compound, the 1,5-diaminonaphthalene compound, and the graphene film may be the same as described above.

When the bioprobe specifically or selectively binding to the anti-stress hormone includes the carbene compound or the 1,5-diaminonaphthalene compound as a linker and is bound (immobilized) to the graphene film, on the graphene film The linker bound to may form a linker layer in the form of a single layer, and the bioprobe specifically or selectively binding to the anti-stress hormone immobilized on the carbene compound also forms a bioprobe layer in the form of a single layer. can do.

When the linker layer is formed as a single layer, graphene not only has excellent charge mobility, transparency and/or flexibility, but also has the effect of blocking noise signals due to the approach of external non-specific charges.

In addition, the linker layer may have a thickness of 0.1 to 2 nm. When the thickness of the linker layer is thinner than 0.1 nm, there is a problem in that resistance increases, and when the thickness of the linker layer is thicker than 2 nm, there is a problem in that the interaction between the linkers increases, thereby reducing the immobilization efficiency of the bioprobe.

3. Biosensor

Another aspect of the present invention provides a biosensor including the graphene transistor described above.

The biosensor according to the present invention uses a semiconductor characteristic in which a current flowing in a graphene film between a source and a drain electrode changes due to an electric field effect.

Specifically, when a bioprobe that specifically or selectively binds to the anti-stress hormone formed on the surface of the graphene film reacts with cortisol or serotonin, a change in the surrounding electric field occurs, which causes a gap between the source electrode and the drain electrode. The value of the current flowing through the graphene film changes together, and the target can be detected by measuring the change in the current.

This biosensor is excellent in sensitivity, specificity, speed and/or portability by using the graphene transistor as described above. It has excellent sensitivity and real-time detection performance, and accordingly, the detection limit of the anti-stress hormone generated by the metabolic process of the human body is improved, thereby enabling detection with high sensitivity and reproducibility.

In addition, when the linker layer is formed on the graphene film in the graphene transistor as described above and a bioprobe layer that specifically or selectively binds to the anti-stress hormone bound thereto is present in the channel region of the graphene transistor, the sensor Not only the sensitivity is further improved, but also the doping treatment and the attachment of a bioprobe that specifically or selectively binds to the anti-stress hormone can be performed at the same time, thereby simplifying the process.

Furthermore, the aforementioned graphene transistor is manufactured in the form of a SIM chip as shown in FIGS. 1 and 2 and can be applied to a miniaturized biosensor (portable electronic gas sensor, etc.) It can be identified very simply and accurately.

Hereinafter, the present invention will be described in more detail with reference to preferred embodiments.

However, these examples are for explaining the present invention in more detail, and the scope of the present invention is not limited thereto.

<Production Example>

<Production Example 1>

According to the above reaction scheme, carbene compound 1 was prepared.

¹ H-NMR spectrum of the carbene compound 1 is shown in FIG. 3 .

<Preparation Example 2>

According to the above reaction scheme, carbene compound 2 was prepared.

¹ H-NMR spectrum of the carbene compound 2 is shown in FIG. 4 .

<Production Example 3>

According to the above reaction scheme, carbene compound 3 was prepared.

¹ H-NMR spectrum of the carbene compound 3 is shown in FIG. 5 .

<Production Example 4>

According to the above reaction scheme, carbene compound 4 was prepared.

¹ H-NMR spectrum of the carbene compound 4 is shown in FIG. 6 .

<Preparation Example 5>

According to the above reaction scheme, carbene compound 5 was prepared.

¹ H-NMR spectrum of the carbene compound 5 is shown in FIG. 7 .

<Preparation Example 6>

The nucleotide sequence encoding the 2Q1V receptor having the amino acid sequence reported as Genbank Accession 2Q1V_A is synthesized, amplified by PCR, and purified by gel extraction. After the amplified genes are inserted into the pENTR vector, they are inserted and cultured in E. coli, and the strain into which the vector has been properly introduced is selected with kanamycin.

In the selected strain, the gene inside the pENTR vector is transferred to the pDEST42 vector using a genetic recombination method, which is again inserted into E. coli and cultured, and then selected with ampicillin to select the strain into which the vector is properly introduced.

After culturing the selected strain to amplify the pDEST42 vector, it was again inserted into E. coli BL21 (DE3), and expression was induced with IPTG (Isopropyl β-D-1-thiogalactopyranoside) to produce 2Q1V receptor.

<Production Example 7>

A gene of 5-HT _1E receptor having a nucleotide sequence reported as Genbank Accession NM_000856 was synthesized, and 5-HT _1E receptor was produced in the same manner as in Example 6.

<Example 1> Preparation of graphene transistor for detecting cortisol

1-1. Formation of a graphene film on a substrate

A copper foil was placed in the chamber, heated to 1,000° C., held at H ₂ 90 mTorr and 8 sccm for 30 min (20 min pre-annealing and 10 min stabilization), and then CH ₄ was heated to 20 sccm 40 A single-layer graphene film (graphene layer) was formed on the copper foil by applying a total pressure of 560 mTorr for minutes, then cooling it to 200°C at 35°C/min, and cooling the furnace to room temperature. formed.

Next, a polymethyl methacrylate (PMMA, MicroChem Corp, 950 PMMA A4, 4% in anisole) solution was spin-coated at a speed of 6,000 rpm per minute on the graphene film formed on the copper foil, and an etchant (etchant) ) was used to separate the PMMA-coated graphene film from the copper foil, and the graphene film separated from the copper foil as described above was immersed in deionized distilled water for 10 minutes to remove the remaining film on the graphene film. The shunt ions were removed.

The graphene film is formed on the substrate by transferring the cleaned graphene film to a silicon wafer as a substrate, and then dropping the PMMA solution on the graphene film to remove the PMMA coating the graphene film. did At this time, transparency was maintained at 97.8%.

A positive photoresist (AZ5214, Clariant Corp) was spin-coated on the graphene film formed on the substrate, and then the graphene film was patterned through UV exposure, baking and development processes.

1-2. electrode formation

A pattern electrode (width/length=W/L=1, L=100 μm channel length) was applied to both ends of the graphene film that was patterned and aligned as described above through RIE (Reactive Ion Etching) (oxygen plasma treatment) method. After forming, a graphene transistor in which an electrode (W/L=5, L=100 μm channel length) is formed on a part of the graphene film through the process of image inversion, thermal deposition, and lift-off prepared.

1-3. Formation of a linker layer

A linker layer was formed on the graphene film formed as described above in the same manner as shown in FIG. 8 . Specifically, the carbene compound 1 (50 mg) of Preparation Example 1 was dissolved in a THF solvent (10 mL), added to the graphene film, mixed with 2 mM tetrabutylammonium fluoride, and reacted at room temperature for 30 minutes. Then, it was washed with THF and the solvent was removed to form a linker layer.

1-4. Immobilization of the anti-stress hormone receptor (2Q1V)

After dissolving 1 mg each of EDC (1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide HCl) and N-hydroxysuccinimide (NHS) reagent in pH 4.5 MES buffer, the EDC-NHS solution was placed on a graphene transistor at room temperature at room temperature. After 30 minutes of treatment, washing, the 2Q1V receptor of Preparation Example 6 was placed and reacted at room temperature for 4 hours to prepare a graphene transistor in which the 2Q1V receptor was immobilized.

<Example 2> Preparation of graphene transistor for serotonin detection

In Example 1-4., 5-HT _1E receptor was prepared in the same manner as in Example 1, except that the 5-HT _1E receptor of Preparation 7 was used instead of the 2Q1V receptor of Preparation Example 6. An immobilized graphene transistor was fabricated.

<Example 3> Preparation of graphene transistor for simultaneous detection of cortisol and serotonin

In Example 1-4., instead of the 2Q1V receptor of Preparation Example 6, the 2Q1V receptor of Preparation Example 6 and the 5-HT _1E receptor of Preparation Example 7 were used together. In the same manner, a graphene transistor in which both the 2Q1V receptor and the 5-HT _1E receptor were immobilized was prepared.

<Experimental Example 1>

Using Keithley equipment as shown in FIG. 9, a cortisol detection experiment of the graphene transistor of Example 1 was performed, and the results are shown in FIG. 10, and the serotonin detection experiment of the graphene transistor of Example 2 was carried out and the results are shown in FIG. 11 .

According to FIG. 10, in the case of the graphene transistor of Example 1, it was not detected in a serotonin 10 μM solution, but was detected in a cortisol 100 nM, 1 μM, 10 μM solution, so it was confirmed that there was cortisol selectivity.

In addition, according to FIG. 11, in the case of the graphene transistor of Example 2, although it was not detected in a 1 μM solution of cortisol, it was confirmed that serotonin selectivity was found in that it was detected in a 10 nM, 10 μM solution of serotonin.

<Experimental Example 2>

Using the graphene transistor of Example 3, a detection experiment of cortisol derived from human saliva (saliva) was performed, the results are shown in FIG. 12, and a detection experiment of serotonin in human saliva (saliva) was performed Thus, the results are shown in FIG. 13 .

According to FIG. 12, when human saliva (undiluted saliva) is injected, it can be confirmed that cortisol and/or serotonin in saliva is detected by a graphene transistor, and artificial saliva (Insung Chromatek Co., Ltd., Cat. No. 1700-0303) ), it can be confirmed that cortisol can be detected using the graphene transistor of the present invention even when cortisol is diluted to 10 ^-4 M, 10 ^-2 M concentrations.

In addition, according to FIG. 13, when serotonin was diluted to a concentration of 10 ^-3 M, 10 ^-2 M, and 10 ^-1 M in artificial saliva (Inseong Chromatek Co., Ltd., Cat. No. 1700-0303), the present invention It can be confirmed that serotonin can be detected using the graphene transistor of

<Experimental Example 3>

A solution was prepared by diluting the concentration of cortisol to 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, and 1 μM using a PBS buffer, and a detection experiment using the graphene transistor of Example 3 and the results are shown in FIG. 14, and a solution obtained by diluting the concentration of serotonin to 10 pM, 100 pM, 1 nM, 10 nM, and 100 nM using a PBS buffer was prepared, and the graphene transistor of Example 3 A detection experiment was performed using , and the results are shown in FIG. 15 .

14 and 15, in the case of the graphene transistor of the present invention, cortisol and serotonin can be detected separately by concentration, and furthermore, it can be confirmed that both cortisol and serotonin can be precisely detected at a level of 10 pM. can

<Experimental Example 4>

Using the graphene transistor of Example 3, cortisol and serotonin were detected in serum from an actual patient.

Specifically, a total of 23 patients with depression and patients without depression were provided with serum samples from St. Vincent's Hospital, and these samples were diluted 100-fold with PBS buffer and processed on the graphene transistor of Example 3 above. The detection of cortisol and serotonin was confirmed.

As a result, as shown in FIG. 16 , it was confirmed that among 23 patients, patients with particularly high cortisol (A) and/or serotonin (B) were clearly distinguished.

In addition, cortisol and serotonin were detected by processing a sample in which serum derived from patient A suffering from depression was diluted 100 times with PBS buffer among the 23 patients with the graphene transistor of Example 3.

As a result, as shown in FIG. 17 , it was confirmed that both peaks for cortisol (A) and serotonin (B) were significantly increased after the sample was processed.

From the above results, it can be seen that the sensor according to the present invention can be effectively applied to easily, quickly, and clearly quantitatively and qualitatively analyze the level of anti-stress hormone from a serum sample derived from an actual patient.

Claims (13)

A graphene film and an anti-stress hormone immobilized on the graphene film comprising a bio-probe specifically binding to the graphene film. The method according to claim 1,
The anti-stress hormone-specifically binding bio-probe is at least one selected from the group consisting of an antibody, an aptamer, and a receptor, graphene channel member. 3. The method according to claim 2,
The anti-stress hormone is at least one selected from the group consisting of cortisol and serotonin, graphene channel member. 4. The method according to claim 3,
The receptor is a graphene channel member comprising at least one selected from a 2Q1V receptor and a 5-HT _1E receptor.
The method according to claim 1,
The bio-probe is immobilized on the graphene film via a linker as a medium, the graphene channel member. 6. The method of claim 5,
The linker is a carbene compound represented by Formula 1 or Formula 2; and
1,5-diaminonaphthalene (1,5-diaminonaphthalene, DAN); will include at least one selected from the group consisting of, the graphene channel member.
[Formula 1]

[Formula 2]

In Formulas 1 and 2,
R1, R2, R5 and R6 are the same as or different from each other and each independently represent hydrogen, an alkyl group having 1 to 20 carbon atoms, a cycloalkyl group having 3 to 20 carbon atoms, an aryl group having 6 to 30 carbon atoms, or a heteroaryl group having 2 to 30 carbon atoms; ,
R3, R4, R7, R8, R9 and R10 are the same as or different from each other and each independently represent hydrogen, an alkyl group having 1 to 20 carbon atoms, a cycloalkyl group having 3 to 20 carbon atoms, an aryl group having 6 to 30 carbon atoms, or an aryl group having 2 to 30 carbon atoms. A heteroaryl group or a structure represented by the following Chemical Formula 3, or two or more substituents adjacent to each other among R7 to R10 combine to form a hydrocarbon ring,
At least one of R3 and R4 is a structure represented by the following formula (3),
At least one of R7 to R10 is a structure represented by the following formula (3), or when two or more substituents adjacent to each other of R7 to R10 combine to form a hydrocarbon ring, at least one of the hydrogens bonded to the carbon forming the hydrocarbon ring is substituted with a structure represented by the following formula (3),
[Formula 3]

In Formula 3,
n is an integer from 1 to 30 as the number of repeating units in parentheses,
A is an alkyl group having 1 to 20 carbon atoms including a nitrogen (N) atom or a heteroaryl group having 2 to 30 carbon atoms including a nitrogen (N) atom. 7. The method of claim 6,
Wherein A is at least one selected from azide, phthalimide, and amine. 7. The method of claim 6,
The carbene compound is covalently bonded to the graphene film,
The 1,5-diaminonaphthalene (1,5-diaminonaphthalene, DAN) is the graphene film and pi-to which the graphene channel member is coupled by a pi interaction. The method according to claim 1,
The graphene film is a single-layer or multi-layer (multi-layer), the graphene channel member. The method according to claim 1,
The graphene film is a patterned, graphene channel member. The method according to claim 1,
The graphene film has a thickness of 0.1 to 1 nm, the graphene channel member. Board;
The graphene channel member of any one of claims 1 to 11; and
a pair of electrodes;
Including, graphene transistor. A biosensor comprising the graphene transistor of claim 12 .

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