JP7472609B2 - Electrode for enzyme sensor and enzyme sensor - Google Patents
Electrode for enzyme sensor and enzyme sensor Download PDFInfo
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- JP7472609B2 JP7472609B2 JP2020066574A JP2020066574A JP7472609B2 JP 7472609 B2 JP7472609 B2 JP 7472609B2 JP 2020066574 A JP2020066574 A JP 2020066574A JP 2020066574 A JP2020066574 A JP 2020066574A JP 7472609 B2 JP7472609 B2 JP 7472609B2
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- Apparatus Associated With Microorganisms And Enzymes (AREA)
Description
本発明は、酵素センサー用電極及び酵素センサーに関する。 The present invention relates to an electrode for an enzyme sensor and an enzyme sensor.
血液や汗等の生体試料や食品等に含まれる特定成分を、簡便に計測する酵素センサーが実用化されている。例えば、血液中のグルコースを電気化学的な手段により検出、あるいは定量化する血糖値センサー等が挙げられる。これは血中に含まれるグルコースに対し酵素の基質特異性により選択的に酸化し、メディエーターを介して、あるいは直接電極に電荷が到達して電流が発生、その電流値あるいは電荷量からグルコース濃度を定量することができる。
実用化されている酵素センサーにおける電極部分は、スパッタやめっき等により金属層が形成されたものが用いられることがある（例えば特許文献１）。しかし、その測定感度は未だ十分とは言い難い状況であり、高価な金属を使用しないことは腐食やコストの観点からも好ましい。
Enzyme sensors that can easily measure specific components contained in biological samples such as blood and sweat, and in foods, etc., have been put to practical use. For example, there is a blood glucose sensor that detects or quantifies glucose in blood by electrochemical means. In this sensor, the glucose contained in blood is selectively oxidized by the substrate specificity of the enzyme, and a current is generated when a charge reaches an electrode via a mediator or directly, and the glucose concentration can be quantified from the current value or charge amount.
In the electrode portion of enzyme sensors that have been put to practical use, a metal layer is sometimes formed by sputtering, plating, or the like (see, for example, Patent Document 1). However, the measurement sensitivity is still far from sufficient, and not using expensive metals is preferable from the viewpoints of corrosion and cost.
本発明の目的は、感度に優れる酵素センサー用電極および酵素センサーを提供することである。 The object of the present invention is to provide an enzyme sensor electrode and an enzyme sensor with excellent sensitivity.
本発明者らは、前記の諸問題を解決するために研究を重ねた結果、本発明に至った。
すなわち本発明は、基材上に設置された、作用極と対極を有する酵素センサー用電極であって、少なくとも作用極が導電性炭素材料を含みかつ水に対する接触角が１１０度以下である酵素センサー用電極に関する。
The present inventors have conducted extensive research to solve the above-mentioned problems and have arrived at the present invention.
In other words, the present invention relates to an electrode for an enzyme sensor having a working electrode and a counter electrode placed on a substrate, in which at least the working electrode contains a conductive carbon material and has a contact angle with water of 110 degrees or less.
また、本発明は作用極中に含まれる導電性炭素材料の比率が６０％以上である前記酵素センサー用電極に関する。 The present invention also relates to an electrode for an enzyme sensor, in which the ratio of conductive carbon material contained in the working electrode is 60% or more.
また、本発明は、導電性炭素材料が少なくとも黒鉛を含む前記酵素センサー用電極に関する。 The present invention also relates to an electrode for an enzyme sensor, in which the conductive carbon material contains at least graphite.
また、本発明は、前記電極中に親水性化合物を含む前記酵素センサー用電極に関する。 The present invention also relates to an electrode for an enzyme sensor, the electrode containing a hydrophilic compound.
また、本発明は、親水性化合物が、ポリアクリル酸、ポリビニルアルコール、ポリビニルピロリドン、カルボキシメチルセルロース、ヒドロキシエチルセルロース、界面活性剤の中から少なくとも一種を含む前記酵素センサー用電極に関する。 The present invention also relates to the enzyme sensor electrode, in which the hydrophilic compound contains at least one of polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethyl cellulose, hydroxyethyl cellulose, and a surfactant.
また、本発明は、更に酵素および／またはメディエーターを含む、前記酵素センサー用電極を用いた酵素センサーに関する。 The present invention also relates to an enzyme sensor using the enzyme sensor electrode, which further contains an enzyme and/or a mediator.
本発明によれば、導電性炭素材料を含む酵素センサー用電極において、センシングの応答性に優れかつ感度の良好な酵素センサーを提供することができる。更に、主に炭素材料から構成される電極であるため、腐食に強く低コストで使い捨て可能な電極を提供することができる。 According to the present invention, an enzyme sensor having excellent sensing response and good sensitivity can be provided using an electrode for an enzyme sensor containing a conductive carbon material. Furthermore, since the electrode is mainly composed of a carbon material, it is possible to provide an electrode that is resistant to corrosion, low-cost, and disposable.
本発明の酵素センサー用電極は、少なくとも作用極と対極とを有し、必要に応じて参照極を有していてもよい。これらの電極は、基材上に形成された導電層からなり、例えば、基材に、導電性炭素材料とバインダーと、必要に応じて溶剤等を含有する酵素センサー電極形成用組成物を塗工、必要に応じてプレス処理等を行って、導電層を形成することで得ることができる。また、必要に応じて酸化還元酵素やメディエーターを担持して電極を得ることができる。
本発明の酵素センサー用電極は、少なくとも作用極を構成する導電層が導電性炭素材料を含みかつ水に対する接触角が１１０度以下である。
The enzyme sensor electrode of the present invention has at least a working electrode and a counter electrode, and may have a reference electrode as necessary. These electrodes are made of a conductive layer formed on a substrate, and can be obtained, for example, by coating a substrate with a composition for forming an enzyme sensor electrode, which contains a conductive carbon material, a binder, and, if necessary, a solvent, and the like, and then performing a pressing process or the like as necessary to form a conductive layer. In addition, an electrode can be obtained by supporting an oxidoreductase or a mediator as necessary.
In the electrode for an enzyme sensor of the present invention, at least the conductive layer constituting the working electrode contains a conductive carbon material and has a contact angle with water of 110 degrees or less.
＜導電層＞
（導電性炭素材料）
本発明において、測定に有効な表面積が大きく、耐腐食性に優れ、更に使い捨てや生体適合性等の観点から、導電性材料として導電性の炭素材料を用いる。導電性の炭素材料としては、導電性に優れた導電層を得られることから、黒鉛、カーボンブラック、導電性炭素繊維（カーボンナノチューブ、カーボンナノファイバー、カーボンファイバー）、グラフェン、フラーレン等が挙げられる。これらを単独で、もしくは２種類以上併せて使用することが出来る。導電性炭素材料は、黒鉛を含むことが好ましく、特に、作用極および／または対極の導電層は黒鉛を含むことが好ましい。
<Conductive Layer>
(Conductive carbon material)
In the present invention, a conductive carbon material is used as the conductive material from the viewpoints of a large surface area effective for measurement, excellent corrosion resistance, disposability, biocompatibility, etc. Examples of the conductive carbon material include graphite, carbon black, conductive carbon fiber (carbon nanotube, carbon nanofiber, carbon fiber), graphene, fullerene, etc., because they can provide a conductive layer with excellent conductivity. These can be used alone or in combination of two or more kinds. The conductive carbon material preferably contains graphite, and in particular, the conductive layer of the working electrode and/or the counter electrode preferably contains graphite.
黒鉛としては、例えば人造黒鉛や天然黒鉛等を使用することが出来る。人造黒鉛としては、無定形炭素の熱処理により、不規則な配列の微小黒鉛結晶の配向を人工的に行わせたものであり、一般的には石油コークスや石炭系ピッチコークスを主原料として製造される。天然黒鉛としては、球形黒鉛、鱗片状黒鉛、塊状黒鉛、土状黒鉛等を使用することが出来る。また、鱗片状黒鉛を化学処理等した膨張黒鉛（膨張性黒鉛ともいう）や、膨張黒鉛を熱処理して膨張化させた後、微細化やプレスにより得られた膨張化黒鉛等を使用することも出来る。これらの黒鉛の中でも、導電性基材の導電層に用いる場合は、導電性の観点から、天然黒鉛が好ましく、球形黒鉛、鱗片状黒鉛、膨張化黒鉛、および薄片化黒鉛等の薄片状黒鉛が好ましい。 As graphite, for example, artificial graphite and natural graphite can be used. Artificial graphite is made by artificially aligning irregularly arranged micrographite crystals by heat treatment of amorphous carbon, and is generally produced using petroleum coke or coal-based pitch coke as the main raw material. As natural graphite, spherical graphite, scaly graphite, lump graphite, clay-like graphite, etc. can be used. In addition, expanded graphite (also called expandable graphite) obtained by chemically treating scaly graphite, and expanded graphite obtained by heat treating expanded graphite to expand it and then refining or pressing it, etc. can also be used. Among these graphites, when used in the conductive layer of the conductive substrate, natural graphite is preferred from the viewpoint of conductivity, and flaky graphite such as spherical graphite, scaly graphite, expanded graphite, and flaky graphite are preferred.
また、用いる黒鉛の平均粒径は、０．５～５００μｍが好ましく、特に、２～１００μｍが好ましい。 The average particle size of the graphite used is preferably 0.5 to 500 μm, and more preferably 2 to 100 μm.
本発明でいう平均粒径とは、体積粒度分布において、粒子径の細かいものからその粒子の体積割合を積算していったときに、５０％となるところの粒子径（Ｄ５０）であり、一般的な粒度分布計、例えば、動的光散乱方式の粒度分布計（日機装社製「マイクロトラックＵＰＡ」）等で測定される。 The average particle size in this invention is the particle size (D50) at which the volumetric percentage of particles is 50% when the volumetric percentage of particles is accumulated from the finest particle size in the volumetric particle size distribution, and is measured using a general particle size distribution meter, such as a dynamic light scattering particle size distribution meter (Microtrack UPA, manufactured by Nikkiso Co., Ltd.).
市販の黒鉛としては、例えば、薄片状黒鉛として、日本黒鉛工業社製のＣＭＸ、ＵＰ－５、ＵＰ－１０、ＵＰ－２０、ＵＰ－３５Ｎ、ＣＳＳＰ、ＣＳＰＥ、ＣＳＰ、ＣＰ、ＣＢ－１５０、ＣＢ－１００、ＡＣＰ、ＡＣＰ－１０００、ＡＣＢ－５０、ＡＣＢ－１００、ＡＣＢ－１５０、ＳＰ－１０、ＳＰ－２０、Ｊ－ＳＰ、ＳＰ－２７０、ＨＯＰ、ＧＲ－６０、ＬＥＰ、Ｆ＃１、Ｆ＃２、Ｆ＃３、中越黒鉛社製のＣＸ－３０００、ＦＢＦ、ＢＦ、ＣＢＲ、ＳＳＣ－３０００、ＳＳＣ－６００、ＳＳＣ－３、ＳＳＣ、ＣＸ－６００、ＣＰＦ－８、ＣＰＦ－３、ＣＰＢ－６Ｓ、ＣＰＢ、９６Ｅ、９６Ｌ、９６Ｌ－３、９０Ｌ－３、ＣＰＣ、Ｓ－８７、Ｋ－３、ＣＦ－８０、ＣＦ－４８、ＣＦ－３２、ＣＰ－１５０、ＣＰ－１００、ＣＰ、ＨＦ－８０、ＨＦ－４８、ＨＦ－３２、ＳＣ－１２０、ＳＣ－８０、ＳＣ－６０、ＳＣ－３２、伊藤黒鉛工業社製のＥＣ１５００、ＥＣ１０００、ＥＣ５００、ＥＣ３００、ＥＣ１００、ＥＣ５０、西村黒鉛社製の１００９９Ｍ、ＰＢ－９９等が挙げられる。球形黒鉛としては、日本黒鉛工業社製のＣＧＣ－２０、ＣＧＣ－５０、ＣＧＢ－２０、ＣＧＢ－５０が挙げられる。土状黒鉛としては、日本黒鉛工業社製の青Ｐ、ＡＰ、ＡＯＰ、Ｐ＃１、中越黒鉛社製のＡＰＲ、Ｓ－３、ＡＰ－６、３００Ｆが挙げられる。人造黒鉛としては、日本黒鉛工業社製のＰＡＧ－６０、ＰＡＧ－８０、ＰＡＧ－１２０、ＰＡＧ－５、ＨＡＧ－１０Ｗ、ＨＡＧ－１５０、中越黒鉛社製のＲＡ－３０００、ＲＡ－１５、ＲＡ－４４、ＧＸ－６００、Ｇ－６Ｓ、Ｇ－３、Ｇ－１５０、Ｇ－１００、Ｇ－４８、Ｇ－３０、Ｇ－５０、ＳＥＣカーボン社製のＳＧＰ－１００、ＳＧＰ－５０、ＳＧＰ－２５、ＳＧＰ－１５、ＳＧＰ－５、ＳＧＰ－１、ＳＧＯ－１００、ＳＧＯ－５０、ＳＧＯ－２５、ＳＧＯ－１５、ＳＧＯ－５、ＳＧＯ－１、ＳＧＸ－１００、ＳＧＸ－５０、ＳＧＸ－２５、ＳＧＸ－１５、ＳＧＸ－５、ＳＧＸ－１が挙げられる。 Examples of commercially available graphite include flake graphite such as CMX, UP-5, UP-10, UP-20, UP-35N, CSSP, CSPE, CSP, CP, CB-150, CB-100, ACP, ACP-1000, ACB-50, ACB-100, ACB-150, SP-10, SP-20, J-SP, SP-270, HOP, GR-60, LEP, F#1, F#2, and F#3 manufactured by Nippon Graphite Industries Co., Ltd.; and CX-3000, FBF, BF, CBR, SSC-3000, SSC-600, and SS manufactured by Chuetsu Graphite Co., Ltd. Examples of the graphite include C-3, SSC, CX-600, CPF-8, CPF-3, CPB-6S, CPB, 96E, 96L, 96L-3, 90L-3, CPC, S-87, K-3, CF-80, CF-48, CF-32, CP-150, CP-100, CP, HF-80, HF-48, HF-32, SC-120, SC-80, SC-60, SC-32, EC1500, EC1000, EC500, EC300, EC100, EC50 manufactured by Ito Graphite Industries Co., Ltd., 10099M and PB-99 manufactured by Nishimura Graphite Co., Ltd. Examples of the spherical graphite include CGC-20, CGC-50, CGB-20, and CGB-50 manufactured by Nippon Graphite Industries Co., Ltd. Examples of amorphous graphite include Ao-P, AP, AOP, and P#1 manufactured by Nippon Graphite Industries Co., Ltd., and APR, S-3, AP-6, and 300F manufactured by Chuetsu Graphite Co., Ltd. Examples of artificial graphite include PAG-60, PAG-80, PAG-120, PAG-5, HAG-10W, and HAG-150 manufactured by Nippon Graphite Industries Co., Ltd.; RA-3000, RA-15, RA-44, GX-600, G-6S, G-3, G-150, G-100, G-48, G-30, and G-50 manufactured by Chuetsu Graphite Co., Ltd.; and SGP-100, SGP-50, SGP-25, SGP-15, SGP-5, SGP-1, SGO-100, SGO-50, SGO-25, SGO-15, SGO-5, SGO-1, SGX-100, SGX-50, SGX-25, SGX-15, SGX-5, and SGX-1 manufactured by SEC Carbon Co., Ltd.
黒鉛以外の導電性の炭素材料は特に限定されないが、コストや導電性などの観点から、カーボンブラックや導電性炭素繊維を用いることが好ましい。 There are no particular limitations on the conductive carbon material other than graphite, but from the standpoint of cost and conductivity, it is preferable to use carbon black or conductive carbon fiber.
カーボンブラックとしては、気体もしくは液体の原料を反応炉中で連続的に熱分解し製造するファーネスブラック、特にエチレン重油を原料としたケッチェンブラック、原料ガスを燃焼させて、その炎をチャンネル鋼底面にあて急冷し析出させたチャンネルブラック、ガスを原料とし燃焼と熱分解を周期的に繰り返すことにより得られるサーマルブラック、特にアセチレンガスを原料とするアセチレンブラックなどの各種のものを単独で、もしくは２種類以上併せて使用することができる。また、通常行われている酸化処理されたカーボンブラックや、中空カーボン等も使用できる。
カーボンの酸化処理は、カーボンを空気中で高温処理したり、硝酸や二酸化窒素、オゾン等で二次的に処理したりすることより、例えばフェノール基、キノン基、カルボキシル基、カルボニル基の様な酸素含有極性官能基をカーボン表面に直接導入（共有結合）する処理であり、カーボンの分散性を向上させるために一般的に行われている。
Carbon black may be any of a variety of types, such as furnace black, which is produced by continuously pyrolyzing a gaseous or liquid raw material in a reactor, particularly ketjen black, which is made from ethylene heavy oil, channel black, which is produced by burning a raw material gas and applying the flame to the bottom surface of a channel steel to rapidly cool and precipitate, thermal black, which is obtained by periodically repeating combustion and pyrolysis of a gas raw material, particularly acetylene black, which is made from acetylene gas, and may be used alone or in combination of two or more types. Conventionally oxidized carbon black and hollow carbon may also be used.
Carbon oxidation is a process in which oxygen-containing polar functional groups such as phenol groups, quinone groups, carboxyl groups, and carbonyl groups are directly introduced (covalently bonded) to the carbon surface by treating the carbon at high temperatures in air or by secondary treatment with nitric acid, nitrogen dioxide, ozone, etc., and is commonly performed to improve the dispersibility of carbon.
用いるカーボンブラックの比表面積は、値が大きいほど、カーボンブラック粒子どうしの接触点が増えるため、電極の内部抵抗を下げるのに有利となる。具体的には、窒素の吸着量から求められる比表面積（ＢＥＴ）で、２０ｍ２／ｇ以上、好ましくは５０ｍ２／ｇ以上、更に好ましくは１００ｍ２／ｇ以上のものを使用することが望ましい。比表面積が２０ｍ２／ｇを下回るカーボンブラックを用いると、十分な導電性を得ることが難しくなる場合がある。
また、用いるカーボンブラックの粒径は、一次粒子径で０．００５～１μｍが好ましく、特に、０．０１～０．２μｍが好ましい。ただし、ここでいう一次粒子径とは、電子顕微鏡などで測定された粒子径を平均したものである。
The larger the specific surface area of the carbon black used, the more contact points between the carbon black particles there are, which is advantageous for lowering the internal resistance of the electrode. Specifically, it is desirable to use carbon black with a specific surface area (BET) calculated from the amount of nitrogen adsorption of 20 m 2 /g or more, preferably 50 m 2 /g or more, and more preferably 100 m 2 /g or more. If carbon black with a specific surface area of less than 20 m 2 /g is used, it may be difficult to obtain sufficient conductivity.
The particle size of the carbon black used is preferably 0.005 to 1 μm, particularly preferably 0.01 to 0.2 μm, in terms of primary particle size, where the primary particle size is the average particle size measured with an electron microscope or the like.
市販のカーボンブラックとしては、例えば、東海カーボン社製のトーカブラック＃４３００、＃４４００、＃４５００、＃５５００、デグサ社製のプリンテックスＬ、コロンビヤン社製のＲａｖｅｎ７０００、５７５０、５２５０、５０００ＵＬＴＲＡIII、５０００ＵＬＴＲＡ、ＣｏｎｄｕｃｔｅｘＳＣ ＵＬＴＲＡ、Ｃｏｎｄｕｃｔｅｘ ９７５ ＵＬＴＲＡ、ＰＵＥＲＢＬＡＣＫ１００、１１５、２０５、三菱化学社製の＃２３５０、＃２４００Ｂ、＃２６００Ｂ、＃３０５０Ｂ、＃３０３０Ｂ、＃３２３０Ｂ、＃３３５０Ｂ、＃３４００Ｂ、＃５４００Ｂ、キャボット社製のＭＯＮＡＲＣＨ１４００、１３００、９００、ＶｕｌｃａｎＸＣ－７２Ｒ、ＢｌａｃｋＰｅａｒｌｓ２０００、イメリス・グラファイト＆カーボン社製のＥｎｓａｃｏ２５０Ｇ、Ｅｎｓａｃｏ２６０Ｇ、Ｅｎｓａｃｏ３５０Ｇ、ＳｕｐｅｒＰ－Ｌｉ等のファーネスブラック、ライオン・スペシャリティ・ケミカルズ社製のＥＣ－２００Ｌ、ＥＣ－３００Ｊ、ＥＣ－６００ＪＤ等のケッチェンブラック、デンカ社製のデンカブラック、デンカブラックＨＳ－１００、ＦＸ－３５等のアセチレンブラックが挙げられるが、これらに限定されるものではなく、２種以上を組み合わせて用いても良い。 Examples of commercially available carbon black include Tokai Carbon's Toka Black #4300, #4400, #4500, and #5500; Degussa's Printex L; and Colombian's Raven 7000, 5750, 5250, 5000 ULTRA III, 5000 ULTRA, Conductex SC ULTRA, and Conductex 975. ULTRA, PUERBLACK 100, 115, 205, Mitsubishi Chemical Corporation #2350, #2400B, #2600B, #3050B, #3030B, #3230B, #3350B, #3400B, #5400B, Cabot Corporation MONARCH 1400, 1300, 900, Vulcan XC-72R, BlackPearls 2000, Imerys Graphite & Carbon Corporation Ensaco 250G, E Examples of such blacks include furnace blacks such as Ensaco 260G, Ensaco 350G, and Super P-Li; ketjen blacks such as EC-200L, EC-300J, and EC-600JD manufactured by Lion Specialty Chemicals; and acetylene blacks such as Denka Black, Denka Black HS-100, and FX-35 manufactured by Denka Co., Ltd., but are not limited to these, and two or more types may be used in combination.
導電性炭素繊維としては石油由来の原料から焼成して得られるものが良いが、植物由来の原料からも焼成して得られるものも用いることが出来る。また、カーボンナノチューブには、グラフェンシートが一層でナノメートル領域の直径を有するチューブを形成する単層カーボンナノチューブと、グラフェンシートが多層である多層カーボンナノチューブがある。そのため、多層カーボンナノチューブの直径は、典型的な単層カーボンナノチューブの０．７－２．０ｎｍに対して、３０ｎｍと大きい値を示す。 Conductive carbon fibers obtained by firing petroleum-derived raw materials are preferable, but those obtained by firing plant-derived raw materials can also be used. Carbon nanotubes are classified into single-walled carbon nanotubes, which form tubes with a diameter in the nanometer range from a single graphene sheet, and multi-walled carbon nanotubes, which have multiple graphene sheets. Therefore, the diameter of multi-walled carbon nanotubes is large, at 30 nm, compared to the 0.7-2.0 nm of typical single-walled carbon nanotubes.
市販の導電性炭素繊維やカーボンナノチューブとしては、昭和電工社製のＶＧＣＦ等の気相法炭素繊維、名城ナノカーボン社製のＥＣ１．０，ＥＣ１．５，ＥＣ２．０，ＥＣ１．５－Ｐ等の単層カーボンナノチューブ、ＣＮａｎｏ社製のＦｌｏＴｕｂｅ９０００、ＦｌｏＴｕｂｅ９１００、ＦｌｏＴｕｂｅ９１１０、ＦｌｏＴｕｂｅ９２００、Ｎａｎｏｃｙｌ社製のＮＣ７０００、Ｋｎａｎｏ社製の１００Ｔ等が挙げられる。 Commercially available conductive carbon fibers and carbon nanotubes include vapor grown carbon fibers such as VGCF manufactured by Showa Denko K.K., single-walled carbon nanotubes such as EC1.0, EC1.5, EC2.0, and EC1.5-P manufactured by Meijo Nano Carbon Co., Ltd., FloTube 9000, FloTube 9100, FloTube 9110, and FloTube 9200 manufactured by CNano Corporation, NC7000 manufactured by Nanocyl Corporation, and 100T manufactured by Knano Corporation.
導電層中に含まれる導電性炭素の比率は、導電性や感度の観点から６０％以上含有していることが好ましく、更には７０％以上が好ましい、また更には７５％以上が好ましい。特に、作用極を構成する導電層に含まれる導電性炭素材料の比率が６０％以上であることが好ましい。 From the viewpoint of conductivity and sensitivity, the ratio of conductive carbon contained in the conductive layer is preferably 60% or more, more preferably 70% or more, and even more preferably 75% or more. In particular, it is preferable that the ratio of conductive carbon material contained in the conductive layer constituting the working electrode is 60% or more.
＜親水化処理＞
作用極の導電層の接触角を１１０度以下にする方法、すなわち親水性を付与するための方法は特に限定されないが、導電層を親水化処理する方法、あるいは親水性化合物を用いる方法が挙げられる。親水化処理としては、プラズマ処理、オゾン処理、ＵＶや電子線によるラジカル活性化処理等の表面改質等が挙げられる。
また、親水性化合物を含んだ電極や基材に対して更に親水化処理を行ってもよい。親水化処理は、電極を設置する前後は問わず、いずれの工程で実施してもよい。
更に対極、参照極、および前記電極間の基材表面の少なくとも一部が親水性であることが好ましい。
接触角は、１１０度以下が好ましい。更に好ましくは、４０度～１１０度である。
<Hydrophilic Treatment>
The method for making the contact angle of the conductive layer of the working electrode 110 degrees or less, i.e., the method for imparting hydrophilicity, is not particularly limited, but examples thereof include a method of subjecting the conductive layer to hydrophilic treatment or a method using a hydrophilic compound. Examples of hydrophilic treatment include surface modification such as plasma treatment, ozone treatment, and radical activation treatment using UV or electron beams.
Furthermore, the electrodes and the substrate containing the hydrophilic compound may be further subjected to a hydrophilization treatment. The hydrophilization treatment may be performed in any step, regardless of whether it is before or after the electrodes are provided.
Furthermore, it is preferable that at least a part of the surface of the counter electrode, the reference electrode, and the base material between the electrodes is hydrophilic.
The contact angle is preferably 110 degrees or less, and more preferably 40 degrees to 110 degrees.
（親水性化合物）
親水性化合物を用いる方法としては特に限定されないが、少なくとも作用極に親水性化合物が含まれることが好ましく、更に好ましくは対極や参照極、また更には作用極－対極－参照極の間の基材に存在することが好ましい。これにより、水あるいはイオン伝導体やセンシング対象等を含む水溶液が浸潤しやすくなり、酵素センサーの起動や安定化が容易になる。親水性化合物を電極や基材に含ませる方法としては、電極を形成する組成物に予め混合し塗工・乾燥する方法、電極を形成してから親水性化合物を含有する溶液を電極や基材に滴下・乾燥する方法等が挙げられる。
親水性化合物としては水溶性低分子化合物や水溶性高分子等の水溶性化合物、親水性粒子が挙げられ、単独でも併用して用いてもよい。
水溶性低分子化合物としては、水に溶解するものであれば特に限定はなく、界面活性剤、グルコース等の糖、クエン酸等の酸、塩化ナトリウム等の塩が例として挙げられる。
水溶性高分子としては、ポリアクリル酸、ポリビニルアルコール、ポリビニルピロリドン、カルボキシメチルセルロース、ヒドロキシエチルセルロース等が例として挙げられ、更に共重合体でもよい。また、水溶性高分子は水に対し完全に溶解しなくともよく、水を含むゲルとなるもの等も適用できる。
親水性粒子としては、ポリスチレン、シリカ、アルミナ等の微粒子をスルホン化等の親水処理を施したものが例として挙げられる。
親水性化合物は、センサーとしての測定に影響を及ぼし難いものが好ましく、ポリアクリル酸、ポリビニルアルコール、ポリビニルピロリドン、カルボキシメチルセルロース、ヒドロキシエチルセルロース、界面活性剤が好ましい。特に応答性を高めるために分子量は小さいものが好ましく、平均分子量で１０００００以下が好ましい。
前記の通り、導電層においては導電性炭素の比率は高い方が好ましいが、一方で測定対象を含む電解液の電極表面および内部への濡れが難しくなる。ＵＶ照射など電極形成後の表面改質は特に電極内部を均一に親水化することは難しいが、親水性化合物を予め電極形成用組成物に混合・分散する場合は、電極内部にも均質な親水性を得ることができる。
(hydrophilic compound)
Although the method of using the hydrophilic compound is not particularly limited, it is preferable that the hydrophilic compound is contained at least in the working electrode, and more preferably in the counter electrode or reference electrode, or further in the substrate between the working electrode, counter electrode, and reference electrode. This makes it easier for water or an aqueous solution containing an ion conductor or a sensing target to infiltrate, making it easier to start and stabilize the enzyme sensor. Examples of methods for incorporating the hydrophilic compound into the electrode or substrate include a method in which the hydrophilic compound is mixed in advance with a composition that forms the electrode and then coated and dried, and a method in which a solution containing the hydrophilic compound is dropped onto the electrode or substrate after forming the electrode and then dried.
Examples of the hydrophilic compound include water-soluble compounds such as water-soluble low molecular weight compounds and water-soluble polymers, and hydrophilic particles, and these may be used alone or in combination.
The water-soluble low molecular weight compound is not particularly limited as long as it dissolves in water, and examples thereof include surfactants, sugars such as glucose, acids such as citric acid, and salts such as sodium chloride.
Examples of the water-soluble polymer include polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethyl cellulose, hydroxyethyl cellulose, etc., and may further be copolymers. In addition, the water-soluble polymer does not have to be completely dissolved in water, and those that form gels containing water can also be used.
Examples of hydrophilic particles include fine particles of polystyrene, silica, alumina, etc., which have been subjected to hydrophilic treatment such as sulfonation.
The hydrophilic compound is preferably one that does not easily affect the measurement as a sensor, and is preferably polyacrylic acid, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethyl cellulose, hydroxyethyl cellulose, or a surfactant. In particular, in order to increase the responsiveness, it is preferable that the molecular weight is small, and the average molecular weight is preferably 100,000 or less.
As described above, the conductive layer preferably has a high ratio of conductive carbon, but on the other hand, it becomes difficult for the electrolyte solution containing the object to be measured to wet the surface and inside of the electrode. Surface modification after electrode formation, such as UV irradiation, is particularly difficult to make the inside of the electrode uniformly hydrophilic, but when a hydrophilic compound is mixed and dispersed in advance in the electrode-forming composition, uniform hydrophilicity can be obtained even inside the electrode.
（バインダー）
バインダーの種類は、導電性炭素材料の分散性や基材および炭素材料への密着性、基材の可とう性および導電性組成物の安定性を付与できるものであれば特に制限されず、樹脂等が挙げられる。
(binder)
The type of binder is not particularly limited as long as it can impart dispersibility of the conductive carbon material, adhesion to the substrate and the carbon material, flexibility of the substrate, and stability of the conductive composition, and examples of the binder include resins.
バインダーとしては、ポリウレタン系樹脂、ポリアミド系樹脂、アクリロニトリル系樹脂、アクリル系樹脂、ブタジエン系樹脂、ポリビニル系樹脂、ポリビニルブチラール系樹脂、ポリオレフィン系樹脂、ポリエステル系樹脂、ポリスチレン系樹脂、ＥＶＡ系樹脂、ポリフッ化ビニリデン系樹脂、ポリテトラフルオロエチレン系樹脂、シリコーン系樹脂、ポリエーテル系樹脂、カルボキシメチルセルロース等のセルロース系樹脂等からなる群から選ばれる１種類以上を含むことができる。ただし、これらの樹脂に限定されるわけではない。バインダー樹脂は１種単独で用いても良く、２種以上併用しても良い。 The binder may include one or more types selected from the group consisting of polyurethane resins, polyamide resins, acrylonitrile resins, acrylic resins, butadiene resins, polyvinyl resins, polyvinyl butyral resins, polyolefin resins, polyester resins, polystyrene resins, EVA resins, polyvinylidene fluoride resins, polytetrafluoroethylene resins, silicone resins, polyether resins, cellulose resins such as carboxymethyl cellulose, etc. However, the binder is not limited to these resins. One type of binder resin may be used alone, or two or more types may be used in combination.
バインダー樹脂は、バインダー樹脂が基材に適用された後に、硬化（架橋）反応を受ける、硬化性樹脂を用いることもできる。
バインダー樹脂は、水系または非水系溶剤に溶解する溶解性樹脂や分散型樹脂微粒子を用いることもできる。分散型樹脂微粒子は、樹脂微粒子が水系または非水系の分散媒中で溶解せずに、微粒子の状態で存在するもので、その分散体は、一般的にエマルジョンとも呼ばれる。これらは１種単独で用いても良く、２種以上併用しても良い。
The binder resin may be a curable resin that undergoes a curing (crosslinking) reaction after the binder resin is applied to a substrate.
The binder resin may be a soluble resin that dissolves in an aqueous or non-aqueous solvent or a dispersion-type resin fine particle. Dispersion-type resin fine particles are those in which the resin fine particles are not dissolved in an aqueous or non-aqueous dispersion medium and exist in the form of fine particles, and the dispersion is generally called an emulsion. These may be used alone or in combination of two or more.
分散型樹脂微粒子の粒子構造は、多層構造、いわゆるコアシェル粒子にすることもできる。例えば、コア部、またはシェル部に官能基を有する単量体を主に重合させた樹脂を局在化させたり、コアとシェルによってＴｇや組成に差を設けたりすることにより、硬化性、乾燥性、成膜性、バインダーの機械強度を向上させることができる。
樹脂微粒子の平均粒子径は、結着性や粒子の安定性の観点から、１０～１０００ｎｍであることが好ましく、１０～３００ｎｍであることが好ましい。なお、本発明における平均粒子径とは、体積平均粒子径のことを表し、動的光散乱法により測定できる。
動的光散乱法による平均粒子径の測定は、以下のようにして行うことができる。樹脂微粒子の固形分に応じて、分散液と同じ分散媒で２００～１０００倍に希釈しておく。該希釈分散液約５ｍｌを測定装置（日機装社製マイクロトラック）のセルに注入し、サンプルに応じた分散媒および樹脂の屈折率条件を入力後、測定を行う。この時得られた体積粒子径分布データ（ヒストグラム）のピークによって測定することができる。
The particle structure of the dispersion type resin fine particles can be a multi-layer structure, i.e., a so-called core-shell particle. For example, by localizing a resin obtained by mainly polymerizing a monomer having a functional group in the core or shell, or by providing a difference in Tg or composition between the core and the shell, it is possible to improve the curability, drying property, film-forming property, and mechanical strength of the binder.
From the viewpoint of binding property and particle stability, the average particle diameter of the resin fine particles is preferably 10 to 1000 nm, and more preferably 10 to 300 nm. Note that the average particle diameter in the present invention refers to the volume average particle diameter, and can be measured by a dynamic light scattering method.
Measurement of the average particle size by dynamic light scattering can be performed as follows. Depending on the solid content of the resin fine particles, the resin fine particles are diluted 200 to 1000 times with the same dispersion medium as the dispersion liquid. Approximately 5 ml of the diluted dispersion liquid is poured into the cell of a measuring device (Microtrac, manufactured by Nikkiso Co., Ltd.), and the dispersion medium and resin refractive index conditions according to the sample are input, and then measurement is performed. The average particle size can be measured from the peak of the volume particle size distribution data (histogram) obtained at this time.
グルコースオキシダーゼ（ＧＯｘ）などの酵素は水系溶液に分散したものを担持する場合が多く、グルコースなどのセンシング対象は水系溶液に溶解されたものとする場合が多いことから、濡れ性や浸透性などの観点から、バインダーとしては、水系溶剤に溶解可能な水溶性樹脂や水系の分散媒中で溶解せずに、微粒子の状態で存在する水分散樹脂微粒子を使用することが好ましい。 Enzymes such as glucose oxidase (GOx) are often supported in a state dispersed in an aqueous solution, and sensing targets such as glucose are often dissolved in an aqueous solution. From the standpoint of wettability and permeability, it is therefore preferable to use, as the binder, a water-soluble resin that can be dissolved in an aqueous solvent, or water-dispersed resin microparticles that do not dissolve in an aqueous dispersion medium and exist in the form of microparticles.
バインダーに用いる水溶性樹脂とはしては、ポリビニル系樹脂やポリウレタン系樹脂、ポリエステル系樹脂、ポリエーテル系樹脂、カルボキシメチルセルロース等のセルロース系樹脂、ホルマリン縮合物、シリコーン系樹脂、及びこれらの複合系樹脂等が挙げられる。更に、これら２種類以上を併用してもよい。また、バインダーに水溶性樹脂を使用する場合は、親水性化合物としての機能も兼ねるが、重量平均分子量は５００００以上であることが好ましい。 Examples of water-soluble resins used in the binder include polyvinyl resins, polyurethane resins, polyester resins, polyether resins, cellulose resins such as carboxymethyl cellulose, formalin condensates, silicone resins, and composite resins of these. Two or more of these may be used in combination. When a water-soluble resin is used in the binder, it also functions as a hydrophilic compound, and it is preferable that the weight average molecular weight is 50,000 or more.
バインダーに用いる水分散樹脂微粒子としては、（メタ）アクリル系エマルション、ニトリル系エマルション、ウレタン系エマルション、ポリオレフィン系エマルション、フッ素系エマルション（ポリフッ化ビニリデン（ＰＶＤＦ）やポリテトラフルオロエチレン（ＰＴＦＥ）など）、スチレン―ブタジエン系樹脂等が挙げられる。
なお、（メタ）アクリルは、メタクリルまたはアクリルを意味する。
Examples of the water-dispersed resin fine particles used as the binder include (meth)acrylic emulsions, nitrile emulsions, urethane emulsions, polyolefin emulsions, fluorine-based emulsions (such as polyvinylidene fluoride (PVDF) and polytetrafluoroethylene (PTFE)), styrene-butadiene resins, and the like.
Incidentally, (meth)acrylic means methacryl or acrylic.
（液状媒体）
酵素センサー電極形成用組成物は、任意の液状媒体を含むことができる。液状媒体としては特に限定されず、公知のものを用いることができる。ペースト組成物の分散性向上、並びに、非導電性基材上へのペースト組成物の塗工性向上のために、複数種の液状媒体を混ぜて使用してもよい。
液状媒体としては、アルコール類、グリコール類、セロソルブ類、アミノアルコール類、アミン類、ケトン類、カルボン酸アミド類、リン酸アミド類、スルホキシド類、カルボン酸エステル類、リン酸エステル類、エーテル類、ニトリル類等の有機溶剤、及び水等が挙げられる。
(Liquid medium)
The composition for forming an enzyme sensor electrode may contain any liquid medium. The liquid medium is not particularly limited, and any known liquid medium may be used. In order to improve the dispersibility of the paste composition and the applicability of the paste composition on a non-conductive substrate, a mixture of multiple liquid media may be used.
Examples of the liquid medium include organic solvents such as alcohols, glycols, cellosolves, amino alcohols, amines, ketones, carboxylic acid amides, phosphoric acid amides, sulfoxides, carboxylic acid esters, phosphoric acid esters, ethers, and nitriles, as well as water.
（分散剤）
酵素センサー電極形成用組成物は分散剤を使用することができる。分散剤は、炭素材料等に対して分散剤として有効に機能し、その凝集を緩和することができる。分散剤は炭素材料に対して凝集を緩和する効果が得られれば特に限定されるものではない。
(Dispersant)
The composition for forming an enzyme sensor electrode may use a dispersant. The dispersant functions effectively as a dispersant for carbon materials and the like, and can reduce the aggregation. The dispersant is not particularly limited as long as it has the effect of reducing the aggregation of carbon materials.
更に、酵素センサー電極形成用組成物には、増粘剤、成膜助剤、硬化剤、消泡剤、レベリング剤、防腐剤、ｐＨ調整剤などを必要に応じて配合できる。 In addition, the composition for forming an enzyme sensor electrode can contain thickeners, film-forming aids, hardeners, defoamers, leveling agents, preservatives, pH adjusters, etc., as needed.
＜基材＞
基材としては、非導電性基材としては、ポリエチレンテレフタレート、ポリエチレンナフタレート等のポリエステル樹脂、ポリメチルメタクリレート等のアクリル樹脂、ポリエチレン、ポリプロピレン、ポリ塩化ビニル、ポリスチレン、ポリカーボネート、ポリイミド、ポリテトラフルオロエチレン、パーフルオロアルコキシアルカン、パーフルオロエチレンプロペンコポリマー、エチレンテトラフルオロエチレンコポリマー、ポリビニリデンフルオライド、ポリクロロトリフルオロエチレン、エチレンクロロトリフルオロエチレンコポリマー等の樹脂フィルムが例示できる。また、樹脂フィルム以外にも紙や布等も挙げられる。
また、カーボンペーパーやカーボンクロス等の導電性基材も挙げられる。
<Substrate>
Examples of the non-conductive substrate include polyester resins such as polyethylene terephthalate and polyethylene naphthalate, acrylic resins such as polymethyl methacrylate, and resin films such as polyethylene, polypropylene, polyvinyl chloride, polystyrene, polycarbonate, polyimide, polytetrafluoroethylene, perfluoroalkoxyalkane, perfluoroethylenepropene copolymer, ethylenetetrafluoroethylene copolymer, polyvinylidene fluoride, polychlorotrifluoroethylene, and ethylenechlorotrifluoroethylene copolymer. In addition to resin films, paper and cloth can also be used.
Other examples include conductive substrates such as carbon paper and carbon cloth.
（導電層の形成）
導電層は、前記の導電性基材あるいは非導電性基材に酵素センサー電極形成用組成物を塗工・印刷、必要に応じてプレス処理等を行って形成することができる。非導電性基材上に導電性組成物を塗工・印刷する方法としては、特に制限はなく、例えばスクリーン印刷、インクジェット印刷、グラビア印刷、ナイフコーター、バーコーター、ブレードコーター、スプレー、ディップコーター、スピンコーター、ロールコーター、ダイコーター、カーテンコーター等の一般的な方法を適用できる。
(Formation of Conductive Layer)
The conductive layer can be formed by coating/printing the composition for forming an enzyme sensor electrode on the conductive substrate or non-conductive substrate, and, if necessary, subjecting it to a press treatment, etc. The method for coating/printing the conductive composition on the non-conductive substrate is not particularly limited, and general methods such as screen printing, inkjet printing, gravure printing, knife coater, bar coater, blade coater, spray, dip coater, spin coater, roll coater, die coater, curtain coater, etc. can be applied.
また、塗布後に平版プレスやカレンダーロール等による圧延処理を行ってもよく、導電層を軟化させてプレスしやすくするため、加熱しながら行ってもよい。導電層の厚みは、一般的には０．１μｍ以上、１ｍｍ以下であり、好ましくは１μｍ以上、２００μｍ以下である。 After coating, the conductive layer may be rolled using a lithographic press or a calendar roll, or may be heated to soften the conductive layer and make it easier to press. The thickness of the conductive layer is generally 0.1 μm or more and 1 mm or less, and preferably 1 μm or more and 200 μm or less.
＜酵素センサー＞
酵素センサーは、少なくとも作用極及び対極、あるいは作用極、対極及び参照極で構成される。また、必要に応じて血液などの試料を電極へ導入するスペーサーやカバー等を設けてもよい。
酵素センサーに用いる複数の電極は、異なる基材上に導電層をそれぞれ形成することで作製する場合や、同一の非導電性基材上にそれぞれの電極について導電層を形成する場合や、同一の非導電性基材上に導電層を設置した後に非導電部位を形成することで電極を作製してもよい。また予め、非導電性基材に金属スパッタなどで金属層を形成した上に、各電極の導電層を形成して電極を作製してもよい。参照極を設置する場合は、例えば導電層の上部へ更に銀や塩化銀などを積層することによって作製される。各電極のリード部は、金属スパッタなどで金属層を形成する方法、導電層を延長して用いる方法、延長した導電層の上部や下部に金属スパッタなどで金属層を更に形成する方法等、が例示できる。
酸化還元酵素やメディエーターを設置する方法としては、これらの電極上部、あるいは作用極の上部および／または内部に、酸化還元酵素や必要に応じてメディエーターを含ませる方法や、酸化還元酵素や必要に応じてメディエーターを加えた混合物層を形成させる方法等が挙げられる。酸化還元酵素やメディエーターを含む混合物層を形成する場合、親水性化合物を混合してもよい。
<Enzyme sensor>
The enzyme sensor is composed of at least a working electrode and a counter electrode, or a working electrode, a counter electrode and a reference electrode. If necessary, a spacer or a cover for introducing a sample such as blood to the electrodes may be provided.
The electrodes used in the enzyme sensor may be prepared by forming conductive layers on different substrates, by forming conductive layers for each electrode on the same non-conductive substrate, or by forming a non-conductive portion after placing a conductive layer on the same non-conductive substrate. Alternatively, the electrodes may be prepared by forming a metal layer on the non-conductive substrate in advance by metal sputtering or the like and then forming a conductive layer for each electrode. When a reference electrode is to be placed, it is prepared, for example, by further laminating silver or silver chloride on the top of the conductive layer. Examples of the lead portion of each electrode include a method of forming a metal layer by metal sputtering or the like, a method of extending the conductive layer, and a method of further forming a metal layer on the top or bottom of the extended conductive layer by metal sputtering or the like.
Methods for disposing the oxidoreductase or mediator include a method of incorporating the oxidoreductase or, if necessary, the mediator on the upper part of the electrode or on and/or inside the working electrode, a method of forming a mixture layer to which the oxidoreductase or, if necessary, the mediator is added, etc. When forming a mixture layer containing the oxidoreductase or the mediator, a hydrophilic compound may be mixed.
酵素センサーは、前記の通り血液等の生体試料や食品等に含まれる特定成分を、酵素の基質特異性により選択的に酸化あるいは還元し、その電流値等から定性あるいは定量するものである。酵素センサーの用途としては、例えば、各種有機物を対象とした有機物センサー、血液や汗、尿、便、涙、唾液、間質液、呼気などの生体試料中の有機物や体液を対象とした生体センサー、水分を対象にした水分センサー、果物や食品中の糖等を対象にした食品用センサー、ＩｏＴセンサー、大気や河川、土壌など環境中の有機物を対象にした環境センサー、動物や昆虫、植物を対象にした動植物センサー等が挙げられる。生体センサーとしては、例えば、血液中の糖をセンシングする血糖値センサーや、尿中の糖をセンシングする尿糖値センサー、汗中の乳酸値をセンシングする疲労度センサーや熱中症センサー、汗や尿中の水分をセンシングする発汗センサーや排尿センサー等が挙げられる。また、生体向けのウェアラブルセンサーとしての用途として、例えば、おむつ内にセンサーを仕込んだ排尿センサーや尿糖値センサー、経皮貼付型の発汗、熱中症センサー、穿刺型での間質液の糖センサー、などが挙げられる。 As described above, an enzyme sensor selectively oxidizes or reduces specific components contained in biological samples such as blood and foods, etc., using the substrate specificity of the enzyme, and qualitatively or quantitatively determines the amount of the components from the current value, etc. Examples of uses of enzyme sensors include organic matter sensors for various organic matter, biosensors for organic matter and body fluids in biological samples such as blood, sweat, urine, stool, tears, saliva, interstitial fluid, and breath, moisture sensors for moisture, food sensors for sugar in fruits and foods, IoT sensors, environmental sensors for organic matter in the environment such as the atmosphere, rivers, and soil, and animal and plant sensors for animals, insects, and plants. Examples of biosensors include blood glucose level sensors that sense sugar in blood, urine glucose level sensors that sense sugar in urine, fatigue level sensors and heat stroke sensors that sense lactic acid levels in sweat, and sweat sensors and urination sensors that sense moisture in sweat and urine. In addition, examples of applications as wearable sensors for living organisms include urination sensors and urine glucose level sensors with sensors installed inside diapers, perspiration and heat stroke sensors that are attached to the skin, and puncture-type interstitial fluid glucose sensors.
＜酸化還元酵素＞
本発明における酵素としては、反応により電子を授受できる酵素であれば特に制限はなく、検出対象に応じて適宜選択される。糖や有機酸などのオキシダーゼやデヒドロゲナーゼなどが利用できる。中でも、人体の血液や尿などの生体試料に含まれるグルコースを検出対象にできるグルコースオキシダーゼやグルコースデヒドロゲナーゼが好ましい場合がある。その他、フルクトースを検出対象にできるフルクトースオキシダーゼやフルクトースデヒドロゲナーゼ、乳酸を検出対象にできる乳酸オキシダーゼや乳酸デヒドロゲナーゼが好ましい場合がある。
用いられる酵素は１種類でも２種類以上であってもよい。また、センシング対象を加水分解等により酸化あるいは還元可能な状態にする酵素等の触媒と、酸化あるいは還元を促進する酵素との組み合わせであってもよい。
<Oxidoreductase>
The enzyme in the present invention is not particularly limited as long as it can donate and receive electrons through a reaction, and is appropriately selected depending on the detection target. Oxidases and dehydrogenases of sugars and organic acids can be used. Among them, glucose oxidase and glucose dehydrogenase, which can detect glucose contained in biological samples such as human blood and urine, may be preferable. In addition, fructose oxidase and fructose dehydrogenase, which can detect fructose, and lactate oxidase and lactate dehydrogenase, which can detect lactate, may be preferable.
The enzyme to be used may be one kind or two or more kinds. In addition, a catalyst such as an enzyme that makes the sensing target oxidizable or reducible by hydrolysis or the like and an enzyme that promotes oxidation or reduction may be used in combination.
＜メディエーター＞
酵素には電極に直接電子を伝達できる直接電子移動型（ＤＥＴ型）酵素と直接電子を伝達できない酵素が存在する。ＤＥＴ型以外の酵素の場合には、基質の酸化によって生じた電子を酵素から電極に伝達する役割を担うメディエーターを併用する必要がある。メディエーターとしては、電極に電子を伝達できる酸化還元物質であれば特に制限はなく、従来公知のものを使用できる。メディエーターの使用方法としては、電極に担持させる方法や電解液に溶解させて使用する方法等がある。メディエーターとしては、テトラチアフルバレン、ハイドロキノンや１，４‐ナフトキノン等のキノン類などの非金属化合物、フェロセン、フェリシアン化物、オスミウム錯体、及びこれら化合物を修飾したポリマー等が例示できる。メディエーターは、電極を形成する組成物中に予め含ませてもよく、また電極形成後に単独、あるいは酵素などと共に電極上部および／または内部に含ませてもよい。
<Mediator>
Enzymes include direct electron transfer (DET) enzymes that can transfer electrons directly to the electrode, and enzymes that cannot transfer electrons directly. In the case of enzymes other than DET, it is necessary to use a mediator that transfers electrons generated by the oxidation of the substrate from the enzyme to the electrode. There is no particular limitation on the mediator as long as it is an oxidation-reduction substance that can transfer electrons to the electrode, and any known mediator can be used. The mediator can be used by supporting it on the electrode or by dissolving it in an electrolyte. Examples of mediators include non-metallic compounds such as tetrathiafulvalene, quinones such as hydroquinone and 1,4-naphthoquinone, ferrocene, ferricyanide, osmium complexes, and polymers modified with these compounds. The mediator may be included in the composition that forms the electrode in advance, or may be included alone or together with the enzyme on the upper part and/or inside the electrode after the electrode is formed.
＜イオン伝導体＞
本発明におけるイオン伝導体として電極の間でイオンの伝導を行うものを用いてもよい。イオン伝導体の形態はイオン伝導性を有するものであれば特に限定されるものではない。イオン伝導体としては、液体に溶ける電解質や固体のポリマー電解質などを使用しても良い。
<Ionic Conductor>
The ion conductor in the present invention may be one that conducts ions between electrodes. The form of the ion conductor is not particularly limited as long as it has ion conductivity. As the ion conductor, an electrolyte that dissolves in a liquid or a solid polymer electrolyte may be used.
以下に、実施例により本発明をさらに具体的に説明するが、以下の実施例は本発明の権利範囲を何ら制限するものではない。尚、特に断らない限り、実施例および比較例における「部」は「質量部」を、「％」は「質量％」を表す。 The present invention will be described in more detail below with reference to examples, but the following examples are not intended to limit the scope of the invention. In the examples and comparative examples, "parts" refers to "parts by mass" and "%" refers to "% by mass" unless otherwise specified.
［実施例１］
＜酵素センサー電極形成用組成物の作製＞
イオン交換水５００質量部に水溶性樹脂（ＣＭＣダイセル＃１２４０（ダイセル化学工業社製、固形分１００質量％））３質量部を溶解させ、黒鉛（球状化黒鉛 ＣＧＢ－５０（日本黒鉛社製））７１質量部と黒鉛以外の炭素材料（ライオナイト ＥＣ－２００Ｌ（ライオン・スペシャリティ・ケミカルズ社製））９質量部を添加しミキサーに入れて混合した後、サンドミルにて分散を行った。
次に水分散性樹脂微粒子（アクリル樹脂水分散液 Ｗ－１６８（トーヨーケム社製 固形分５０質量％））３４質量部、親水性化合物（ポリビニルピロリドン Ｋ－１５ 平均分子量１００００）５部を添加し、適宜イオン交換水を加えてミキサーで混合し、表１に示す酵素センサー電極形成用組成物（１）を得た。
[Example 1]
<Preparation of composition for forming enzyme sensor electrode>
3 parts by mass of a water-soluble resin (CMC Daicel #1240 (manufactured by Daicel Chemical Industries, Ltd., solid content 100% by mass)) was dissolved in 500 parts by mass of ion-exchanged water, 71 parts by mass of graphite (spheroidized graphite CGB-50 (manufactured by Nippon Graphite Co., Ltd.)) and 9 parts by mass of a carbon material other than graphite (Lionite EC-200L (manufactured by Lion Specialty Chemicals)) were added, and the mixture was mixed in a mixer and then dispersed using a sand mill.
Next, 34 parts by mass of water-dispersible resin particles (acrylic resin water dispersion W-168 (manufactured by Toyochem Co., Ltd., solid content 50% by mass)) and 5 parts of a hydrophilic compound (polyvinylpyrrolidone K-15, average molecular weight 10,000) were added, and ion-exchanged water was added appropriately and mixed with a mixer to obtain a composition (1) for forming an enzyme sensor electrode shown in Table 1.
＜酵素センサー用電極および酵素センサーの作製＞
基材として厚さ１００μｍのＰＥＴ基材（ルミラー（東レ社製））上に、酵素センサー電極形成用組成物（１）を２×３０ｍｍの開口部を３か所備えたメタルマスクを用いて塗布後、加熱乾燥した。その後、導電層の一部に絶縁性のレジストインキをメタルマスクを用いて塗工、その後加熱乾燥した。更に、銀／塩化銀を分散したインキを絶縁性レジストで囲われた開口部内の電極の内１つに塗工し、加熱乾燥を経て、図１に示す酵素センサー用電極（１）を得た。得られた電極に対して、作用極あるいは対極となる部分にイオン交換水を滴下し、接触角をθ／２法により測定した（ＤＭｅ－２１０ 協和界面科学社製）。
<Preparation of enzyme sensor electrode and enzyme sensor>
The enzyme sensor electrode forming composition (1) was applied to a 100 μm thick PET substrate (Lumirror (manufactured by Toray Industries, Inc.)) using a metal mask with three openings of 2 × 30 mm, and then heated and dried. Then, an insulating resist ink was applied to a part of the conductive layer using a metal mask, and then heated and dried. Furthermore, an ink in which silver/silver chloride was dispersed was applied to one of the electrodes in the opening surrounded by the insulating resist, and after heating and drying, the enzyme sensor electrode (1) shown in FIG. 1 was obtained. Ion-exchanged water was dropped on the part of the obtained electrode that was to become the working electrode or the counter electrode, and the contact angle was measured by the θ/2 method (DMe-210, manufactured by Kyowa Interface Science Co., Ltd.).
前記、酵素センサー用電極（１）の絶縁性レジストで囲われた開口部内に、メディエーターであるフェリシアン化カリウムと酵素であるグルコースオキシダーゼを０．１Ｍリン酸緩衝（ｐＨ７）で溶解した水溶液を滴下、自然乾燥させてメディエーターと酵素を担持し、酵素センサーを得た。 An aqueous solution of potassium ferricyanide (mediator) and glucose oxidase (enzyme) dissolved in 0.1 M phosphate buffer (pH 7) was dropped into the opening of the enzyme sensor electrode (1) surrounded by the insulating resist, and the solution was allowed to dry naturally to support the mediator and enzyme, yielding an enzyme sensor.
表１に示す組成比を変更した以外は、実施例１と同様の方法により、それぞれ酵素センサー電極形成用組成物（２）～（１６）を得た。 Except for changing the composition ratio shown in Table 1, compositions (2) to (16) for forming enzyme sensor electrodes were obtained in the same manner as in Example 1.
実施例１と同様の方法によって実施例２～１４および比較例１～２の酵素センサー用電極を得た。 The electrodes for the enzyme sensors of Examples 2 to 14 and Comparative Examples 1 and 2 were obtained by the same method as in Example 1.
＜電気化学評価＞
上記作製した酵素センサー用電極に銀／塩化銀が塗工された電極を参照極、他は作用極と対極として、電解液に０．１Ｍリン酸緩衝液（ｐＨ７．０）中に、反応基質（センシング対象物）としてＤ－グルコースを１０ｍＭとなるように添加し、０．５Ｖ（ｖｓＡｇ／ＡｇＣｌ）の電位を印加して１０秒後の電流値を測定した。表１に示すように、比較例１における電位印加１０秒後の電流値に対する、各実施例における同電流値の百分率（％）で比較した。
〇：１２０％以上
△：１００％以上１２０％未満
×：１００％未満（比較例１より悪い）
<Electrochemical evaluation>
The electrode prepared above for the enzyme sensor was coated with silver/silver chloride as a reference electrode, and the other electrodes were used as working and counter electrodes. D-glucose was added to the electrolyte solution (0.1 M phosphate buffer (pH 7.0) to a concentration of 10 mM as a reaction substrate (sensing target), and a potential of 0.5 V (vs Ag/AgCl) was applied to measure the current value after 10 seconds. As shown in Table 1, the current value after 10 seconds of potential application in each example was compared with the current value in Comparative Example 1 in terms of percentage (%).
◯: 120% or more △: 100% or more and less than 120% ×: Less than 100% (worse than Comparative Example 1)
いずれの実施例においても、比較例より電流値が高く得られたため、本発明により高感度な酵素センサーとして利用できる。また、金属を含まないため腐食に強く、低コストであり、廃棄の際の分別も容易である。 In all of the examples, a higher current value was obtained than in the comparative examples, and therefore the present invention can be used as a highly sensitive enzyme sensor. In addition, since it does not contain metal, it is resistant to corrosion, is low cost, and is easy to separate when disposed of.
実施例１の酵素センサーについて銀／塩化銀が塗工された電極を参照極に、他は作用極と対極として接続し、５ｍＭ、１０ｍＭ、２０ｍＭのグルコースを含む各０．１Ｍりん酸緩衝液を滴下、それぞれ０．５Ｖ（ｖｓＡｇ／ＡｇＣｌ）の電位を印加して１０秒後の電流値を測定したところ、グルコース濃度と電流値に相関が見られた。基質濃度による電流値変化が得られるため、センサーとして活用が可能である。 For the enzyme sensor of Example 1, the silver/silver chloride coated electrode was connected as the reference electrode, and the other electrodes were connected as the working electrode and counter electrode. 0.1 M phosphate buffer solutions containing 5 mM, 10 mM, and 20 mM glucose were dropped, and a potential of 0.5 V (vs Ag/AgCl) was applied to each electrode, and the current value was measured after 10 seconds. A correlation was found between the glucose concentration and the current value. Since the change in current value due to the substrate concentration can be obtained, it can be used as a sensor.
１ 酵素センサー
２ 基材
３ 導電層
４ 絶縁性レジスト
５ 開口部
６ 参照極
1 Enzyme sensor 2 Substrate 3 Conductive layer 4 Insulating resist 5 Opening 6 Reference electrode
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| JP2000131264A (en) | 1998-10-23 | 2000-05-12 | Techno Medica:Kk | Enzyme sensor |
| JP2006292495A (en) | 2005-04-08 | 2006-10-26 | Toray Ind Inc | Carbon nanotube composition, biosensor and manufacturing method of them |
| US20150182155A1 (en) | 2008-09-30 | 2015-07-02 | Abbott Diabetes Care Inc. | Analyte Sensors Comprising Leveling Agents |
| JP2011069727A (en) | 2009-09-25 | 2011-04-07 | National Institute Of Advanced Industrial Science & Technology | Redox protein immobilization nanostructure electrode |
| JP2013034422A (en) | 2011-08-05 | 2013-02-21 | Jnc Corp | Enzyme modified electrode, electrochemical reactor using the electrode, and method for producing chemical substance using the reactor |
| JP2018037394A (en) | 2016-08-29 | 2018-03-08 | 東レ株式会社 | Conductive laminate |
| JP2019038869A (en) | 2017-08-22 | 2019-03-14 | 東洋インキＳｃホールディングス株式会社 | Aqueous conductive dispersion, biosensor, and method for manufacturing biosensor |
| JP2019038870A (en) | 2017-08-22 | 2019-03-14 | 東洋インキＳｃホールディングス株式会社 | Aqueous conductive dispersion liquid, biosensor, and method for manufacturing biosensor |
| JP2019038868A (en) | 2017-08-22 | 2019-03-14 | 東洋インキＳｃホールディングス株式会社 | Aqueous conductive dispersion liquid for electrode formation for biosensor, biosensor, and method for manufacturing biosensor |
| JP2019084519A (en) | 2017-11-10 | 2019-06-06 | 東洋インキＳｃホールディングス株式会社 | Aqueous conductive dispersion body, production method for aqueous conductive dispersion body, biosensor and production method for biosensor |
| WO2019189808A1 (en) | 2018-03-29 | 2019-10-03 | 東洋紡株式会社 | Nanocarbon electron transfer action |
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