WO2021194207A1 - Graphene channel member comprising nanovesicle containing trpa1, and biosensor - Google Patents

Abstract

The present invention relates to a graphene channel member comprising a graphene film and a nanovesicle (containing TRPA1) immobilized in the graphene film; a graphene transistor comprising a substrate, the graphene channel member, and a pair of electrodes; and a biosensor comprising the graphene transistor. In addition, the present invention relates to a method of detecting a biocide from a sample, the method comprising the steps of: processing the sample in the biosensor; and measuring an electric signal in the biosensor.

Description

Graphene channel member including nanovesicles including TRPA1, and biosensor

The present invention relates to a graphene channel member that can be used for detecting a biocide, and a graphene transistor and a biosensor including the same.

A transistor is a device capable of amplifying an electrical signal, and based on this, is widely applied in the sensor industry for the purpose of detecting a trace amount of a target material. Graphene is one of the carbon allotropes made of carbon atoms, and while having electrically semi-metallic properties, since the charge acts as a zero effective mass particle therein, it has very high electrical conductivity (intrinsic electron mobility of ^{20,000 cm 2 /Vs).} ) is known to have In particular, since it was reported that there is a field effect characteristic when graphene composed of two-dimensional carbon atoms having a hexagonal structure by mechanical exfoliation of graphite is used in a transistor, graphene is similar to conventional silicon. It is in the spotlight as a material that can replace semiconductor materials.

On the other hand, among the sensors that can detect target substances, various types of sensors have been developed and commercialized in the environmental sector such as odor detection, pollutant detection, and decay detection, but they are included in everyday chemical products. The development of sensors to detect possible biocides has not yet progressed, and only the presence of biocides is detected through cell experiments.

A biocide is a product used to kill or incapacitate living things such as pests, bacteria, and mice that can harm humans, livestock, crops, etc. Since it is composed of ingredients showing effects, it is known that when such biocides are introduced into the human body, they can adversely affect human health. In particular, the dangers and awareness of biocides are increasing day by day due to fatal accidents caused by biocides contained in humidifier disinfectants that have occurred in Korea. The need to develop a technology that can detect it is further emphasized. In addition, waste generated in the process of manufacturing products containing biocides or soil and water pollution by biocides may occur in the process of disposing of the products, so biocides may threaten not only people but also the ecosystem. is a substance in Accordingly, development and research of an efficient biocide sensor is urgent, but the development of a sensor capable of detecting it through a quick and simple method while having high selectivity and sensitivity to the biocide is still insufficient.

TRPA1 (Transient receptor potential cation channel, subfamily A, member 1) is a type of receptor that exists in animal cell membranes. Somatosensory receptors known to function in response. As a ligand capable of detecting the binding of TRPA1, various substances such as phenolic compounds of olive oil, thymol, and menthol are known, but biocides such as those described above, for example, PHMG (polyhexamethyleneguanidine), etc. It has not been reported or disclosed that it can be used as a sensor capable of detecting it with high sensitivity by selectively binding to biological agents.

Accordingly, the inventor of the present invention newly found that TRPA1 can bind with biocides including PHMG with high selectivity and sensitivity, and implements an environment similar to in vivo cells in the sensor for detecting it, The present invention was completed by developing a novel biosensor capable of rapidly and sensitively detecting a biocide.

An object of the present invention is to provide a sensor capable of detecting a biocide such as polyhexamethyleneguanidine (PHMG) with high sensitivity, and a graphene channel member capable of selectively reacting with a biocide so that it can be used in the sensor, and the same An object of the present invention is to provide a graphene transistor comprising.

In order to achieve the above object, one aspect of the present invention is a graphene channel member including a graphene film and a nanovesicle immobilized on the graphene film, wherein the nanovesicle is a TRPA1 (Transient receptor potential cation channel, It provides a graphene channel member that includes subfamily A, member 1).

In addition, in order to achieve the above object, another aspect of the present invention is the graphene film; And a pair of electrodes; it provides a graphene transistor comprising a.

In addition, in order to achieve the above object, another aspect of the present invention is to provide a biosensor including the graphene transistor, and processing a sample in the biosensor; and measuring an electrical signal of the biosensor.

The graphene channel member of the present invention and the graphene transistor and biosensor including the same have selective and specific detection ability for various types of biocides such as PHMG, OIT, CMIT, and MIT by using TRPA1, It can detect even a very small amount of a biocide with a concentration of about 10 μg/L, which has the effect of being used as a highly sensitive biocide sensor.

Furthermore, in the present invention, it is simpler by using a graphene channel member including a graphene film having improved stability of attachment and fixation of nanovesicles through surface modification and excellent electrical conductivity, and a miniaturized graphene transistor using the same. There is an advantage in that it is possible to detect biocides and provide a graphene transistor with excellent portability.

1 shows the results of fluorescence microscopy to determine whether TRPA1 fused with GFP is present in the membrane of nanovesicles prepared by transforming and expressing a gene encoding TRPA1 in HEK-293 cells. Green fluorescence is shown.

Figure 2 shows the results of confirming the presence or absence of TRPA1 through western blotting by expressing the gene encoding TRPA1 in HEK-293 cells after transformation. The leftmost part means the result of the marker, and the middle part means the result of the control group, and it can be seen that a band is formed in the 151 kDa region, which means that TRPA1 is expressed and exists.

3 shows the results of confirming the nanovesicles containing TRPA1 prepared from HEK-293 cells with a scanning electron microscope (SEM).

4 is a photograph of a graphene transistor surface-modified with a poly-di-lysine linker.

5 is a schematic diagram illustrating a graphene channel member in which nanovesicles including TRPA1 are immobilized on a graphene film and a graphene transistor including the same.

6 is a graph showing the value of the change amount of the current measured for the graphene transistor of the present invention. In the graph, Nanovesicle/PDL/Gr indicates a transistor in which nanovesicles containing TRPA1 are immobilized on a graphene film through PDL (poly-di-lysine), and PDL/Gr indicates only the PDL linker layer without immobilizing the nanovesicles. A transistor formed on a graphene film, pristine graphene means a transistor made of a graphene film in which the PDL linker layer is not formed. Vds is a source/drain voltage, and Vg is a gate voltage.

7 is a graphene transistor (Nanovesicle with receptor) of the present invention and a graphene transistor (Nanovesicle w/p receptor) immobilized with a nanovesicle not containing TRPA1 by injecting PHMG by concentration to measure the value of change in current This is the graph shown.

8 is a graph showing the measurement of the change amount of current by sequentially inputting OIT, CMIT, and PHMG at the same concentration to the graphene transistor of the present invention.

9 is a graph showing the amount of change in current by inputting BNP, OIT, MIT, CA, CBDZ, and SPO to the graphene transistor of the present invention for each concentration.

10 is a graph showing the amount of change in current by sequentially introducing a biocide of a combination of CMIT + OIT, CMIT + PHMG, OIT + PHMG, and CMIT + OIT + PHMG to the graphene transistor of the present invention.

11 is a graphene transistor of the present invention MIT + OIT, MIT + IPBC, MIT + CA, MIT + DDAC, DDAC + IPBC and DDAC + CA combinations of biocides by concentration, respectively, by measuring the amount of change in the current. It is a graph expressed as a sensitivity (sensitivity) numerical value.

12 is a graph showing the amount of change in current as a sensitivity value by injecting samples of six commercially available household chemical products into the transistor of the present invention. The time of injection of each sample was indicated by an arrow.

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 nanovesicle immobilized on the graphene film, and the nanovesicle includes a transient receptor potential channel, subfamily A, member 1 (TRPA1).

The nanovesicle of the present invention refers to a nanometer-sized vesicle having a shape surrounded by a membrane composed of a phospholipid bilayer, and may be used interchangeably with 'nanovesicles'. Nanovesicles may have the same size and structure as exosomes, which are membrane-structured vesicles secreted from various types of cells, and are released to the outside of the cell and bind to other cells and tissues, and then protein in the vesicle Unlike exosomes, which serve to deliver substances such as , RNA, etc., nanovesicles are artificially obtained from cells. The nanovesicle has the form of a vesicle surrounded by a membrane composed of a lipid bilayer, and the lipid bilayer membrane may be one or more.

The nanovesicle includes a transient receptor potential cation channel (TRPA1, subfamily A, member 1). The TRPA1 may be located on the membrane surface of the nanovesicle, and other proteins, glycoproteins, cholesterol, etc. may be bound to the membrane surface in addition to TRPA1. Because nanovesicles have a cell-like structure, they can provide an environment similar to the actual intracellular environment and cell membrane. Therefore, as TRPA1, which originally exists on the surface of the cell membrane in vivo, is included in the nanovesicle, there is an advantage that TRPA1 can function identically or similarly to that of TRPA1 in vivo. There is an advantage in using a nanovesicle containing TRPA1 as a sensor by measuring a change in an electrical signal as a result of realizing a difference in ion concentration inside and outside the nanovesicle by inflow/outflow.

The TRPA1 is a kind of ion channel protein also called 'transient receptor potential ankyrin 1'. It is located on the surface of biological membranes of animal cells including humans, detects physical and chemical stress, and is a receptor related to somatosensory sensations such as pain, cold, itch. .

TRPA1 contained in the nanovesicles of the present invention can react selectively to biocides. A biocide refers to an agent capable of killing or incapacitating living organisms such as bacteria and insects, and may include preservatives, fungicides, insecticides, rodenticides, preservatives, and the like. In particular, it may be used for the purpose of exterminating pathogens, pests, mice, etc. that damage people, crops, and livestock, but it may be toxic to people, animals, plants, etc. It can cause disease, disability, and even death. Specifically, the biocide is PHMG (polyhexamethyleneguanidine), BNP (2-bromo-2-nitropropane-1,3-diol), OIT (2-Octyl-3 (2H)-isothiazolone), CMIT (Chloromethylisothiazolinone), MIT (Methylisothiazolinone), CA (Citric acid), CBDZ (Carbendazim), SPO (Sodium 2-pyridinethiol 1-oxide), IPBC (iodopropynyl butylcarbamate) and DDAC (didecyldimethylammonium chloride) may be at least one selected from the group consisting of, and The same biocide may be contained in household chemicals or processed foods. The TRPA1 may have the highest selectivity for PHMG among the biocides listed above. As the TRPA1 selectively binds to the biocide, the structure is changed and channels are opened so that ions existing outside the nanovesicles, such as calcium ions, can move into the nanovesicles. Accordingly, a change in the potential inside and outside the nanovesicle membrane may occur, and an electrical change may occur according to the potential difference before/after TRPA1 binds to the biocide. Therefore, by measuring a change in an electrical signal according to a change in the structure of TRPA1, the TRPA1 can be used for the purpose of confirming the presence of a biocide.

The TRPA1 may be a protein comprising the amino acid sequence of SEQ ID NO: 1, and the TRPA1 binds selectively to the biocide and does not affect the detectable activity, deletion or insertion of amino acid residues , may be variants or fragments of amino acids having different sequences by substitution or a combination thereof. Amino acid exchange at the peptide level that selectively binds to the biocide and does not entirely alter its detectable activity is known in the art, and includes, for example, phosphorylation, sulfation, acrylation ( acrylation), glycosylation, methylation, farnesylation, and the like. Accordingly, the present invention includes a protein comprising an amino acid sequence substantially identical to the protein comprising the amino acid sequence of SEQ ID NO: 1, and a variant thereof or an active fragment thereof. The substantially identical protein means an amino acid sequence having 75% or more, for example, 80% or more, 90% or more, 95% or more sequence homology to the amino acid sequence of SEQ ID NO: 1, respectively. In addition, the protein may further include a targeting sequence, a tag, a labeled residue, an amino acid sequence prepared for a specific purpose to increase half-life or protein stability.

In addition, the TRPA1 of the present invention can be obtained by various methods well known in the art. As an example, it may be prepared using polynucleotide recombination and protein expression systems, or synthesized in vitro through chemical synthesis such as peptide synthesis, and cell-free protein synthesis.

In addition, to obtain better chemical stability, enhanced pharmacological properties (half-life, absorption, potency, potency, etc.), altered specificity (e.g., broad spectrum of biological activity), reduced antigenicity, the N-terminal or A protecting group may be attached to the C-terminus. For example, the protecting group may be an acetyl group, a fluorenyl methoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or polyethylene glycol (PEG). As long as it is a possible component, it may be included without limitation. The 'stability' refers to storage stability (eg, room temperature storage stability) as well as in vivo stability that protects the protein of the present invention from attack by a protein cleaving enzyme in vivo.

The nanovesicles containing TRPA1 may be prepared by producing and separating animal cells. Specifically, the animal cells are treated with a substance that expresses TRPA1 from animal cells transformed with the gene encoding the TRPA1 protein and reduces the stability of the cell membrane, such as cytochalasin B, and then centrifuged. Nanovesicles can be obtained through Transforming the gene encoding the TRPA1 protein may use any technique and method known in the art that can be used to transform a specific gene in an animal cell, for example, the TRPA1 protein into an expression vector. By cloning the coding gene, it may be transformed into animal cells through treatment with a lipofectamine solution.

The diameter of the nanovesicles including TRPA1 may be 100 nm to 200 nm, specifically, 120 nm to 200 nm, 140 nm to 180 nm, 120 nm to 180 nm, or 140 nm to 160 nm. . When the diameter of the nanovesicle is smaller than the above range, the amount of TRPA1 that can be included in the nanovesicle may be reduced than the target value, and if the diameter is larger than the above, in the process of immobilizing the nanovesicle to the graphene film can be an obstacle

The nanovesicles including the TRPA1 may be immobilized on the graphene film by chemical bonding. The immobilization refers to fixing the nanovesicles so that they do not move at one position of the graphene film, and the structural change of TRPA1 caused by the binding of TRPA1 contained in the nanovesicles with a biocide and the resulting electrical signal. As long as the change is fixed so that it can be transmitted to the graphene film, it can be fixed through any method.

The chemical bond is from the group consisting of poly-D-lysine (or PDL), poly-L-lysine and poly-L-ornithine. It may be immobilized by using any one selected as a linker, but it is not limited thereto, and if there is a property that an electron carrier (electron or hole) can move through it and can bind to the graphene film and the nanovesicle, respectively, any It can be used in any form. The poly-di-lysine is a polymer in which a plurality of D-lysine is linked, and forms ionic bonds on the surfaces of graphene films and nanovesicles, respectively, by using the properties of cations represented by lysine. can do. Therefore, the surface of the graphene film is modified by coating the graphene film with poly-di-lysine, and the nanovesicles of the present invention can be immobilized on the graphene film by binding the nanovesicles to the poly-di-lysine. have.

The graphene film may be a single layer or a bi-layer. When the two-layer graphene film is used, it is more preferable to include a single-layer graphene film in that the sensitivity of the graphene transistor may be lowered due to a decrease in surface resistance.

The graphene film may be patterned, specifically, micro-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). The nanovesicles including the TRPA1 may be immobilized on the surface of the patterned graphene film. In this case, as described above, the nanovesicles may be immobilized on the graphene film using poly-di-lysine as a linker.

The graphene channel member of the present invention includes a graphene film. As above, when graphene is used as the channel member, a high current flows even in the OFF state where no voltage is applied to the gate, so the on/off ratio of the operating current is very low. It has the advantage of being able to manufacture high-performance transistors.

In this case, the thickness of the graphene film may be 0.1 to 1 nm, specifically 0.2 to 0.8 nm, 0.3 to 0.8 nm, or 0.5 to 0.7 nm. The thickness of the graphene film refers to the thickness of a single layer of graphene, and when the thickness of the graphene film is within the above range, it exhibits high conductivity and high charge mobility. Available.

2. Graphene Transistor

Another aspect of the present invention provides a graphene transistor.

The graphene transistor may include a substrate; The graphene channel member of the present invention; 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 may be used, and when an insulating substrate is used, a silica (SiO ₂ ) substrate or a silicon wafer is preferable from the viewpoint of excellent affinity with the graphene channel member.

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 and 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 a 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 Thus, it is preferable that the chemical vapor deposition method is LPCVD in view of possible deposition.

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 in 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, from a graphite crystal composed of multiple layers, a physical exfoliation method to make graphene by peeling one layer off with 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 material having conductivity, 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, and 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 one in which nanovesicles including TRPA1 are immobilized on the graphene film through a chemical bond, for example, poly-di-lysine. Description of the graphene channel member, TRPA1, nanovesicle, poly-di-lysine, and graphene film is described in '1. It is the same as described in 'Graphene Channel Member'.

When the nanovesicle containing the TRPA1 is immobilized on the graphene film, the graphene transistor including a graphene channel member in which TRPA1 is bonded to the graphene film in a form not included in the nanovesicle (ie, TRPA1 is Compared to a graphene transistor immobilized on a graphene film or the like by a single physical or chemical bond), the sensitivity is higher and the detection limit is improved. As described above, since nanovesicles have a cell-like structure, when TRPA1 contained in nanovesicles is used, TRPA1 can function identically or similarly to that of TRPA1 actually functioning in vivo, and the structural changes of TRPA1 It is possible to realize the difference in ion concentration inside and outside the nanovesicle by the inflow/outflow of ions that are generated accordingly. Therefore, when compared to a transistor or biosensor that uses only protein without using a nanovesicle, it is not possible to measure only the structural change of the protein itself or the resistance change according to it, but it is also possible to measure the movement of ions and the ion gradient inside and outside the nanovesicle. Since the electrical difference formed according to the measurement can be measured, the change of the electrical signal can be measured to be larger, and thus a higher sensitivity can be exhibited. In addition, in the case of graphene, since it has high charge mobility compared to materials having other semiconductor characteristics, it is possible to measure changes in electrical signals faster and with high sensitivity, which has the advantage that the detection limit can be improved accordingly.

Poly-D-lysine bound to the graphene film may form a linker layer in the form of a single layer, and the nanovesicles immobilized on the poly-D-lysine may also form an acceptor layer in the form of a single layer. can

When the linker layer is formed as a single layer, graphene not only has excellent charge mobility, transparency and/or flexibility, but also has an 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 it is thicker than 2 nm, there is a problem in that transparency is reduced.

3. Biosensor and biocide detection method using the same

Another aspect of the present invention provides a biosensor and a method for detecting a biocide using the same.

The biosensor includes the graphene transistor of the present invention. 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 is changed by an electric field effect.

The biosensor may be for detecting a biocide. The description of the biocide that can be detected by the biosensor of the present invention is described in '1. It is the same as described in 'No graphene channel, for example, the biocide is PHMG (polyhexamethyleneguanidine), BNP (2-bromo-2-nitropropane-1,3-diol), OIT (2-Octyl-3 (2H)- from the group consisting of isothiazolone), CMIT (Chloromethylisothiazolinone), MIT (Methylisothiazolinone), CA (Citric acid), CBDZ (Carbendazim), SPO (Sodium 2-pyridinethiol 1-oxide), IPBC (iodopropynyl butylcarbamate) and DDAC (didecyldimethylammonium chloride) There may be one or more selected, and the biocide as described above may be included in household chemical products or processed foods.

Specifically, when TRPA1 contained in the nanovesicle immobilized on the surface of the graphene film reacts with a biocide, a change in the surrounding electric field may occur due to its structural change and resistance change, and the structure of TRPA1 As the movement of ions becomes possible due to the change, a change in potential inside and outside the nanovesicle may occur, and accordingly, the value of the current flowing in the graphene film between the source electrode and the drain electrode changes together, and this change in current is measured. In this way, the target biocide can be detected.

The biosensor can detect even when the concentration of the biocide is 10 g/l or less, for example, 1 g/l or less, 100 mg/l or less, 10 mg/l or less, 1 mg/l or less, 100 µg/l or less A biocide of less than or equal to l, or less than or equal to 10 μg/l can be detected.

Such a biosensor is excellent in sensitivity, specificity, speed and/or portability by using the graphene transistor as described above, and in particular, due to the high charge carrier mobility and conductivity characteristics of graphene by using a graphene film as a channel layer. It has excellent sensitivity and real-time detection performance, thereby improving the detection limit of biocides that may be included in household chemicals or processed foods, thereby enabling detection with high sensitivity and reproducibility.

In addition, when nanovesicles including TRPA1 immobilized thereon are present in the channel region of the graphene transistor by forming the linker layer on the graphene film in the graphene transistor as described above, the sensitivity of the sensor is further improved and the doping It has the effect of simplifying the process by being able to perform the treatment and the nanovesicle attachment at the same time.

Furthermore, the above-mentioned graphene transistor is manufactured in the form of a SIM chip and can be applied to a miniaturized biosensor (portable electronic biocide sensor, etc.) It can be accurately identified in real time, and it can be used in various food industries and environmental evaluation industries.

The method for detecting a biocide from the sample includes: processing the sample with the biosensor of the present invention; and measuring an electrical signal of the biosensor.

The detection means confirming the presence of a target substance, and includes quantifying or semi-quantifying the concentration of a biocide that is a target substance.

The sample means any mixture or solution that contains or is suspected to contain a biocide, which is a target substance to be detected, and thus needs detection. It may be water, food, household products, by-products generated from household products, biological samples obtained from humans or animals, or processed products thereof that contain or are believed to contain biocides, such as household chemical products or processed foods. can The household chemical product may be, for example, an insecticide, a deodorant, and the like, but is not limited thereto.

The measurement of the electrical signal may be to measure the amount of change in the current, and specifically, by measuring the value obtained by dividing the value of the change in the current with time by the initial amount of current, the presence or absence of the biocide in the sample and / or the amount of the biocide Concentration and quantity can be detected.

The method of detecting the biocide from the sample may further include determining that the biocide is present in the sample when it is measured that the electrical signal of the biosensor changes.

The description of the graphene channel member, TRPA1, nanovesicle, poly-di-lysine, graphene film, graphene transistor, etc. is described in '1. Graphene Channel Absence' and '2. It is the same as described in 'Graphene Transistor'.

Hereinafter, the present invention will be described in detail by way of Examples.

[Production Example 1]

Preparation of Nanovesicles Containing TRPA1

[1-1] Transformation of TRPA1 gene and preparation of nanovesicles

In order to express the TRPA1 of the present invention and prepare it to be included in the nanovesicles, the TRPA1 gene was transformed into HEK-293 cells, a type of mammalian cells, and the formation of nanovesicles was induced.

First, in order to prepare an expression vector capable of expressing TRPA1, PCR is performed using the TRPA1 gene as a template to amplify it, and then rho-tag, an import sequence that induces the expression protein to move to the surface of the cell membrane. was fused with the PCR-amplified TRPA1 gene, and cloned into a mammalian expression vector, pCMV6-AC-GFP (CAT#: PS100010, OriGene). The pCMV6-AC-GFP vector includes an ampicillin resistance gene, a CMV promoter, and a gene sequence encoding GFP, a green fluorescent protein. Then, 0.5 μg of the expression vector was diluted with 100 μl of Opti-MEM (Reduced Serum Media ^{) and mixed with 0.75 μl to 1.75 μl of a lipofectamine solution (Lipofectamine LTX TM} , Invitrogen), followed by DNA-lipofectamine complex. was reacted at room temperature for 30 minutes to form Prior to introducing the expression vector into HEK-293 cells, the HEK-293 cells were humidified at 37 °C in DMEM medium (Dulbecco's Modified Eagle Medium, 4 mM L-glutamine, 10% FBS, 1% penicillin-streptomycin added). 5% CO ₂ ^{Culturing using an incubator, and aliquoted to contain 0.5 to 1.24 × 10 5} cells per 0.5 ml of medium in a 24-well cell plate, the expression vector prepared as above and lipofect After the tamine complex was introduced into each well by treatment with 100 ml, the cells were additionally cultured for 18 to 24 hours in a _{5% CO 2 incubator at 37 ° C.}

In order to express TRPA1 protein from HEK-293 cells transformed with the TRPA1 gene through the above method and to prepare a nanovesicle containing the same, the transformed HEK-293 cells were treated with 10 μg/ml of cytochalasin B (cytochalasin B). B, Sigma, USA) was stirred in DMEM medium at 37 °C and 300 rpm for 30 minutes. Cytochalasin B is a type of toxin (mycotoxin) capable of penetrating cell membranes, and can reduce the stability of cell membranes by interfering with the formation of contractile microfibers. Cells and nanovesicles were separated by centrifugation for 1 minute, and nanovesicles were obtained by centrifuging the centrifuged supernatant at 15,000 g for 30 minutes. The precipitate settled through the centrifugation was resuspended in PBS (pH 7.4, Sigma-Aldrich, USA) containing a protease inhibitor to obtain nanovesicles and stored at -80 ° C for use in additional experiments. was used.

[1-2] Confirmation of expression of TRPA1 and formation of nanovesicles

It was confirmed whether the TRPA1 gene was successfully expressed from the HEK-293 cells transformed through the method of Preparation Example 1-1 to synthesize the TRPA1 protein, and whether TRPA1 was included in the resulting nanovesicles, and the The shape of the nanovesicle was confirmed.

First, it was confirmed through a fluorescence microscope whether the protein expressed from the transformed HEK-293 cells was successfully expressed and contained in the nanovesicles prepared in Preparation Example 1-1. Since the GFP gene is fused together in the expression vector prepared to introduce TRPA1 into HEK-293 cells, it can be confirmed whether the protein expressed from the expression vector is present in the membrane of the nanovesicle by observing the green fluorescence of GFP. can As a result of observing the nanovesicles prepared in Preparation Example 1-1 with a Z-axis scan through a confocal fluorescence microscope (LSM 800, ZEISS), it was confirmed that green fluorescence appeared as shown in the figure of FIG. 1 . Since this means that GFP emitting green fluorescence is present in the nanovesicle, the expression vector was successfully introduced into HEK-293 cells and transformed, which means that the protein expressed therefrom is contained in the nanovesicle. . Since TRPA1 is encoded in the expression vector, it can be interpreted that TRPA1 fused with GFP is expressed in the nanovesicle through the above results.

In addition, in order to accurately confirm the expression of TRPA1 in the nanovesicles, Western blotting was performed on the TRPA1 protein according to a method commonly used in the art. For the proteins isolated from the nanovesicles, SDS-PAGE was performed using a 10% polyacrylamide gel, and then, the proteins separated in the gel were transferred to a PDFV membrane (Bio-Rad, USA) to anti- After reacting with GFP mouse antibody (Santa Cruz Biotechnology), the ECL reaction was performed by washing with a secondary antibody (anti-mouse antibody, Santa Cruz Biotechnology) bound to horseradish peroxidase (HRP). As a marker, a product from Bio-Rad was used, and as a control, a protein that was not solubilized by treatment with a surfactant was used. As a result of Western blotting, as shown in FIG. 2, a band was identified in the 151 kDa portion corresponding to the size of the TRPA1 protein (the combined size of 124 kDa TRPA1 and the 27 kDA GFP tag bound thereto), and TRPA1 was expressed and present. could confirm that

Furthermore, in order to confirm the form in which the nanovesicles were formed, the nanovesicles prepared by the method of Preparation Example 1-1 were observed using a scanning electron microscope (Field Emission SEM, Magellan400). As a result, it was confirmed that the nanovesicles formed a spherical shape as shown in FIG. 3 . Combining the above measurement results, it can be seen that TRPA1 in a fused form with GFP is expressed and located on the surface film of the nanovesicle formed in the spherical shape of the present invention prepared by the method of Preparation Example 1-1. .

[Production Example 2]

Fabrication of Graphene Transistors

[2-1] Formation of a graphene channel layer on a substrate

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

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 layer formed on the copper foil, and an etchant was used. The PMMA-coated graphene layer was separated from the copper foil using were removed.

The graphene layer washed as described above was transferred to a silicon wafer as a substrate, and then a PMMA solution was dropped on the graphene layer to remove the PMMA coating the graphene layer, thereby forming a graphene channel layer on the substrate. At this time, transparency was maintained at 97.8%.

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

[2-2] electrode formation

A pattern electrode (width/length=W/L=1, L=100 μm channel length) was formed on both ends of the graphene channel layer that was patterned and aligned as described above through an RIE (oxygen plasma treatment) method, A graphene transistor having an electrode (W/L=5, L=100 μm channel length) formed on the graphene channel layer was manufactured through the process of image inversion, thermal deposition, and lift-off.

[2-3] Formation of linker layer

Prior to fixing the nanovesicles containing TRPA1 to the graphene transistors prepared as described above, a linker layer serving as a medium for fixing the graphene transistors and the nanovesicles was formed. Specifically, as the linker, poly-D-lysine (PDL) was used, the surface of the graphene layer was coated with a PDL solution of 0.1 mg/ml concentration, and the linker was reacted at 25° C. for 2 hours. layer was formed.

[2-4] Immobilization of nanovesicles containing TRPA1

By adding 1 μl of a nanovesicle (1 mg/ml) solution containing TRPA1 prepared according to the method of Preparation Example 1-1 to the linker layer to the surface of the PDL-coated linker layer, the nanoveg of the present invention A graphene transistor with a fixed claw was fabricated.

[Experimental Example 1]

Check the difference in the measurement of electrical properties of graphene transistors before and after fixing nanovesicles with TRPA1

The electrical characteristics appearing in the graphene transistor in the form of a fixed nanovesicle containing TRPA1 prepared in Preparation Example 2 were measured using a source meter (Keithly 2412 sourcemteter) and a potentiometer (Wonatech 3000 potentiostat), Graphene transistor before fixing the nanovesicles (graphene transistors prepared up to Preparation Example 2-3, in which only a linker is coupled), graphene transistors before forming a linker layer (graphene transistors of Preparation Example 2-2) It is shown in FIG. 6 as compared with the measurement result of the electrical properties of .

As a result of measuring the amount of current according to the change in voltage with respect to the graphene transistor, when compared to a graphene transistor in which only the PDL linker is bonded and the nanovesicle is not bonded and the graphene transistor to which the PDL linker is not bonded, the nano of the present invention In the graphene transistor with a fixed vesicle, the slope of the resistance increased. This is a result of resistance generated as nanovesicles containing TRPA1 are immobilized on the surface of graphene through a linker. It was confirmed that the immobilization of the nanovesicles was performed on the vesicles.

[Experimental Example 2]

Confirmation of the detection effect on the biocide of the graphene transistor of the present invention

[2-1] Confirmation of detection effect according to the concentration of biocide PHMG

PHMG (polyhexamethyleneguanidine), a kind of biocide, was added to the graphene transistor of the present invention at various concentrations, and the sensing effect on PHMG was confirmed by measuring the change in current thereof.

Specifically, 10 μg/L, 100 μg/L, 1 mg/L, 10 mg/L, and 10 mg/L of PHMG were added to the graphene transistor in which the nanovesicles containing TRPA1 prepared in Preparation Example 2 were fixed, respectively. They were sequentially added at concentrations of 100 mg/L, 1 g/L, and 10 g/L. By measuring the electrical characteristics in the same manner as in Experimental Example 1, the value of the amount of change in current with time (ΔI/I ₀ = (II ₀ )/I ₀ , I is the current value measured in real time, I ₀ is the first measured value current values) are shown in FIG. 7 . As a control group, a graphene transistor made by fabricating and using a nanovesicle without TRPA1 was used.

As a result, as the concentration of the added PHMG increased, the value of the change amount of current increased. Accordingly, the graphene transistor of the present invention has the effect of detecting PHMG according to the amount of PHMG, and a very high concentration of 10 μg/L It was confirmed that detection and detection were possible even at a small concentration.

[2-2] Confirmation of detection selectivity for biocide PHMG

In the graphene transistor of the present invention, various biocides including PHMG, the detection effect of which was confirmed in Experimental Example 2-1, were added, and the current change thereof was measured to determine whether any of the biocides had the highest selectivity. did.

Specifically, OIT (2-Octyl-3(2H)-isothiazolone), CMIT (Chloromethylisothiazolinone) and PHMG as a biocide in a graphene transistor in which the nanovesicles containing TRPA1 prepared in Preparation Example 2 are fixed. were sequentially added at a concentration of 100 μg/L, respectively. In addition, the electrical characteristics were measured in the same manner as in Experimental Example 1, and the value of the change amount of the current with time is shown in FIG. 8 .

As a result, a change in current appeared according to the addition of OIT and CMIT, but the largest electrical signal was detected when PHMG was added. Therefore, the graphene transistor including the nanovesicles including TRPA1 of the present invention can detect and detect OIT and CMIT, but in particular, it has excellent detection effect on PHMG, and thus it can be confirmed that the selectivity is very large.

[2-3] Confirmation of the detection effect according to the concentration of various biocides

Various types of biocides were added at various concentrations to the graphene transistor of the present invention, and the sensing effect of each biocide was confirmed by measuring the change in current thereof.

Specifically, BNP, OIT, MIT, CA, CBDZ, and SPO were respectively added to the nanovesicle-fixed graphene transistor comprising TRPA1 prepared in Preparation Example 2 at 10 μg/L and 100 μg/L, respectively. , 1 mg/L, 10 mg/L, 100 mg/L, 1 g/L, and 10 g/L were sequentially added. By measuring the electrical characteristics in the same manner as in Experimental Example 1, the value of the amount of change of current with time (ΔI/I ₀ = (II ₀ )/I ₀ , I is the current value measured in real time, I ₀ is the first measured value current values) are shown in FIG. 9 .

As a result, as can be seen in FIG. 9 , the magnitude of the change value of the current was different for each biocide, and the degree of electrical signal generation according to the concentration was also different, but when a small amount of the biocide was treated In the case of using the graphene transistor of the present invention, it was confirmed that their detection and detection were possible with high sensitivity.

[2-4] Confirmation of detection effect by treatment of complex biocides

As can be seen from the results of the above experimental examples, it was confirmed that the graphene transistor of the present invention has a detection effect on various types of biocides. It was checked whether

The biocides of CMIT + OIT, CMIT + PHMG, OIT + PHMG, and CMIT + OIT + PHMG were added to the graphene transistor of the present invention so that the total amount was 10 mg/L, respectively, and the same method as in Experimental Example 1 was used. 10 shows the value of the change in current with time (ΔI/I ₀ = (II ₀ )/I ₀ , I is the current value measured in real time, I ₀ is the first measured current value) by measuring the electrical characteristics through It was.

As a result, even when several types of biocides were compounded, electrical signals could be observed, and as confirmed in Experimental Example 2-2, the detection effect was the best when PHMG was treated. . Therefore, when the graphene transistor on which the nanovesicles containing TRPA1 of the present invention are immobilized is used as a biosensor, it is possible to detect them with high sensitivity even if they are complexly mixed in various combinations as well as a single biocide. could confirm that there was

In addition, the detection effect of the transistor of the present invention was confirmed for a combination of different types of biocides. As biocides, MIT, IPBC (iodopropynyl butylcarbamate), CA and DDAC (didecyldimethylammonium chloride) were used, and these were MIT + OIT, MIT + IPBC, MIT + CA, MIT + DDAC, DDAC + IPBC and DDAC + CA combinations, respectively. Each of the biocides was injected into the transistors of the present invention, and electrical characteristics were measured in the same manner as in Experimental Example 1, and the sensitivity measured based on the value of the change in current with time is shown in FIG. 11 .

As a result, it was confirmed that an electrical signal was generated depending on the concentration of the biocide when the MIT, IPBC, CA and DDAC biocides were treated in combination. It was confirmed once again that they could be detected with high sensitivity.

[2-5] Confirmation of the detection effect of biocides in household chemical products using the transistor of the present invention

It was confirmed whether the transistor of the present invention can detect whether a component acting as a biocide is contained in a commercially available household chemical product. 5 μl of a total of 6 kinds of household chemical samples was put into the transistor of the present invention to measure electrical characteristics, and the measured sensitivity based on the value of the change in current with time is shown in FIG. 12 . The household chemical products were measured by injecting a liquid sample collected by spraying the disinfectant and deodorant products in the form of spraying into the transistor of the present invention.

As a result, it was measured that the sensitivity value was changed due to a change in electrical characteristics at the time when 6 kinds of household chemical products were injected. Therefore, it was confirmed that the transistor of the present invention can detect the biocide contained in household chemical products, and it can be confirmed that the transistor of the present invention can be used to quickly and conveniently detect the biocide present in the product. could

In the above, the manufacturing examples and experimental examples of the present invention have been exemplarily described, but the scope of the present invention is not limited only to the specific preparation examples and experimental examples as described above, and those of ordinary skill in the art may claim the present invention. Appropriate changes may be made within the scope described in the scope.

Claims (13)

1. As a graphene channel member comprising a graphene film and nanovesicles immobilized on the graphene film,

   The nanovesicle is TRPA1 (Transient receptor potential cation channel, subfamily A, member 1) will include, the graphene channel member.
2. The method according to claim 1,

   The nanovesicle is a graphene channel member that is immobilized by a chemical bond to the graphene film.
3. 3. The method according to claim 2,

   Wherein the chemical bond is immobilized using poly-D-lysine as a linker, graphene channel member.
4. The method according to claim 1,

   The TRPA1 is a graphene channel member that is located on the membrane surface of the nanovesicle.
5. The method according to claim 1,

   The TRPA1 will selectively respond to a biocide, graphene channel member.
6. 6. The method of claim 5,

   The biocide is PHMG (polyhexamethyleneguanidine), BNP (2-bromo-2-nitropropane-1,3-diol), OIT (2-Octyl-3 (2H)-isothiazolone), CMIT (Chloromethylisothiazolinone), MIT (Methylisothiazolinone) , CA (Citric acid), CBDZ (Carbendazim), SPO (Sodium 2-pyridinethiol 1-oxide), IPBC (iodopropynyl butylcarbamate) and DDAC (didecyldimethylammonium chloride) to one or more selected from the group consisting of, the graphene channel member.
7. The method according to claim 1,

   The graphene film is a single layer or a bi-layer (bi-layer), the graphene channel member.
8. The method according to claim 1,

   The graphene film is a patterned, graphene channel member.
9. The method according to claim 1,

   The graphene film has a thickness of 0.1 to 1 nm, the graphene channel member.
10. Board;

    The graphene channel member of any one of claims 1 to 9; and

    A pair of electrodes; including, a graphene transistor.
11. A biosensor comprising the graphene transistor of claim 10 .
12. 12. The method of claim 11,

    The biosensor is for the detection of a biocide, the biosensor.
13. processing the sample in the biosensor of claim 12; and

    Measuring the electrical signal of the biosensor; A method of detecting a biocide from a sample, comprising a.

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