Biosensors for capturing and measuring substances in the gas phase

Abstract

The present invention provides a gas-phase substance capture material for capturing substances in the gas phase, preferably volatile organic compounds (VOCs), which offers significantly simplified production and utilization processes, can be manufactured in a short time, requires no on-demand preparation, can be manufactured in a short time, and can be stored for a long period of time. [Solution] We have discovered that by solidifying and processing a solution of a polymer material and a functional molecule (e.g., an enzyme) in an appropriate manner, and by embedding the functional molecule in the polymer material, it is possible to express the unique functions of the functional molecule that normally does not show activity in the gas phase. Based on this, we have created a gas-phase substance capture material as a composite material in which a functional molecule is embedded in the polymer material. Specifically, we provide a gas-phase substance capture material for capturing substances in the gas phase, which is formed of a water-soluble polymer containing a functional protein that retains its ability to bind to the substance, and the functional protein captures the substance by exhibiting its ability to bind to the substance.

Description

[Technical Field]

[0001] The present invention relates to a gas-phase substance capture material formed from a polymer mixed with functional proteins for capturing a target substance in the gas phase, and to a gas-phase substance capture method for capturing a target substance in the gas phase using the gas-phase substance capture material. [Background technology]

[0002] Living organisms release various volatile organic compounds (VOCs) as biological gases in conjunction with their life activities. In a broad sense, biological gases include gases emitted by plants and other organisms, but in a narrow sense, they refer specifically to gases emitted by humans. These biological gases in the narrow sense can be broadly classified into gases derived from the skin, breath, intestines, blood, and urine.

[0003] Of these biological gases, exhaled breath is frequently used in clinical applications. This is because exhaled breath contains 200-300 different components, it provides useful biological information distinct from blood components, it allows for non-invasive sampling, and it can be easily measured even by patients without medical knowledge. Examples of breath analyzers currently in use include EtOH (ethanol) for alcohol detection checkpoints and for H. pylori infection testing in the stomach. 13 Examples include CO2 (stable isotope), NO (nitric oxide) as a monitor for asthma treatment, H2 (hydrogen) as a monitor for lactose intolerance, and CO (carbon monoxide) as a smoking monitor.

[0004] Therefore, a device for measuring EtOH in exhaled breath has been developed (Patent Document 1). In this device, alcohol dehydrogenase (ADH) is immobilized on a cotton mesh by physical adsorption, and the coenzyme nicotinamide adenine dinucleotide (NAD) is added to it. +A gas-phase substance capture material is used, to which ) is added and made visible. When this cotton mesh is exposed to ethanol (EtOH) gas contained in skin gases, etc., the substrate EtOH is broken down into acetaldehyde by the enzymatic activity reaction of ADH, and consequently NAD is released. + It is reduced to NADH. This NADH is excited by ultraviolet light at a wavelength of 340 nm and emits fluorescence at a wavelength of 490 nm. By monitoring this fluorescence, the EtOH gas can be measured.

[0005] However, this cotton mesh-based exhaled EtOH measurement device has several drawbacks: (1) its manufacture requires a multi-step enzyme immobilization process that takes several hours, making mass production difficult; and (2) the user must add the coenzyme solution to the enzyme membrane immediately before use, i.e., it requires on-demand preparation. Therefore, there is a need for the development of a gas-phase substance capture material that significantly simplifies the production and usage processes, eliminates the need for on-demand preparation, can be manufactured in a short time, and can be stored for a long period of time. [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] International public brochure WO2019 / 103130A1 [Overview of the project] [Problems that the invention aims to solve]

[0007] The present invention aims to provide a gas-phase substance capture material for capturing substances in the gas phase, preferably volatile organic compounds (VOCs), that significantly simplifies the production and utilization processes, can be manufactured in a short time, does not require on-demand preparation, can be manufactured in a short time, and can be stored for a long period of time. [Means for solving the problem]

[0008] As a result of intensive research, the inventors have found that by solidifying and processing a solution in which a water-soluble polymer material and a functional molecule such as a protein (for example, an enzyme) are mixed in an appropriate manner, and incorporating the functional molecule into the water-soluble polymer material, it is possible to express the functions inherent to the functional molecule that usually do not exhibit activity in the gas phase. Thus, a gas-phase substance capturing material as a composite material in which a functional molecule is incorporated into a water-soluble polymer material has been created.

[0009] Specifically, the present invention provides a gas-phase substance capturing material for capturing a substance in a gas phase, which is formed of a water-soluble polymer incorporating a functional protein in which the binding ability to the substance is retained, and the functional protein captures the substance by exerting the binding ability to the substance.

[0010] In the gas-phase substance capturing material of the present invention, the function of the functional protein in the water-soluble polymer may be retained.

[0011] In the gas-phase substance capturing material of the present invention, the functional protein may be an enzyme or an antibody.

[0012] In the gas-phase substance capturing material of the present invention, the substance may be a volatile organic compound (VOCs).

[0013] In the gas-phase substance capturing material of the present invention, when the functional protein is an enzyme, the water-soluble polymer may be further mixed with a cofactor.

[0014] In the gas-phase substance capturing material of the present invention, the water-soluble polymer may be selected from polyvinyl alcohol (PVA), polyethylene oxide (PEO), polyacrylamide (PAAm), polyvinylpyrrolidone (PVP), dextran or polyethylene glycol (PEG).

[0015] In the gas-phase substance scavenging material of the present invention, the form of the water-soluble polymer may be a microfiber or a thin film.

[0016] In the gas-phase substance scavenging material of the present invention, the volatile organic compound is ethanol, the enzyme is alcohol dehydrogenase, and the cofactor is NAD + It may be (oxidized nicotinamide adenine dinucleotide).

[0017] Furthermore, the present invention provides a biosensor for capturing and measuring substances in the gas phase using the aforementioned gas phase substance capturing material.

[0018] In the biosensor of the present invention, the substance may be a volatile organic compound.

[0019] Furthermore, the present invention provides a method for capturing a substance in the gas phase, wherein the method is formed from a water-soluble polymer containing a functional protein, and the functional protein captures the substance by exhibiting binding ability to the substance.

[0020] In the method of the present invention, the substance may be a volatile organic compound. [Effects of the Invention]

[0021] The present invention significantly simplifies the production and utilization processes for capturing substances such as volatile organic compounds (VOCs) in the gas phase, and provides a gas-phase substance capture material that can be manufactured in a short time, does not require on-demand preparation, can be manufactured in a short time, and can be stored for a long period of time. [Brief explanation of the drawing]

[0022] [Figure 1] This diagram illustrates the detection principle of EtOH gas by the reaction of EtOH with alcohol dehydrogenase (ADH) in a gas-phase substance capture material, for capturing and detecting EtOH in the gas phase. [Figure 2]A schematic diagram showing a method for manufacturing a gas-phase substance capture material to capture EtOH in the gas phase. [Figure 3] This diagram shows the experimental setup for EtOH gas measurement (top) and the settings for the distance between the excitation light source and the excited object (i.e., the gas phase substance trapping material) and the distance between the Pasteur (gas outlet) and the enzyme membrane (i.e., the gas phase substance trapping material) (bottom). [Figure 4] This fluorescence image shows the fluorescence (excitation wavelength: 340 nm, synchrotron radiation wavelength: 490 nm) observed when air containing EtOH is loaded onto a microfiber mesh of polyvinyl alcohol (PVA) mixed with ADH and NAD+. [Figure 5] The figure shows the time course of fluorescence intensity (excitation wavelength: 340 nm, synchrotron radiation wavelength: 490 nm) when (1) a microfiber mesh made of polyvinyl alcohol (PVA) mixed with ADH and NAD+ (PVA / ADH / NAD+ mesh) is loaded with air containing 200 ppm of EtOH, and as a negative control, (2) a PVA / ADH / NAD+ mesh is loaded with air that does not contain EtOH, and (3) a microfiber mesh made of polyvinyl alcohol mixed with NAD+ but without ADH (PVA / NAD+ mesh) is loaded with air containing EtOH. [Figure 6] This figure shows the change in the differential value of the time-dependent change in fluorescence intensity, i.e., the fluorescence change rate, when air containing 200 ppm of EtOH is loaded onto a PVA / ADH / NAD+ mesh at various flow rates. [Figure 7] This figure shows the relationship between the change in the differential value of the change in fluorescence intensity over time (fluorescence change rate) and the amount of EtOH loaded per unit time when air containing EtOH is loaded onto a PVA / ADH / NAD+ mesh at various flow rates. [Figure 8A] Scanning electron microscope images of PVA / ADH / NAD+ mesh on the day of fabrication, from an experiment investigating the storage stability of PVA / ADH / NAD+ mesh. [Figure 8B]Scanning electron microscope image of PVA / ADH / NAD+ mesh after 2 weeks of storage at 4°C following an experiment to investigate the storage stability of PVA / ADH / NAD+ mesh. [Figure 9] This figure shows the response (change in fluorescence intensity over time) to EtOH loading (loaded for 20 seconds) on the day of preparation and after 2 weeks of storage at 4°C in an experiment to investigate the storage stability of PVA / ADH / NAD+ mesh. [Figure 10] This figure shows the time course of fluorescence intensity (excitation wavelength: 340 nm, synchrotron radiation wavelength: 490 nm) when a microfiber mesh containing ADH and NAD+ mixed with various water-soluble polymers is exposed to air containing 200 ppm of EtOH for 20 seconds. PVA represents polyvinyl alcohol, PAAm represents polyacrylamide, PEO represents polyethylene oxide, and PVP represents polyvinylpyrrolidone. [Modes for carrying out the invention]

[0023] 1. Gas-phase substance capture material, and biosensor using the gas-phase substance capture material One embodiment of the present invention is a gas-phase substance scavenging material. This gas-phase substance scavenging material can be manufactured by solidifying and processing a solution obtained by mixing a water-soluble polymer material with a functional molecule such as a protein (e.g., an enzyme) in an appropriate manner, thereby imbuing the water-soluble polymer material with the functional molecule. It is a gas-phase substance scavenging material that is a composite material capable of exhibiting the intrinsic functions of functional molecules that normally do not show activity in the gas phase.

[0024] Typically, functional molecules such as enzymes and antibodies exhibit their functions in the liquid phase. In this specification, "gas-phase substance capture material" refers to a composite material in which a functional polymer is embedded in a water-soluble polymer material, which can capture target substances such as organic compound molecules including volatile organic compound molecules or viruses by binding the target substance in the gas phase to the functional molecule, based on the unique function of the functional molecule in the solid phase rather than the liquid phase. In the case of a functional molecule such as an enzyme, the gas-phase substance capture material of the present invention also includes cases where the functional molecule not only binds to and captures the target substance with the enzyme, but also exhibits the catalytic activity of the enzyme.

[0025] Examples of gaseous phases include, but are not limited to, general air containing target molecules, as well as biological gases such as exhaled breath and skin gases released from living organisms.

[0026] In this specification, "retaining function" is used in a qualitative sense, meaning that the same function is present before and after the comparison. That is, it means not only that the same function is maintained to the same degree or higher, but also that even if the function is quantitatively reduced, it is still able to perform the same function without becoming inactive.

[0027] Generally, when functional molecules such as proteins are mixed with polymer materials, the intrinsic function of the functional molecules is lost or significantly weakened. The inventors of this invention have found that by using a water-soluble polymer as the polymer material, the function of functional molecules such as proteins is preserved, and that when this water-soluble polymer material containing functional polymers is used, target substances in the gas phase can be effectively captured. This invention is based on this finding.

[0028] More specifically, the gas-phase substance capture material of the present invention is a gas-phase substance capture material for capturing a substance in the gas phase, and is formed of a water-soluble polymer containing a functional protein that maintains the ability to bind to the substance, wherein the functional protein captures the substance by exhibiting its ability to bind to the substance.

[0029] Furthermore, this gas-phase substance capture material can be used, but is not limited to, as a gas sensor, particularly a biosensor, for capturing and measuring a target substance in the gas phase.

[0030] Herein, in this specification, "biosensor" refers to a sensor that detects or measures target substances by utilizing the molecular recognition function of living organisms such as enzymes, microorganisms, and antibodies.

[0031] The gas-phase substance capture material of the present invention can target volatile organic compounds (VOCs) as an example of substances to be captured. For example, VOCs in biological gases such as exhaled breath and skin gases emitted from living organisms can be captured using the gas-phase substance capture material of the present invention and used as a biosensor to measure the amount of captured VOCs, which can be used for diagnosing the physiological state of living organisms and diseases.

[0032] For example, the concentration of EtOH in exhaled breath is 70-2000 ppb, acetaldehyde is 3-90 ppb, methanol is 100-2300 ppb, acetone is 200-900 ppb, isopropanol is 50-250 ppb, formaldehyde is 48-83 ppb, ammonia is 400-1350 ppb, and dimethyl sulfide is 50 ppb or less.

[0033] Examples of biosensors using the gas-phase substance capture material of the present invention include, but are not limited to, a biosensor for measuring ethanol (EtOH) for the treatment of alcohol dependence, a biosensor for measuring acetaldehyde for risk assessment of oral and esophageal cancer, a biosensor for measuring methanol for evaluation of the intestinal environment, a biosensor for measuring acetone for evaluation of diabetes and lipid metabolism, a biosensor for measuring isopropanol for evaluation of diabetes and lipid metabolism, a biosensor for measuring formaldehyde for the diagnosis of lung cancer, a biosensor for measuring ammonia for the diagnosis of liver disease, and a biosensor for measuring dimethyl sulfide for evaluation of bad breath and oral environment.

[0034] In addition to its use as a gas sensor or biosensor, the gas-phase substance capture material of the present invention can be used to purify spaces by capturing substances suspended in the air. Furthermore, the gas-phase substance capture material of the present invention can be used to recover desired substances by selectively capturing substances suspended in the air. By capturing, recovering, and analyzing substances suspended in the air, the cleanliness and contamination levels of the gas phase of the space can be measured. Moreover, by capturing pathogenic substances suspended in the air, it is possible to prevent illness.

[0035] In the gas-phase substance scavenging material of the present invention, examples of the functional protein include enzymes or antibodies, with enzymes being more preferred.

[0036] In the gas-phase substance scavenging material of the present invention, when the functional protein is an enzyme, the polymer may be further mixed with a cofactor to prepare the material. A coenzyme is a preferred example of a cofactor.

[0037] In the gas-phase substance scavenging material of the present invention, examples of the water-soluble polymer can be selected from, but are not limited to, polyvinyl alcohol (PVA), polyethylene oxide (PEO), polyacrylamide (PAAm), polyvinylpyrrolidone (PVP), dextran, or polyethylene glycol (PEG).

[0038] In the gas-phase substance capture material of the present invention, when a gas phase such as exhaled breath or skin gas containing the VOCs to be measured is loaded onto the gas-phase substance capture material, the form of the water-soluble polymer is preferably a microfiber or a thin film in order to increase the contact area with which the VOCs come into contact. By using a microfiber or thin film that can increase the contact area, the VOCs to be measured can be detected sensitively and with high accuracy. Furthermore, for example, when the form of the gas-phase substance capture material is used as a microfiber, by using it as a microfiber mesh, good permeability of the gas containing VOCs can be ensured, thereby capturing even minute amounts of VOCs contained in the gas phase with the gas-phase substance capture material.

[0039] The gas-phase substance capture material of the present invention can be manufactured as a microfiber mesh by spinning a polymer material mixed with a functional polymer, or a mixture of a water-soluble polymer further mixed with a cofactor, using various methods. Electrospinning is preferred as the spinning method, but the invention is not limited thereto.

[0040] Electrospinning allows for the fabrication of three-dimensional structures composed of microfibers containing proteins, cofactors, or both, which also function as gas-phase substance capture agents. The advantages of using a three-dimensional structure include: (1) improved capture efficiency by increasing the occupied space of the gas-phase substance capture agent; and (2) while sheet-like capture agents used as sensors are limited to two-dimensional data for measuring the distribution of gas-phase components, a three-dimensional structure allows for simultaneous measurement of three-dimensional data. In this case, using a microfiber three-dimensional structure makes it possible to create a three-dimensional structure with good permeability suitable for measuring gas-phase components.

[0041] An example of the use of the gas-phase substance scavenging material of the present invention is as a biosensor targeting ethanol, a type of VOC, with alcohol dehydrogenase (ADH) as the enzyme and NAD as the cofactor. + Examples of water-soluble polymer materials containing (oxidized nicotinamide adenine dinucleotide) include water-soluble polymer materials.

[0042] This ADH and NAD + A biosensor that uses a gas-phase substance capture material for EtOH detection, formed from a water-soluble polymer mixture of ADH and NAD, detects EtOH binding to ADH and the dehydrogenation of EtOH into acetaldehyde during the process. + EtOH can be detected with high sensitivity by measuring the fluorescence of NADH generated from it (excitation light wavelength: 340 nm, synchrotron radiation wavelength: 490 nm) (see Figure 1).

[0043] The gas-phase substance capture material used in this EtOH detection biosensor is, for example, freeze-dried ADH powder and NAD + By using an aqueous solution of a water-soluble polymer material mixed with freeze-dried powder, and applying a high voltage while transporting the solution using the electrospinning method, microfibers can be formed on the collector electrode and manufactured as a microfiber mesh (see Figure 2).

[0044] This ADH and NAD+ By loading a gas containing EtOH onto a microfiber mesh formed of a water-soluble polymer incorporating + and measuring the fluorescence of the generated NADH, it can be used as a biosensor for detecting EtOH in the gas phase.

[0045] As methods for analyzing the measured fluorescence, (1) a method of measuring and analyzing the change in fluorescence intensity over time (see Examples and Fig. 5), and (2) a method of analyzing the fluorescence change rate, which is the amount of change per unit time of the change in fluorescence intensity over time, that is, a method of analyzing the differential value of the change in fluorescence intensity over time in the former (1) can be used. The differential value of the change in fluorescence intensity over time in the latter (2) means the amount of NADH generated per unit time, and this analysis method can more sensitively reflect the detected amount of EtOH (see Examples and Fig. 6).

[0046] This ADH and NAD + A substance capturing material for substances in the gas phase using EtOH as a target substance, formed of a water-soluble polymer incorporating + and + , and a biosensor for measuring EtOH using this, + Unlike a biosensor for detecting EtOH using a cotton mesh (Patent Document 1) that requires continuous supply during on-demand preparation and detection of the NAD solution, it can be used immediately after being manufactured as a microfiber mesh. Also, as shown in the following examples, it has excellent storage stability and does not require on-demand preparation.

[0047] 2. Method for capturing substances in the gas phase Another embodiment of the present invention is a method for capturing substances in the gas phase. More specifically, it is a method for capturing substances in the gas phase, which is formed of a water-soluble polymer incorporating a functional protein having a function of binding to a target substance, and the functional protein captures the substance in the gas phase by exerting its binding ability with the substance.

[0048] The present invention provides a capture method in which a functional molecule having the ability to bind to a target substance is embedded in a water-soluble polymer material, and by maintaining this binding ability, a gas containing the target substance is loaded onto the water-soluble polymer material, causing the target substance in the gas phase to be captured by the functional molecule embedded in the water-soluble polymer material.

[0049] In the capture method of the present invention, examples of the gas phase include biological gases such as exhaled breath and skin gases released from living organisms. Examples of the substances include volatile organic compounds (VOCs), and examples of functional proteins include enzymes and antibodies.

[0050] In the capture method of the present invention, examples of the water-soluble polymer include polyvinyl alcohol (PVA), polyethylene oxide (PEO), polyacrylamide (PAAm), polyvinylpyrrolidone (PVP), dextran, or polyethylene glycol (PEG).

[0051] The capture method of the present invention can be used, for example, in a biosensor for detecting VOCs in biological gases for the purpose of diagnosing the physiological state or diseases of living organisms. Specifically, examples of biosensors using the capture method of the present invention include, but are not limited to, a biosensor for measuring ethanol (EtOH) for the treatment of alcohol dependence, a biosensor for measuring acetaldehyde for risk assessment of oral and esophageal cancer, a biosensor for measuring methanol for evaluation of the intestinal environment, a biosensor for measuring acetone for evaluation of diabetes and lipid metabolism, a biosensor for measuring isopropanol for evaluation of diabetes and lipid metabolism, a biosensor for measuring formaldehyde for the diagnosis of lung cancer, a biosensor for measuring ammonia for the diagnosis of liver disease, and a biosensor for measuring dimethyl sulfide for evaluation of bad breath and oral environment.

[0052] As a biosensor for detecting ethanol (EtOH), a VOC, the enzyme alcohol dehydrogenase (ADH) and the coenzyme NAD are used. +One example of a biosensor is one that uses a gas-phase substance capture material formed by spinning an aqueous solution of a water-soluble polymer mixed with (oxidized nicotinamide adenine dinucleotide).

[0053] This ADH and NAD + A gas-phase substance scavenger formed from a water-soluble polymer containing a mixture of the above, as described above, dehydrogenates EtOH to acetaldehyde with ADH and NAD + By observing the fluorescence of NADH generated from this (excitation light wavelength: 340 nm, synchrotron radiation wavelength: 490 nm), the amount of EtOH in the gas phase can be measured.

[0054] The gas-phase substance capture material used in the method of the present invention is preferably in the form of a microfiber or thin film, from the viewpoint of having a large contact area with the gas phase and being able to capture substance molecules in the gas phase effectively. Furthermore, a microfiber mesh form is more preferable from the viewpoint of good gas-phase permeability.

[0055] Furthermore, as described above, the biosensor utilizing the method of the present invention does not require the preparation of reagents on a case-by-case basis, for example, ADH and NAD + By using a water-soluble polymer aqueous solution, which is a mixture of [components], and spinning it using the electrospinning method to produce a microfiber mesh, it can be used immediately. Furthermore, this microfiber mesh biosensor also has the characteristic of excellent storage stability. [Examples]

[0056] All references made herein are incorporated herein by reference in their entirety. The examples described herein are illustrative of embodiments of the present invention and should not be construed as limiting the scope of the invention.

[0057] [Example 1] Alcohol dehydrogenase (ADH) and NAD +Detection of EtOH in the gas phase by visualization using a microfiber mesh containing polyvinyl alcohol (PVA). 1. Materials and equipment (1) Laboratory reagents and equipment The following experimental reagents, equipment, and apparatus were used. • Terumo Injection Needle 22G 1 1 / 2 RB (Terumo Corporation, NN-2238R) • Disposable syringe 1 mL (Terumo Corporation, SS-01T) • Polyvinyl alcohol (PVA) solution (Yamato Co., Ltd., Arabic Yamato® liquid glue) ·ADH;Alcohol Dehydrogenase from Saccharomyces cerevisiae (Sigma-Aldrich, Cat No. A7011) · NAD + β-Nicotinamide-adenine dinucleotide oxidized form (Oriental Yeast Co., Ltd., Cat No. 44056000) • Sodium hydroxide (Fujifilm Wako Pure Chemical Industries, Ltd., Cat No. 198-13765) • MilliQ Water • Ethanol (Fujifilm Wako Pure Chemical Industries, Ltd., Cat No. 056-06967)

[0058] (2) Experimental apparatus • Spinning device (MEC Corporation, NANON-03) • Magnetron sputtering system (Vacuum Devices Co., Ltd., MSP-10) • Scanning electron microscope (Keyence Corporation, VE-9800) • Benchtop pH / water quality analyzer (Horiba, Ltd., LAQUA, pH / ION METER F-72) • Monochrome CMOS camera (Thorlabs Inc., CS235MU)

[0059] 2. ADH and NAD +Fabrication of a microfiber mesh containing (1) 50 mL of Milli-Q water was placed in a beaker and stirred with a stirrer. 2 g of sodium hydroxide was added in small increments to prepare 1 M NaOH.

[0060] (2) The 1M NaOH prepared in (1) was added to the PVA solution (approximately pH 4) to adjust the pH to 8. A benchtop pH / water quality analyzer corrected with three pH standard solutions (pH 4, 7, and 9) was used for this purpose.

[0061] (3) ADH concentration; 6.2 mg / mL, NAD + Concentration: Add ADH and NAD directly to the pH 8 PVA solution prepared in (2) to a concentration of 13.3 mg / mL. + The following was added. The solution concentration was calculated based on an ADH amount of 100 units and NAD when the amount of yarn spun per sheet was 50 μL. + The solution was prepared to a volume of 1 μmol. Table 1 shows the detailed composition. Additionally, NAD was prepared as a negative control using the same procedure. + A polyvinyl alcohol solution with a concentration of 13.3 mg / mL was prepared. [Table 1]

[0062] (4) Using the solution prepared in (3) above, fibers were spun by electrospinning to produce a microfiber mesh. The electrospinning conditions are shown in Table 2. A collector consisting of a resin base with 1.5 cm square holes covered with aluminum foil was used. [Table 2]

[0063] 3. Visualization experiment of EtOH gas (5) The fiber prepared in (4) above was placed in front of the camera lens, air containing EtOH was loaded, and the change in fluorescence due to NADH produced by ADH based on the EtOH molecules captured by the microfiber mesh was captured as a video using a video camera, thereby quickly visualizing the EtOH gas.

[0064] A schematic diagram of the experimental setup is shown in Figure 3. The distance between the excitation light source and the object to be excited was set to 14.2 mm, and the distance between the Pasteur tube for gas loading and the enzyme membrane was set to 2 mm. There were three types of gas loading conditions shown in Table 3, and the time for each condition was taken from the start of video recording. The EtOH gas concentration was set to 200 ppm (preparation conditions: diffusion tube type; D-05, constant temperature bath temperature; 40℃, flow rate; 0.6 L / min), and the flow rate of EtOH gas and dry gas was set to 100 mL / min. The camera shutter speed was set to 33.33 ms, the gain to 5.0, and the black level to 5.0, and video was recorded for 180 seconds at 30 fps. [Table 3]

[0065] (6) Two types of numerical analysis were performed on the acquired video images. One was a method for calculating the change in fluorescence intensity over time, and the other was a method for calculating the fluorescence change rate, i.e., the NADH production rate. The region of interest (ROI) for the numerical analysis was set to 250 × 250 pixels.

[0066] 4. Experimental Results PVA / ADH / NAD + Microfiber mesh formed with blended fibers (hereinafter referred to as "PVA / ADH / NAD") + When EtOH gas was applied to the microfiber mesh (described as "microfiber mesh"), the fluorescence intensity ΔI increased (Figures 4 and 5), while no increase in ΔI was observed when dry gas was applied (Figure 5). Furthermore, the enzyme-free PVA / NAD +In the Blend fiber, no increase in ΔI was observed even when the substrate EtOH gas was loaded (Figure 5). From this, it can be concluded that PVA / ADH / NAD + The increase in ΔI observed when EtOH gas was loaded onto Blend fibers was shown to be due to an enzymatic reaction.

[0067] [Example 2] Examination of quantitative aspects PVA / ADH / NAD prepared using the same method as in Example 1 + The quantitative analysis of EtOH in the gas phase was investigated using a microfiber mesh.

[0068] 1. Materials and equipment The materials and apparatus used were the same as those in Example 1.

[0069] 2. Fabrication of microfiber mesh (1) 50 mL of Milli-Q water was placed in a beaker and stirred with a stirrer. 2 g of sodium hydroxide was added in small increments to prepare 1 M NaOH.

[0070] (2) The 1 M NaOH prepared in (1) was added to the PVA solution to adjust the pH to 8.

[0071] (3) ADH concentration; 3.125 mg / mL, NAD + Concentration: Add ADH and NAD directly to the pH 8 PVA solution prepared in (2) to a concentration of 132.5 mg / mL. + The following was added. Note that the solution concentration was determined by the amount of ADH when the amount of yarn spun per sheet is 50 μL, NAD + The solution was prepared to a volume of 10 μmol. Details are shown in Table 4. [Table 4]

[0072] (4) Microfiber mesh was fabricated by spinning microfibers using the solution prepared in (3) by electrospinning. The electrospinning conditions are shown in Table 5. A collector consisting of a resin base with 1.5 cm square holes covered with aluminum foil was used. [Table 5]

[0073] 3. Evaluation of the output response to changes in the flow rate of the load gas (amount of EtOH moles loaded). (5) The microfiber mesh prepared in (4) was placed in front of the camera lens, and a gas containing EtOH was loaded. The fluorescence change due to NADH produced based on the reaction between EtOH molecules trapped in the microfiber mesh and ADH was visualized and recorded with a video camera to measure the EtOH gas. At this time, the distance between the excitation light source and the excited object was set to 13 mm, and the distance between the Pasteur tube for gas loading and the enzyme membrane was set to 2 mm. The concentration of EtOH gas was set to 200 ppm (diffusion tube; D-05, temperature; 40℃, dilution gas flow rate; 0.6 L / min), and the flow rates of EtOH gas and dry gas were set to four different values ​​of 20, 50, 100, and 200 ml / min, respectively (corresponding to EtOH loading amounts of 2.7, 6.8, 13.6, and 27.3 nmol / sec). EtOH gas was loaded for 20 seconds starting 20 seconds after the start of video recording, and then the video was switched to dry gas and recorded until 180 seconds. At this time, the camera's shutter speed was set to 33.33 ms, the gain to 5.0, and the black level to 5.0, and the video was recorded at 30 fps.

[0074] (6) Two types of numerical analysis were performed on the acquired video images. One was a method for calculating the change in fluorescence intensity over time, and the other was a method for calculating the fluorescence change rate, i.e., the NADH production rate. However, the region of interest (ROI) for the numerical analysis was set to 250 × 250 pixels.

[0075] 4. Experimental Results PVA / ADH / NAD were tested using four different EtOH gas flow rates of 200 ppm: 20, 50, 100, and 200 ml / min (corresponding to EtOH loads of 2.7, 6.8, 13.6, and 27.3 nmol / sec, respectively). + Figure 6 shows the change in fluorescence rate when a microfiber mesh is loaded, i.e., the change in the derivative per unit time of the change in fluorescence intensity due to the generated NADH over time.

[0076] PVA / ADH / NAD + Figure 7 shows the relationship between the amount of EtOH loaded onto the microfiber mesh per unit time (2.7 to 27.3 nmol / sec) and the change in fluorescence rate. The relationship between the amount of EtOH loaded per unit time and the change in fluorescence rate showed good linearity, and the PVA / ADH / NAD + The microfiber mesh was shown to have excellent quantitative accuracy for determining the EtOH content in the gas phase.

[0077] [Example 3] PVA / ADH / NAD + Evaluation of storage stability of microfiber mesh Next, PVA / ADH / NAD + The storage stability of microfiber mesh was evaluated.

[0078] 1. Materials and equipment The materials and apparatus used were the same as those in Examples 1 and 2.

[0079] 2. PVA / ADH / NAD + Fabrication of microfiber mesh (1) 50 mL of Milli-Q water was placed in a beaker and stirred with a stirrer. 2 g of sodium hydroxide was added in small increments to prepare 1 M NaOH.

[0080] (2) The 1 M NaOH prepared in (1) was added to the PVA solution to adjust the pH to 8.

[0081] (3) ADH concentration; 3.125 mg / mL, NAD + Concentration: Add ADH and NAD directly to the pH 8 PVA solution prepared in (2) to a concentration of 132.5 mg / mL. + The following was added. Note that the solution concentration was determined by the amount of ADH when the amount of yarn spun per sheet is 50 μL, NAD + The solution was prepared to a volume of 10 μmol. Details are shown in Table 6. [Table 6]

[0082] (4) The fibers were spun using the solution prepared in (3) above. The spinning conditions are shown in Table 7. The collector used was a resin base with a 1.5 cm square hole covered with aluminum foil. [Table 7]

[0083] (5) Morphological observation using a scanning electron microscope (SEM) PVA / ADH / NAD + PVA / ADH / NAD on the day the blend fiber was spun and after 14 days of storage at 4°C + The morphology of the microfiber mesh was observed using a scanning electron microscope (SEM).

[0084] (6) Evaluation of the conservation of enzyme activity of enzyme membranes As a control, EtOH gas visualization (positive control) and dry gas loading (negative control) were performed on fibers immediately after fabrication. The excitation light source-excitation target distance was set to 13 mm, and the Pasteur tube-enzyme membrane distance for gas loading was set to 2 mm. The EtOH gas concentration was set to 200 ppm (diffusion tube; D-05, temperature; 40°C, dilution gas flow rate; 0.6 L / min), and the flow rates of both EtOH gas and dry gas were set to 100 mL / min. EtOH gas was loaded for 20 seconds starting 20 seconds after the start of video recording, and then the video was switched to dry gas and recorded for 180 seconds. At this time, the camera shutter speed was set to 33.33 ms, gain to 5.0, and black level to 5.0, and the video was recorded at 30 fps. Furthermore, the sample to be measured two weeks later was placed in a φ35 mm dish, which was then placed in a resealable plastic bag and sealed, and stored in a refrigerator at 4°C. Two weeks later, EtOH gas was visualized using the same procedure with newly prepared fibers (control) and stored fibers.

[0085] (7) Two types of numerical analysis were performed on the acquired video images. One was a method for calculating the change in fluorescence intensity over time, and the other was a method for calculating the fluorescence change rate, i.e., the NADH production rate. However, the region of interest (ROI) for the numerical analysis was set to 250 × 250 pixels.

[0086] 3. Experimental Results PVA / ADH / NAD + Scanning electron microscope images of the Blend fiber mesh on the day of fabrication (Figure 8A) and 14 days later showed no changes (Figure 8B).

[0087] PVA / ADH / NAD immediately after preparation + Blend fiber mesh, and polymer B / ADH / NAD stored in a 4°C refrigerator for 2 weeks after preparation. + The change in fluorescence intensity when EtOH gas is visualized using a Blend fiber mesh is shown (Figure 9). PVA / ADH / NAD +The Blend fiber mesh showed a good change in fluorescence intensity based on the EtOH capture effect even after being stored at 4°C for 14 days after fabrication.

[0088] This is a characteristic not found in conventional methods using ADH-immobilized cotton mesh (Patent Document 1) (in the case of cotton mesh, the enzyme becomes inactive the next day even when stored in a 4°C refrigerator, making visualization impossible), and is considered a major advantage for the industrial application of the molecular supplement of the present invention.

[0089] [Example 4] ADH and NAD for various water-soluble polymers + Fabrication and evaluation of microfiber mesh containing [specific material / component] In addition to PVA, ADH and NAD are also used for various other water-soluble polymers besides PVA. + A microfiber mesh was fabricated by mixing [material A] and [material B], and the visualization method for measuring EtOH gas was investigated. The application of various water-soluble polymer materials to gas-phase substance capture materials was also evaluated.

[0090] 1. Materials and equipment Other than those listed below, the same materials and equipment as in Examples 1-3 were used. • PVP; Polyvinylpyrrolidone K-90, Mv: 360,000, Nacalai Tesque, CAS No. 9003-39-8 ·PEO; Poly(ethylene oxide), Mv: ~900,000, Sigma-Aldrich, CAS No. 25322-68-3 Dextran 70, Mw: ca. 70,000, Cas No. 9004-54-0 ·PAAm ; Polyacrylamide, Mn: 150,000, ALDRICH (PAAm (low molecular weight))

[0091] 2. Experimental Method (1) Preparation of aqueous solutions of water-soluble polymers The aqueous solutions of PVP, PEO, dextran, and PAAM (low molecular weight) were adjusted in concentration to achieve a viscosity similar to that of the PVA solution (the undiluted solution of Arabic Yamato® liquid glue). (i) Preparation of PVA aqueous solution As the PVA aqueous solution, Arabic Yamato® liquid glue was used, as in Examples 1-3. (ii) Preparation of PVP aqueous solution A 25 w / w % PVP aqueous solution was prepared by placing PVP in a 20 mL vial, adding MilliQ water, and stirring overnight or longer under light-shielding conditions. (iii) Preparation of PEO aqueous solution A 6 w / w % PEO aqueous solution was prepared by placing PEO in a 20 mL vial, adding MilliQ water, and stirring overnight or longer under light-shielding conditions. (iv) Preparation of dextran aqueous solution A 50 w / w % dextran aqueous solution was prepared by adding dextran to a 20 mL vial, adding MilliQ water, and stirring overnight or longer under light-shielding conditions. (v) Preparation of PAAm (low molecular weight) aqueous solution A 15 w / w % PAAm (low molecular weight) aqueous solution was prepared by placing PAAm (low molecular weight) in a 20 mL vial, adding MilliQ water, and stirring overnight or longer under light-shielding conditions.

[0092] Next, aqueous solutions of PVA, PVP, PEO, dextran, and PAAM (low molecular weight) were prepared by adding 0.1N NaOH aqueous solution to each solution until the pH approached 8. (2) Preparation of raw material aqueous solutions for various microfiber meshes The above-mentioned aqueous solutions of various water-soluble polymers (pH 8) 50 μL / mesh (preparation volume: 0.8 mL), ADH 50 Units / mesh (preparation volume: 2.56 mg), and NAD + Aqueous solutions of raw materials for various microfiber meshes were prepared by mixing 10 μL / mesh (preparation amount: 106 mg). (3) Fabrication of microfiber mesh by electrospinning Using the raw material aqueous solutions of the various microfiber meshes prepared in (2) above, electrospinning was performed under the conditions shown in Table 7 to produce microfiber meshes of various water-soluble polymers. [Table 8]

[0093] (4) Visualization experiment using EtOH gas loading The various water-soluble polymers prepared above, along with ADH and NAD + We evaluated whether the activity of ADH is maintained in water-soluble polymers other than PVA by measuring the fluorescence intensity at 490 nm after loading a microfiber mesh prepared by mixing and loading with 200 ppm EtOH gas for 20 seconds and irradiating it with excitation light at 340 nm.

[0094] Specifically, the fiber prepared in (3) above was placed in front of the camera lens, EtOH gas was loaded, and the EtOH gas was quickly visualized and measured. The EtOH gas concentration was set to 200 ppm, and the flow rate of EtOH gas and dry gas was set to 100 mL / min. The camera's shutter speed was set to 33.33 ms, the gain to 5.0, and the black level to 5.0, and a video was recorded for 180 seconds at 30 fps.

[0095] 3. Experimental Results Figure 10 shows the change in fluorescence intensity in 490 nm synchrotron radiation emitted by various water-soluble polymer microfiber meshes when EtOH gas is loaded onto the mesh and irradiated with 340 nm excitation light.

[0096] As shown in Figure 10, PEO and PAAm (low molecular weight) showed a change in fluorescence intensity similar to that of PVA microfiber mesh upon EtOH gas loading. On the other hand, PVP and dextran showed an increase in fluorescence intensity, although the change in fluorescence intensity was small.

[0097] These results demonstrate that the activity of ADH present in various water-soluble polymer materials other than PVA is maintained. [Industrial applicability]

[0098] The gas-phase substance capture material of the present invention can measure gas-phase substances such as VOCs, and therefore can be used as various gas sensors.

[0099] Examples of gas sensors include biosensors. For instance, biosensors can be used to measure ethanol (EtOH) for the treatment of alcohol dependence, acetaldehyde for risk assessment of oral and esophageal cancer, methanol for assessment of the gut environment, acetone for assessment of diabetes and lipid metabolism, isopropanol for assessment of diabetes and lipid metabolism, formaldehyde for the diagnosis of lung cancer, ammonia for the diagnosis of liver disease, and dimethyl sulfide for assessment of bad breath and oral environment.

[0100] In addition to its use as a gas center, the gas-phase substance capture material of the present invention can be used to purify spaces by capturing substances suspended in the air. Furthermore, the gas-phase substance capture material of the present invention can be used to recover desired substances by selectively capturing substances suspended in the air. By capturing, recovering, and analyzing substances suspended in the air, the cleanliness and contamination levels of the gas phase of the space can be measured. Moreover, by capturing pathogenic substances suspended in the air, it is possible to prevent people from contracting diseases.

Claims

[Claim 1] A gas-phase substance capture material for capturing a substance in the gas phase, characterized in that it is formed of a water-soluble polymer containing a functional protein that maintains the ability to bind to the substance, and the functional protein captures the substance by exhibiting its ability to bind to the substance.

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