WO2012004862A1 - Selectively permeable membrane using molecular recognition macromolecules - Google Patents

Selectively permeable membrane using molecular recognition macromolecules Download PDF

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WO2012004862A1
WO2012004862A1 PCT/JP2010/061505 JP2010061505W WO2012004862A1 WO 2012004862 A1 WO2012004862 A1 WO 2012004862A1 JP 2010061505 W JP2010061505 W JP 2010061505W WO 2012004862 A1 WO2012004862 A1 WO 2012004862A1
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molecular recognition
permeable membrane
membrane
porous support
mips
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PCT/JP2010/061505
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French (fr)
Japanese (ja)
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大森唯義
数坂昭夫
濱田辰夫
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株式会社センシングネットワーク
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Priority to PCT/JP2010/061505 priority Critical patent/WO2012004862A1/en
Publication of WO2012004862A1 publication Critical patent/WO2012004862A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/08—Polysaccharides
    • B01D71/12—Cellulose derivatives
    • B01D71/20—Esters of inorganic acids, e.g. cellulose nitrate
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B01D67/0081—After-treatment of organic or inorganic membranes
    • B01D67/0093—Chemical modification
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/14—Dynamic membranes
    • B01D69/141—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes
    • B01D69/142—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes with "carriers"
    • B01D69/144—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes with "carriers" containing embedded or bound biomolecules

Definitions

  • the present invention relates to a selective permeable membrane using a molecular recognition polymer and a method for producing the same, such as biological fluids such as raw milk, trace amounts of specific antibiotics, specific saccharides, specific organic compounds, etc. contained in secretions.
  • biological fluids such as raw milk
  • trace amounts of specific antibiotics, specific saccharides, specific organic compounds, etc. contained in secretions are included in secretions.
  • an operation for extracting a specific drug component or organic compound component from an aqueous solution containing a large amount of a small amount of drug or organic compound also requires advanced techniques. The same applies to the separation and analysis of trace amounts of toxic components contained in an aqueous solution. For this reason, in the food chemistry field, the pharmaceutical field, and the medical field, it is required to separate, isolate, and quantify specific antibiotics, organic drugs, saccharides, amino acids, toxic substances, and the like by an inexpensive and simple technique.
  • molecularly imprinted polymers MolecularlymersImprinted Polymers, sometimes referred to as molecular recognition resins, hereinafter referred to as “MIPs”.
  • MIPs molecular recognition resins
  • Japanese Patent Application Laid-Open No. 2005-232205 describes an invention relating to “molecularly imprinted cellulose and a method for producing the same”, which can be bonded to cellulose by hydrogen bonding, electrostatic bonding, hydrophobic bonding, or the like.
  • molecularly imprinting a template molecule molecularly imprinted cellulose of the molecule is formed.
  • the template molecule is extracted to form cellulose imprinted with the template of the molecule. Is.
  • the imprint cellulose is applied to a substrate such as paper.
  • Japanese Patent Application Laid-Open No. 2005-205333 describes an invention relating to a “sheet having a molecular adsorption function”.
  • an ethylene-acrylic copolymer is used as an imprint polymer, and this is supported on paper or glass fiber.
  • Japanese Patent Application No. 2004-520873 Japanese Patent Publication No. 2005-533146
  • This invention is a block in which styrene and an acrylic monomer are mixed with a template molecule, a crosslinking agent, etc. Polymerization is performed, and the template molecule, unreacted polymer, and cross-linking agent are removed with a solvent to produce a porous film having a thickness of 60 micrometers having a template of a specific molecule.
  • all of the above inventions utilize the adsorption characteristics (affinity) between the molecular template used for the imprint polymer and the template molecule, and adsorb the template molecule to the imprint polymer to thereby contain the template contained in the sample solution.
  • the purpose is to remove the template molecule from the sample solution by utilizing the concentration of the molecule in the imprinted polymer and the concentration effect.
  • a complicated process for desorbing and purifying the template molecule adsorbed on the imprint polymer again is necessary.
  • the present invention has been proposed in order to cope with such various situations, and is a biological fluid or secretory fluid containing compound molecules to be separated and isolated, for example, various types of antibiotics and organic compounds.
  • a simple and inexpensive MIPs molecule recognition polymer that can easily separate, isolate, and extract specific single species of antibiotics or specific single species of useful organic compounds from aqueous solutions It is an object of the present invention to provide a selective permeable membrane used and a method for producing the same.
  • the invention according to claim 1 is characterized in that a fractionation / isolation is carried out on a porous support having a large number of pores having an inner diameter capable of sufficiently permeating a specific compound molecule to be fractionated / isolated.
  • An extremely thin MIPs permeable membrane imprinted with the template of the specific compound molecule to be isolated is formed, the specific compound molecule in the sample solution is selectively permeated by the MIPs permeable membrane, and the porous The compound molecules permeate and migrate from the pores of the porous support into the solution on the side of the support.
  • the invention described in claim 2 is characterized in that, in claim 1, the base material of the porous support is nitrocellulose or other cellulose derivatives.
  • the invention according to claim 3 is characterized in that the base material of the porous support is various synthetic resins such as fluorine, nylon, polyethylene, polypropylene, polyvinyl alcohol, polycarbonate and polyamide.
  • the invention according to claim 4 is characterized in that, in claim 1, the base material of the porous support is various inorganic compounds such as various glass fibers, ceramics, and zeolites.
  • the MIPs permeable membrane has a thickness of 1 to 5 micrometers.
  • the invention according to claim 6 is a polymer comprising a template of the specific compound molecule on a porous support having a large number of pores having an inner diameter capable of sufficiently permeating the specific compound molecule to be separated and isolated. And a step of evaporating and drying the organic solvent solution of the polymer to form an extremely thin MIPs film.
  • the present invention is characterized in that it aims at fractionation and isolation using the selective permeation ability of a template molecule, which is another characteristic of molecularly imprinted polymers (hereinafter referred to as “MIPs”).
  • MIPs molecularly imprinted polymers
  • the separation / isolation is performed on a porous support having a large number of pores having an inner diameter capable of easily permeating a specific compound molecule to be separated / isolated.
  • the MIPs film on which the template of a specific compound molecule to be subjected to imprinting is imprinted is formed. This reinforces the MIPs membrane, which is extremely thin and difficult to handle, and improves the mechanical strength of the selective permeable membrane using the molecular recognition polymer.
  • the properties required as a base material for the porous support are an infinite number of micrometer-level pores and a thin film or thin plate with a thickness of 0.1 mm to 1 mm, which can easily be used for antibiotics such as penicillin G and tetracycline. It is necessary to be a material that can permeate substances and organic compound molecules, has mechanical strength, is chemically stable, and does not deteriorate.
  • the cellulose, the synthetic resin, and the inorganic compound in the present inventions according to claims 2 to 4 satisfy such properties, and mechanically by attaching an extremely thin film such as a MIPs film to these supports.
  • the fragile MIPs membrane can be strengthened, and it has practicality as a selective permeable membrane using a molecular recognition polymer.
  • an organic solvent solution of a polymer containing a template of a specific compound molecule to be separated and isolated is applied onto a porous support, and the solvent is evaporated to dryness.
  • a MIPs film is formed.
  • a selective permeable membrane using a molecular recognition polymer according to one embodiment of the present invention is formed on a gold thin film (about 0.1 mm), and the permeable membrane is slightly inclined by a scanning electron microscope (SEM). It is the figure which showed the image of the microscope image
  • a selective permeable membrane using a molecular recognition polymer according to one embodiment of the present invention is formed on a gold thin film (about 0.1 mm), and the permeable membrane is scanned by a scanning electron microscope (SEM).
  • FIG. 1 shows a schematic configuration of a measuring apparatus for quantifying penicillin G by permeation from a sample of raw milk using a selective permeable membrane using a selective molecular recognition polymer using a molecular recognition polymer according to one embodiment of the present invention.
  • FIG. It is a graph which shows the experimental result which confirmed that penicillin G could be fractionated by permeation from raw milk containing penicillin G and tetracycline by the selective permeation membrane using the molecular recognition polymer for penicillin G of the present invention.
  • Penicillin G is permeated from a raw milk sample containing a small amount of penicillin G and ampicillin having a molecular structure very similar to penicillin G by the selective permeation membrane using the molecular recognition polymer for penicillin G of the present invention, and is separated and isolated. It is a graph which shows the experimental result at the time of doing. The graph which shows the experimental result at the time of performing the experiment which permeate
  • MIPs membrane / porous support membrane 11 MIPs membrane 12 Porous support membrane (nitrocellulose permeable membrane) 21A Sample introduction tube 21B Sample discharge tube 22 Measurement sample cell 23 Silicone plate 24 Electrochemical cell 25 Electrolyte introduction / discharge tube 26 Platinum counter electrode 27 Electrochemical sensor electrode 28 Reference electrode
  • FIG. 1 is a schematic view showing the structure of a selective permeable membrane using a molecular recognition polymer according to one embodiment of the present invention.
  • FIG. 2 shows an image of a microscope in which a permeable membrane according to the present embodiment is formed on a gold thin film (about 0.1 mm), and the permeable membrane is photographed from slightly above with a scanning electron microscope (SEM).
  • FIG. 3 is a diagram showing an image of the plane of the permeable membrane by a microscope.
  • the selective permeable membrane 1 using the molecular recognition polymer of this embodiment is a laminated structure comprising an extremely thin MIPs membrane 11 having a thickness of 1 to 5 micrometers and a porous support 12.
  • the MIPs membrane is formed on a porous support (for example, a nitrocellulose membrane having a thickness of 0.1 mm and a pore diameter of 0.025 ⁇ m).
  • the porous support has a large number of pores having an inner diameter through which a specific compound molecule to be separated and isolated can permeate sufficiently.
  • the MIPs film 11 is an acrylic urea resin imprinted with a template of a specific compound molecule to be separated and isolated, and recognizes a specific compound molecule complementary to the template.
  • the compound molecules that can be selectively permeated through the MIPs membrane 11 further permeate the pores of the porous support. As a result, the compound molecules can be separated and isolated by the selective permeable membrane 1 using a molecular recognition polymer.
  • the MIPs film 11 is formed on a gold carrier in order to make the MIPs film 11 visible.
  • the thickness of the MIPs film is 3 ⁇ m. It was measured that.
  • the MIPs film 11 is formed with a permeation hole that allows a specific compound molecule to permeate. However, because of the fine hole, the hole cannot be seen in the microscope image of FIG. I could not do it.
  • the selective permeable membrane 1 using the molecular recognition polymer of this embodiment is produced as follows. First, 0.1 to 1 (ml) of a saturated solution of MIPs in acetonitrile is pipetted and uniformly applied onto the porous support 12, and the solvent is evaporated and dried overnight at room temperature. The resulting MIPs-porous support membrane (substrate) is stored at room temperature. By producing the MIPs-porous support membrane (substrate), the MIPs membrane 11 which is thin and difficult to handle has a reinforced property, so that selective permeation using a molecular recognition polymer is performed. The mechanical strength of the film 1 is improved and it becomes suitable for practical use.
  • Example 1 Using the MIPs membrane for penicillin G of the present invention described above, an experiment was conducted in which only penicillin G was permeated from a raw milk sample containing a small amount of penicillin G and tetracycline, and was separated and isolated.
  • the selective permeable membrane using the molecular recognition polymer used in this example is a MIPs-nitrocellulose support membrane containing a template of penicillin G as a compound to be separated and isolated, and is described in detail in the embodiment of the invention. It is formed by the manufacturing process.
  • FIG. 4 is a diagram showing a schematic configuration of a measuring apparatus 20 that quantifies penicillin G contained in a raw milk sample.
  • the measuring device 20 is composed of two measurement sample cells 22 made of polystyrene, an electrochemical cell 24, and a 3 mm-thick plate 23 made of silicone rubber for partitioning between them. ing.
  • the central part of the silicone plate is cut out into a 13 mm ⁇ 13 mm square shape, and a selective permeable membrane (MIPs membrane 11 and porous support 12) 1 using the molecular recognition polymer of the present invention is silicone-based on the upper part.
  • MIPs membrane 11 and porous support 12 selective permeable membrane 11 and porous support 12
  • a selective permeable membrane using a molecular recognition polymer has a rectangular shape with a size of 15 mm ⁇ 15 mm.
  • an electrochemical sensor electrode 27 for measuring antibiotics, a platinum counter electrode 26, and a silver / silver chloride reference electrode 28 are inserted, and the potential of the sensor electrode is set to each electrode terminal.
  • a potentiostat (not shown) for measuring the current flowing between the sensor electrode and the counter electrode is connected.
  • the electrochemical sensor electrode is an electrochemical biosensor for histamine measurement that has an enzyme (histamine dehydrogenase), gold microparticles, tetrathiafulvalene, Nafion resin, and cross-linked ceratin on a metal plate. The activity of histamine oxidation of the enzyme in the presence of antibiotics. Antibiotics are quantitatively measured using a drop in current due to a decrease in function.
  • the silicone plate 23 to which the selective permeable membrane 1 using a molecular recognition polymer is fixed is fixed between a sample cell and an electrochemical measurement cell by a silicone-based adhesive. ing.
  • FIG. 5 shows the experimental results confirming that penicillin G can be separated from a raw milk sample containing penicillin G and tetracycline by the selective permeable membrane 1 using the molecular recognition polymer for penicillin G of the present invention. Shown in In FIG. 5, the vertical axis represents current (nA) and the horizontal axis represents time (min).
  • the vertical axis represents current (nA) and the horizontal axis represents time (min).
  • 4 ml of raw milk is put into the sample cell 22 from the sample introduction tube 21A, the electrochemical measurement cell 24 is filled with a phosphate buffer solution (pH 7.1) containing 100 ppb histamine, and the potential of the electrochemical sensor electrode is set to 0.4. Set to V (vs. Ag / AgCl).
  • tetracycline is added to the raw milk in the sample cell to a concentration of 4 ppb, and the histamine oxidation current is observed (32).
  • tetracycline is added to a concentration of 40 ppb and the histamine oxidation current is observed (33).
  • penicillin G is added so that the concentration becomes 4 ppb, and the histamine oxidation current is observed (34).
  • Example 2 A small amount of penicillin G using a selective permeable membrane using the molecular recognition polymer for penicillin G of the present invention. And penicillin G were permeated from a raw milk sample containing ampicillin having a molecular structure very similar to that of penicillin G, and were subjected to an experiment for separation and isolation.
  • the selective permeable membrane using a molecular recognition polymer is for penicillin G, and the measuring apparatus is the same as that in FIG.
  • the measurement operation is also the same as the above-described fractionation / isolation experiment of penicillin G and tetracycline.
  • FIG. 4 ml of raw milk is placed in a sample cell, and the electrochemical measurement cell 24 is filled with a phosphate buffer solution containing 100 ppb histamine.
  • the histamine oxidation current value becomes constant (background current (41))
  • ampicillin is added to the raw milk in the measurement sample to a concentration of 4 ppb, and the histamine oxidation current is observed (42).
  • the concentration of ampicillin is set to 20 ppb, and the histamine oxidation current is observed (43).
  • penicillin G is added to the raw milk to a concentration of 4 ppb, and the histamine oxidation current is observed (44).
  • ampicillin did not change the histamine oxidation current value of the electrochemical sensor electrode until the concentration reached 20 ppb as in the case of tetracycline (42) (43).
  • Subsequent addition of 4 ppb penicillin G significantly reduced the histamine oxidation current, confirming that penicillin G in the raw milk sample was selectively permeated and could be separated and isolated.
  • penicillin G was added at a concentration of 4 ppb, the penicillin G molecule in the template of the MIPs film for penicillin G decreased in the histamine oxidation current after the addition.
  • Example 3 Experiments were conducted to separate and isolate only tetracycline from a raw milk sample containing penicillin G and a small amount of tetracycline using the selective permeable membrane 1 using the molecular recognition polymer for tetracycline of the present invention.
  • the measuring device is the same as in FIG.
  • the measurement operation is also the same as that in the first and second embodiments. Details of the implementation and results are shown in FIG. Take 4 ml of raw milk in the sample cell and fill the electrochemical measurement cell with phosphate buffer solution containing 100 ppb histamine.
  • penicillin G does not permeate even at a concentration of 40 ppb in the selective permeable membrane using the molecular recognition polymer for tetracycline, and no change is observed in the histamine oxidation current value of the electrochemical sensor electrode.
  • the oxidation current decreases remarkably depending on the concentration, and tetracycline in raw milk is selectively permeated and transferred, and tetracycline is separated and isolated from raw milk. It was confirmed that
  • the selective permeable membrane using the selective molecular recognition polymer using the molecular recognition polymer of the present invention described above has the following effects. 1. As a selective permeable membrane using a molecular recognition polymer, an extremely thin molecular recognition polymer containing a large number of specific antibiotic or organic compound molecule template molecules is used, so that only the antibiotic or organic compound molecule is selective. And can be easily separated and isolated from other antibiotics or organic compounds. In addition, the molecular recognition polymer maintains a permeation function for a long time when fractionating and isolating antibiotics or organic compound molecules with an extremely thin film thickness of 1 to 5 micrometers. It is easy to use as a selective permeable membrane using a molecular recognition polymer, and the durability is greatly improved. 2.
  • an apparatus for separating and isolating antibiotics or organic compound molecules used for permeation requires an electrochemical sensor for histamine measurement, a potential setting of the sensor, and a potentiostat for current measurement. It can be manufactured inexpensively and is easy to operate. It can also be miniaturized for portable use. 3. Regarding the production of the MIPs film, since the film itself is extremely thin, the consumption of MIPs as a material and the solvent is small, and the production is inexpensive and easy.
  • acetonitrile is used as a solvent for preparing the MIPs saturated solution.
  • ketones such as acetone, ethers, dimethylformamide, or an aqueous solution containing an organic solvent may be used as appropriate. Can be used.
  • a nitrocellulose membrane is used as the base material for the porous support, but the present invention is not limited to this, and a large number of pores having an inner diameter through which a specific compound molecule to be separated and isolated can permeate sufficiently are provided.
  • acetyl cellulose, cellulose ether, cellulose ester, or the like can be used as a base material for the porous support (membrane or substrate).
  • various polymer compounds can be used in addition to cellulose. Examples of the polymer compound include fluororesin, nylon, polyethylene, polypropylene, polyvinyl alcohol, polycarbonate, and polyamide.
  • an inorganic compound can be used as the base material of the porous support, and various glass fibers, ceramics, zeolites and the like are included.
  • MIPs-porous support membrane or substrate
  • the nitrocellulose membrane is used as the porous support membrane 12, it is not limited to this, and it has a large number of micropores having an inner diameter capable of permeating specific compound molecules to be separated and isolated.
  • acetyl cellulose, cellulose acetate, cellulose nitrate, cellulose sulfate, cellulose ethers, cellulose esters, and the like can be used.
  • identification is made from biological fluids containing various types of antibiotics, organic compounds, etc., secretion fluids, and aqueous solutions containing various types of organic compounds that are subject to separation and isolation. It is possible to easily separate and isolate compound molecules such as a single kind of antibiotics and other organic compounds.

Abstract

Disclosed is a selectively permeable membrane using selective molecular recognition macromolecules using molecular recognition macromolecules which use an extremely thin molecular recognition resin membrane which, by allowing the permeation of a specified single kind of component from a mixed aqueous solution of various antibiotics, saccharides, amino acids, organic toxic substances and other organic compounds, is easily capable of fractional extraction or fractional removal, fractional isolation or detection, or quantitative analysis. A molecular recognition macromolecule membrane (11), whereon a template molecule of a specified compound molecule which is the object of fractionation or isolation has been molecularly imprinted, is formed on a porous support membrane (12) provided with a plurality of micropores having an internal diameter through which the specified compound molecule which is the object of fractionation or isolation can permeate, and the specified compound molecule is selectively permeated by the molecular recognition resin membrane (11), and the compound molecule is permeated from the micropores of the porous support membrane (12).

Description

分子認識高分子を用いた選択性透過膜Selective permeable membrane using molecular recognition polymer
　本発明は、分子認識高分子を用いた選択性透過膜及びその製造方法に係わり、例えば生乳などの生物体液や、分泌液中に含まれる微量の特定抗生物質、特定糖類、特定有機化合物等の分析、生物体液および分泌液以外に有機化合物水溶液中に含まれる多種の有機化合物から微量に含まれる特定の単一有機化合物を分別・単離、定量等する際に利用される選択性透過膜及びその製造方法に関する。 The present invention relates to a selective permeable membrane using a molecular recognition polymer and a method for producing the same, such as biological fluids such as raw milk, trace amounts of specific antibiotics, specific saccharides, specific organic compounds, etc. contained in secretions. Selective permeable membranes used for analysis, separation, isolation, quantification, etc. of specific single organic compounds contained in trace amounts from various organic compounds contained in organic compound aqueous solutions in addition to biological fluids and secretions It relates to the manufacturing method.
多種類にわたる有機化合物を含む生物体液又は分泌液から、その中に含まれる微量の特定の抗生物質や特定の糖類、その他の有機化合物を分別・単離、定量する場合には複雑な前処理操作を必要とする。例えば脂肪、蛋白、糖類その他の多量かつ多種類の有機化合物を含有する生乳中に微量のペニシリンＧとテトラサイクリンが含まれている場合、高速液体クロマトグラフ質量分析計を用いた方法（例えばM.Kozono等　Seikatu Eisei 49巻, 220-226頁2005年）が有効であるが、その定量測定にはこれらの含有有機化合物の除去のための遠心分離、濾過、溶出など、何段階にも及ぶ熟練を要する前処理操作が必要であり、測定装置も非常に高価である。 Complex pretreatment procedures for the separation, isolation, and quantification of trace amounts of specific antibiotics, specific sugars, and other organic compounds from biological fluids or secretions containing a wide variety of organic compounds Need. For example, when a small amount of penicillin G and tetracycline are contained in raw milk containing a large amount and many kinds of organic compounds such as fat, protein, saccharide and the like, a method using a high performance liquid chromatograph mass spectrometer (for example, M. Kozono) Seikatu Eisei Vol.49, 220-226 (2005)) is effective, but its quantitative measurement requires many steps such as centrifugation, filtration and elution to remove these organic compounds. Pretreatment operations are required, and the measuring device is very expensive.
　また、多種類にわたる微量の薬剤や有機化合物を含む水溶液から、特定の薬剤成分や有機化合物成分を抽出する操作も高度な技術を要する。水溶液中に含まれる微量の毒物成分の分別除去や分析も同様である。このため、食品化学分野、製薬分野、医療分野では、特定抗生物質、有機薬剤、糖類、アミノ酸、毒物等を、安価で簡便な手法によって分別・単離、定量することが求められている。 In addition, an operation for extracting a specific drug component or organic compound component from an aqueous solution containing a large amount of a small amount of drug or organic compound also requires advanced techniques. The same applies to the separation and analysis of trace amounts of toxic components contained in an aqueous solution. For this reason, in the food chemistry field, the pharmaceutical field, and the medical field, it is required to separate, isolate, and quantify specific antibiotics, organic drugs, saccharides, amino acids, toxic substances, and the like by an inexpensive and simple technique.
最近、有機化合物を除去する手段として、分子インプリントポリマー(Molecularly Imprinted Polymers、分子認識樹脂と称されることもある、以下「ＭＩＰｓ」と称する。)を用いた方法が公知である。　特開2005-232205号公報には、「分子インプリントセルロース及びその製造方法」に関する発明が記載され、同発明はセルロース等に水素結合、静電的結合、疎水性結合等によりセルロースと結合可能な鋳型分子を分子インプリントすることによって、該分子の分子インプリントセルロースを形成するものである。具体的には、セルロースまたはセルロース誘導体、および鋳型分子を含む有機溶媒溶液から有機溶媒を除去して固化させた後、鋳型分子を抽出することによって、該分子の鋳型をインプリントしたセルロースを形成するものである。該インプリントセルロースは紙などの基盤に塗布される。 Recently, as a means for removing organic compounds, a method using molecularly imprinted polymers (MolecularlymersImprinted Polymers, sometimes referred to as molecular recognition resins, hereinafter referred to as “MIPs”) is known. Japanese Patent Application Laid-Open No. 2005-232205 describes an invention relating to “molecularly imprinted cellulose and a method for producing the same”, which can be bonded to cellulose by hydrogen bonding, electrostatic bonding, hydrophobic bonding, or the like. By molecularly imprinting a template molecule, molecularly imprinted cellulose of the molecule is formed. Specifically, after removing an organic solvent from an organic solvent solution containing cellulose or a cellulose derivative and a template molecule and solidifying it, the template molecule is extracted to form cellulose imprinted with the template of the molecule. Is. The imprint cellulose is applied to a substrate such as paper.
特開2005-205333号公報には、「分子吸着機能を有するシート」に関する発明が記載されている。当該発明はエチレンーアクリル共重合体をインプリントポリマーとして用い、これを紙、ガラス繊維に担持している。
特願2004―520873号（特表2005-533146号公報）には、「分子インプリントポリマー材料」に関する発明があるが、該発明はスチレン、アクリルモノマーを鋳型分子、架橋剤等と混合し、ブロック重合させ、鋳型分子、未反応ポリマー、架橋剤を溶剤で除去し、特定分子の鋳型を有する厚さ60マイクロメーターの多孔性フイルムを作製する。 Japanese Patent Application Laid-Open No. 2005-205333 describes an invention relating to a “sheet having a molecular adsorption function”. In the present invention, an ethylene-acrylic copolymer is used as an imprint polymer, and this is supported on paper or glass fiber.
Japanese Patent Application No. 2004-520873 (Japanese Patent Publication No. 2005-533146) has an invention relating to “molecularly imprinted polymer material”. This invention is a block in which styrene and an acrylic monomer are mixed with a template molecule, a crosslinking agent, etc. Polymerization is performed, and the template molecule, unreacted polymer, and cross-linking agent are removed with a solvent to produce a porous film having a thickness of 60 micrometers having a template of a specific molecule.
特開2005-232205号公報JP-A-2005-232205 特開2005-205333号公報JP 2005-205333 A 特表2005-533146号公報Special Table 2005-533146
しかしながら、上記発明はいずれもインプリントポリマーに用いた分子鋳型と鋳型分子との吸着特性（親和性）を利用し、インプリントポリマーに該鋳型分子を吸着させることによって、試料溶液に含有する該鋳型分子のインプリントポリマー中への濃縮や濃縮効果を利用した該鋳型分子の試料溶液からの除去を目的にしたものである。しかしながらこの方法によって該鋳型分子を分別し、単離するためには該インプリントポリマーに吸着した鋳型分子を再度脱離、精製するための複雑なプロセスが必要である。 However, all of the above inventions utilize the adsorption characteristics (affinity) between the molecular template used for the imprint polymer and the template molecule, and adsorb the template molecule to the imprint polymer to thereby contain the template contained in the sample solution. The purpose is to remove the template molecule from the sample solution by utilizing the concentration of the molecule in the imprinted polymer and the concentration effect. However, in order to separate and isolate the template molecule by this method, a complicated process for desorbing and purifying the template molecule adsorbed on the imprint polymer again is necessary.
　本発明は、このような諸事情に対処するために提案されたものであって、分別、単離の対象となる化合物分子、例えば多種類の抗生物質や有機化合物などを含む生物体液、分泌液および水溶液から、特定の単一種の抗生物質、または特定の単一種の有用な有機化合物分子を容易に分別・単離、抽出することができる、構造が簡単で安価な、ＭＩＰｓ分子認識高分子を用いた選択性透過膜およびその製造方法を提供することを目的とする。 The present invention has been proposed in order to cope with such various situations, and is a biological fluid or secretory fluid containing compound molecules to be separated and isolated, for example, various types of antibiotics and organic compounds. A simple and inexpensive MIPs molecule recognition polymer that can easily separate, isolate, and extract specific single species of antibiotics or specific single species of useful organic compounds from aqueous solutions It is an object of the present invention to provide a selective permeable membrane used and a method for producing the same.
上記目的を達成するために、請求項１記載の発明は、分別・単離の対象となる特定の化合物分子を充分透過可能な内径を有する孔を多数備えた多孔質支持体上に、分別・単離の対象となる前記特定の化合物分子の鋳型をインプリントした極めて薄いＭＩＰｓ透過膜を形成し、該ＭＩＰｓ透過膜によって試料溶液中の該特定の化合物分子を選択的に透過させ、且つ前記多孔質支持体の孔から支持体側面の溶液中に該化合物分子を透過・移行させるようにしたことを特徴とする。 In order to achieve the above object, the invention according to claim 1 is characterized in that a fractionation / isolation is carried out on a porous support having a large number of pores having an inner diameter capable of sufficiently permeating a specific compound molecule to be fractionated / isolated. An extremely thin MIPs permeable membrane imprinted with the template of the specific compound molecule to be isolated is formed, the specific compound molecule in the sample solution is selectively permeated by the MIPs permeable membrane, and the porous The compound molecules permeate and migrate from the pores of the porous support into the solution on the side of the support.
　請求項２記載の発明は、請求項１において、前記多孔質支持体の基材はニトロセルロース又は、その他のセルロース誘導体であることを特徴とする。 The invention described in claim 2 is characterized in that, in claim 1, the base material of the porous support is nitrocellulose or other cellulose derivatives.
請求項３記載の発明は、前記多孔質支持体の基材はフッ素、ナイロン、ポリエチレン、ポリプロピレン、ポリビニルアルコール、ポリカーボネート、ポリアミド等の各種合成樹脂であることを特徴とする。 The invention according to claim 3 is characterized in that the base material of the porous support is various synthetic resins such as fluorine, nylon, polyethylene, polypropylene, polyvinyl alcohol, polycarbonate and polyamide.
　請求項４記載の発明は、請求項１において、前記多孔質支持体の基材は各種グラスファイバー、セラミック、ゼオライト等、各種無機化合物であることを特徴とする。
  The invention according to claim 4 is characterized in that, in claim 1, the base material of the porous support is various inorganic compounds such as various glass fibers, ceramics, and zeolites.
請求項５記載の発明は、請求項１～４のうち、いずれか１項において、前記ＭＩＰｓ透過膜は、膜厚が１～５マイクロメーターであることを特徴とする。 According to a fifth aspect of the present invention, in any one of the first to fourth aspects, the MIPs permeable membrane has a thickness of 1 to 5 micrometers.
請求項６記載の発明は、分別・単離の対象となる特定の化合物分子を充分透過可能な内径を有する孔を多数備えた多孔質支持体上に、前記特定の化合物分子の鋳型を含むポリマーの有機溶媒溶液を塗布する工程と、前記ポリマーの有機溶媒溶液を蒸発乾燥させて、極めて薄いＭＩＰｓ膜を形成する工程とを具備したことを特徴とする。 The invention according to claim 6 is a polymer comprising a template of the specific compound molecule on a porous support having a large number of pores having an inner diameter capable of sufficiently permeating the specific compound molecule to be separated and isolated. And a step of evaporating and drying the organic solvent solution of the polymer to form an extremely thin MIPs film.
本発明は分子インプリントポリマー（以下「ＭＩＰｓ」とする）のもう一つの特性である、鋳型分子の選択的透過能力を利用して、分別、単離することを目的とすることに特徴がある。　　　　　　　　
要するに、各請求項に記載の発明によれば、分別・単離の対象となる特定の化合物分子を容易に透過可能な内径を有する孔を多数備えた多孔質支持体上に、分別・単離の対象となる特定の化合物分子の鋳型がインプリントされたＭＩＰｓ膜を形成するようにしている。これによってきわめて薄く取り扱いが困難なＭＩＰｓ膜が補強され、分子認識高分子を用いた選択性透過膜の機械的強度を向上させることが可能になる。 The present invention is characterized in that it aims at fractionation and isolation using the selective permeation ability of a template molecule, which is another characteristic of molecularly imprinted polymers (hereinafter referred to as “MIPs”). .
In short, according to the invention described in each claim, the separation / isolation is performed on a porous support having a large number of pores having an inner diameter capable of easily permeating a specific compound molecule to be separated / isolated. The MIPs film on which the template of a specific compound molecule to be subjected to imprinting is imprinted is formed. This reinforces the MIPs membrane, which is extremely thin and difficult to handle, and improves the mechanical strength of the selective permeable membrane using the molecular recognition polymer.
　特に、多孔質支持体の基材として要求される性質は、マイクロメータレベルの孔を無数に有し、膜厚が０．１ｍｍ～１ｍｍの薄膜あるいは薄板で、容易にペニシリンＧやテトラサイクリンなどの抗生物質や有機化合物分子を透過することのできる素材であり、機械的な強度を有し、化学的に安定で、変質しない材質であることが必要である。
今回の請求項２乃至４記載の発明におけるセルロース、合成樹脂、無機化合物は、このような性質を満たし、ＭＩＰｓ膜のような極薄の膜をこれらの支持体に貼付することによって、機械的に脆弱なＭＩＰｓ膜を強化することができ、分子認識高分子を用いた選択性透過膜としての実用性を有するものとなる。 In particular, the properties required as a base material for the porous support are an infinite number of micrometer-level pores and a thin film or thin plate with a thickness of 0.1 mm to 1 mm, which can easily be used for antibiotics such as penicillin G and tetracycline. It is necessary to be a material that can permeate substances and organic compound molecules, has mechanical strength, is chemically stable, and does not deteriorate.
The cellulose, the synthetic resin, and the inorganic compound in the present inventions according to claims 2 to 4 satisfy such properties, and mechanically by attaching an extremely thin film such as a MIPs film to these supports. The fragile MIPs membrane can be strengthened, and it has practicality as a selective permeable membrane using a molecular recognition polymer.
特に、請求項６記載の発明によれば、多孔質支持体上に、分別・単離をしようとする特定の化合物分子の鋳型を含むポリマーの有機溶媒溶液を塗布し、溶媒を蒸発乾燥させることによってＭＩＰｓ膜を形成するようにしている。これにより、分子認識高分子を用いた選択性透過膜の作製を短時間で容易、且つ安価に行うことが可能である。 In particular, according to the invention described in claim 6, an organic solvent solution of a polymer containing a template of a specific compound molecule to be separated and isolated is applied onto a porous support, and the solvent is evaporated to dryness. By doing so, a MIPs film is formed. As a result, it is possible to easily and inexpensively produce a selective permeable membrane using a molecular recognition polymer.
本発明の一つの実施形態に係る分子認識高分子を用いた選択性透過膜の構造を示した概略図である。It is the schematic which showed the structure of the selective permeable membrane using the molecular recognition polymer | macromolecule which concerns on one Embodiment of this invention. 本発明の一つの実施形態に係る分子認識高分子を用いた選択性透過膜を金の薄膜（０．１ｍｍ程度）上に形成し、当該透過膜を走査型電子顕微鏡（ＳＥＭ）によって、やや斜め上方から撮影した顕微鏡の画像を示した図である。A selective permeable membrane using a molecular recognition polymer according to one embodiment of the present invention is formed on a gold thin film (about 0.1 mm), and the permeable membrane is slightly inclined by a scanning electron microscope (SEM). It is the figure which showed the image of the microscope image | photographed from upper direction. 同じく、本発明の一つの実施形態に係る分子認識高分子を用いた選択性透過膜を金の薄膜（０．１ｍｍ程度）上に形成し、当該透過膜を走査型電子顕微鏡（ＳＥＭ）によって、撮影した透過膜の平面の画像を示した図である。Similarly, a selective permeable membrane using a molecular recognition polymer according to one embodiment of the present invention is formed on a gold thin film (about 0.1 mm), and the permeable membrane is scanned by a scanning electron microscope (SEM). It is the figure which showed the image of the plane of the picked-up permeable membrane. 本発明の一つの実施形態に係る分子認識高分子を用いた選択性分子認識高分子を用いた選択性透過膜を使用して生乳のサンプルから透過によってペニシリンＧを定量する計測装置の概略構成を示した図である。1 shows a schematic configuration of a measuring apparatus for quantifying penicillin G by permeation from a sample of raw milk using a selective permeable membrane using a selective molecular recognition polymer using a molecular recognition polymer according to one embodiment of the present invention. FIG. 本発明のペニシリンＧ用の分子認識高分子を用いた選択性透過膜によってペニシリンＧ、テトラサイクリンを含む生乳から、透過によってペニシリンＧの分別が可能であることを確認した実験結果を示すグラフである。It is a graph which shows the experimental result which confirmed that penicillin G could be fractionated by permeation from raw milk containing penicillin G and tetracycline by the selective permeation membrane using the molecular recognition polymer for penicillin G of the present invention. 本発明のペニシリンＧ用の分子認識高分子を用いた選択性透過膜によって微量のペニシリンＧとペニシリンＧときわめて類似した分子構造を持つアンピシリンを含む生乳試料からペニシリンＧを透過し、分別・単離した場合の実験結果を示すグラフである。Penicillin G is permeated from a raw milk sample containing a small amount of penicillin G and ampicillin having a molecular structure very similar to penicillin G by the selective permeation membrane using the molecular recognition polymer for penicillin G of the present invention, and is separated and isolated. It is a graph which shows the experimental result at the time of doing. 本発明のテトラサイクリン用の分子認識高分子を用いた選択性透過膜を用いて微量のペニシリンＧとテトラサイクリンを含む生乳からテトラサイクリンだけを透過し、分別する実験を行った場合の実験結果を示すグラフである。The graph which shows the experimental result at the time of performing the experiment which permeate | transmits only a tetracycline from raw milk containing a trace amount of penicillin G and tetracycline using the selective permeable membrane using the molecular recognition polymer | macromolecule for tetracycline of this invention, and is fractionated. is there.
　　　　　１　分子認識高分子を用いた選択性透過膜(ＭＩＰｓ膜／多孔質支持体膜)
         
１１　ＭＩＰｓ膜
 　　　　
１２　多孔質支持体膜（ニトロセルロース透過膜）
２１Ａ　試料導入管
２１Ｂ　試料排出管
２２　測定試料用セル
２３　シリコーン板
２４　電気化学セル
２５　電解液導入・排出管
２６　白金製対極
２７　電気化学センサ電極
２８　参照電極 1 Selective membrane using molecular recognition polymer (MIPs membrane / porous support membrane)

11 MIPs membrane
12 Porous support membrane (nitrocellulose permeable membrane)
21A Sample introduction tube 21B Sample discharge tube 22 Measurement sample cell 23 Silicone plate 24 Electrochemical cell 25 Electrolyte introduction / discharge tube 26 Platinum counter electrode 27 Electrochemical sensor electrode 28 Reference electrode
　以下、本発明に係わる分子認識高分子を用いた選択性透過膜及びその製造方法の好適な実施形態について、添付図面を参照して説明する。
図１は本発明の一つの実施形態に係わる分子認識高分子を用いた選択性透過膜の構造を示した概略図である。また、図２は金の薄膜（０．１ｍｍ程度）に本実施形態に係わる透過膜を形成し、当該透過膜を走査型電子顕微鏡（ＳＥＭ）によって、やや斜め上方から撮影した顕微鏡の画像を示した図、図３は透過膜の平面の顕微鏡による画像を示した図である。 Hereinafter, preferred embodiments of a selective permeable membrane using a molecular recognition polymer and a method for producing the same according to the present invention will be described with reference to the accompanying drawings.
FIG. 1 is a schematic view showing the structure of a selective permeable membrane using a molecular recognition polymer according to one embodiment of the present invention. FIG. 2 shows an image of a microscope in which a permeable membrane according to the present embodiment is formed on a gold thin film (about 0.1 mm), and the permeable membrane is photographed from slightly above with a scanning electron microscope (SEM). FIG. 3 is a diagram showing an image of the plane of the permeable membrane by a microscope.
図１に示されるように、本実施形態の分子認識高分子を用いた選択性透過膜１は、厚さ１～５マイクロメーターの極めて薄いＭＩＰｓ膜１１と、多孔質支持体１２からなる積層構造を有している。ＭＩＰｓ膜は多孔質支持体（例えば厚さ０．１ｍｍ、孔径０．０２５μｍのニトロセルロース膜）上に形成される。該多孔質支持体は分別・単離の対象となる特定の化合物分子が充分透過可能な内径を有する孔を多数備えている。
ＭＩＰｓ膜１１は、分別・単離の対象となる特定の化合物分子の鋳型がインプリントされたアクリルーウレア樹脂で、該鋳型と相補的な関係にある特定の化合物分子を認識して、これを選択的に透過させることができるようになっており、ＭＩＰｓ膜１１を透過した化合物分子は、さらに多孔質支持体の孔を透過する。これによって、分子認識高分子を用いた選択性透過膜１による該化合物分子の分別・単離が可能になっている。 As shown in FIG. 1, the selective permeable membrane 1 using the molecular recognition polymer of this embodiment is a laminated structure comprising an extremely thin MIPs membrane 11 having a thickness of 1 to 5 micrometers and a porous support 12. have. The MIPs membrane is formed on a porous support (for example, a nitrocellulose membrane having a thickness of 0.1 mm and a pore diameter of 0.025 μm). The porous support has a large number of pores having an inner diameter through which a specific compound molecule to be separated and isolated can permeate sufficiently.
The MIPs film 11 is an acrylic urea resin imprinted with a template of a specific compound molecule to be separated and isolated, and recognizes a specific compound molecule complementary to the template. The compound molecules that can be selectively permeated through the MIPs membrane 11 further permeate the pores of the porous support. As a result, the compound molecules can be separated and isolated by the selective permeable membrane 1 using a molecular recognition polymer.
図２及び図３は、ＭＩＰｓ膜１１を可視的に現すために、金の担体上に、ＭＩＰｓ膜１１を形成したものであり、特に図２に示されるように、ＭＩＰｓ膜の膜厚は３μｍであることが計測された。また、ＭＩＰｓ膜１１には、特定の化合物分子を透過させることが可能な透過孔が形成されているのであるが、微細な孔のため、図３の顕微鏡による画像では、その孔は見ることは出来なかった。 2 and 3 show a case where the MIPs film 11 is formed on a gold carrier in order to make the MIPs film 11 visible. In particular, as shown in FIG. 2, the thickness of the MIPs film is 3 μm. It was measured that. In addition, the MIPs film 11 is formed with a permeation hole that allows a specific compound molecule to permeate. However, because of the fine hole, the hole cannot be seen in the microscope image of FIG. I could not do it.
本実施形態の分子認識高分子を用いた選択性透過膜１は以下のように作製される。まず、ＭＩＰｓのアセトニトリル飽和溶液０．１～１（ｍｌ）をピペットにとり、多孔質支持体１２上に一様に塗布し、溶媒を室温で一昼夜蒸発乾燥させ、作製する。得られたＭＩＰｓ-多孔質支持体膜（基板）は室温で保存される。該ＭＩＰｓ-多孔質支持体膜（基板）を作製することによって、薄く、取り扱いが困難であったＭＩＰｓ膜１１が補強された性状を有するようになるので、分子認識高分子を用いた選択性透過膜１の機械的強度が向上し、実用に適するようになる。 The selective permeable membrane 1 using the molecular recognition polymer of this embodiment is produced as follows. First, 0.1 to 1 (ml) of a saturated solution of MIPs in acetonitrile is pipetted and uniformly applied onto the porous support 12, and the solvent is evaporated and dried overnight at room temperature. The resulting MIPs-porous support membrane (substrate) is stored at room temperature. By producing the MIPs-porous support membrane (substrate), the MIPs membrane 11 which is thin and difficult to handle has a reinforced property, so that selective permeation using a molecular recognition polymer is performed. The mechanical strength of the film 1 is improved and it becomes suitable for practical use.
（実施例１）
　上記において説明した本発明のペニシリンＧ用ＭＩＰｓ膜を用いて微量のペニシリンＧとテトラサイクリンを含む生乳試料 からペニシリンＧだけを透過し、分別・単離する実験を行った。本実施例で用いる分子認識高分子を用いた選択性透過膜は、分別・単離する化合物としてのペニシリンＧの鋳型を包有するＭＩＰｓ－ニトロセルロース支持体膜で、発明の実施の形態で詳述した製造工程によって形成されるものである。 Example 1
Using the MIPs membrane for penicillin G of the present invention described above, an experiment was conducted in which only penicillin G was permeated from a raw milk sample containing a small amount of penicillin G and tetracycline, and was separated and isolated. The selective permeable membrane using the molecular recognition polymer used in this example is a MIPs-nitrocellulose support membrane containing a template of penicillin G as a compound to be separated and isolated, and is described in detail in the embodiment of the invention. It is formed by the manufacturing process.
図４は生乳試料中に含有するペニシリンＧを定量する計測装置２０の概略構成を示した図である。図４（Ａ）に示されるように、計測装置２０は、二つのポリスチロール製の測定試料用セル２２、電気化学セル２４と、その間を仕切るシリコーンゴム製の厚さ３ｍｍの板２３から構成されている。シリコーン板の中央部は、サイズ13mm×13mmの方形状にくり抜かれ、その上部に本発明の分子認識高分子を用いた選択性透過膜（ＭＩＰｓ膜１１及び多孔質支持体１２）１がシリコーン系接着剤によって固定される。分子認識高分子を用いた選択性透過膜はサイズ15mm×15mmの方形状である。 FIG. 4 is a diagram showing a schematic configuration of a measuring apparatus 20 that quantifies penicillin G contained in a raw milk sample. As shown in FIG. 4 (A), the measuring device 20 is composed of two measurement sample cells 22 made of polystyrene, an electrochemical cell 24, and a 3 mm-thick plate 23 made of silicone rubber for partitioning between them. ing. The central part of the silicone plate is cut out into a 13 mm × 13 mm square shape, and a selective permeable membrane (MIPs membrane 11 and porous support 12) 1 using the molecular recognition polymer of the present invention is silicone-based on the upper part. Fixed with adhesive. A selective permeable membrane using a molecular recognition polymer has a rectangular shape with a size of 15 mm × 15 mm.
電気化学セル２４の内側には、抗生物質測定用の電気化学センサ電極２７、白金製対極２６、及び銀／塩化銀参照電極28が挿入され、各電極端子にはセンサ電極の電位を設定し、センサ電極と対極間を流れる電流を計測するポテンシオスタット（図示せず）が接続される。電気化学センサ電極は金板に酵素(ヒスタミンデヒドロゲナーゼ)、金微粒子、テトラチアフルバレン、ナフィオン樹脂、架橋セラチンを担持したヒスタミン計測用電気化学バイオセンサで、抗生物質存在下で、酵素のヒスタミン酸化の活性機能の低下による電流の降下を利用して、抗生物質を定量測定するものである。図２(B)に示されるように、分子認識高分子を用いた選択性透過膜１を固定したシリコーン板２３は、試料用セル、電気化学測定セルとの間にシリコーン系接着剤によって固定されている。 Inside the electrochemical cell 24, an electrochemical sensor electrode 27 for measuring antibiotics, a platinum counter electrode 26, and a silver / silver chloride reference electrode 28 are inserted, and the potential of the sensor electrode is set to each electrode terminal. A potentiostat (not shown) for measuring the current flowing between the sensor electrode and the counter electrode is connected. The electrochemical sensor electrode is an electrochemical biosensor for histamine measurement that has an enzyme (histamine dehydrogenase), gold microparticles, tetrathiafulvalene, Nafion resin, and cross-linked ceratin on a metal plate. The activity of histamine oxidation of the enzyme in the presence of antibiotics. Antibiotics are quantitatively measured using a drop in current due to a decrease in function. As shown in FIG. 2 (B), the silicone plate 23 to which the selective permeable membrane 1 using a molecular recognition polymer is fixed is fixed between a sample cell and an electrochemical measurement cell by a silicone-based adhesive. ing.
(ペニシリンＧの選択的分別・単離の検証)
以下に本発明のペニシリンＧ用の分子認識高分子を用いた選択性透過膜１によって、ペニシリンＧ、テトラサイクリンを含む生乳試料から、ペニシリンＧの分別が可能であることを確認した実験結果を図５に示す。図５において、縦軸は電流 (nA)、横軸は時間（min）である。
まず、試料セル２２内に試料導入管２１Ａから生乳４ｍｌを入れるとともに、電気化学測定セル２４内を100ppbのヒスタミンを含有するリン酸緩衝溶液（pH 7.1）で満たし、電気化学センサ電極の電位を0.4V (vs. Ag/AgCl)に設定する。ヒスタミン酸化の電流値が一定になった後（バックグランド電流（３１））、試料用セル中の生乳に濃度4ppb となるようにテトラサイクリンを加え、ヒスタミン酸化電流を観測する（３２）。次に濃度40ppbとなるようにテトラサイクリンを加えてヒスタミン酸化電流を観測する(３３)。その後、濃度4ppbとなるように、ペニシリンＧを加えヒスタミン酸化電流を観測する(３４)。　 (Verification of selective fractionation and isolation of penicillin G)
FIG. 5 shows the experimental results confirming that penicillin G can be separated from a raw milk sample containing penicillin G and tetracycline by the selective permeable membrane 1 using the molecular recognition polymer for penicillin G of the present invention. Shown in In FIG. 5, the vertical axis represents current (nA) and the horizontal axis represents time (min).
First, 4 ml of raw milk is put into the sample cell 22 from the sample introduction tube 21A, the electrochemical measurement cell 24 is filled with a phosphate buffer solution (pH 7.1) containing 100 ppb histamine, and the potential of the electrochemical sensor electrode is set to 0.4. Set to V (vs. Ag / AgCl). After the current value of histamine oxidation becomes constant (background current (31)), tetracycline is added to the raw milk in the sample cell to a concentration of 4 ppb, and the histamine oxidation current is observed (32). Next, tetracycline is added to a concentration of 40 ppb and the histamine oxidation current is observed (33). Thereafter, penicillin G is added so that the concentration becomes 4 ppb, and the histamine oxidation current is observed (34).
 図５に示されるように、生乳試料中のテトラサイクリンの添加は濃度40ppbまでヒスタミン酸化電流に変化は認められない(３３)。一方、引き続き行った4ppbのペニシリンＧの添加では該酸化電流は著しく低下した。後述の実施例３で示すように、テトラサイクリンはペニシリンＧと同様、該電気化学センサ電極のヒスタミン酸化電流を低下させる。したがって図３の観測結果は生乳試料中のペニシリンＧだけが、選択的にペニシリンＧ用の分子認識高分子を用いた選択性透過膜を通過することを示している。　 As shown in FIG. 5, the addition of tetracycline in the raw milk sample shows no change in the histamine oxidation current up to a concentration of 40 ppb (33). On the other hand, the subsequent addition of 4 ppb penicillin G significantly reduced the oxidation current. As shown in Example 3 described later, tetracycline, like penicillin G, reduces the histamine oxidation current of the electrochemical sensor electrode. Therefore, the observation results in FIG. 3 indicate that only penicillin G in the raw milk sample selectively passes through the selective permeable membrane using the molecular recognition polymer for penicillin G. *
(実施例２)
本発明のペニシリンＧ用の分子認識高分子を用いた選択性透過膜を用いて微量のペニシリンＧ
とペニシリンＧにきわめて類似した分子構造を持つアンピシリンを含む生乳試料からペニシリンＧを透過し、分別・単離する実験を行った。分子認識高分子を用いた選択性透過膜はペニシリンＧ用で、計測装置は図４と同一である。測定操作も上記のペニシリンＧ、テトラサイクリンの分別・単離実験と同様である。 (Example 2)
A small amount of penicillin G using a selective permeable membrane using the molecular recognition polymer for penicillin G of the present invention.
And penicillin G were permeated from a raw milk sample containing ampicillin having a molecular structure very similar to that of penicillin G, and were subjected to an experiment for separation and isolation. The selective permeable membrane using a molecular recognition polymer is for penicillin G, and the measuring apparatus is the same as that in FIG. The measurement operation is also the same as the above-described fractionation / isolation experiment of penicillin G and tetracycline.
以下に実験内容及び結果の詳細を図６に示す。
試料用セルに生乳４mlをとり、電気化学測定セル２４を100ppbのヒスタミンを含むリン酸緩衝溶液で満たす。ヒスタミン酸化電流値が一定になった後（バックグランド電流（４１））、測定試料中の生乳に濃度4ppbになるようにアンピシリンを加え、ヒスタミン酸化電流を観測する (４２)。 次にアンピシリンの濃度を20ppbにし、ヒスタミン酸化電流を観測する（４３）。さらにこの生乳に濃度４ppbになるようにペニシリンＧを加え、ヒスタミン酸化電流を観測する（４４）。 The details of the experiment and the results are shown in FIG.
4 ml of raw milk is placed in a sample cell, and the electrochemical measurement cell 24 is filled with a phosphate buffer solution containing 100 ppb histamine. After the histamine oxidation current value becomes constant (background current (41)), ampicillin is added to the raw milk in the measurement sample to a concentration of 4 ppb, and the histamine oxidation current is observed (42). Next, the concentration of ampicillin is set to 20 ppb, and the histamine oxidation current is observed (43). Further, penicillin G is added to the raw milk to a concentration of 4 ppb, and the histamine oxidation current is observed (44).
図６に示されるように、アンピシリンは上記テトラサイクリンの場合と同様に濃度20ppbにいたるまで電気化学センサ電極のヒスタミン酸化電流値に変化を与えなかった（４２）（４３）。
引き続き行った4ppbのペニシリンＧの添加ではヒスタミン酸化電流は著しく低下し、生乳試料中のペニシリンＧが選択的に透過され、分別・単離が可能なことが確認された。濃度4ppbのペニシリンＧの添加では添加後、ヒスタミン酸化電流の低下に、ペニシリンＧ用のＭＩＰｓ膜の鋳型におけるペニシリンＧ分子の、ペニシリンＧ
と分子構造が酷似するアンピシリン分子による透過妨害とみられる50～60分間のインダクションピリオドが観測された（４４）。 As shown in FIG. 6, ampicillin did not change the histamine oxidation current value of the electrochemical sensor electrode until the concentration reached 20 ppb as in the case of tetracycline (42) (43).
Subsequent addition of 4 ppb penicillin G significantly reduced the histamine oxidation current, confirming that penicillin G in the raw milk sample was selectively permeated and could be separated and isolated. When penicillin G was added at a concentration of 4 ppb, the penicillin G molecule in the template of the MIPs film for penicillin G decreased in the histamine oxidation current after the addition.
An induction period of 50 to 60 minutes, which is thought to be permeation by ampicillin molecules that closely resemble the molecular structure, was observed (44).
（実施例３）
本発明のテトラサイクリン用の分子認識高分子を用いた選択性透過膜１を用いてペニシリンＧと微量のテトラサイクリンを含む生乳試料からテトラサイクリンだけ分別・単離する実験を行った。
計測装置は図４と同一である。測定操作も実施例１、実施例２の操作と同一である。実施内容及び結果の詳細を図７に示す。
試料用セルに4mlの生乳をとり、電気化学測定セルを100ppbのヒスタミンを含むリン酸緩衝溶液で満たす。ヒスタミン酸化の電流値が一定になった後（バックグランド電流（５１））、試料用セル中の生乳に濃度40ppbになるようにペニシリンＧを加え、ヒスタミン酸化電流を観測する（５２）。次にこの生乳試料にテトラサイクリンを濃度0.4ppbになるように加え、ヒスタミン酸化電流を観測し（５３）、さらに濃度4ppbになるようにテトラサイクリンを追加し、ヒスタミン酸化電流を観測する（５４）。 (Example 3)
Experiments were conducted to separate and isolate only tetracycline from a raw milk sample containing penicillin G and a small amount of tetracycline using the selective permeable membrane 1 using the molecular recognition polymer for tetracycline of the present invention.
The measuring device is the same as in FIG. The measurement operation is also the same as that in the first and second embodiments. Details of the implementation and results are shown in FIG.
Take 4 ml of raw milk in the sample cell and fill the electrochemical measurement cell with phosphate buffer solution containing 100 ppb histamine. After the current value of histamine oxidation becomes constant (background current (51)), penicillin G is added to the raw milk in the sample cell to a concentration of 40 ppb, and the histamine oxidation current is observed (52). Next, tetracycline is added to the raw milk sample to a concentration of 0.4 ppb, and the histamine oxidation current is observed (53). Tetracycline is further added to a concentration of 4 ppb, and the histamine oxidation current is observed (54).
図７に示されるように、テトラサイクリン用分子認識高分子を用いた選択性透過膜ではペニシリンＧは濃度40ppb においても透過せず、電気化学センサ電極のヒスタミン酸化電流値に変化が観測されない。
他方、引き続き行った0.4ppb及び4ppbのテトラサイクリンの添加では、その濃度に応じて酸化電流は顕著に低下し、生乳中のテトラサイクリンが選択的に透過・移行し、生乳からテトラサイクリンが分別・単離されたことが確認された。 As shown in FIG. 7, penicillin G does not permeate even at a concentration of 40 ppb in the selective permeable membrane using the molecular recognition polymer for tetracycline, and no change is observed in the histamine oxidation current value of the electrochemical sensor electrode.
On the other hand, with the subsequent addition of 0.4 ppb and 4 ppb tetracycline, the oxidation current decreases remarkably depending on the concentration, and tetracycline in raw milk is selectively permeated and transferred, and tetracycline is separated and isolated from raw milk. It was confirmed that
以上説明した本発明の分子認識高分子を用いた選択性分子認識高分子を用いた選択性透過膜は下記の効果を有する。
１．分子認識高分子を用いた選択性透過膜として、多数の特定抗生物質または有機化合物分子の鋳型分子を包有する極めて薄い分子認識高分子を用いるため、該抗生物質または該有機化合物分子だけが選択的に透過することが可能になり、容易に他の抗生物質または有機化合物から分別・単離できる。
　また、分子認識高分子は、膜厚が１～５マイクロメーターと、極めて薄い膜構成である抗生物質又は有機化合物分子を分別・単離する際に、分子に対する透過機能を長時間に亘って維持することができ、分子認識高分子を用いた選択性透過膜として使い易く、耐久性が大幅に向上している。
２．透過に用いられる抗生物質又は有機化合物分子を分別・単離装置は、図２に示すように、ヒスタミン計測用の電気化学センサと該センサの電位設定と電流測定するためのポテンシオスタットが必要なだけであり、安価に製作でき、操作も容易である。また携帯用に小型化することも可能である。
３．ＭＩＰｓ膜の製作に関しても膜自体が極めて薄いために、材料となるＭＩＰｓ及び溶媒の消費が少量で済み、安価で、且つ製作も容易である。 The selective permeable membrane using the selective molecular recognition polymer using the molecular recognition polymer of the present invention described above has the following effects.
1. As a selective permeable membrane using a molecular recognition polymer, an extremely thin molecular recognition polymer containing a large number of specific antibiotic or organic compound molecule template molecules is used, so that only the antibiotic or organic compound molecule is selective. And can be easily separated and isolated from other antibiotics or organic compounds.
In addition, the molecular recognition polymer maintains a permeation function for a long time when fractionating and isolating antibiotics or organic compound molecules with an extremely thin film thickness of 1 to 5 micrometers. It is easy to use as a selective permeable membrane using a molecular recognition polymer, and the durability is greatly improved.
2. As shown in FIG. 2, an apparatus for separating and isolating antibiotics or organic compound molecules used for permeation requires an electrochemical sensor for histamine measurement, a potential setting of the sensor, and a potentiostat for current measurement. It can be manufactured inexpensively and is easy to operate. It can also be miniaturized for portable use.
3. Regarding the production of the MIPs film, since the film itself is extremely thin, the consumption of MIPs as a material and the solvent is small, and the production is inexpensive and easy.
　なお、本実施形態では、ＭＩＰｓ飽和溶液を作製する溶剤として、アセトニトリルを用いているが、ＭＩＰｓの樹脂構成によっては、アセトン等ケトン類一般、エーテル類、ジメチルホルムアミド、あるいは有機溶媒を含む水溶液等適宜用いることができる。 In this embodiment, acetonitrile is used as a solvent for preparing the MIPs saturated solution. However, depending on the resin configuration of MIPs, ketones such as acetone, ethers, dimethylformamide, or an aqueous solution containing an organic solvent may be used as appropriate. Can be used.
また、多孔質支持体として、ニトロセルロース膜を基材として使用しているが、これに限らず、分別・単離の対象となる特定の化合物分子が充分透過可能な内径を有する孔を多数備えている多孔質の支持体（膜または基板）であれば、アセチルセルロース、セルロースエーテル、セルロースエステルなどを基材として用いることが可能である。
さらに、多孔質支持体の基材として、セルロースの他、各種高分子化合物を用いることができる。高分子化合物としてはフッ素樹脂、ナイロン、ポリエチレン、ポリプロピレン、ポリビニルアルコール、ポリカーボネート、ポリアミド等が該当する。
加えて、多孔質支持体の基材として、無機化合物を用いることもでき、各種グラスファイバー、セラミック、ゼオライト等が含まれる。
分子認識高分子を用いた選択性透過膜の用途や利用方法を勘案し、セルロース、各種合成樹脂、無機化合物のうち、適切な基材を選択することにより、耐久性に富み、分別・単離が容易なＭＩＰｓ-多孔質支持体（膜又は基板）を安価に製作することができるようになる。また、多孔質支持体膜１２として、ニトロセルロース膜を使用しているが、これに限らず、分別・単離の対象となる特定の化合物分子を透過可能な内径を有する微細孔を多数備えている多孔質の支持体膜であれば、アセチルセルロース、酢酸セルロース、硝酸セルロース、硫酸セルロース、セルロースエーテル類、セルロースエステルなどを用いることも可能である。 In addition, a nitrocellulose membrane is used as the base material for the porous support, but the present invention is not limited to this, and a large number of pores having an inner diameter through which a specific compound molecule to be separated and isolated can permeate sufficiently are provided. For example, acetyl cellulose, cellulose ether, cellulose ester, or the like can be used as a base material for the porous support (membrane or substrate).
Furthermore, as a base material of the porous support, various polymer compounds can be used in addition to cellulose. Examples of the polymer compound include fluororesin, nylon, polyethylene, polypropylene, polyvinyl alcohol, polycarbonate, and polyamide.
In addition, an inorganic compound can be used as the base material of the porous support, and various glass fibers, ceramics, zeolites and the like are included.
Considering the use and usage of selective permeable membranes using molecular recognition polymers, it is highly durable and can be separated and isolated by selecting an appropriate base material from cellulose, various synthetic resins, and inorganic compounds. MIPs-porous support (membrane or substrate) can be manufactured at low cost. Moreover, although the nitrocellulose membrane is used as the porous support membrane 12, it is not limited to this, and it has a large number of micropores having an inner diameter capable of permeating specific compound molecules to be separated and isolated. As long as the porous support film is used, acetyl cellulose, cellulose acetate, cellulose nitrate, cellulose sulfate, cellulose ethers, cellulose esters, and the like can be used.
以上説明したように、本発明によれば、分別・単離の対象となる例えば多種類の抗生物質、有機化合物などを含む生物体液、分泌液および多種類の有機化合物を含有する水溶液から、特定の単一種の抗生物質、その他の有機化合物等の化合物分子を容易に分別・単離することができる。

  As described above, according to the present invention, identification is made from biological fluids containing various types of antibiotics, organic compounds, etc., secretion fluids, and aqueous solutions containing various types of organic compounds that are subject to separation and isolation. It is possible to easily separate and isolate compound molecules such as a single kind of antibiotics and other organic compounds.

Claims (6)

  1. 　分別・単離の対象となる特定の化合物分子を充分透過可能な内径を有する孔を多数備えた多孔質支持体上に、分別・単離の対象となる前記特定の化合物分子の鋳型をインプリントした極めて薄いＭＩＰｓ透過膜を形成し、該ＭＩＰｓ透過膜によって試料溶液中の該特定の化合物分子を選択的に透過させ、且つ前記多孔質支持体の孔から支持体側面の溶液中に該化合物分子を透過・移行させるようにしたことを特徴とする分子認識高分子を用いた選択性透過膜。 Imprinting the template of the specific compound molecule to be separated / isolated on a porous support having a large number of pores having an inner diameter that can sufficiently permeate the specific compound molecule to be separated / isolated Forming a very thin MIPs permeable membrane, allowing the specific compound molecules in the sample solution to selectively permeate through the MIPs permeable membrane, and the compound molecules from the pores of the porous support to the solution on the side of the support A permselective membrane using a molecule-recognizing polymer, characterized in that it permeates and migrates.
  2. 　前記多孔質支持体の基材はニトロセルロース、又はその他のセルロース誘導体であることを特徴とする請求項１に記載の分子認識高分子を用いた選択性透過膜。 The selective permeable membrane using a molecular recognition polymer according to claim 1, wherein the substrate of the porous support is nitrocellulose or other cellulose derivative.
  3. 前記多孔質支持体の基材はフッ素、ナイロン、ポリエチレン、ポリプロピレン、ポリビニルアルコール、ポリカーボネート、ポリアミド等の各種合成樹脂であることを特徴とする請求項１に記載の分子認識高分子を用いた選択性透過膜。 2. The selectivity using a molecular recognition polymer according to claim 1, wherein the substrate of the porous support is various synthetic resins such as fluorine, nylon, polyethylene, polypropylene, polyvinyl alcohol, polycarbonate, and polyamide. Permeable membrane.
  4. 前記多孔質支持体の基材は各種グラスファイバー、セラミック、ゼオライト等、各種無機化合物であることを特徴とする請求項１記載の分子認識高分子を用いた選択性透過膜。 2. The selective permeable membrane using a molecular recognition polymer according to claim 1, wherein the substrate of the porous support is various inorganic compounds such as various glass fibers, ceramics, zeolites and the like.
  5. 　前記ＭＩＰｓ透過膜は、膜厚が１～５マイクロメーターであることを特徴とする請求項１～４のうち、いずれか１項に記載の分子認識高分子を用いた選択性透過膜。 5. The selective permeable membrane using a molecular recognition polymer according to any one of claims 1 to 4, wherein the MIPs permeable membrane has a thickness of 1 to 5 micrometers.
  6. 分別・単離の対象となる特定の化合物分子を充分透過可能な内径を有する孔を多数備えた多孔質支持体上に、前記特定の化合物分子の鋳型を含むポリマーの有機溶媒溶液を塗布する工程と、前記ポリマーの有機溶媒溶液を蒸発乾燥させて、極めて薄いＭＩＰｓ膜を形成する工程とを具備したことを特徴とする分子認識高分子を用いた選択性透過膜の製造方法。
    　　　　　　　　　　　     A step of applying an organic solvent solution of a polymer containing a template of the specific compound molecule on a porous support having a large number of pores having an inner diameter that can sufficiently permeate the specific compound molecule to be separated and isolated And a method of evaporating and drying an organic solvent solution of the polymer to form an extremely thin MIPs membrane, and a method for producing a selective permeable membrane using a molecular recognition polymer.
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JP2890469B2 (en) * 1989-05-25 1999-05-17 東レ株式会社 Method for producing porous separation membrane
JP2005205333A (en) * 2004-01-23 2005-08-04 Hokuetsu Paper Mills Ltd Sheet having molecular adsorbing function
JP2005264013A (en) * 2004-03-19 2005-09-29 Japan Science & Technology Agency Polymer composition having a function of molecular recognition/capturing and manufacturing process of polymer molding using the composition
JP2005533146A (en) * 2002-07-13 2005-11-04 クランフィールド ユニヴァーシティー Molecularly imprinted polymer material
JP3921530B2 (en) * 2002-10-30 2007-05-30 独立行政法人産業技術総合研究所 DNA molecule identification material
JP2008508952A (en) * 2004-08-04 2008-03-27 アスピラ バイオシステムズ， インコーポレイテッド Capturing and removing biomolecules from body fluids using partial molecular imprinting
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2890469B2 (en) * 1989-05-25 1999-05-17 東レ株式会社 Method for producing porous separation membrane
JP2005533146A (en) * 2002-07-13 2005-11-04 クランフィールド ユニヴァーシティー Molecularly imprinted polymer material
JP3921530B2 (en) * 2002-10-30 2007-05-30 独立行政法人産業技術総合研究所 DNA molecule identification material
JP2005205333A (en) * 2004-01-23 2005-08-04 Hokuetsu Paper Mills Ltd Sheet having molecular adsorbing function
JP2005264013A (en) * 2004-03-19 2005-09-29 Japan Science & Technology Agency Polymer composition having a function of molecular recognition/capturing and manufacturing process of polymer molding using the composition
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