Structure for real-time nicotine detection biosensor, the biosensor comprising the same, and real-time nicotine analysis method in saliva using the biosensor

Abstract

The present invention relates to a structure for a biosensor, a biosensor including the same, and a method for real-time nicotine analysis in saliva using the biosensor. According to the present invention, a structure connected to an electrode of a biosensor forms a structure of a crystalline carbon membrane / calcium ion-selective membrane / TRPA1-containing nanovesicle. When nicotine is contained in saliva containing calcium ions, the nanovesicle having a TRPA1 ion channel in the outermost layer of the structure specifically reacts with the nicotine contained in the saliva to introduce calcium ions. The resulting change in calcium ion concentration changes the surface potential of the crystalline carbon membrane in contact with the calcium ion-selective membrane through the calcium ion-selective membrane. By measuring the change in electrical conductivity, such as the change in open-circuit voltage, nicotine can be detected even in minute amounts of 1 pM, thereby allowing for the quantitative determination of the nicotine concentration in a smoker's saliva in real time.

Description

Technology Field

[0001] The present invention relates to a biosensor, and more specifically to an electrochemical biosensor. Background Technology

[0002] Reports on various diseases caused by smoking are continuously being published. Specifically, the fact that inhaled tobacco smoke is a major environmental carcinogen has been proven in several epidemiological studies, and for example, in the case of lung cancer, it has been reported that the incidence rate is more than 10 times higher in male smokers than in non-smokers.

[0003] In addition, 30-40% of cancers occurring in the current population are tobacco-induced cancers (lung, oral, esophageal, kidney, bladder, and pancreatic), and in the case of lung cancer in particular, it has been found that 83% of the causes are due to smoking. Furthermore, it is known that smoking is associated with heart disease, represented by coronary artery disease; respiratory diseases such as chronic bronchitis; various complications of pregnancy; and peptic ulcers.

[0004] Meanwhile, secondhand smoke refers to a situation where a person near a smoker inhales tobacco smoke indirectly against their will, even without smoking directly; in this case, toxic chemical substances in sidestream smoke—the raw smoke burning at the tip of the cigarette—can cause serious harm to secondhand smokers.

[0005] The harmful effects of smoking are even more severe for adolescents because their physical development is not yet complete, making all their cells and tissues weak.

[0006] Therefore, accurately assessing smoking status is crucial in research and patient care related to smoking and smoking cessation. There are various methods to determine smoking status. Among these, self-reporting via questionnaires has low reliability, so it is important to identify smoking status through testing for specific substances.

[0007] Smoking status can be measured by detecting components or metabolites of tobacco smoke in body fluids such as urine, blood, saliva, and sweat, or in hair. Among these, nicotine is a substance specific to tobacco and is theoretically the most desirable indicator for determining whether smoking has occurred. However, since the nicotine has a short half-life of about 2 hours, it is converted into metabolites after 2 hours, which makes accurate detection difficult.

[0008] Consequently, conventional methods utilized the detection of cotinine, a nicotine metabolite; however, measuring cotinine in urine presents problems such as the inconvenience of having to use the restroom, potential human rights issues, and the possibility of subjects switching or manipulating the samples. In response, a method for detecting cotinine through saliva was proposed, but this approach had the drawback of not being able to measure in real time.

[0009] Accordingly, there is a need for technology that can determine smoking status simply and in real-time through saliva. Prior art literature

[0010] Republic of Korea Registered Patent No. 10-1493164 The problem to be solved

[0011] The problem that the present invention aims to solve is to provide a biosensor capable of quantitatively analyzing nicotine concentration in real time through saliva in a simple manner. means of solving the problem

[0012] To solve the above problem, one aspect of the present invention provides a structure for a real-time nicotine detection biosensor. The structure comprises a crystalline carbon membrane; a calcium ion-selective membrane formed on the crystalline carbon membrane; and a nanovesicle comprising TRPA1 (Transient receptor potential channel, subfamily A, member 1) formed on the calcium ion-selective membrane.

[0013] The nanovesicles containing the above TRPA1 can be immobilized on the calcium ion-selective membrane by chemical bonding.

[0014] The above chemical bond can be immobilized using poly-d-lysine as a linker.

[0015] In addition, another aspect of the present invention provides a real-time nicotine detection biosensor comprising the above structure.

[0016] The above real-time nicotine detection biosensor is characterized by quantitatively detecting the nicotine concentration in saliva.

[0017] The above real-time nicotine detection biosensor can quantitatively detect nicotine concentration by detecting changes in calcium ion concentration that occur as calcium ions in saliva flow into nanovesicles containing TRPA1, as nicotine in saliva selectively reacts with TRPA1 on a structure within the sensor.

[0018] The above biosensor for real-time nicotine detection may have a nicotine detection limit of 1 pM.

[0019] Furthermore, another aspect of the present invention provides a method for real-time nicotine analysis in saliva using the biosensor. The analysis method comprises the steps of: processing a saliva sample to be measured into the biosensor; and measuring an electrical signal of the biosensor.

[0020] The measurement of the above electrical signal may be a measurement of a change in open circuit voltage (OCV).

[0021] The above analysis method may further include a step of quantitatively analyzing the nicotine concentration based on the magnitude of the change in the electrical signal of the biosensor.

[0022] The above analysis method is characterized by having a nicotine detection limit of 1 pM. Effects of the invention

[0023] According to the present invention, a structure connected to an electrode of a biosensor forms a structure of a crystalline carbon membrane, a calcium ion-selective membrane, and a nanovesicle containing TRPA1. When nicotine is contained in saliva containing calcium ions, the nanovesicle having a TRPA1 ion channel in the outermost layer of the structure specifically reacts with the nicotine contained in the saliva to introduce calcium ions. The resulting change in calcium ion concentration changes the surface potential of the crystalline carbon membrane in contact with the calcium ion-selective membrane through the calcium ion-selective membrane. Consequently, electrical conductivity changes, and the concentration of nicotine can be quantitatively analyzed by measuring the change in electrical conductivity, such as the change in open-circuit voltage. In particular, since the biosensor containing the structure according to the present invention can detect nicotine even at an ultra-trace amount of 1 pM, the concentration of nicotine in a smoker's saliva can be quantitatively determined in real time. In addition, the biosensor comprising the structure according to the present invention is economical because the materials are easy to obtain, the manufacturing method is simple, and mass production is possible, so it can be rapidly applied in actual field settings such as public health centers. Brief explanation of the drawing

[0024] FIG. 1 is a schematic diagram of a structure for a real-time nicotine detection biosensor according to one embodiment of the present invention. FIG. 2 shows a method for manufacturing a structure for a real-time nicotine detection biosensor according to one embodiment of the present invention. FIG. 3 is a schematic diagram showing the operating principle when actual saliva from a smoker is introduced into a biosensor containing a nanovesicle structure containing CNT / ISM / TRPA1 according to one embodiment of the present invention. Figure 4 shows the energy dispersive X-ray spectroscopy (EDX) spectrum and field emission scanning electron microscope (FE-SEM) image of a CNT film and a CNT / ISM film according to one comparative example and one embodiment of the present invention. FIG. 5 is a graph showing the change in open-circuit voltage (OCV) over time when electrodes with attached CNT membranes and CNT / ISM membranes according to one comparative example and one embodiment of the present invention are placed in artificial saliva containing calcium ions and exposed to a 1% concentration of tobacco smoke extract. FIG. 6 is a graph showing the change in open circuit voltage (OCV) when electrodes with attached CNT membranes and CNT / ISM membranes according to one comparative example and one embodiment of the present invention are placed in artificial saliva containing calcium ions, and 2 mM MgCl2, 2 mM KCl, 2 mM NaCl, and 2 mM CaCl2 are added to the artificial saliva. Figure 7 is a graph showing the change in fluorescence intensity of cells over time when a calcium ion-sensitive fluorescent dye is added to HEK-293T cells mock-infected with TRPA1 or an empty vector and a tobacco smoke extract (CSE) solution is applied. Figure 8 is a graph showing the change in fluorescence intensity of nanovesicles over time when a calcium ion-sensitive fluorescent dye is added to nanovesicles isolated from HEK-293T cells mock-infected with TRPA1-containing nanovesicles or empty vectors, and a tobacco smoke extract (CSE) solution is applied. FIG. 9 is a graph showing the sensing performance according to nicotine concentration of a biosensor including a nanovesicle structure containing CNT / ISM / TRPA1 according to one embodiment of the present invention. FIG. 10 is a graph showing the sensing performance according to the nicotine concentration in actual saliva of a biosensor comprising a nanovesicle structure containing CNT / ISM / TRPA1 according to one embodiment of the present invention. Specific details for implementing the invention

[0025] Hereinafter, embodiments of the present invention will be described in detail with reference to the attached drawings. In describing the present invention, specific descriptions of related known functions or configurations will be omitted if it is determined that such detailed descriptions may unnecessarily obscure the essence of the present invention. Furthermore, the terms used in this specification are used to appropriately express preferred embodiments of the present invention, and these may vary depending on the intentions of the user or operator, or the conventions of the field to which the present invention belongs. Accordingly, the definitions of these terms should be based on the content throughout this specification.

[0026] Identical reference numerals in each drawing indicate identical components.

[0027] Throughout the specification, when it is stated that a component is located "on" another component, this includes not only cases where a component is in contact with another component, but also cases where another component exists between the two components.

[0028] Throughout the specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0030] [Structure for Real-time Nicotine Detection Biosensor]

[0031] One aspect of the present invention provides a structure for a real-time nicotine detection biosensor.

[0032] FIG. 1 is a schematic diagram of a structure for a real-time nicotine detection biosensor according to one embodiment of the present invention.

[0033] Referring to FIG. 1, a structure for a real-time nicotine detection biosensor according to the present invention is characterized by comprising a crystalline carbon membrane (10), a calcium ion-selective membrane (ISM) (20) formed on the crystalline carbon membrane, and a nanovesicle (40) containing TRPA1 (Transient receptor potential channel, subfamily A, member 1) formed on the calcium ion-selective membrane.

[0034] The crystalline carbon film (10) has high strength, chemical or biological inertness with surrounding cells, and excellent electrical properties such as conductivity, so when reacting with biomolecules in a biosensor, high-sensitivity signal conversion is possible through a change in the surface potential of the crystalline carbon film. For such a crystalline carbon film, a carbon material with high electrical conductivity, such as carbon nanotubes (CNT) or graphene, can be used.

[0035] The calcium ion-selective membrane (20) is positioned on the crystalline carbon membrane (10) and has a polymer that maintains the shape of the membrane as a matrix, and includes an ion-selective material (ionophore) that binds to a specific ion, namely calcium ions, to cause charge separation, a plasticizer which is a non-volatile organic solvent, and a lipophilic additive. The calcium ion-selective membrane (20) acts as a physical barrier that blocks interference caused by various ion species contained in cigarette smoke in the smoker's saliva by means of the lipophilic additive, and at the same time serves to deliver a specific ion, namely calcium ions, to the crystalline carbon membrane through the ionophore.

[0036] The above calcium ion-selective membrane (20) was selected considering that the TRPA1 ion channel within the TRPA1-containing nanovesicle (hereinafter, 'TRPA1-containing nanovesicle') described later reacts with nicotine in cigarette smoke to introduce calcium ions, thereby causing a change in calcium ions, and through this, the reaction and concentration of nicotine can be detected by measuring the change in calcium ion concentration.

[0037] Nanovesicles (40) containing TRPA1 are formed on the calcium ion-selective membrane (20) above.

[0038] In the present invention, 'nanovesicle' refers to a nanometer-sized vesicle having a form surrounded by a membrane composed of a phospholipid bilayer, and may be used interchangeably with 'nanovesicle' or 'nanovesicle'. Nanovesicles may have the same or similar size and structure as exosomes, which are membrane-structured vesicles secreted from various types of cells; however, unlike exosomes, which are released outside the cell, bind to other cells and tissues, and serve to deliver substances such as proteins and RNA within the vesicle, nanovesicles are artificially obtained from cells. The nanovesicles have the form of vesicles surrounded by a membrane composed of a lipid bilayer, and the lipid bilayer membrane may be one or more.

[0039] The above-mentioned nanovesicle contains TRPA1. TRPA1 may be located on the membrane surface of the nanovesicle, and other proteins, glycoproteins, cholesterol, etc., in addition to TRPA1, may be bound to the membrane surface. Since the nanovesicle has a structure similar to a cell, it can provide an environment similar to an actual intracellular environment or a cell membrane. Therefore, as TRPA1, which is originally present on the cell membrane surface in vivo, is included in the nanovesicle, there is an advantage that it can function identically or similarly to how TRPA1 functions in vivo.

[0040] The above TRPA1 is a type of ion channel protein also known as 'Transient receptor potential ankyrin 1'. It is located on the surface of the biological membranes of human and animal cells, detects physical and chemical stress, and is a receptor associated with somatic sensations such as pain, cold, and itching. As TRPA1 selectively binds to nicotine, its structure changes and the channel opens, allowing ions present outside the nanovesicle, such as calcium ions, to move into the interior of the nanovesicle. Accordingly, TRPA1 can be used to confirm the presence of nicotine by measuring changes in calcium ion concentration.

[0041] The TRPA1 of the present invention can be obtained by various methods widely known in the field. For example, it can be produced by a method of producing it using a polynucleotide recombination and protein expression system, or by synthesizing it in vitro through chemical synthesis such as peptide synthesis, and by cell-free protein synthesis methods.

[0042] Additionally, to obtain better chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), modified specificity (e.g., broad spectrum of biological activity), and reduced antigenicity, a protecting group may be attached to the N-terminus or C-terminus of the protein. 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), but may be included without limitation as long as it is a component capable of modifying the protein, particularly enhancing the stability of the protein. The term 'stability' refers not only to stability in vivo that protects the protein of the present invention from attack by protein-cleaving enzymes in vivo, but also to storage stability (e.g., room temperature storage stability).

[0043] The nanovesicles containing TRPA1 described above may be prepared by producing and isolating them from animal cells. Specifically, TRPA1 may be expressed in animal cells transformed with a gene encoding the TRPA1 protein, and then the animal cells may be treated with a substance that reduces cell membrane stability, such as cytochalasin B, followed by centrifugation to obtain the nanovesicles. Transforming the gene encoding the TRPA1 protein may utilize any technique or method known in the art of the present invention to be used for transforming a specific gene into animal cells; for example, this may involve cloning the TRPA1-coding gene into an expression vector and transforming it into animal cells through treatment with a lipofectamine solution.

[0044] The diameter of the nanovesicle containing the 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 within the above range, the nanovesicle can be effectively immobilized on a calcium ion-selective membrane.

[0045] The nanovesicles containing TRPA1 may be immobilized on the calcium ion-selective membrane by chemical bonding. The immobilization means fixing the nanovesicles containing TRPA1 so that they do not move from one position on the calcium ion-selective membrane.

[0046] The above chemical bond may be immobilized using a linker (30) selected from the group consisting of poly-D-lysine (or PDL), poly-L-lysine, and poly-L-ornithine, but is not limited thereto; any form may be utilized as long as it has the characteristic of allowing electron carriers (electrons or holes) to move through it and to bind to each of the calcium ion-selective membrane (20) and the nanovesicle (40) containing TRPA1. The above poly-D-lysine is a polymer in which multiple D-lysine units are linked, and can bind by forming ionic bonds on the surfaces of the calcium ion-selective membrane and the nanovesicle, respectively, by utilizing the cation characteristics exhibited by lysine. Therefore, the surface of the calcium ion-selective membrane can be modified by coating it with poly-di-lysine, and the TRPA1-containing nanovesicles can be immobilized on the calcium ion-selective membrane by binding the TRPA1-containing nanovesicles to the poly-di-lysine.

[0048] FIG. 2 shows a method for manufacturing a structure for a real-time nicotine detection biosensor according to one embodiment of the present invention.

[0049] Referring to FIG. 2, the method for manufacturing a structure for a real-time nicotine detection biosensor according to the present invention is as follows:

[0050] Step of forming a crystalline carbon film;

[0051] A step of forming a calcium ion-selective film on the crystalline carbon film above;

[0052] A step of immobilizing a linker on the calcium ion-selective membrane; and

[0053] The method includes the step of immobilizing a nanovesicle containing TRPA1 to the above linker.

[0055] The above crystalline carbon film can be formed on a substrate or electrode commonly used in biosensors in the industry.

[0056] The above substrate may be a rigid substrate or a flexible substrate. For example, the above substrate may be a rigid substrate such as an insulating inorganic substrate such as a Si substrate, a glass substrate, a GaN substrate, or a silica (SiO2) substrate, a metal substrate such as Ni, Cu, or W, or a plastic substrate, or at least one of a flexible substrate that can be folded or bent, such as polyimide (PI), polypropylene (PP), polyester (PET), polyethylene (PE), polyvinyl chloride (PVC), polyamide (PA), stretched polypropylene (OPP), ethylene vinyl acetate copolymer (EVA), polyurethane (PU), polydimethylsiloxane (PDMS), silicone, or latex, but is not limited thereto.

[0057] The above electrode can be electrically connected and may include a conductive material, and may be formed, for example, a metal, a metal alloy, a conductive metal oxide, a conductive metal nitride, a carbon electrode, etc.

[0058] The formation of the above crystalline carbon film and calcium ion-selective film can be performed using a solution process or a deposition process.

[0059] The above solution processes may include spin coating, drop casting, bar coating, slot die coating, gravure printing, nozzle printing, inkjet printing, screen printing, electrohydrodynamic jet printing, electrospray, etc.

[0060] The above deposition processes may include vacuum deposition (evaporation), thermal deposition, flash deposition, laser deposition, chemical vapor deposition, atomic layer deposition, physical vapor deposition, physical-chemical coevaporation deposition, sequential vapor deposition, solution process-assisted thermal deposition, etc.

[0061] The above linker may be selected from the group consisting of poly-D-lysine (or PDL), poly-L-lysine, and poly-L-ornithine, but is not limited thereto, and may be used in any form as long as it has the characteristic that electron carriers (electrons or holes) can move through it and can bind to each of the calcium ion-selective membrane (20) and the nanovesicle (40) containing TRPA1.

[0062] For example, the surface of the calcium ion-selective membrane can be modified by coating it with poly-di-lysine, and the TRPA1-containing nanovesicles can be immobilized on the calcium ion-selective membrane by binding the TRPA1-containing nanovesicles to the poly-di-lysine.

[0064] [Real-time Nicotine Detection Biosensor]

[0065] In addition, another aspect of the present invention provides a real-time nicotine detection biosensor comprising the above structure.

[0066] In one embodiment of the present invention, the biosensor is characterized in that the structure is formed on an electrode or the structure is formed to be connected to an electrode.

[0067] The above biosensor may be a two-electrode type biosensor comprising an operating electrode and a counter electrode, or a three-electrode type biosensor comprising an operating electrode, a counter electrode, and a reference electrode. In one embodiment of the present invention, the biosensor used a three-electrode type biosensor comprising an operating electrode (1), a counter electrode (2), and a reference electrode (3) as shown in FIG. 2, but is not limited thereto. Additionally, the biosensor may be a transistor type biosensor.

[0069] FIG. 3 is a schematic diagram showing the operating principle when actual saliva from a smoker is introduced into a biosensor including a structure according to one embodiment of the present invention.

[0070] Referring to FIG. 3, the structure connected to the electrode of the biosensor according to the present invention forms a structure of a crystalline carbon membrane / calcium ion-selective membrane / TRPA1-containing nanovesicle, so that when the actual saliva of a smoker is introduced into the biosensor, if nicotine is contained in the saliva containing calcium ions, the nanovesicle having a TRPA1 ion channel in the outermost layer of the structure connected to the electrode of the biosensor does not react with other components in the saliva, but specifically reacts with the nicotine contained in the saliva to introduce calcium ions, and the change in calcium ion concentration caused by this changes the surface potential of the crystalline carbon membrane in contact with the calcium ion-selective membrane through the calcium ion-selective membrane, thereby detecting the change in electrical conductivity, such as a change in open circuit voltage, so that the nicotine concentration can be quantitatively detected in real time.

[0071] The above biosensor has a nicotine detection limit of 1 pM, making it useful for detecting low concentrations of nicotine present in saliva. Since it can detect nicotine in real time, it can be rapidly applied in actual field settings such as public health centers.

[0073] [Real-time Nicotine Analysis Method in Saliva]

[0074] In addition, another aspect of the present invention provides a method for real-time nicotine analysis in saliva using the biosensor.

[0075] The above method includes the step of processing a saliva sample of a measurement target into the real-time nicotine detection biosensor; and the step of measuring the electrical signal of the real-time nicotine detection biosensor.

[0076] The above detection means confirming the presence of a target substance and includes quantifying or semi-quantifying the concentration of nicotine, which is the target substance.

[0077] The subjects of measurement may include smokers or non-smokers, and specifically, may target smokers.

[0078] The measurement of the above electrical signal may be a measurement of a change in open circuit voltage (OCV), and by measuring the change in OCV, the presence of nicotine in a saliva sample and / or the concentration and amount of nicotine may be detected.

[0079] The method for real-time nicotine analysis in saliva using the above biosensor may further include a step of quantitatively analyzing the nicotine concentration according to the magnitude of the change in the electrical signal of the biosensor.

[0080] As the description of the above biosensor is as previously stated, a detailed explanation is omitted.

[0081] The real-time nicotine analysis method in saliva using the above biosensor is characterized by a nicotine detection limit of 1 pM.

[0083] Hereinafter, preferred manufacturing examples and experimental examples are presented to aid in understanding the present invention. However, the following manufacturing examples and experimental examples are intended only to aid in understanding the present invention, and the present invention is not limited by the following manufacturing examples and experimental examples.

[0085] [Preparation Example 1: Preparation of an electrode containing a nanovesicle structure containing CNT / ISM / TRPA1]

[0086] (1) Preparation of nanovesicles containing TRPA1

[0087] HEK-293T cells were placed in DMEM (Dulbecco's Modified Eagle Medium) supplemented with 10% fetal bovine serum (FBS) and 100 μg / ml penicillin and cultured at 37°C and 5% CO2. The cells were placed in 100mm dishes. 6 Cells were dispensed at a density of cells / ml. Subsequently, TRPA1 was introduced into the cells using lipofectamine, and the cells were cultured for 48 hours.

[0088] Next, the cells were cultured in serum-free DMEM containing cytocalcin B (10 μg / ml) at 37°C for 30 minutes. Cytocalcin B is a type of mycotoxin capable of penetrating cell membranes and can reduce the stability of cell membranes by interfering with the formation of contractile microfilaments. After vigorously stirring the cell suspension for 30 seconds, TRPA1 nanovesicles were isolated from the cells through two stages of centrifugation (500xg for 10 minutes and 15000xg for 30 minutes).

[0089] The above nanovesicles were subsequently redispersed in PBS containing a protease inhibitor cocktail and stored in a frozen state at minus 80°C.

[0091] (2) Preparation of calcium ion-selective membrane solution

[0092] 1 g of a mixture (weight ratio of 65.45 / 33 / 0.55 / 1) of DOS (dioctylsebacate) as a plasticizer, PVC (polyvinyl chloride) as a matrix, Na-TFPB (Sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate) as a lipophilic additive, and calcium ionophor II as an ion-selective substance was dissolved in 6.6 mL of tetrafuran to [contain] calcium ions (Ca 2+ A selective membrane solution was prepared. The above calcium ion (Ca 2+ The selective membrane solution was stored at 4 ℃.

[0094] (3) Preparation of an electrode formed with a nanovesicle structure containing CNT / ISM / TRPA1

[0095] As shown in FIG. 2, a working electrode, a counter electrode, and a reference electrode were printed with silver ink. Among these, a CNT film (10) was printed with CNT ink on the working electrode (1), and calcium ions (Ca) prepared in (2) were printed thereon thereon. 2+ A CNT (10) / ISM (20) membrane was prepared by drop-casting a selective membrane solution.

[0096] To confirm whether a calcium ion-selective film was successfully formed on the above CNT film, an untreated CNT film and a CNT / ISM film were elementally analyzed using energy-dispersive X-ray spectroscopy (EDX), and the results are shown in Figure 4.

[0097] Figure 4 shows the energy dispersive X-ray spectroscopy (EDX) spectrum and field emission scanning electron microscope (FE-SEM) image of a CNT film and a CNT / ISM film according to one comparative example and one embodiment of the present invention.

[0098] As shown in Fig. 4, Ca 2+The Cl content on the CNT surface (b) coated with an ion-selective membrane (ISM) is 32.09%, indicating an increase compared to the untreated CNT surface (2.47%) (a). The Cl appears to be a component derived from polyvinyl chloride (PVC), and since the polyvinyl chloride is the main component of the calcium ion-selective membrane (ISM), this leads to Ca in the CNT / ISM membrane. 2+ It can be confirmed that the ISM has been successfully attached.

[0099] Next, the TRPA1-containing nanovesicles (TRPA1-NV) prepared in (1) above were attached to the CNT / ISM membrane. Specifically, a poly-d-lysine (PDL) solution (0.1 mg / ml in PBS) was introduced onto the CNT / ISM membrane and left for 1 hour. Afterward, the TRPA1-containing nanovesicles were dropped onto the poly-d-lysine layer so that the poly-d-lysine layer (30) and the TRPA1 nanovesicles (40) were attached by positive-negative charge interaction, thereby producing an electrode in which a CNT (10) / ISM (20) / TRPA1-containing nanovesicle (40) structure was formed.

[0101] [Preparation Example 2: Preparation of a biosensor containing a nanovesicle structure containing CNT / ISM / TRPA1]

[0102] A biosensor was fabricated using a working electrode formed with a nanovesicle structure containing CNT / ISM / TRPA1 prepared in Preparation Example 1, a counter electrode screen-printed with CNT ink, and a reference electrode screen-printed with silver ink.

[0104] [Comparative Example 1]

[0105] A biosensor was fabricated using a working electrode and a counter electrode screen-printed with CNT ink, and a reference electrode screen-printed with silver ink.

[0107] [Comparative Example 2]

[0108] A biosensor was prepared in the same manner as in Preparation Example 1, except that the TRPA1-containing nanovesicles were cultured in PBS containing 5 μM of HC-030031 for 10 minutes to produce nanovesicles with inhibited TRPA1 activity.

[0110] [Experimental Example 1: Effect of a Calcium Ion Selective Membrane on a CNT Film on Tobacco Smoke Extract Detection]

[0111] In order to investigate the effect of a calcium ion-selective membrane formed on a CNT membrane on the detection of tobacco smoke extract in a biosensor structure according to the present invention, the following experiment was performed.

[0112] The untreated CNT membrane of Comparative Example 1 and the CNT / ISM membrane prepared in Preparation Example 1 were attached to electrodes and placed in artificial saliva containing calcium ions (containing 10 mM KCl, 7.4 mM NaCl, 2 mM CaCl2, 6.4 mM Na2HPO4, and 2.5 mM NaHCO3 at pH 7.4), and after being exposed to tobacco smoke (CSE) at a concentration of 1% for 1 minute, the change in open-circuit voltage (OCV) was measured and is shown in Fig. 5.

[0113] FIG. 5 is a graph showing the change in open circuit voltage (OCV) over time when electrodes with attached CNT membranes and CNT / ISM membranes according to one comparative example and one embodiment of the present invention are placed in artificial saliva containing calcium ions and exposed to 1% concentration of tobacco smoke (CSE).

[0114] As shown in Figure 5, the OCV of the electrode attached to the untreated CNT film was nonspecifically altered by exposure to cigarette smoke (CSE). It is believed that various ion species in the cigarette smoke acted as a resistor, changing the surface potential of the CNT film, which is a cause of noise signals. However, the electrode attached to the CNT film coated with a calcium ion-selective membrane (ISM) did not exhibit a nonspecific response to exposure to cigarette smoke (CSE), indicating that the calcium ion-selective membrane (ISM) on the CNT surface acts as a physical barrier that blocks interference caused by various ion species contained in the cigarette smoke. At this time, since the cigarette smoke does not contain calcium ions, no change in calcium ions occurred, and thus no change in OCV occurred. Through this, it was confirmed that coating an ion-selective membrane on the CNT film is essential to block noise signals.

[0116] Additionally, Ca of the CNT / ISM membrane 2+ To determine the selectivity for changes, changes in OCV were measured while adding 2 mM MgCl2, 2 mM KCl, 2 mM NaCl, and 2 mM CaCl2 to artificial saliva, respectively, and the results are shown in Figure 6.

[0117] FIG. 6 is a graph showing the change in open circuit voltage (OCV) when electrodes with attached CNT membranes and CNT / ISM membranes according to one comparative example and one embodiment of the present invention are placed in artificial saliva containing calcium ions, and 2 mM MgCl2, 2 mM KCl, 2 mM NaCl, and 2 mM CaCl2 are added to the artificial saliva.

[0118] As shown in Fig. 6, Ca 2+ The ISM-coated CNT film is Mg 2+ , K + , Na + It was confirmed that the OCV selectively increases only in response to changes in added calcium ions without reacting to other cations such as the back.

[0119] Accordingly, the biosensor according to the present invention comprises a structure in which a calcium ion-selective membrane is formed on a CNT membrane, thereby enabling high-sensitivity detection of a target that changes calcium ions without interference or noise from other elements.

[0121] [Experimental Example 2: Effect of TRPA1 Ion Channel on Tobacco Smoke Detection]

[0122] In order to investigate the effect of the TRPA1 ion channel within the nanovesicle formed in the outermost layer of the structure on tobacco smoke detection in a biosensor comprising a CNT / ISM / TRPA1-containing nanovesicle structure according to the present invention, the following experiment was performed.

[0123] Specifically, after mock-infecting HEK-293T cells with TRPA1 or an empty vector, calcium ions (Ca 2+ After adding a responsive fluorescent dye, a 1% tobacco smoke extract (CSE) solution, in which 1% tobacco smoke was absorbed, was applied to artificial saliva containing calcium ions, and a series of images were captured over time for 150 seconds to measure changes in fluorescence intensity, which are shown in Fig. 7. At this time, intracellular calcium ions (Ca 2+ The higher the inflow, the greater the fluorescence intensity.

[0124] Figure 7 is a graph showing the change in fluorescence intensity of cells over time when a calcium ion-sensitive fluorescent dye is added to HEK-293T cells mock-infected with TRPA1 or an empty vector and a tobacco smoke extract (CSE) solution is applied.

[0125] As shown in Figure 7, in cells expressing the TRPA1 ion channel, the fluorescence intensity increased in a time-dependent manner over time upon application of the tobacco smoke extract (CSE) solution and reached saturation at 120 seconds. This increase in fluorescence intensity is thought to be due to the TRPA1 ion channel reacting with the tobacco smoke extract to introduce calcium ions into the cell.

[0126] However, in the case of cells mock-infected with an empty vector, that is, cells lacking TRPA1, no fluorescence reaction occurred within the cells even upon exposure to tobacco smoke extract.

[0127] From this, it was confirmed that the TRPA1 ion channel changes the calcium ion concentration by introducing calcium ions in response to tobacco smoke extract.

[0129] Additionally, to determine whether TRPA1-containing nanovesicles isolated from the above HEK-293T cells also respond to the tobacco smoke extract, the following experiment was performed.

[0130] Specifically, regarding the TRPA1-containing nanovesicles prepared in (1) of Preparation Example 1 and the nanovesicles isolated from HEK-293T cells mock-infected with an empty vector, a calcium ion-sensitive fluorescent dye was added, and a 3% tobacco smoke extract (CSE) solution, in which 3% tobacco smoke was absorbed into artificial saliva containing calcium ions, was applied. A series of images were captured over time for 300 seconds to measure the change in fluorescence intensity, which is shown in Fig. 8. At this time, calcium ions (Ca 2+ The higher the inflow, the greater the fluorescence intensity.

[0131] Figure 8 is a graph showing the change in fluorescence intensity of nanovesicles over time when a calcium ion-sensitive fluorescent dye is added to nanovesicles isolated from HEK-293T cells mock-infected with TRPA1-containing nanovesicles or empty vectors, and a tobacco smoke extract (CSE) solution is applied.

[0132] As shown in Figure 8, in the case of TRPA1-containing nanovesicles containing TRPA1 ion channels, the fluorescence intensity increased over time when a tobacco smoke extract (CSE) solution was applied, even though they were isolated from cells. This indicates that TRPA1-containing nanovesicles isolated from cells also respond to the tobacco smoke extract and take in calcium ions at a level similar to that of cells.

[0133] From this, it was confirmed that TRPA1-containing nanovesicles also change the calcium ion concentration by reacting with tobacco smoke extract and introducing calcium ions.

[0135] [Experimental Example 3: Measurement of Nicotine Detection Performance of a Biosensor Containing a CNT / ISM / TRPA1-Containing Nanovesicle Structure According to the Present Invention]

[0136] Nicotine in cigarette smoke primarily activates TRPA1, and the activated TRPA1 changes the calcium ion concentration by moving calcium ions into the cell membrane. However, since nicotine has a short half-life and is converted into metabolites after 2 hours, rapid real-time detection of even small amounts of nicotine in saliva is required to detect nicotine.

[0137] Accordingly, the following experiment was performed to measure the nicotine detection performance of a biosensor comprising a nanovesicle structure containing CNT / ISM / TRPA1 according to the present invention.

[0138] Specifically, nicotine was added to artificial saliva containing 10 mM KCl, 7.4 mM NaCl, 2 mM CaCl2, 6.4 mM Na2HPO4, and 2.5 mM NaHCO3 at pH 7.4, and then dropped onto the biosensor prepared in Preparation Example 1 to measure the change in open-circuit voltage (OCV).

[0139] A biosensor without TRPA1 of Comparative Example 1 was used as a control group (CTL), and a biosensor with inhibited TRPA1 activity of Comparative Example 2 was used as a control group.

[0140] The measurement results are shown in Fig. 9.

[0141] FIG. 9 is a graph showing the sensing performance according to nicotine concentration of a biosensor including a nanovesicle structure containing CNT / ISM / TRPA1 according to one embodiment of the present invention.

[0142] As shown in FIG. 9, a biosensor comprising a nanovesicle structure containing CNT / ISM / TRPA1 according to the present invention is 10 -12 Nicotine at a concentration of M (i.e., 1 pM) is detected by showing a change in OCV, and the change in OCV is linear in proportion to the nicotine concentration, whereas in the control biosensor without a TRPA1 channel and the biosensor in which the activity of TRPA1 is blocked by a TRPA1 inhibitor (HC-030031), it was found that there is almost no change in OCV even with an increase in nicotine concentration.

[0143] Accordingly, a biosensor comprising a nanovesicle structure containing CNT / ISM / TRPA1 according to the present invention can rapidly and quantitatively detect minute concentrations of nicotine in artificial saliva.

[0145] [Experimental Example 4: Measurement of nicotine detection performance in actual saliva of a biosensor containing a CNT / ISM / TRPA1-containing nanovesicle structure according to the present invention]

[0146] In order to determine whether the biosensor comprising a CNT / ISM / TRPA1-containing nanovesicle structure according to the present invention can measure the nicotine concentration in actual saliva in real time in the field, the following experiment was performed.

[0147] Specifically, as shown in FIG. 3, the actual saliva of a smoker was continuously dropped onto a biosensor containing a nanovesicle structure containing CNT / ISM / TRPA1 of Preparation Example 1 at a rate of once every 10 seconds, and the change in OCV was measured in real time and is shown in FIG. 10.

[0148] FIG. 10 is a graph showing the sensing performance according to the nicotine concentration in actual saliva of a biosensor comprising a nanovesicle structure containing CNT / ISM / TRPA1 according to one embodiment of the present invention.

[0149] As shown in Fig. 10, the biosensor containing a nanovesicle structure containing CNT / ISM / TRPA1 according to the present invention exhibits a change in OCV within 10 seconds depending on the nicotine concentration in actual saliva, thereby allowing for the quantitative detection of nicotine concentration in real saliva in real time, and it was confirmed that it can be used in actual field conditions.

[0151] The present invention is not limited to the above embodiments and can be manufactured in various different forms, and those skilled in the art will understand that the invention can be implemented in other specific forms without changing the technical concept or essential features of the invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. Explanation of the symbols

[0152] 1: Working electrode 2: Counter electrode 3: Reference electrode 10: Carbon nanotube (CNT) membrane 20: Calcium ion-selective membrane 30: Linker (Poly-d-lysine (PDL)) 40: TRPA1-containing nanovesicles

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 A biosensor comprising a structure including a crystalline carbon membrane; a calcium ion-selective membrane formed on the crystalline carbon membrane; and a nanovesicle comprising a calcium ion channel protein formed on the calcium ion-selective membrane, wherein the calcium ion channel protein is TRPA1 (Transient receptor potential channel, subfamily A, member 1), and detects the nicotine concentration in saliva, wherein the nicotine in the saliva reacts with TRPA1 so that calcium ions in the saliva flow into the nanovesicle through TRPA1, and the nicotine concentration is quantitatively detected by detecting the change in the concentration of calcium ions in the saliva accordingly, and the detection limit of nicotine is 1 pM. Claim 6 delete Claim 7 In claim 5, the biosensor is a biosensor that exhibits a change in open-circuit voltage (OCV) that changes linearly in proportion to the nicotine concentration. Claim 8 delete Claim 9 delete Claim 10 A method for analyzing nicotine in saliva, comprising: a step of treating a saliva sample to be measured in a biosensor having a structure comprising a crystalline carbon membrane; a calcium ion-selective membrane formed on the crystalline carbon membrane; and a nanovesicle comprising a calcium ion channel protein, TRPA1 (Transient receptor potential channel, subfamily A, member 1), formed on the calcium ion-selective membrane; and a step of measuring an electrical signal of the biosensor, wherein the electrical signal is attributable to a change in the concentration of calcium ions in the saliva sample, wherein nicotine in the saliva sample reacts with TRPA1, causing calcium ions in the saliva sample to flow into the nanovesicle through TRPA1, and the detection limit of nicotine is 1 pM. Claim 11 A method for analyzing nicotine in saliva, wherein, in claim 10, the measurement of the electrical signal is characterized by measuring a change in open circuit voltage (OCV). Claim 12 A method for analyzing nicotine in saliva according to claim 10, wherein the above analysis method further comprises the step of quantitatively analyzing the nicotine concentration according to the magnitude of the change in the electrical signal of the biosensor. Claim 13 A method for analyzing nicotine in saliva, wherein, in claim 11, the change in the open-circuit voltage (OCV) changes linearly in proportion to the nicotine concentration.

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