JP2010117308A - Detection method of fluorescence quenching material in gaseous phase - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple method hardly influenced by a noise caused by nonspecific adsorption, for detecting a fluorescence quenching material in a gaseous phase. <P>SOLUTION: In this method for detecting the fluorescence quenching material in the gaseous phase, fluorescence quenching is measured by bringing in the gaseous phase, the fluorescence quenching material into contact with a substrate on which a bonded material is immobilized, which comprises molecules interacting with the fluorescence quenching material and a fluorescent component quenched by the fluorescence quenching material. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for detecting a fluorescence quenching substance in a gas phase, and a gas phase sensor for performing the detection method.

  Fluorescence quenching immunoassay is a method in which a fluorescence quenching substance is brought into contact with a conjugate of a molecule that interacts with the fluorescence quenching substance and a fluorescent component that is quenched by the fluorescence quenching substance. This is a method for analyzing a fluorescence quenching substance by measurement, and is described in, for example, Patent Document 1 and Non-Patent Documents 1 and 2. However, the methods described in these documents do not relate to the antigen-antibody reaction in the gas phase, which utilizes the antigen-antibody reaction in the liquid phase.

  Non-Patent Document 3 describes a gas-phase sensing method using surface acoustic waves (SAWS). This method is based on the principle that a quencher is brought into contact with a fluorescently labeled immobilized antibody. Is different.

  Furthermore, Patent Document 2 describes a method of reducing the background using a quencher in a method of detecting fluorescence using a solid phase carrier on which a probe molecule to be detected is immobilized. . The method of Patent Document 2 is intended to reduce the background of the DNA microarray and is not related to a method of detecting a fluorescence quenching substance.

  On the other hand, in the case of monitoring a quenching antigen present in the air, it is necessary to make an evaluation via a solution or a dispersion, and there is a problem that accuracy and convenience are poor. In addition, there is a problem that noise is increased due to being easily influenced by non-specific adsorption components. Furthermore, it is necessary to fix the antibody on a special piezo substrate, and there is a problem that it takes time and cost.

J. Phys. Chem. B, 109, 19604 (2005) JACS, 127,6744 (2005) Anal.Chem.Acta, 217, 111 (1989) JP 2002-365289 A JP 2003-84002 A

  An object of the present invention is to solve the above-described problems of the prior art. That is, the present invention provides a method for detecting a fluorescence quenching substance in the gas phase, which is simple to be hardly affected by noise due to non-specific adsorption, and a gas phase sensor for use in the above method. Was a problem to be solved.

  As a result of diligent studies to solve the above problems, the present inventors have attempted to apply a fluorescence quenching assay performed in a liquid phase, which is a homogeneous reaction system, to a gas phase system. It was found that the interaction between two kinds of molecules can be detected in the gas phase by measuring the extinction. The present invention has been completed based on these findings.

  That is, according to the present invention, in a method for detecting a fluorescence quenching substance in a gas phase, a binding substance between a molecule that interacts with the fluorescence quenching substance and a fluorescence component that is quenched by the fluorescence quenching substance is fixed. There is provided the above method, which comprises contacting the fluorescence quenching substance with a fluorinated substrate in a gas phase and measuring fluorescence quenching.

Preferably, the molecule that interacts with the fluorescence quencher is an antibody against the fluorescence quencher.
Preferably, the fluorescent component quenched by the fluorescence quenching substance is Eu3 + cryptate, allophycocyanin, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC). ), R-phycoetrin (R-PE), Cy3, or Cy5.

Preferably, the fluorescence quenching substance aerosol is brought into contact with the substrate in the gas phase.
Preferably, the gas containing the fluorescence quenching substance is brought into contact with the substrate under a constant air volume.

Furthermore, according to the present invention, the method of the present invention as described above, comprising a gas permeable substrate on which a combined product of a molecule that interacts with a fluorescence quenching substance and a fluorescent component quenched by the fluorescence quenching substance is immobilized. A gas phase sensor for use in is provided.
Preferably, the gas permeable substrate is a fiber.

  According to the present invention, there is provided a method for detecting a fluorescence quenching substance in the gas phase, which is not easily affected by noise due to non-specific adsorption, and a gas phase sensor for use in the above method. Became possible. Further, according to the present invention, after conducting the evaluation in the gas phase, a competitive reaction in the liquid phase is further performed, so that the fluorescence quenching substance and the molecule that interacts with the fluorescence quenching substance interact with each other. The effect can also be reconfirmed.

Hereinafter, the present invention will be described in more detail.
The present invention relates to a method for detecting a fluorescence quenching substance in a gas phase, on a substrate on which a combined substance of a molecule that interacts with the fluorescence quenching substance and a fluorescence component quenched by the fluorescence quenching substance is immobilized. The above-described method includes contacting the fluorescence quenching substance in a gas phase and measuring fluorescence quenching. A schematic diagram of the principle of the present invention is shown in FIG.

  A conventional fluorescence quenching immunoassay is a method performed in a liquid phase. In the present invention, quantitative and qualitative sensing is performed by bringing a flow of air (including a fluorescence quenching substance) into contact with a substrate at a predetermined flow rate. Is possible. In the present invention, since the fluorescence quenching substance is detected in the gas phase, the fluorescence quenching substance in the gas phase can be directly sensed as it is. On the other hand, when performing evaluation in the conventional liquid phase, there is a problem that it takes time and is a batch type, so that continuous measurement cannot be performed and the speed is inferior. That is, when performing evaluation in the liquid phase, it is necessary to collect the aerosol in the liquid with an impinger or the like and then inject it into a fluorescent label-immobilized microplate to measure the fluorescence quenching. On the other hand, in the present invention, the fluorescence quenching substance can be measured only by measuring the fluorescence quenching of the recovered sample, and can be continuously measured until saturation.

  Furthermore, in the present invention, it is preferable that the gas containing the fluorescence quenching substance is brought into contact with the substrate under a constant air volume. Thereby, the gas passage amount can be made uniform with respect to the surface carrying the fluorescently labeled antibody. Specifically, for example, a labeled antibody can be uniformly supported on a gas-permeable substrate, and an air stream introduced into a cylindrical shape as shown in FIG.

  In the present invention, a molecule that interacts with a fluorescence quenching substance is used. A combination of a fluorescence quenching substance and a molecule that interacts with the fluorescence quenching substance is not particularly limited. For example, antigen and antibody, avidin and biotin, enzyme and substrate, host and guest, complex and metal, etc. Can be mentioned. Of these, antigens and antibodies are particularly preferable. In the above, the fluorescence quenching substance may be any of an antigen and an antibody, any of avidin and biotin, any of an enzyme and a substrate, any of a host and a guest, and any of a complex and a metal. .

  When the fluorescence quenching substance or the molecule that interacts with the quenching substance is an antigen, the type of the antigen is not particularly limited as long as it has fluorescence quenching properties. For example, explosives (TNT (trinitrotoluene), picrin Acids (2,4,6-trinitrophenol), nitro compounds, etc., insecticides / pesticides (parathion, malathion, etc.), bacteria (staphylococci, micrococcus, anthrax, cereus, bacillus subtilis, acne, Pseudomonas aeruginosa, Serratia, Sephacia, pneumococci, Legionella, tuberculosis, etc., mold (yeast, Aspergillus, Penicillius, Cladosporium, etc.), virus (influenza virus, adenovirus, coronavirus, rhinovirus, norovirus) Allergens (pollen, mite allergens, mold spores, cat allergens, etc.), Ten (biotoxins such as verotoxin, mitotoxin, ciguatoxin, penicillin, benzopyrenes, dioxins, steroids, pharmaceuticals, narcotics, poisonous gases, fragrances, etc. contained in cigarette smoke, dust, and automobile exhaust) Can be mentioned. As the antibody, an antibody capable of recognizing and binding to the above antigen can be used. Details of the antibody will be described later in this specification.

A molecule that interacts with the fluorescence quenching substance is labeled with a fluorescent component that is quenched by the fluorescence quenching substance. The fluorescent component used in the present invention is not particularly limited as long as it is quenched by a fluorescence quenching substance. For example, Eu ³⁺ cryptate, allophycocyanin (APC), CFP, YFP, R-phycoetrin (R-PE), Cy3 / Cy5 (Cyanine series), anilinonaphthalenesulfonic acids (ANS, MANS, TNS, etc.), aminonaphthalenes (DNS-Cl, IAEDANS, etc.), pyrene, stilbene, coumarin derivatives (DACM, DCIA, etc.), NBD (nitrobenzo) -2-oxa-1,3-diazole), fluorescein derivatives (such as fluorescein isothiocyanate (FITC)), eosin, erythrosine, rhodamine derivatives (tetramethylrhodamine, tetramethylrhodamine isothiocyanate (TRI) C), etc.), BODIPY derivatives, anthracene derivatives, benzofuran derivatives, Purufirin derivatives, Alexa Fluor, mention may be made DyLight, HiLyte Fluor, Oyster, MegaStokes Dye, and IRDye fluorescent dye.

  In the present invention, the fluorescence quenching substance is brought into contact in a gas phase with a substrate on which a combined substance of a molecule that interacts with the fluorescence quenching substance and a fluorescent component quenched by the fluorescence quenching substance is immobilized, By measuring fluorescence quenching, the fluorescence quenching substance can be detected in the gas phase.

  The fluorescence quenching can be measured by a conventional method, for example, using a commercially available fluorescence measuring device. Various optical devices such as fluorescent scanners, CCD camera type imagers, and microtiter plate readers can be used as long as optical quantification is ensured as the fluorescent device to be used. However, it is desirable to use a microtiter plate reader. . Furthermore, in order to eliminate the influence of background, an apparatus with a time-resolved fluorescence detection function can be used.

  According to the present invention, there is further provided a gas phase sensor comprising a gas permeable substrate on which a conjugate of a molecule that interacts with a fluorescence quenching substance and a fluorescent component that is quenched by the fluorescence quenching substance is immobilized. . The type of gas permeable substrate can be appropriately selected according to the use of the gas phase sensor. For example, when monitoring a target substance in the air (indoor ventilation efficiency monitor, dust drop test, airflow monitor, etc.) A non-woven fabric can be used as the gas permeable substrate, and when performing a performance test of an air filter, a porous substrate according to the size and adsorbability of the substance to be captured can be used. In the case of detecting an order or more), a mesh substrate can be used, and in the case of quantification, an adsorptive porous substrate such as activated carbon can be used.

  As the gas permeable substrate used in the present invention, fibers are preferred. As the main fiber of the fiber used in the present invention, a fiber mainly composed of at least one of cellulose ester, vinylon, acrylic and polyurethane is preferable.

  The cellulose ester in the present invention refers to a cellulose derivative in which the hydroxyl group of cellulose is esterified with an organic acid. Examples of organic acids used for esterification include fatty carboxylic acids such as acetic acid, propionic acid and butyric acid, and aromatic carboxylic acids such as benzoic acid and salicylic acid. It may be used alone or in combination. Although there is no restriction | limiting in particular about the ester group substitution rate of the hydroxyl group of a cellulose, It is preferable that it is 60% or more.

  Cellulose acylate fibers are desirable among the group of main materials forming a fine fiber aggregate having a fiber diameter of 1 μm or less in the present invention. Cellulose acylate refers to a cellulose ester in which some or all of the hydrogen atoms constituting the hydroxyl group of cellulose are substituted with acyl groups. Examples of the acyl group include an acetyl group, a propionyl group, and a butyryl group. These groups may be constituted by replacing only one kind, or two or more kinds of acyl groups may be mixed and substituted. The total acyl group substitution degree is preferably 2.0 to 3.0, more preferably 2.1 to 2.8, and particularly preferably 2.2 to 2.7. Among these, cellulose acetate, cellulose acetate propionate, or cellulose acetate butyrate that satisfies this degree of substitution is preferable, and cellulose acetate is most preferable. Cellulose acylate is generally known to have different solvents depending on the degree of esterification, but after preparing a fine fiber aggregate with a fiber diameter of 1 μm or less with cellulose acylate with a high esterification rate, alkali hydrolysis treatment Etc. may be performed to make the surface hydrophilic.

  Although it is possible to form a sufficiently practical substrate using only cellulose acylate fiber, polyester fiber, polyolefin fiber, polyamide fiber, acrylic, etc. for the purpose of further improving strength and dimensional stability. A fine fiber aggregate having a fiber diameter of 1 μm or less may be formed from a blended fiber with a base fiber or the like. When blended fiber is used, the mass fraction of the cellulose acylate fiber is preferably 50% or more, and more preferably 70% or more.

  Among the main group of materials forming the fine fiber aggregate having a fiber diameter of 1 μm or less in the present invention, it is also desirable that the fiber is a polyamide fiber.

  The polyamide in the present invention refers to a fiber made of a linear polymer having an amide bond in a chemical structural unit.

  Among polyamides, a linear chain that is a combination of an aliphatic diamine such as ethylenediamine, 1-methylethylenediamine, 1,3-propylenediamine, and hexamethylenediamine and an aliphatic dicarboxylic acid such as malonic acid, succinic acid, and adipic acid. Type aliphatic polyamides are preferred. Nylon 66 is particularly preferable.

  Fats using lactams such as ε-caprolactam and laurolactam, aminocarboxylic acids such as aminocaproic acid and aminoundecanoic acid, para-aminomethylbenzoic acid and the like alone or as a copolymer component in addition to the diamine and dicarboxylic acid. A group polyamide can also be used. In particular, nylon 6 produced by using ε-caprolactam alone is preferable.

  In addition to these, some or all of the aliphatic diamines used as raw materials are alicyclic diamines such as cyclohexanediamine, 1,3-bis (aminomethyl) cyclohexane, and 1,4-bis (aminomethyl) cyclohexane. An aliphatic polyamide using an alicyclic dicarboxylic acid such as 1,4-cyclohexanedicarboxylic acid, hexahydroterephthalic acid, hexahydroisophthalic acid, etc. may be used as an aromatic polyamide and / or a part or all of the dicarboxylic acid. .

  Furthermore, by using aromatic diamines such as aliphatic paraxylylenediamine (PXDA) and metaxylylenediamine (MXDA), and aromatic dicarboxylic acids such as terephthalic acid as partial raw materials, water absorption is reduced and elastic modulus is improved. Also included is a polyamide that achieves the above. Further, it may be a polymer having an amide bond in a side chain such as polyacrylic acid amide, poly (N-methylacrylic acid amide), poly (N, N-dimethylacrylic acid amide) and the like.

  Most preferred of the polyamides is nylon 66 or nylon 6. Appropriate hygroscopicity derived from amide bonds, easy to orient the molecular chain consisting of long chain fatty acids of appropriate length, relatively high stretchability, high heat of fusion and large heat capacity It is difficult to melt in terms of kinetics (melt resistance), the flexibility of molecular chains consisting of long-chain fatty chains, and the property that fibrillation and kink bands do not easily occur due to the formation of hydrogen bonds between amide bonds. This is because it is possible to utilize performances preferable for the substrate of the present invention, such as flexibility.

  A polyamide having an amide bond in the chemical structural unit in the side chain instead of the main chain can also be preferably used. Polyacrylamide such as poly (N-isopropylacrylamide), poly (N, N′-dimethylacrylamide), and poly (N-hexylacrylamide) can be used. In general, polymers with amide bonds in the side chain are highly hydrophilic and easily swell and deform, so they are hydrophobized by methods such as forming physical crosslinks or introducing alkyl groups using gelation. It is desirable.

  Similarly, for the purpose of improving strength and dimensional stability, the substrate may be reinforced with other appropriate structural materials such as metal, polymer material, ceramics and the like. These reinforcing materials are desirably used for portions other than the substantially outermost surface of the side surface for supplying the substrate (for example, used for an opposite surface of the side surface or a core material).

  The vinylon in the present invention is a fiber composed of a linear polymer containing 65% by mass or more of vinyl alcohol units, and having a moisture content of less than 7% after being left for 1 week or longer in an environment of temperature 20 ° C. and humidity 65%. . It may be a formalized hydroxyl group of vinyl alcohol, but it is a non-formalized fiber that has been subjected to water resistance treatment by a polymer such as a boric acid-crosslinked hydroxyl group or a known alkali spinning method or cooling gel spinning method. It may be. Components other than vinyl alcohol units may contain ethylene chains, vinyl acetate chains, etc., but fibers formed from vinyl alcohol alone are preferred. Furthermore, it is most desirable to be a non-formalized fiber by cooling gel spinning in that it is homogeneous and has a high degree of orientation and crystallinity, so that excellent mechanical properties and reliability can be obtained.

  Vinylon generally has high strength, high elastic modulus, moderate hydrophilicity, weather resistance, chemical resistance, adhesion and the like with respect to other fibers, and utilizes these preferable performances as a substrate of the present invention. be able to.

  The acrylic system in the present invention refers to a fiber containing 40% or more of acrylonitrile group repeating units by mass ratio, for example, a homopolymer of acrylonitrile, a nonionic monomer such as an acrylate ester, a methacrylate ester, or vinyl acetate; Examples include copolymers of acrylonitrile, copolymers of anionic monomers such as vinylbenzene sulfonic acid and allyl sulfonic acid and acrylonitrile, and copolymers of cationic monomers such as vinyl pyridine and methyl vinyl pyridine and acrylonitrile. So-called promix fibers formed from acrylonitrile and milk casein are also included in this category.

  In general, acrylic fibers are often produced by an organic wet spinning method. In this method, when the spinning stock solution forms a coagulated yarn in the coagulation bath, water as a coagulant enters the spinning stock solution spun from the nozzle, while the spinning solvent diffuses from the spun stock solution to the outside. At this time, the water and the organic solvent (DMF, DMAc, etc.) are mutually diffused, so that a polymer is precipitated and a coagulated yarn having a structure in which innumerable cavities are connected in a network form is formed. In addition, it is characterized by deformation of the fiber cross section formed by volume shrinkage due to diffusion of the solvent into the coagulation bath during the coagulation process and formation of irregularities by forming a macrofibril structure on the surface. These fine structures are preferable as the structure of the substrate used in the present invention in terms of improving the specific surface area and ease of carrying the antibody.

  The acrylic fiber used in the present invention varies depending on the composition of the raw material polymer, the spinning method, the post-treatment conditions in the production process, etc., but generally, it is easy to obtain a bulky fiber with moderate hydrophilicity and high weather resistance. There are advantages.

  The polyurethane used in the present invention refers to a fiber composed of a linear synthetic polymer in which the bonding portion between monomers or the bonding portion between basic base polymers is mainly a urethane bond. It is desirable that the polyurethane segment contains 85% or more by mass ratio. It is desirable to be a block copolymer of a segmented polyurethane composed of a soft segment having a low melting point and a soft molecular weight of up to several thousand, and a hard segment having a high melting point and rigidity and high cohesion. Polyethers such as polypropylene glycol and polytetramethylene glycol can be used as the soft segment, and urethane groups formed from 4,4'-diphenylmethane diisocyanate, m-xylene diisocyanate, and the like can be used as the hard segment. Polyurethane is generally characterized by high elasticity. It varies depending on the temporary structure of the polymer chain, such as the chemical structure and distribution of both segments, and on the difference in secondary structure resulting from differences in the spinning conditions, but it stretches well. It has characteristics such as high resilience, resistance to aging compared to rubber materials, and thin fibers can be obtained, and these properties can be utilized even when used as a substrate of the present invention.

  Regarding the mechanical properties and dimensional stability of the fibers constituting the substrate, it is desirable that the elongation at drying is 25% or more. Here, the elongation at drying refers to the breaking elongation in a tensile test at 20 ° C. of the fiber dried over a sufficiently long time. In general, it is preferable that the elongation at drying is 10% or more and that it is suitable for processing such as cloth making is 25% or more in order to prevent filter processing and breakage during practical use (leading to a decrease in filtration efficiency). % Or more is more preferable, and 35% or more is most preferable.

  The fiber used in the present invention can be produced by a general production method such as melt spinning, wet spinning, dry spinning, wet drying spinning, or physical treatment (for example, strong mechanical shearing treatment using an ultra-high pressure homogenizer). In order to ensure stable quality, it is preferable to use dry spinning or wet dry spinning. In order to produce uniform fibers with an average fiber diameter of 100 nm or less, further processing techniques, 2005, 40, No. 2, 101, and 167; Polymer International, 1995, 36, 195- 201; Polymer Preprints, 2000, 41 (2), 1193; Journal of Macromolecular Science: Physics, 1997, B36, 169, etc. It is preferable to employ the electrospinning method.

  Solvents used for spinning include chlorinated solvents such as methylene chloride, chloroform and dichloroethane, amide solvents such as dimethylformamide, dimethylacetamide and N-methylpyrrolidone, and ketones such as acetone, ethyl methyl ketone, methyl isopropyl ketone and cyclohexanone. Any solvent that dissolves the resin used for the synthetic resin fiber, such as a solvent, an ether solvent such as THF or diethyl ether, or an alcohol solvent such as methanol, ethanol, or isopropanol can be used. These solvents may be used alone or in combination of two or more.

  When the electrospinning method is employed, a salt such as lithium chloride, lithium bromide, potassium chloride, or sodium chloride may be further added to the resin solution.

  The fibers constituting the substrate of the present invention desirably have a structure in which a three-dimensional network is formed by partial adhesion. By adopting such a structure, it is possible to improve processing and practical mechanical resistance, and to improve the reliability of the gas phase sensor. Further, the retention characteristics of the antibody of the present invention can be improved. The adhesion between fibers can be observed by a method such as SEM. The density of the adhesion points between fibers is preferably 10 or more per 1 mm square with respect to the projected surface area of the gas phase sensor, and more preferably 100 or more.

  As a method for forming an adhesion point, it may be formed by an adhesion formed by a dry spinning method or a fusion point formed by a melt spinning method, or after spinning, addition of an adhesive, a plasticizing solvent, etc. You may perform the adhesion point formation process by. From the viewpoint of production cost, it is preferable to form adhesion points by a dry spinning method using an appropriate solution formulation.

  In the present invention, an antibody against the fluorescence quenching substance can be preferably used as a molecule that interacts with the fluorescence quenching substance. The antibody used in the present invention is a protein that specifically reacts with a specific fluorescence quenching substance (antigen) (antigen-antibody reaction), has a molecular size of 7 to 8 nm, and has a Y-shaped molecular form. Have Of the Y-shaped molecular structure of an antibody, a pair of branch parts is called Fab and a trunk part is called Fc, and of these, the antigen is captured by the Fab part.

The type of the antibody corresponds to the type of antigen (fluorescence quenching substance) that can be captured.
Examples of the method for producing the antibody include a method in which an antigen is administered to an animal such as a goat, horse, sheep, ostrich, and rabbit, and a polyclonal antibody is purified from the blood. Spleen cells and cultured cancer cells of the animal to which the antigen is administered And the method of purifying monoclonal antibodies from the culture fluid or the body fluid of the animal in which the fused cells are implanted (ascites, etc.), the genetically modified bacteria introduced with the antibody-producing gene, plant cells, and the antibody from the culture solution of animal cells And a method of purifying a chicken egg antibody from egg yolk powder obtained by administering an antigen to a chicken to produce an immunized egg and sterilizing and spray-drying the egg yolk liquid. Among these, the method for obtaining an antibody from a chicken egg can easily obtain a large amount of the antibody and can reduce the cost.

  The antibody used in the present invention is preferably a chicken egg antibody or an ostrich antibody.

  The substrate in the present invention is preferably subjected to antibacterial processing such as coating with an antibacterial agent and / or antifungal processing such as coating with an antifungal agent. Antibodies are basically proteins, especially chicken egg antibodies are food, and may be accompanied by proteins other than antibodies, which are good food for bacteria and fungi to grow, but they are antibacterial on the substrate. If processing and / or anti-mold processing is performed, the growth of such bacteria and molds is suppressed, and long-term storage can be performed.

  Examples of antibacterial / antifungal agents include organic silicon quaternary ammonium salts, organic quaternary ammonium salts, biguanides, polyphenols, chitosan, silver-supporting colloidal silica, and zeolite-supporting silver. In addition, as a processing method, a post-processing method in which an antibacterial / antifungal agent is impregnated or applied to a substrate made of fibers, or a raw yarn raw cotton that is kneaded with an antibacterial / antifungal agent at the synthesis stage of fibers constituting the substrate. There is a quality law.

  As a method for immobilizing an antibody on the substrate, the substrate is silanized using γ-aminopropyltriethoxysilane or the like, and then an aldehyde group is introduced onto the substrate surface with glutaraldehyde or the like, and the aldehyde group and the antibody are combined. A method of covalent bonding, a method in which an untreated substrate is immersed in an antibody aqueous solution and the antibody is immobilized on the substrate by ionic bonding, an aldehyde group is introduced into a substrate having a specific functional group, and the aldehyde group and the antibody are combined. A method of covalently bonding, a method of ionically binding an antibody to a substrate having a specific functional group, a method of introducing an aldehyde group after coating the substrate with a polymer having a specific functional group, and a method of covalently bonding the aldehyde group and the antibody I can give you.

Here, examples of the specific functional group include an NHR group (R is any alkyl group other than H, methyl, ethyl, propyl, and butyl), NH ₂ group, C ₆ H ₅ NH ₂ group, and CHO group. , COOH group, and OH group.

  Further, there is a method in which the functional group on the substrate surface is converted into another functional group using BMPA (N-β-Maleimidopropionic acid) or the like, and then the functional group and the antibody are covalently bonded (SH group in BMPA). Are converted to COOH groups).

  Further, there is a method in which a molecule (Fc receptor, protein A / G, etc.) that selectively binds to the Fc portion of the antibody is introduced onto the substrate surface, and the antibody Fc is bound thereto. In this case, the Fab that captures the fluorescence quenching substance is directed outward with respect to the substrate, and the probability of contact of the fluorescence quenching substance with the Fab increases, so that the fluorescence quenching substance can be efficiently captured.

The antibody may be supported on a substrate via a linker. In this case, the degree of freedom of the antibody on the substrate is increased and access to the fluorescence quenching substance is facilitated, so that high removal performance can be obtained. Examples of the linker include bi- or higher-valent cross-linking reagents, specifically maleimide, NHS (N-Hydroxysuccinimidyl) ester, imide ester, EDC (1-Ethyl-3- [3-dimethylaminopropyl] carbodiimido), There is PMPI (N- [p-Maleimidophenyl] isocyanate), which is selective to target functional groups (SH group, NH ₂ group, COOH group, OH group) and non-selective. Moreover, the distance (space arm) between crosslinks also changes for every crosslink reagent, and it can select in the range of about 0.1 nm-3.5 nm according to the target antibody. From the viewpoint of efficiently capturing the fluorescence quenching substance, a substance that binds to the Fc of the antibody as a linker is preferable.

  As a method for introducing a linker, either a method in which a linker is bound to an antibody and then further bound to the antibody, or a method in which a linker is bound to a substrate and the antibody is bound to the linker on the substrate is possible. is there.

  The features of the present invention will be described more specifically with reference to examples and comparative examples. The materials, amounts used, ratios, processing details, processing procedures, and the like shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[Example 1] Nitro compound sensor
FITC-labeled anti-Trinitrophenol antibody (American Research Products, Goat), FITC-labeled anti-Mouse IgG (H + L) antibody (manufactured by Goat), Nunc microplate (# 437111, PS flat bottom, maxi soap treatment・ Three fixed black plates (N = 10) were prepared. Next, each of the above plates is introduced into a nitrogen-filled glove box (volume: 210L, with circulation fan), and a 0.1% ethanol solution of 2,4,6-Trinitrophenol (manufactured by Wako Pure Chemical Industries) is used as a nebulizer (OMRON). The product was sprayed with 0.5 mL of NE-U22) and allowed to stand for 1 minute. Next, a blank sample (1) in which only ethanol was similarly sprayed in the glove box as well as a blank sample (2) for nothing were prepared as comparative controls.

  Next, the plate sample was measured for fluorescence of the three plates by a plate reader (PerVision Elmer, EnVision) (fluorescence intensity was monitored with bandpass filters of excitation 488 nm and 520 nm. The measurement results are shown in Table 1. Specific quenching for TNP was observed.

The numerical value in the parenthesis was N = 10 average value of the number of counts, and the background was 235.

[Example 2] Pesticide / insecticide sensor A malathion (manufactured by Cerilliant Corporation) -ovalbumin (derived from hen's egg) complex was prepared by the method described in JP-T-8-510231. Next, immunized eggs laid by chickens administered with the complex were purified to prepare malathion antibodies (IgY antibodies). FITC labeling was performed using the standard protocol of the Sigma Fluoro Tag FITC Conjugation Kit. Non-immune IgY was used as a control, and FITC-labeled non-immune IgY was prepared. Next, both antibody samples were fixed to a nylon mesh (NY-50-HD), and then attached to the entire surface of a DC axial fan (manufactured by Sanyo Denki Co., Ltd.).

  After operating the DC axial flow fan in the glove box, 0.5 mL of a 0.1% methanol solution of malathion was sprayed with a nebulizer (NE-U22 manufactured by OMRON Corporation) and allowed to stand for 1 minute. Next, a nylon mesh sample was cut out and allowed to stand on the bottom of a Nunc microplate (# 437111, PS flat bottom, maxi soap treatment, black) with the exposed surface facing up. A nylon mesh not fixed as a blank was also prepared. A similar experiment was conducted with a 0.1% methanol solution of the similar compound fenitrothion (AccuStandard).

  Next, the plate sample was measured for fluorescence of the three plates by a plate reader (PerVision Elmer, EnVision) (fluorescence intensity was monitored with bandpass filters of excitation 488 nm and 520 nm. Table 2 shows the measurement results. A specific quenching phenomenon for malathion was observed.

The numerical value in the parenthesis was an average value of count number N = 10, and the background was 1235.

[Example 3] Sensor for carcinogen substance Using a cellulose acetate (manufactured by Aldrich, total substitution degree 2.4, number average molecular weight 30,000) in acetone: water (97: 3) solution (10% by mass), a nanofiber production apparatus (Cato Was used for electrospinning at a syringe feed rate of 0.05 mm / min and an applied voltage of 15 kV, and further dried in a vacuum at 80 ° C. for 8 hours to prepare a fine nonwoven fabric sample having a thickness of 50 μm. The average fiber diameter measured by SEM was 80 nm. Abcam anti-Benzopyrene antibody (Rat) was labeled with a standard protocol using Alexa Fluor555 ^R monoclonal antibody labeling kit (Molecular Probes).

  The labeled antibody was dissolved in phosphate buffered saline (PBS), adjusted to an antibody concentration of 100 ppm, and filtered through a 0.45 μm filter to prepare a carrier solution. The nonwoven fabric sample was immersed in the supporting liquid at room temperature for 16 to 24 hours to support the antibody and the compound of the present invention on the fiber surface, thereby obtaining a filter sample.

 The antibody-immobilized filter sample was fixed to two acrylic pipes with a diameter of 10 mm, and the outlet side was connected to the suction port of the metering pump.

  After spraying 0.5 mL of a 0.01% methanol solution of Benzopyrene (MB Biomedicals) with a nebulizer (NE-U22 manufactured by OMRON) in a glove box, the metering pump of the acrylic pipe experimental device was operated (flow rate of 0.3 m) See Table 3 for / s and ventilation time). Thereafter, the filter was recovered and allowed to stand on the bottom of a Nunc microplate (# 437111, PS flat bottom, maxi soap treatment, black) with the exposed surface facing up. The fluorescence of the above three plates was measured with a plate reader (PerVision Elmer, EnVision) (fluorescence intensity was monitored with bandpass filters of excitation 320 nm and 560 nm. As a control, (1) Abcam anti-Mouse IgM heavy chain (Rat The same experiment was carried out in the case of (1) loading, (2) no antibody loading, and the aeration time was changed as shown in Table 3.

  The measurement results are shown in Table 3 and FIG. Specific quenching could be confirmed in an almost linear relationship with the amount of antigen passing, indicating that it was useful as a quantitative sensor.

The numbers in the table are the average values of count number N = 5

FIG. 1 shows a schematic diagram of the principle of the present invention. FIG. 2 shows a state in which the airflow introduced into the cylindrical shape is passed in the direction perpendicular to the plane. FIG. 3 shows the measurement results of Example 3.

Claims (7)

In the method for detecting a fluorescence quenching substance in a gas phase, the fluorescence quenching substance is attached to a substrate on which a combined substance of a molecule that interacts with the fluorescence quenching substance and a fluorescence component that is quenched by the fluorescence quenching substance is immobilized. The method as described above, which comprises contacting a fluorescent substance in a gas phase and measuring fluorescence quenching. The method according to claim 1, wherein the molecule that interacts with the fluorescence quencher is an antibody against the fluorescence quencher. Fluorescent components quenched by the fluorescence quenching substance are Eu3 + cryptate, allophycocyanin, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), R The method according to claim 1 or 2, which is any one of phycoetrin (R-PE), Cy3, and Cy5. The method according to any one of claims 1 to 3, wherein the aerosol of the fluorescence quenching substance is brought into contact with the substrate in the gas phase. The method according to any one of claims 1 to 4, wherein a gas containing a fluorescence quenching substance is brought into contact with the substrate under a constant air volume. The method according to any one of claims 1 to 5, comprising a gas-permeable substrate on which a combined product of a molecule that interacts with a fluorescence quenching substance and a fluorescent component that is quenched by the fluorescence quenching substance is immobilized. Gas phase sensor for use. The gas phase sensor according to claim 5, wherein the gas permeable substrate is a fiber.