JP4522528B2

JP4522528B2 - Ulcerative colorectal disease measuring electrode, ulcerative colorectal disease measuring device, and ulcerative colorectal disease determining method

Info

Publication number: JP4522528B2
Application number: JP2000081160A
Authority: JP
Inventors: 文代楠; 健介荒井; 明小谷; 毅西田; 英明橋本; 靖林
Original assignee: Panasonic Corp; Matsushita Electric Industrial Co Ltd
Current assignee: Panasonic Corp; Panasonic Holdings Corp
Priority date: 2000-03-02
Filing date: 2000-03-23
Publication date: 2010-08-11
Anticipated expiration: 2020-03-23
Also published as: JP2001318071A

Description

【０００１】
【発明の属する技術分野】
本発明は、大腸癌を含む潰瘍性大腸疾患を発見するための潰瘍性大腸疾患測定電極と、これを使用した潰瘍性大腸疾患測定装置、さらにこの潰瘍性大腸疾患を発見する潰瘍性大腸疾患判定方法に関する。
【０００２】
【従来の技術】
最近、世界中でもっとも恐れられている疾病は悪性の腫瘍、癌である。癌は発病したとしても、初期段階ではあまり自覚症状がなく進行するので手遅れになり易い。中でも大腸癌は胃癌や食道癌、十二指腸癌などより深刻で、排便中に出血が起こるまでほとんど症状があらわれない。そして、この出血が起こったときには、大腸癌は既に相当すすんでいるのが通常であり、この時点で発見された場合は、早期発見がなされた場合と比較して処置もかなり限られてしまうことが多い。この点、これが良性の潰瘍性大腸疾患であったとしても同様で、出血が起こったときには病状はかなり進行しているのが通常である。その上、具合の悪いことには、このような出血性の排便があるのは、大腸癌や潰瘍性の大腸疾患にかぎらず、痔ろう等の慢性疾患でも起こりえる。こうした事情から痔ろう患者が多いわが国ではせっかくの大腸癌等発見のチャンスを、患者自身が安易に考えることで治療の機会を失ってしまうことも多かった。従って、出血が起こる前に、大腸癌等の潰瘍性大腸疾患を早期発見する方法の開発、出血があったときに単なる痔ろうと大腸癌等の疾患との区別を簡単、正確に判定できる方法の開発、さらにはそのために使用する潰瘍性大腸疾患測定装置の開発が望まれていた。
【０００３】
ところで、癌も良性のポリープも細胞***によって細胞が増殖する点では同一である。しかし、良性の細胞***が必要な範囲で制限的に行われるのに対して、癌等の悪性腫瘍は無制限に増殖するものである。この悪性腫瘍も多くは遺伝子が傷つくことなどで正常な細胞が形質転換を起こしたにすぎないから、細胞組織上悪性の細胞を良性の細胞から見分けるのは難しい。採取した細胞組織を遺伝子レベルで分析を行えば癌化した細胞か、正常な細胞かは判断することが可能であるが、遺伝子を解析する大掛かりな分析装置と比較的長時間の分析が必要で、日常的に患者自身が行える簡易な判定方法とはなりえないものであった。早期発見を行うためには、手軽で安価な装置でなければ普及は期待できず、また、出血性の排便があったとしても、患者自身が簡単に痔ろうによる出血か、大腸癌等の潰瘍性疾患による出血かを正確に判定できるような装置でなければ、潰瘍性大腸疾患測定装置は意味をなさない。
【０００４】
従来、大腸癌等を検査する方法として排便中の潜血成分（ヘモグロビン）を、採便具により採取して、抗ヘモグロビン抗体を用いて免疫学的測定方法により測定する方法が行われている。しかし、この検査方法は手軽ではあるが、排便中のヘモグロビンを検便試料として長期間保存するため、保存中に試料中のヘモグロビンが分解し、ヘモグロビンが正確に測定できないという問題があった。ヘモグロビンの分解を抑制するため、ペニシリンや非ペニシリン系抗生物質を添加してする方法（特開平７−７２１５４号公報）などが開示されているが、測定に長時間を費やす点は如何ともしかたがないし、測定結果の正確性は見込めないものであった。しかも、ヘモグロビンの存在は測定できても、それが痔ろうによる出血なのか、大腸癌等による出血なのか、区別することはできない。要するに、この免疫学的測定方法は単に排便中に人血が存在するのか、人血が存在しないのかを判断するものにすぎない。
【０００５】
このように、遺伝子レベルの細胞組織検査によれば正確な判断ができるが、専門家の知識と専門の分析装置を要し、安価で患者自身が簡便に測定する検査方法とは程遠いものである。また、採便具による検便で出血の有無をみる免疫学的測定方法は、手軽ではあるが、正確性に問題があり、加えて、大腸癌や潰瘍といった緊急性を要する疾患と日常の痔等の慢性病との区別がまったくつかないものであった。その上、免疫学的測定を行うには結局は専門家が必要で、患者自身が判断できるものではないし、基本的には再度正確に別の精密検査が必要になるものであった。こうした理由から、遺伝子解析や血液によらず、大腸に潰瘍ができたことを発見するまったく新しい検査方法の創出が望まれている。
【０００６】
ところで、潰瘍性大腸疾患に関して患者の便中細菌とその代謝産物である短鎖脂肪酸について次のような報告がなされている（日本消化器病学会雑誌第７９巻２号第１９３〜１９８頁）。なお、この短鎖脂肪酸というのは腸内細菌による糖質の代謝物で、酢酸、プロピオン酸、酪酸、バレリル酸、乳酸など炭素数６までのカルボン酸である。
【０００７】
この報告によれば、潰瘍性大腸疾患患者の便中細菌数は健常人と比べ減少する。なかでも嫌気性菌数が減少し、好気性菌数が増加する。この結果、便中細菌数と比例した形で、健常人に比べ代謝産物である短鎖脂肪酸濃度が減少し、非揮発性の短鎖脂肪酸である乳酸濃度は高くなると報告されている。病変が拡大するほど、そして緩解期より活発期にあたるほど乳酸濃度は増加するとも報告されている。
【０００８】
このように、潰瘍性大腸疾患は便中に含まれる短鎖脂肪酸と乳酸と密接な関係があり、健常人では短鎖脂肪酸が多くなって乳酸が少ないため、（乳酸濃度／短鎖脂肪酸濃度）の比率は小さい値をとるが、潰瘍性大腸疾患患者の便では短鎖脂肪酸が少なく乳酸が多いため、（乳酸濃度／短鎖脂肪酸濃度）の比率は相対的に大きな値をとることが分かる。従って、乳酸と短鎖脂肪酸の多寡、もしくは比率を簡易かつ正確に測定できれば、従来の技術とまったく異なる潰瘍性大腸疾患測定装置を得ることができる。
【０００９】
この潰瘍性大腸疾患測定装置を実現するためには、乳酸と短鎖脂肪酸をどのようにして測定するかが問題となる。本発明者らは既に脂肪酸等の有機酸の酸度を簡易に、しかも短時間で測定するためにボルタンメトリーを利用する測定装置を提案した（特開平１０−２８８５９９号公報）。これは有機溶媒と支持電解質にｐ−ベンゾキノン誘導体またはｏ−ベンゾキノン誘導体を混合して、これに測定試料を入れて共存電解液を作って酸度を電気化学的に測定するものである。ｐ−ベンゾキノン誘導体またはｏ−ベンゾキノン誘導体は、プロトンアタック物質であり、解離したプロトンと結合するのはいうに及ばず、非解離状態の酸に含まれているプロトンを引き抜く作用を有する物質のことである。共存電解液に浸漬した作用電極と対極、さらには基準の電位を示す比較電極に通電し、比較電極の電位を基準に作用電極に自然電位よりマイナス側の電圧を印加することで、プロトンアタック物質は自らアニオン化し、酸中のプロトンを引き抜くものである。プロトンアタック物質にはキノン化合物やアゾ化合物があるが、ｐ−ベンゾキノン誘導体またはｏ−ベンゾキノン誘導体は光安定性に優れるとともに、還元電位が溶存酸素のボルタモグラムの電流の立ち上がりの点からずれているため除酸素しなくても測定できるという優れた特徴を有すものである。従って、測定のための面倒な準備をすることなく、作用電極に印加する電位を比較電極の電位を基準に掃引すると、ｐ−ベンゾキン誘導体またはｏ−ベンゾキン誘導体が還元されて還元電流が本ピークを示すよりプラス側で、脂肪酸等の有機酸によってそれらの誘導体が各還元電位で還元されボルタモグラム上プレピークを示すものである。脂肪酸は還元電位が近いとき、プレピークを示す還元電流はこの還元電位で総脂肪酸量すなわち酸の量に比例するという性質を持っている。
【００１０】
また、乳酸を電気化学的に測定するための乳酸測定電極も従来提案されている（特開平７−９２１３８号公報）。この乳酸測定電極は、基板上に導電性カーボンペーストを印刷して作用極と対極を形成する。さらに、この電極の上に乳酸オキシダーゼと乳酸ラセマーゼを混合して反応層を形成した構成となっている。乳酸オキシダーゼは乳酸を酸化させる酵素であり、乳酸ラセマーゼは乳酸をラセミ化させるための酵素である。この乳酸ラセマーゼによって、乳酸オキシダーゼによっても酸化されないＤ−乳酸をＬ−乳酸にラセミ化し、Ｌ−乳酸化した乳酸を乳酸オキシダーゼで酸化して過酸化酸素を発生させ、この過酸化酸素を作用極と対極間に電圧を印加することで酸化するものである。電子伝達体としてフェリシアンイオンを用いることもできる。
【００１１】
このように乳酸と短鎖脂肪酸の比率が簡単かつ正確に解れば、従来の技術とまったく異なる潰瘍性大腸疾患測定装置、そしてそのための潰瘍性大腸疾患測定電極を得ることができるが、ただ脂肪酸と乳酸を測定するのでは誤差が生じるものであった。
【００１２】
【発明が解決しようとする課題】
以上説明したように、排便中の潜血成分（ヘモグロビン）を採便具により採取して、抗ヘモグロビン抗体を用いた免疫学的測定方法により測定する方法は、手軽ではあるが、排便中のヘモグロビンを長期間保存するため、保存中に試料中のヘモグロビンが分解し、ヘモグロビンが正確に測定できないという問題があった。そして、ペニシリンや非ペニシリン系抗生物質を添加してする方法を用いても、測定のために長時間を要すし、それほど測定は正確にはならない。しかも、ヘモグロビンの存在は測定できても、それが痔ろうによる出血なのか、大腸癌や潰瘍等による出血なのか、区別することはできないものであった。
【００１３】
また、有機酸の酸度をプロトンアタック物質を混合した試料液をボルタンメトリーによって測定する従来の測定装置やその測定用の電極は、有機酸の総量濃度を電気化学的に測定できるが、この方法だと乳酸だけの測定は難しく、実質的に乳酸の定量はできないものであった。そこで別途乳酸を定量しようとすると、従来の技術のように乳酸オキシダーゼ等の酵素によって乳酸を特異的に酸化し、過酸化酸素を発生させて測定しなければならならないが、この酵素反応で同時にピルビン酸を生成するため、ピルビン酸と乳酸との還元電位の差で、ボルタンメトリーによる総脂肪酸の濃度の測定を狂わせるという問題があった。電子伝達体を用いた場合でも基本的には同様であった。
【００１４】
そこで本発明は従来のこのような問題を解決するもので、専門家によって血液の検査を行うことなく、短時間、かつ簡単、正確に判定するための潰瘍性大腸疾患測定電極を提供することを目的とする。
【００１５】
また本発明は、専門家によって血液の検査を行うことなく、短時間、かつ簡単、正確に判定できる潰瘍性大腸疾患測定装置を提供することを目的とする。
【００１６】
さらに、本発明は、専門家によって血液の検査を行うことなく、短時間、かつ簡単、正確に判定できる潰瘍性大腸疾患判定方法を提供することを目的とする。
【００１７】
【課題を解決するための手段】
上記課題を解決するために本発明の潰瘍性大腸疾患測定電極は、基台上に形成された総脂肪酸検出部と乳酸検出部を備えた潰瘍性大腸疾患測定電極であって、前記総脂肪酸検出部が、表面にプロトンアタック層が固定化された第１作用電極と、前記第１作用電極と対をなす第１対極と、基準の電位を示す比較電極を有し、
前記乳酸検出部が、第２作用電極と、前記第２作用電極と対をなす第２対極と、前記第２作用電極と前記第２対極の表面に設けられ、乳酸を酸化することができる酵素を含んだ反応層とを有し、
前記反応層の表面には乳酸検出を総脂肪酸検出から切り離すバッファ層が設けられたことを特徴とする。
【００１８】
これにより、専門家によって血液の検査を行うことなく、短時間、かつ簡単、正確に判定することができる。
【００１９】
また、本発明の潰瘍性大腸疾患測定装置は、以上記載した潰瘍性大腸疾患測定電極を備え、前記潰瘍性大腸疾患測定電極に被測定試料液を滴下したとき前記第１作用電極と前記第１対極間に電圧を印加する第１電源と、前記第２作用電極と前記対極間に電圧を印加する第２電源とを有し、
前記第１作用電極と前記比較電極間の電位を掃引するとともに前記第２作用電極と前記対極間の電位を所定の電位に制御する制御部と、前記第１作用電極と前記第１対極間に流れる電流を検知する第１検知部と、前記第２作用電極と前記第２対極間に流れる電流を検知する第２検知部を有し、前記第１検知部と前記第２検知部によって検知された電流値により乳酸量と総脂肪酸量の比を算出する演算部を備えたことを特徴とする。
【００２０】
これにより、専門家によって血液の検査を行うことなく、短時間、かつ簡単、正確に判定できる潰瘍性大腸疾患測定電極を提供することができる。
【００２１】
さらに、本発明の潰瘍性大腸疾患判定方法は、被測定試料液を滴下し、比較電極の電位を基準にしてプロトンアタック層が固定化された第１作用電極の電位を掃引するとともに、前記作用電極と第１対極間に流れる還元電流を測定し、次いで前記被測定試料液を乳酸が酸化される酵素を含んだ反応層に浸透させ、第２作用電極と第２対極間に所定の電圧を印加して酸化電流を測定して、前記還元電流と前記酸化電流との関係から潰瘍性大腸疾患と判定することを特徴とする。
【００２２】
これにより、専門家によって血液の検査を行うことなく、短時間、かつ簡単、正確に判定できる潰瘍性大腸疾患判定方法を提供することができる。
【００２３】
【発明の実施の形態】
請求項１に記載された発明は、基台上に形成された総脂肪酸検出部と乳酸検出部を備えた潰瘍性大腸疾患測定電極であって、
前記総脂肪酸検出部が、表面にプロトンアタック層が固定化された第１作用電極と、前記第１作用電極と対をなす第１対極と、基準の電位を示す比較電極を有し、
前記乳酸検出部が、第２作用電極と、前記第２作用電極と対をなす第２対極と、前記第２作用電極と前記第２対極の表面に設けられ、乳酸を酸化することができる酵素を含んだ反応層とを有し、
前記反応層の表面には乳酸検出を総脂肪酸検出から切り離すバッファ層が設けられたことを特徴とする潰瘍性大腸疾患測定電極であるから、総脂肪酸検出部と乳酸検出部にそれぞれ電圧を印加することにより、プロトンアタック物質で脱プロトンして電気化学的に総脂肪酸濃度を測定した後、バッファ層により切り離され遅れて浸透した反応層で酵素反応で発生した乳酸濃度を測定できる。
【００２４】
請求項２に記載された発明は、前記反応層に乳酸をラセミ化することができる酵素が含まれている請求項１記載の潰瘍性大腸疾患測定電極であるから、乳酸を特異的に酸化できる酵素がＬ−乳酸だけであっても、Ｄ−乳酸をラセミ化して乳酸の総量濃度を正確に測定できる。
【００２５】
請求項３に記載された発明は、前記プロトンアタック物質がキノン化合物またはアゾ化合物であることを特徴とする請求項１〜２のいずれかに記載の潰瘍性大腸疾患測定電極であるから、血液が溶解しづらく、電極への固定化が容易である。
【００２６】
請求項４に記載された発明は、前記キノン化合物がメルカプトキノン化合物であることを特徴とする請求項３記載の潰瘍性大腸疾患測定電極であるから、固定化力が強くなる。
【００２７】
請求項５に記載された発明は、前記基台が基板であることを特徴とする請求項１〜４のいずれかに記載の潰瘍性大腸疾患測定電極であるから、コンパクトであり持ち運びが容易である。
【００２８】
請求項６に記載された発明は、前記第１作用電極、前記第１対極、比較電極、前記第２作用電極、前記第２対極のいずれもが薄膜状に形成されたことを特徴とする請求項１〜５のいずれかに記載の潰瘍性大腸疾患測定電極であるから、各電極を平面化して薄くコンパクトにでき、生産も容易である。
【００２９】
請求項７に記載された発明は、請求項１〜６のいずれかに記載の潰瘍性大腸疾患測定電極を備え、前記潰瘍性大腸疾患測定電極に被測定試料液を滴下したとき前記第１作用電極と前記第１対極間に電圧を印加する第１電源と、前記第２作用電極と前記対極間に電圧を印加する第２電源とを有し、
前記第１作用電極と前記比較電極間の電位を掃引するとともに前記第２作用電極と前記対極間の電位を所定の電位に制御する制御部と、前記第１作用電極と前記第１対極間に流れる電流を検知する第１検知部と、前記第２作用電極と前記第２対極間に流れる電流を検知する第２検知部を有し、前記第１検知部と前記第２検知部によって検知された電流値により乳酸濃度と総脂肪酸濃度の比を算出する演算部を備えたことを特徴とする潰瘍性大腸疾患測定装置であるから、ボルタンメトリーによって総脂肪酸検出部で総脂肪酸濃度を測定するとともに、総脂肪酸濃度を測定後、反応層で生成される乳酸の濃度を乳酸検出部で測定することができ、乳酸測定の際に発生するピルビン酸のために、総脂肪酸の総量濃度の測定を狂わせることがなく測定でき、乳酸濃度と総脂肪酸濃度の比を算出するので潰瘍性大腸疾患を正しく判定できる。
【００３０】
請求項８に記載された発明は、請求項１〜６のいずれかに記載の潰瘍性大腸疾患測定電極を備え、前記潰瘍性大腸疾患測定電極に被測定試料液を滴下したとき前記第１作用電極と前記第１対極間に電圧を印加する第１電源と、前記第２作用電極と前記対極間に電圧を印加する第２電源とを有し、
前記第１作用電極と前記第１対極間を前記比較電極を基準にして所定の第１の電位に制御するとともに前記第２作用電極と前記第２対極間の電位を所定の第２の電位に制御する制御部と、前記第１作用電極と前記第１対極間に流れる電流を検知する第１検知部と、前記第２作用電極と前記第２対極間に流れる電流を検知する第２検知部を有し、前記第１検知部と前記第２検知部によって検知された電流値により乳酸濃度と総脂肪酸濃度の比を算出する演算部を備えたことを特徴とする潰瘍性大腸疾患測定装置であるから、クロノアンペロメトリーによって総脂肪酸検出部で総脂肪酸濃度を測定するとともに、総脂肪酸濃度を測定後、反応層で生成される乳酸を乳酸検出部で測定することができ、乳酸測定の際に発生するピルビン酸のために、総脂肪酸の総量濃度の測定を狂わせることがなく測定でき、乳酸濃度と総脂肪酸濃度の比を算出するので潰瘍性大腸疾患を正しく判定できる。
【００３１】
請求項９に記載された発明は、請求項１〜６のいずれかに記載の潰瘍性大腸疾患測定電極に被測定試料液を滴下し、比較電極の電位を基準にしてプロトンアタック層が固定化された第１作用電極の電位を掃引するとともに、前記作用電極と第１対極間に流れる還元電流を測定し、次いで前記被測定試料液を乳酸が酸化される酵素を含んだ反応層に浸透させ、第２作用電極と第２対極間に所定の電圧を印加して酸化電流を測定して、前記還元電流と前記酸化電流から算出される短鎖脂肪酸の総脂肪酸濃度と乳酸濃度の比（乳酸濃度／短鎖脂肪酸濃度）から潰瘍性大腸疾患と判定することを特徴とする潰瘍性大腸疾患判定方法であるから、総脂肪酸濃度をボルタンメトリーで測定後に、乳酸を酵素反応させて選択的に測定するので、乳酸濃度測定の際発生するピルビン酸で総脂肪酸の濃度が狂うことがない。
【００３２】
請求項１０に記載された発明は、請求項１〜６のいずれかに記載の潰瘍性大腸疾患測定電極に被測定試料液を滴下し、比較電極の電位を基準にしてプロトンアタック層が固定化された第１作用電極の電位を掃引するとともに、前記作用電極と第１対極間に流れる還元電流を測定し、次いで前記被測定試料液を乳酸が酸化される酵素を含んだ反応層に浸透させ、第２作用電極と第２対極間に所定の電圧を印加して酸化電流を測定して、前記還元電流と前記酸化電流から算出される短鎖脂肪酸の総脂肪酸濃度と乳酸濃度の比（乳酸濃度／短鎖脂肪酸濃度）から潰瘍性大腸疾患と判定することを特徴とする潰瘍性大腸疾患判定方法であるから、総脂肪酸をクロノアンペロメトリーで測定後に、乳酸を酵素反応させて選択的に測定するので、乳酸濃度測定の際発生するピルビン酸で総脂肪酸の濃度が狂うことがない。
【００３３】
以下、本発明の一実施の形態について、図面を参照しながら説明する。
【００３４】
（実施の形態１）
図１（ａ）は本発明の実施の形態１における潰瘍性大腸疾患測定電極の全体構成図、図１（ｂ）は本発明の実施の形態１における潰瘍性大腸疾患測定電極の断面図、図２（ａ）は本発明の実施の形態１における潰瘍性大腸疾患測定電極を分解したカバー正面図、図２（ｂ）は本発明の実施の形態１における潰瘍性大腸疾患測定電極を分解したスペーサ正面図、図２（ｃ）は本発明の実施の形態１における潰瘍性大腸疾患測定電極を分解した基板正面図である。
【００３５】
図１及び図２において、１は試料液を滴下して電気化学的に総脂肪酸と乳酸を測定するための潰瘍性大腸疾患測定電極（以下、測定電極）、１ａはポリエチレンテレフタレートなどの絶縁材料からなる測定電極１の基板、２は基板１ａを覆うとともに上側から滴下された試料液を測定電極１に注入することができる有孔平板のカバー、３は基板１ａとカバー２の間に挟まれ液溜めとなる開口が形成された平板のスペーサである。カバー２とスペーサ３もポリエチレンテレフタレートで形成するのが好ましい。本実施の形態１では電極を載せる基台として基板１ａを採用しているが、電極を保持できる絶縁材料であればどのようなものであってもよい。４はカバー２に形成され、総脂肪酸検出部Ａと乳酸検出部Ｂの双方へ試料を分配する試料注入孔であり、５はカバー２の試料注入孔５の直下に位置した連絡溝及びこれに接続される２つの液溜め開口からなる試料保持室形成用開口である。ここで、試料液は排便から採取した便を有機溶媒と電解質を混合して作製する。有機溶媒としてはエタノール、イソプロピルアルコール等が望ましく、電解質は塩化ナトリウム、塩化カリウム、塩化リチウム等が望ましい。試料となる便が２０ｇ／Ｌ〜１００ｇ／Ｌとなるように有機溶媒に溶解し、電解質を５０ｍｇ／Ｌ〜１５０ｍｇ／Ｌ混入して試料液を作製するのが適当である。
【００３６】
まず、測定電極１の総脂肪酸検出部Ａについて説明する。６は総脂肪酸検出部Ａを構成するための第１対極、同じく７は第１作用電極、８は比較電極である。第１作用電極７、比較電極８は本実施の形態１においては矩形形状に形成され、第１対極９はこの２つをモザイク状に所定間隙を置いて凹部内に収容し、全体として略矩形状になるように組み合わされる。
【００３７】
９は総脂肪酸検出部Ａを薄膜化して形成するために、基板１ａにスクリーン印刷で樹脂バインダーを含有させた通電性カーボンペーストを薄く帯状に印刷した第１対極用パターンである。１０は同じく総脂肪酸検出部Ａ形成のために通電性カーボンペーストを薄く帯状に印刷して形成した第１作用電極用パターン、１１は同じく比較電極用パターンである。１２は試料の脂肪酸からプロトンを引き抜き、第１作用電極７で還元して脂肪酸のプレピークを形成するためのプロトンアタック層である。
【００３８】
第１対極６は白金、黒鉛、金、ステンレス、アルミニウムその他の導電性材料から構成され、上記した第１対極パターン９と接続されるが、第１対極パターン９と別材料で構成するのでなく、第１対極用パターン９といっしょに通電性カーボンペーストで一体形成するのでもよい。次に、第１作用電極７はグラッシーカーボンと呼ばれる炭素電極、ＰＦＣと呼ばれるプラスチックフォームを１０００℃〜２０００℃で焼結した炭素材料、あるいは金を蒸着またはスパッタリングなどして形成した薄膜で構成され、その表面にプロトンアタック層１２が形成されている。プロトンアタック層１２はプレプロトンアタック物質をメルカプト化や塗布後に溶媒を乾燥させるなどして固定化したもので、酸から解離したプロトン（Ｈ＋）と結合することに止まらず、酸を構成する非解離状態のプロトンを攻撃して引き抜く作用をもつものである。このプレプロトンアタック物質にはキノン化合物やアゾ化合物等があるが、これらは自然電位におかれた状態のときはプロトンを引き抜く作用は奏さない。すなわち、このプロトンアタック層１２は試料液が滴下されると、それを構成するプレプロトンアタック物質がアニオン化した状態となり、印加電圧で酸が分極してプロトン側がδ＋となって＋電位をもつため、酸中のプロトンを引き抜くが、自然電位においてはこれが起こらないからである。第１作用電極用パターン１０と接続されるが、上記したような別材料で構成するのでなく第１作用電極用パターン１０といっしょに通電性カーボンペーストで一体形成するのでも製造工程が簡略化でき好ましい。キノン化合物としては、ｐ−ベンゾキノン、ｏ−ベンゾキノン、ジフェノキノン、ナフトキノン、アントラキノン、ベンゼンアゾヒドロキノン、さらにこれらキノンの誘導体等がある。メルカプト化したメルカプトキノン化合物が好ましい。また、アゾ化合物には、アゾベンゼン、アゾフェノール、ベンゼンアゾメタン、ベンゼンアゾエタン、アゾナフタリン、アゾトルエン、アゾ安息香酸、アゾアニリン、アゾアニソールメチルアゾベンゼン、１−ベンゼンアゾナフタリン、ベンゼンアゾナフトール、オキシアゾベンゼン、２、４−ジオキシアゾベンゼン等がある。本実施の形態１のプレプロトンアタック物質はｏ−ベンゾキノン誘導体が用いられており、側鎖部分がメルカプト化されて第１作用電極７と結合している。
【００３９】
比較電極８は、第１作用電極７に脂肪酸を還元する所定の還元電位を印加するために基準となる電位を発生するもので、金、炭素、グラッシーカーボンと呼ばれる炭素電極、ＰＦＣと呼ばれるプラスチックフォームを１０００℃〜２０００℃で焼結した炭素材料で構成される。この比較電極８は比較電極用パターン１１と接続される。通電性カーボンペーストで一体形成して作るのが第１作用電極用パターン１０と同様に好ましい。
【００４０】
比較電極用パターン１１と第１対極用パターン９と第１作用電極用パターン１０は、測定電極１の長手方向に平行にこの順で３本沿って形成され、試料注入孔４の付近でそれぞれ第１対極６、第１作用電極７、比較電極８と接続される。各電極が形成されている部分とコネクタと接続する部分以外の部分、すなわち各パターンの表面は絶縁材料で被覆されている。
【００４１】
次に、測定電極１の乳酸検出部Ｂについて説明する。
【００４２】
１３は乳酸検出部Ｂを構成するための第２対極、同じく１４は第２作用電極である。第２作用電極１４は本実施の形態１においては矩形形状に形成され、第２対極１３は内部に矩形状の開口を開けた円形状をしている。第２作用電極１４はモザイク状に所定間隙を置いて第２対極１３の開口内に収容され、全体として円形状になるように組み合わされる。
【００４３】
１５は乳酸検出部Ｂを薄膜化して形成するために、基板１ａにスクリーン印刷で樹脂バインダーを含有させた通電性カーボンペーストを薄く帯状に印刷した第２対極用パターンである。１６は同じく乳酸検出部Ｂ形成のために通電性カーボンペーストを薄く帯状に印刷して形成した第２作用電極用パターンである。１７は試料を滴下したとき、総脂肪酸検出部Ａと乳酸検出部Ｂで測定のため発生する反応を切り離し、総脂肪酸の検出を行った後乳酸を測定するために双方の反応の影響を絶つバッファ層である。バッファ層１７は所定時間経過したら試料液が浸透するように選択される。その材料は酢酸メチルセルロースや寒天などのゲル物質である。浸透時間をどの程度の時間にするかで厚みや材料が決定される。１８はＬ−乳酸をピルビン酸に酸化させるために必要な乳酸オキシダーゼ等の酵素からなる反応層で、第２作用電極１４、第２対極１３の薄膜上に親水性高分子と以下説明する電子伝達体を介して形成される。乳酸にはＤ−乳酸もあるが、Ｄ−乳酸をＬ−乳酸にラセミ化する乳酸ラセマーゼと混合しておけば、どのような乳酸であってもピルビン酸に変化させることができる。なお、この他、反応層１８にはリン酸塩が混合される。親水性高分子としては、アルキレンオキサイド系の高分子が適当であり、電子伝達体はフェロセン、フェリシアン化カリウム、ベンゾキノンが適当である。本実施の形態１ではスルホン酸ナトリウム−ｐ−ベンゾキノンを用いている。このほかの水溶性のベンゾキノン、さらには水溶性のキノン化合物であってもよい。
【００４４】
乳酸検出部Ｂに乳酸の酸化電位が印加されると、乳酸は上記のような酵素と電子伝達体によりプロトンを奪われピルビン酸に酸化され、酵素は活性中心が酸化型から還元型に変化する。この還元型の酵素は酸化型の電子伝達体と反応して再び酸化型の酵素に戻り、さらに電子伝達体は酸化型から電子を放出して還元型に戻るものである。本実施の形態１のように、乳酸オキシダーゼと乳酸ラセマーゼを酵素として使い、電子伝達体としてスルホン酸ナトリウム−ｐ−ベンゾキノンを用いた場合には、スルホン酸ナトリウム−ｐ−ベンゾキノンからスルホン酸ナトリウム−ｐ−ヒドロキシベンゾキノンに変化する。ヒドロキシベンゾキノンからベンゾキノンに酸化するとき流れる酸化電流が試料液中に含まれる乳酸量に比例するため、この酸化電流を測定することにより試料液の乳酸が測定できるものである。
【００４５】
第２対極１３は白金、黒鉛、金、ステンレスその他の導電性材料から構成され、上記した第２対極用パターン１５と接続されるが、第２対極用パターン１５と別材料で構成するのでなく、第２対極用パターン１５といっしょに通電性カーボンペーストで一体形成するのがよい。また、第２作用電極１４も同じく白金、黒鉛、金、ステンレスその他の導電性材料で構成し、第２作用電極１４と接続されるが、第２作用電極用パターン１６といっしょに通電性カーボンペーストで一体形成するのがよい。
【００４６】
第２対極用パターン１５と第２作用電極用パターン１６は、第１対極用パターン９、第１作用電極用パターン１０、第２対極用パターン１５、第２作用電極用パターン１６、比較電極用パターン１１の順で測定電極１の長手方向に平行に沿って形成され、試料注入孔４の付近でそれぞれ第２対極１３、第２作用電極１４と接続される。測定電極１の一端は後述する潰瘍性大腸疾患測定装置の電極挿入コネクタ（図示しない）に差し込まれるため、各電極パターンの端部は端部付近で屈曲されてよい。
【００４７】
このように、本実施の形態１の測定電極は、潰瘍性大腸疾患測定装置の電極挿入コネクタに挿入して資料注入孔４の上から試料液を滴下すると、試料液は連絡溝を通って測定電極１内の総脂肪酸検出部Ａと乳酸検出部Ｂのそれぞれ２つの試料保持室に２分されて導かれる。バッファ層１７が乳酸検出部Ｂに設けられているため、試料液の乳酸検出部Ｂ内への浸透が遅れ、両者の切り離しが行われ、総脂肪酸検出部Ａによる総脂肪酸検出が終了してから乳酸検出が行えることになる。
【００４８】
総脂肪酸の検出は次のように行われる。比較電極８の発生する基準電位からみて、第１作用電極７の電位を脂肪酸の還元電位となるように第１対極６と第１作用電極７間に電圧を印加すると、試料液の滴下した状態ではプレプロトンアタック物質がプロトンアタック物質化し、脂肪酸からプロトンを奪って還元電流が流れる。潰瘍性大腸疾患により大腸内で産生される短鎖脂肪酸は、酢酸、プロピオン酸、酪酸、バレリル酸、乳酸など炭素数６までのカルボン酸であるが、これらのカルボン酸の還元電位は比較的近く、この近傍の還元電位を印加すれば体内で産生される総脂肪酸の濃度が測定できるものである。
【００４９】
第１対極６と第１作用電極７間にかける電圧の印加の方法には代表的な２つの異なる方法がある。１つは第１作用電極７の電位を比較電極８に対して＋８００ｍＶ〜−１０００ｍＶの範囲で掃引する方法である。この方法はボルタンメトリーと呼ばれる。図５（ａ）はボルタンメトリーを行ったときに現れるプレピークの説明図、図５（ｂ）は還元電流値と脂肪酸濃度との関係図である。図５に示すように、掃引したときの電位−還元電流曲線（ボルタモグラム）の中に現れる還元電流のプレピーク値を測定し、このプレピーク値により脂肪酸の総量濃度に比例するのを利用して脂肪酸の濃度を測定するものである。なお、掃引速度としては電極反応を電子移動律速とするために１０ｍＶ／ｓ〜２００ｍＶ／ｓとするのが適当である。
【００５０】
２つめの測定方法は、第１作用電極７の電位を比較電極８に対して＋１００ｍＶ〜−６００ｍＶの範囲の短鎖脂肪酸の還元電位をパルス状またはステップ状に印加する方法である。但し、第１作用電極７が上述の例示した材料と異なる材料の場合、若干の変動がある。この方法はクロノアンペロメトリーと呼ばれる。図６はクロノアンペロメトリーを行ったときに現れるファラデー電流の説明図である。図６に示すように、第１作用電極７上に電気二重層が形成されると、プレプロトンアタック物質はアニオン化し、プロトンアタック物質となって短鎖脂肪酸からプロトンを奪う。本実施の形態１ではｏ−ベンゾキノン誘導体が用いられているから、電子の移動により還元され、ヒドロ化してｏ−ヒドロキシベンゾキノン誘導体となる。このとき急激に流れる還元電流はファラデー電流と呼ばれるが、このファラデー電流値を測定して短鎖脂肪酸の総濃度を測定するものである。
【００５１】
次に乳酸検出は次のように行われる。バッファ層１７が存在するために総脂肪酸検出部Ａが測定を終えるまで乳酸検出は遅延させられる。実施の形態３として後述する潰瘍性大腸疾患測定装置の制御部により所定の時間経過後に第２対極１３、第２作用電極１４との間に乳酸の酸化電位を印加すると、乳酸は、酵素と電子伝達体であるスルホン酸ナトリウム−ｐ−ベンゾキノンによりプロトンを奪われピルビン酸に酸化され、酵素は活性中心が酸化型から還元型に変化する。この還元型の酵素はスルホン酸ナトリウム−ｐ−ベンゾキノンと反応して再び酸化型の酵素に戻り、スルホン酸ナトリウム−ｐ−ベンゾキノンは電子を受入れてスルホン酸ナトリウム−ｐ−ヒドロキシベンゾキノンになり、さらに電極でキノン体に戻る。このとき流れる酸化電流が試料液中に含まれる乳酸量に比例するため、この酸化電流を測定することにより試料液の乳酸が測定できるものである。
【００５２】
このように、本実施の形態１の測定電極１は、試料注入孔４に試料液を滴下することにより簡単に短鎖脂肪酸の総脂肪酸濃度と乳酸濃度を検出できる。これにより両者のバランスをみることができ、潰瘍性大腸疾患の患者と健常人で（乳酸濃度／短鎖脂肪酸濃度）の比率を計算すると、大きな差となり、精度高く潰瘍性大腸疾患を発見することができる。また、電気化学的測定であるから試料が少なくても測定でき、迅速に検出することができる。痔ろうのような慢性病の場合には、出血はあるが（乳酸濃度／短鎖脂肪酸濃度）が低ければ単なる痔ろうで、潰瘍性大腸疾患ではないことになる。
【００５３】
（実施の形態２）
図３（ａ）は本発明の実施の形態２における潰瘍性大腸疾患測定電極を分解した基板Ａ正面図、図３（ｂ）は本発明の実施の形態２おける潰瘍性大腸疾患測定電極を分解したスペーサ正面図、図３（ｃ）は本発明の実施の形態２における潰瘍性大腸疾患測定電極を分解した基板Ｂ正面図、図４は本発明の実施の形態２における潰瘍性大腸疾患測定電極の断面図である。反応や作用は基本的には実施の形態１と同様であり、詳細な説明は実施の形態１に譲ってここでは省略する。
【００５４】
図３及び図４において、１’ａはポリエチレンテレフタレートなどの絶縁材料からなる測定電極１の総脂肪酸検出部Ａを構成する基板Ａ、１’ｂは同じく絶縁材料からなる測定電極１の乳酸検出部Ｂを構成する基板Ｂ、３は試料を測定電極１に注入することができる連絡溝と液溜めとなる開口が形成された平板のスペーサである。１９はスペーサ３に形成され、総脂肪酸検出部Ａと乳酸検出部Ｂの双方へ試料を分配する試料注入孔であり、２０は試料注入孔１９と連絡された液溜めとなる試料保持室形成用開口である。試料は実施の形態１と同様に作られる。
【００５５】
ここで、実施の形態２の測定電極１の総脂肪酸検出部Ａについて説明する。２１は総脂肪酸検出部Ａを構成するための第１対極、同じく２２は第１作用電極、２３は比較電極である。第１作用電極２２、比較電極２３は本実施の形態２においても矩形形状に形成され、第２対極２１はこの２つをモザイク状に所定間隙を置いて凹部内に収容し、全体として略矩形状になるように組み合わされる。
【００５６】
２４は総脂肪酸検出部Ａを薄膜化して形成するために、基板１’ａにスクリーン印刷で樹脂バインダーを含有させた通電性カーボンペーストを薄く帯状に印刷した第１対極用パターンである。２５は同じく総脂肪酸検出部Ａ形成のために通電性カーボンペーストを薄く帯状に印刷して形成した第１作用電極用パターン、２６は同じく比較電極用パターンである。１２は試料の脂肪酸からプロトンを引き抜き、第１作用電極２２で還元して脂肪酸のプレピークを形成するためのプロトンアタック層である。
【００５７】
第１対極２１、第１作用電極２２、比較電極２３は実施の形態１で説明したものと同様である。
【００５８】
比較電極用パターン２６と第１対極用パターン２４と第１作用電極用パターン２５は、測定電極１の長手方向に平行にこの順で３本沿って形成され、試料注入孔１９の付近でそれぞれ第１対極２１、第１作用電極２３、比較電極２３と接続される。
【００５９】
次に、実施の形態２の測定電極１の乳酸検出部Ｂについて説明する。
【００６０】
２７は乳酸検出部Ｂを構成するための第２対極、同じく２８は第２作用電極である。
【００６１】
第２作用電極２８は本実施の形態２においても矩形形状に形成され、第２対極２７は内部に矩形状の開口を開けた円形状をしている。第２作用電極２８はモザイク状に所定間隙を置いて第２対極２７の開口内に収容され、全体として円形状になるように組み合わされる。
【００６２】
２９は乳酸検出部Ｂを薄膜化して形成するために、基板１’ｂにスクリーン印刷で樹脂バインダーを含有させた通電性カーボンペーストを薄く帯状に印刷した第２対極用パターンである。３０は同じく乳酸検出部Ｂ形成のために通電性カーボンペーストを薄く帯状に印刷して形成した第２作用電極用パターンである。１７は実施の形態１と同様のバッファ層である。１８は実施の形態１と同様の酵素からなる反応層である。
【００６３】
第２対極用パターン２９と第２作用電極用パターン２５は、測定電極１の長手方向に平行に沿って形成される。乳酸検出部Ｂは、スペーサ３に形成された試料保持室形成用開口２０の位置で、基板Ａ１’ａの総脂肪酸検出部Ａと対向してスペーサ３を挟んで積層される。
【００６４】
このように、本実施の形態２の測定電極は構成されるから、実施の形態１の測定電極１の奏する作用効果のほかに、２つの基板のそれぞれに総脂肪酸検出部Ａと乳酸検出部Ｂを印刷して形成すればよく、電極や配線パターンの配置がシンプルであり、試料保持室形成用開口は１つでよいからスペーサの構成も簡単になるというものである。
【００６５】
（実施の形態３）
図７は本発明の実施の形態３の潰瘍性大腸疾患測定装置の斜視図、図８は本発明の本実施の形態３の潰瘍性大腸疾患測定装置の制御回路図である。
【００６６】
図７において、３１は潰瘍性大腸疾患測定装置本体、３２は潰瘍性大腸疾患測定装置３１の上面に配設された測定を開始するためのスタート・ストップボタン、３３は潰瘍性大腸疾患測定装置３１の電源を入切する電源ボタン、３４は潰瘍性疾患を検出する測定電極１を挿入して内部の制御回路に接続するためのコネクー、３５は試料の（乳酸濃度／短鎖脂肪酸濃度）の比率を表示し、この比率が所定の閾値を超えていたときには潰瘍性疾患の可能性があることの表示を行うＬＣＤ（表示手段）である。
【００６７】
図８において、３６は制御部であって、タイマとメモリを備えており、測定電極１の各電極に所定の電位を与えるための制御とともに、乳酸濃度と短鎖脂肪酸濃度に相当する電流値から乳酸濃度と短鎖脂肪酸濃度を算出したり、（乳酸濃度／短鎖脂肪酸濃度）の比率を演算したりし、さらにＬＣＤを制御するものである。スタート・ストップボタン３２と電源ボタン３３を押すと、制御部３６は対応するスイッチをＯＮし、動作可能になる。そして、制御部３６が総脂肪酸濃度の検出を開始を指令するとタイマがカウントを開始し、カウントアウトした時点で乳酸を検出するよう指令するものである。３７は比較電極や第１対極、第２対極に所定の電位を印加するためのデータをアナログ信号に変換するＤ／Ａコンバーター、３８は第１対極または第２対極に所定の電位を印加するためのオペアンプ、３９は比較電極側と対極側への出力を切り替えるリレー、４０は第１対極と第２対極への出力を切り替えるリレーである。また、４１は第１作用電極、第２作用電極に所定の電位を印加するためのデータをアナログ信号に変換するＤ／Ａコンバーター、４３は第１作用電極か第２作用電極に所定の電位を印加するためのオペアンプ、４４は第１作用電極と第１対極間、第２作用電極と第２対極間を流れる電流を測定するための抵抗、４５は第１作用電極と第２作用電極とを切り替えるためのリレーである。４６は抵抗４４の両端の電圧を入力され降下電圧を抵抗値で割って増幅して出力する電圧増幅部、４７は電圧増幅部４６で増幅された降下電圧をデータ化して制御部に入力するＡ／Ｄコンバーターである。制御部３６はこのデータを第１作用電極と第１対極間、第２作用電極と第２対極間を流れる電流値として内部のメモリに記憶する。
【００６８】
次に、本実施の形態３の制御回路がどのように動作するのか説明する。測定電極１をコネクター３４に挿入し、試料注入孔４に試料液を滴下して、スタート・ストップボタン３２と電源ボタン３３を押すと、マイクロコンピュータを内蔵した制御部３６は対応するスイッチをＯＮし、動作可能になる。制御部３６はこれを受けて、カウンタにより計時を開始し、脂肪酸を検出するため総脂肪酸検出部Ａを構成する第１対極、第１作用電極、比較電極に通電するためリレー３９をＢ接点側、リレー４０をＡ’接点側に接続し、リレー４５をＡ”側に接続する。その後、制御部３６は比較電極のデータをメモリから読み出し、Ｄ／Ａコンバーター３７でアナログ化してオペアンプ３８に入力する。オペアンプ３８はイマジナリショートを利用して比較電極をデータどおり基準電圧になるように第１対極の電位を制御する。また、制御部３６は第１作用電極のデータをメモリから読み出し、Ｄ／Ａコンバーターでアナログ化してオペアンプ４３に入力する。オペアンプ４３は電流を検出するための抵抗４４で電圧降下が起こり、出力側の第１作用電極の電位に変化がでるのを防止するため、ホロアとなっている。これにより第１作用電極はデータどおりに所定の電位に制御される。
【００６９】
短鎖脂肪酸を検出するためボルタンメトリーで掃引する場合は、制御部３６は第１作用電極の電位を、１０ｍＶ／ｓ〜２００ｍＶ／ｓの掃引速度、＋８００ｍＶ〜−１０００ｍＶの範囲で掃引する。第１対極には、第１作用電極を比較電極の電位を基準にしたとき、掃引する所定の電位になるような電位がオペアンプ３８のイマジナリショートにより印加される。このとき第１作用電極を流れる還元電流値は抵抗４４による電圧降下で検出され、電圧増幅部４６で増幅してからＡ／Ｄコンバーター４７を介してデータ化して制御部３６に入力される。制御部３６は電流値のデータの中でボルタモグラムを構成するデータの中で、プレピーク値となるデータを選択してメモリする。このデータが短鎖総脂肪酸濃度に比例するものである。
【００７０】
ところで、短鎖脂肪酸はボルタンメトリーによって掃引してプレピークを求めて定量するのでなく、所定の電圧を印加してそのとき過渡的に流れる電流を検出して定量するクロノアンペロメトリーでも測定できる。このクロノアンペロメトリーで短鎖脂肪酸を測定する場合は、制御部３６は第１作用電極にパルス状またはステップ状の電圧を印加する。このとき第１作用電極を流れる還元電流値を抵抗４４によって検出し、電圧増幅部４６、Ａ／Ｄコンバーター４７を介してデータ化して制御部３６に入力する。制御部３６は電流値のデータの中でファラデー電流とみられるデータを選択してメモリする。このデータが短鎖総脂肪酸濃度に比例するものである。
【００７１】
制御部３６は総脂肪酸検出部Ａで脂肪酸量を測定した後、タイマがカウントアウトするとリレー３９をＡ接点、リレー４０をＢ’接点、リレー４５をＢ”接点に切り替える。タイマの設定値は測定電極１のバッファ層１７の材料や厚さで適宜選択されているから、カウントアウトした時点では既に試料液は乳酸検出部Ｂ内に浸透し、反応層の酵素の作用で化学反応を起こしている。次いで、制御部３６は第２対極のデータをメモリから読み出し、Ｄ／Ａコンバーター３７でアナログ化してオペアンプ３８に入力する。オペアンプ３８が構成するホロアを介して、第１対極をデータどおりの電位に制御する。制御部３６は乳酸を検出するために第２作用電極に乳酸の酸化電位を印加するデータを読み出し、オペアンプ４３が構成するホロアを介して第２作用電極に乳酸の酸化電位を印加する。酸化電流値は抵抗４４で検出され、制御部３６はこれをデータとしてメモリする。
【００７２】
短鎖脂肪酸と乳酸の電流値検出が終了すると、制御部３６はこれを短鎖脂肪酸濃度と乳酸濃度に換算し、さらに（乳酸濃度／短鎖脂肪酸濃度）の比率を演算してメモリする。この後、制御部３６はＬＣＤにこれらのデータを送って表示させる。
【００７３】
このように、本実施の形態３の潰瘍性大腸疾患測定装置は、ボルタンメトリーによって総脂肪酸検出部Ａで総脂肪酸を測定するとともに、短鎖脂肪酸を測定後反応層で生成される乳酸を乳酸検出部Ｂで測定することができ、乳酸測定の際に発生するピルビン酸のため、短鎖脂肪酸の総量濃度の測定を狂わせることがなく、乳酸濃度と総脂肪酸濃度の比を算出するので潰瘍性大腸疾患を正しく判定できる。クロノアンペロメトリーによって総脂肪酸検出部で総脂肪酸を測定するため、掃引しないで短時間に総脂肪酸を正確に測定できる。
【００７４】
また、短鎖脂肪酸をボルタンメトリーで測定後に、乳酸を酵素反応させて選択的に測定するので、乳酸測定の際発生するプルビン酸で総脂肪酸の濃度が狂うことがない。短鎖脂肪酸をクロノアンペロメトリーで測定した場合でも同様である。
【００７５】
【発明の効果】
以上のように、本発明によれば、以下のような有利な効果が得られる。
【００７６】
請求項１に記載された潰瘍性大腸疾患測定電極によれば、総脂肪酸検出部と乳酸検出部にそれぞれ電圧を印加することにより、プロトンアタック物質で脱プロトンして電気化学的に総脂肪酸濃度を測定した後、バッファ層により遅れて到達した反応層で酵素反応で発生した乳酸濃度を測定できる。
【００７７】
請求項２に記載された潰瘍性大腸疾患測定電極によれば、乳酸を特異的に酸化できる酵素がＬ−乳酸だけであっても、Ｄ−乳酸をラセミ化して乳酸の総量濃度を正確に測定できる。
【００７８】
請求項３に記載された潰瘍性大腸疾患測定電極によれば、血液が溶解しづらく、電極への固定化が容易である。
【００７９】
請求項４に記載された潰瘍性大腸疾患測定電極によれば、固定化力が強くなる。
【００８０】
請求項５に記載された潰瘍性大腸疾患測定電極によれば、コンパクトであり持ち運びが容易である。
【００８１】
請求項６に記載された潰瘍性大腸疾患測定電極によれば、各電極を平面化して薄くコンパクトにでき、生産も容易である。
【００８２】
請求項７に記載された潰瘍性大腸疾患測定装置によれば、ボルタンメトリーによって総脂肪酸検出部で総脂肪酸を測定するとともに、総脂肪酸を測定後反応層で生成される乳酸を乳酸検出部で測定することができ、乳酸測定の際に発生するピルビン酸のために総脂肪酸の総量の測定を狂わせることがなく、乳酸濃度と総脂肪酸濃度の比を算出するので潰瘍性大腸疾患を正しく判定できる。
【００８３】
請求項８に記載された潰瘍性大腸疾患測定装置によれば、クロノアンペロメトリーによって総脂肪酸検出部で総脂肪酸を測定するとともに、総脂肪酸を測定後反応層で生成される乳酸を乳酸検出部で、乳酸測定の際に発生するピルビン酸のために、総脂肪酸の総量の測定を狂わせることがなく、乳酸濃度と総脂肪酸濃度の比を算出するので潰瘍性大腸疾患を正しく判定できる。
【００８４】
請求項９に記載された潰瘍性大腸疾患判定方法によれば、総脂肪酸をボルタンメトリーで測定後に、乳酸を酵素反応させて選択的に測定するので、乳酸測定の際発生するピルビン酸のために総脂肪酸の濃度が狂うことがない。
【００８５】
請求項１０に記載され潰瘍性大腸疾患判定方法によれば、総脂肪酸をクロノアンペロメトリーで測定後に、乳酸を酵素反応させて選択的に測定するので、乳酸測定の際発生するピルビン酸のために総脂肪酸の濃度が狂うことがない。
【図面の簡単な説明】
【図１】（ａ）本発明の実施の形態１における潰瘍性大腸疾患測定電極の全体構成図
（ｂ）本発明の実施の形態１における潰瘍性大腸疾患測定電極の断面図
【図２】（ａ）本発明の実施の形態１における潰瘍性大腸疾患測定電極を分解したカバー正面図
（ｂ）本発明の実施の形態１における潰瘍性大腸疾患測定電極を分解したスペーサ正面図
（ｃ）本発明の実施の形態１における潰瘍性大腸疾患測定電極を分解した基板正面図
【図３】（ａ）本発明の実施の形態２における潰瘍性大腸疾患測定電極を分解した基板Ａ正面図
（ｂ）本発明の実施の形態２おける潰瘍性大腸疾患測定電極を分解したスペーサ正面図
（ｃ）本発明の実施の形態２における潰瘍性大腸疾患測定電極を分解した基板Ｂ正面図
【図４】本発明の実施の形態２における潰瘍性大腸疾患測定電極の断面図
【図５】（ａ）ボルタンメトリーを行ったときに現れるプレピークの説明図
（ｂ）還元電流値と脂肪酸濃度との関係図
【図６】クロノアンペロメトリーを行ったときに現れるファラデー電流の説明図
【図７】本発明の実施の形態３の潰瘍性大腸疾患測定装置の斜視図
【図８】本発明の本実施の形態３の潰瘍性大腸疾患測定装置の制御回路図
【符号の説明】
１潰瘍性大腸疾患測定電極（測定電極）
１ａ基板
１’ａ基板Ａ
１’ｂ基板Ｂ
２カバー
３スペーサ
４、１９試料注入孔
５、２０試料保持室形成用開口
６、２１第１対極
７、２２第１作用電極
８、２３比較電極
９、２４第１対極用パターン
１０、２５第１作用電極用パターン
１１、２６比較電極用パターン
１２プロトンアタック層
１３、２７第２対極
１４、２８第２作用電極
１５、２９第２対極用パターン
１６、３０第２作用電極用パターン
１７バッファ層
１８反応層
３１潰瘍性大腸疾患測定装置本体
３２スタート・ストップボタン
３３電源ボタン
３４コネクー
３５ＬＣＤ
３６制御部
３７、４１Ｄ／Ａコンバーター
３８、４３オペアンプ
３９、４０、４５リレー
４４抵抗
４６電圧増幅部
４７Ａ／Ｄコンバーター[0001]
BACKGROUND OF THE INVENTION
The present invention relates to an ulcerative colorectal disease measuring electrode for detecting ulcerative colorectal disease including colorectal cancer, an ulcerative colorectal disease measuring device using the same, and an ulcerative colorectal disease determination for detecting this ulcerative colorectal disease Regarding the method.
[0002]
[Prior art]
Recently, the most feared disease in the world is malignant tumor, cancer. Even if cancer develops, it tends to be too late because it progresses with little subjective symptoms in the initial stage. Among them, colorectal cancer is more serious than gastric cancer, esophageal cancer, duodenal cancer, etc., and almost no symptoms appear until bleeding occurs during defecation. And when this bleeding occurs, colorectal cancer is usually already proceeding considerably, and if it is discovered at this point, the treatment will be considerably limited compared to the case of early detection. There are many. This is the case even if this is a benign ulcerative colorectal disease, and when the bleeding occurs, the condition is usually very advanced. What is worse, such hemorrhagic defecation can occur not only in colorectal cancer and ulcerative colorectal disease, but also in chronic diseases such as hemorrhoids. Under these circumstances, there are many patients who are wandering in Japan, and patients often lost the opportunity for treatment because they easily considered the chance of discovering colorectal cancer. Therefore, the development of a method for early detection of ulcerative colorectal diseases such as colorectal cancer before bleeding occurs, and a method that can easily and accurately determine the distinction between mere fistula and diseases such as colorectal cancer when there is bleeding. Development and further development of an ulcerative colorectal disease measuring device used for that purpose have been desired.
[0003]
By the way, cancer and benign polyps are the same in that cells proliferate by cell division. However, while benign cell division is limited to the extent necessary, malignant tumors such as cancer grow indefinitely. Many of these malignant tumors are only transformed by normal cells due to gene damage, etc., so it is difficult to distinguish malignant cells from benign cells on the tissue. If the collected tissue is analyzed at the gene level, it is possible to determine whether it is a cancerous cell or a normal cell, but it requires a large analyzer to analyze the gene and a relatively long time analysis. Therefore, it cannot be a simple determination method that can be performed by the patient on a daily basis. In order to make early detection, it cannot be expected to spread unless it is a simple and inexpensive device, and even if there is bleeding defecation, the patient himself can easily experience hemorrhoid bleeding or ulcers such as colon cancer Unless it is a device that can accurately determine whether bleeding is due to a sexual disease, an ulcerative colorectal disease measuring device does not make sense.
[0004]
Conventionally, as a method for examining colon cancer or the like, a method in which a occult blood component (hemoglobin) in a stool is collected by a stool collection instrument and measured by an immunological measurement method using an anti-hemoglobin antibody. However, although this inspection method is easy, since hemoglobin in stool is stored as a stool sample for a long period of time, there is a problem that hemoglobin in the sample is decomposed during storage and hemoglobin cannot be measured accurately. In order to suppress the degradation of hemoglobin, a method of adding penicillin or a non-penicillin antibiotic (Japanese Patent Laid-Open No. 7-72154) has been disclosed. Also, the accuracy of the measurement results could not be expected. Moreover, even if the presence of hemoglobin can be measured, it cannot be distinguished whether it is bleeding due to hemorrhoids or bleeding due to colorectal cancer. In short, this immunoassay is merely a determination of whether human blood is present during defecation or not.
[0005]
In this way, accurate determination can be made by cell-level tissue examination at the gene level, but it requires expert knowledge and specialized analyzers, and is far from inexpensive and easy to measure by the patient himself. . In addition, an immunological measurement method for checking the presence or absence of bleeding by stool collection using a stool collection is easy but has problems with accuracy. In addition, urgent diseases such as colorectal cancer and ulcers and daily habits, etc. It was completely indistinguishable from chronic diseases. In addition, in the end, specialists are required to perform immunological measurements, and patients cannot judge themselves. Basically, another precise examination is required again. For these reasons, it is desired to create a completely new test method for discovering that an ulcer has formed in the large intestine regardless of genetic analysis or blood.
[0006]
By the way, regarding ulcerative colorectal diseases, the following reports have been made on stool bacteria in patients and short-chain fatty acids that are metabolites thereof (Journal of Japanese Society of Gastroenterology 79: 2 pp.193-198). This short-chain fatty acid is a carbohydrate metabolite by intestinal bacteria and is a carboxylic acid having up to 6 carbon atoms such as acetic acid, propionic acid, butyric acid, valeric acid, and lactic acid.
[0007]
According to this report, the number of bacteria in the stool of patients with ulcerative colorectal disease is reduced compared to healthy individuals. Among them, the number of anaerobic bacteria decreases and the number of aerobic bacteria increases. As a result, it has been reported that the concentration of short-chain fatty acids that are metabolites decreases and the concentration of lactic acid that is a non-volatile short-chain fatty acid increases in proportion to the number of bacteria in the stool. It has also been reported that the lactic acid concentration increases as the lesion expands and as it is in the active phase from the remission phase.
[0008]
Thus, ulcerative colorectal disease has a close relationship with short chain fatty acids and lactic acid contained in the stool, and since healthy people have more short chain fatty acids and less lactic acid, (lactic acid concentration / short chain fatty acid concentration) However, the ratio of (lactic acid concentration / short chain fatty acid concentration) is relatively large because the stool of patients with ulcerative colorectal diseases has a small amount of short-chain fatty acids and a large amount of lactic acid. Therefore, if the amount or ratio of lactic acid and short chain fatty acid can be measured easily and accurately, an ulcerative colorectal disease measuring apparatus completely different from the conventional technique can be obtained.
[0009]
In order to realize this ulcerative colorectal disease measuring apparatus, how to measure lactic acid and short chain fatty acid becomes a problem. The present inventors have already proposed a measuring device using voltammetry in order to measure the acidity of an organic acid such as a fatty acid in a simple and short time (Japanese Patent Laid-Open No. 10-288599). This is a method in which a p-benzoquinone derivative or an o-benzoquinone derivative is mixed with an organic solvent and a supporting electrolyte, a measurement sample is put therein, a coexisting electrolyte is prepared, and the acidity is electrochemically measured. A p-benzoquinone derivative or an o-benzoquinone derivative is a proton attack substance, and it is a substance having an action of extracting a proton contained in a non-dissociated acid as well as binding to a dissociated proton. is there. Proton attack substance by applying current to the working electrode and counter electrode soaked in the coexisting electrolyte and the reference electrode showing the reference potential, and applying a negative voltage to the working electrode based on the reference electrode potential. Is anionized by itself and pulls out protons in the acid. Proton attack materials include quinone compounds and azo compounds. However, p-benzoquinone derivatives or o-benzoquinone derivatives are excellent in light stability, and the reduction potential is deviated from the rising point of the dissolved oxygen voltammogram current. It has an excellent feature that it can be measured without oxygen. Therefore, when the potential applied to the working electrode is swept based on the potential of the reference electrode without making troublesome preparation for measurement, the p-benzoquine derivative or o-benzoquine derivative is reduced and the reduction current reaches this peak. On the plus side, the derivative is reduced at each reduction potential by an organic acid such as a fatty acid and shows a pre-peak on the voltammogram. Fatty acids have the property that when the reduction potential is close, the reduction current showing a pre-peak is proportional to the total fatty acid amount, that is, the amount of acid at this reduction potential.
[0010]
A lactic acid measuring electrode for electrochemically measuring lactic acid has also been proposed (Japanese Patent Laid-Open No. 7-92138). This lactic acid measurement electrode forms a working electrode and a counter electrode by printing a conductive carbon paste on a substrate. Further, a reaction layer is formed on this electrode by mixing lactate oxidase and lactate racemase. Lactate oxidase is an enzyme that oxidizes lactic acid, and lactate racemase is an enzyme that racemates lactic acid. By this lactate racemase, D-lactic acid that is not oxidized by lactate oxidase is racemized into L-lactic acid, and L-lactic acid is oxidized with lactate oxidase to generate oxygen peroxide, and this oxygen peroxide is used as the working electrode. It is oxidized by applying a voltage between the counter electrodes. Ferricyan ions can also be used as the electron carrier.
[0011]
Thus, if the ratio of lactic acid to short chain fatty acid is understood easily and accurately, an ulcerative colorectal disease measuring device completely different from the conventional technology and an ulcerative colorectal disease measuring electrode therefor can be obtained. Measurement of lactic acid produced errors.
[0012]
[Problems to be solved by the invention]
As described above, occult blood components (hemoglobin) in stool are collected by a stool collection instrument and measured by an immunological measurement method using an anti-hemoglobin antibody. Since the sample is stored for a long time, hemoglobin in the sample is decomposed during storage, and there is a problem that hemoglobin cannot be measured accurately. Even if a method of adding penicillin or a non-penicillin antibiotic is used, the measurement takes a long time and the measurement is not so accurate. Moreover, even though the presence of hemoglobin can be measured, it cannot be distinguished whether it is bleeding due to hemorrhoids or bleeding from colon cancer or ulcers.
[0013]
In addition, conventional measuring devices that measure the acidity of an organic acid by mixing a proton attack substance with voltammetry and electrodes for the measurement can measure the total concentration of organic acids electrochemically. It was difficult to measure lactic acid alone, and lactic acid could not be quantitatively determined. Therefore, if lactic acid is separately quantified, it must be measured by specifically oxidizing lactic acid with an enzyme such as lactate oxidase and generating oxygen peroxide as in the conventional technique. In order to produce an acid, there was a problem that the measurement of the concentration of total fatty acid by voltammetry was upset by the difference in reduction potential between pyruvic acid and lactic acid. Even when an electron carrier was used, the same was basically true.
[0014]
Therefore, the present invention solves such a conventional problem, and provides an ulcerative colorectal disease measuring electrode for making a simple, accurate determination in a short time without performing a blood test by a specialist. Objective.
[0015]
It is another object of the present invention to provide an apparatus for measuring ulcerative colorectal disease that can be easily and accurately determined in a short time without having a blood test performed by a specialist.
[0016]
Furthermore, an object of the present invention is to provide a method for determining ulcerative colorectal disease, which can be determined easily and accurately in a short time without performing a blood test by a specialist.
[0017]
[Means for Solving the Problems]
In order to solve the above problems, an ulcerative colorectal disease measuring electrode of the present invention is an ulcerative colorectal disease measuring electrode comprising a total fatty acid detection part and a lactic acid detection part formed on a base, wherein the total fatty acid detection The first working electrode having a proton attack layer fixed on the surface, a first counter electrode paired with the first working electrode, and a reference electrode showing a reference potential,
The lactic acid detector is provided on the surface of the second working electrode, the second counter electrode paired with the second working electrode, and the second working electrode and the second counter electrode, and is capable of oxidizing lactic acid And a reaction layer containing
A buffer layer for separating lactic acid detection from total fatty acid detection is provided on the surface of the reaction layer.
[0018]
This makes it possible to make a simple, accurate determination in a short time without performing a blood test by a specialist.
[0019]
The ulcerative colorectal disease measuring device of the present invention comprises the ulcerative colorectal disease measuring electrode described above, and when the sample liquid to be measured is dropped onto the ulcerative colorectal disease measuring electrode, the first working electrode and the first A first power source for applying a voltage between the counter electrodes, and a second power source for applying a voltage between the second working electrode and the counter electrode,
A controller that sweeps the potential between the first working electrode and the comparison electrode and controls the potential between the second working electrode and the counter electrode to a predetermined potential; and between the first working electrode and the first counter electrode A first detector that detects a flowing current; and a second detector that detects a current flowing between the second working electrode and the second counter electrode, and is detected by the first detector and the second detector. And an arithmetic unit for calculating a ratio between the amount of lactic acid and the total amount of fatty acids based on the current value.
[0020]
Thereby, it is possible to provide an ulcerative colorectal disease measuring electrode that can be determined easily and accurately in a short time without performing a blood test by an expert.
[0021]
Furthermore, the ulcerative colorectal disease determination method of the present invention sweeps the potential of the first working electrode to which the proton attack layer is immobilized with reference to the potential of the reference electrode, while dropping the sample liquid to be measured. A reduction current flowing between the electrode and the first counter electrode is measured, and then the sample liquid to be measured is permeated into a reaction layer containing an enzyme that oxidizes lactic acid, and a predetermined voltage is applied between the second working electrode and the second counter electrode. It is characterized by determining the ulcerative colorectal disease from the relationship between the reduction current and the oxidation current by applying and measuring the oxidation current.
[0022]
Thus, it is possible to provide a method for determining ulcerative colorectal disease that can be determined easily and accurately in a short time without performing a blood test by an expert.
[0023]
DETAILED DESCRIPTION OF THE INVENTION
The invention described in claim 1 is an ulcerative colorectal disease measurement electrode comprising a total fatty acid detection part and a lactic acid detection part formed on a base,
The total fatty acid detection unit includes a first working electrode having a proton attack layer immobilized on a surface thereof, a first counter electrode paired with the first working electrode, and a reference electrode indicating a reference potential;
The lactic acid detector is provided on the surface of the second working electrode, the second counter electrode paired with the second working electrode, and the second working electrode and the second counter electrode, and is capable of oxidizing lactic acid And a reaction layer containing
Since the reaction layer is provided with a buffer layer for separating lactic acid detection from total fatty acid detection, a voltage is applied to each of the total fatty acid detection unit and the lactic acid detection unit. Thus, after deprotonating with a proton attack substance and measuring the total fatty acid concentration electrochemically, the concentration of lactic acid generated by the enzymatic reaction can be measured in the reaction layer that is separated by the buffer layer and penetrates with delay.
[0024]
The invention described in claim 2 is the ulcerative colorectal disease measuring electrode according to claim 1, wherein the reaction layer contains an enzyme capable of racemizing lactic acid, so that lactic acid can be specifically oxidized. Even if the enzyme is only L-lactic acid, the total concentration of lactic acid can be accurately measured by racemizing D-lactic acid.
[0025]
The invention described in claim 3 is the ulcerative colorectal disease measuring electrode according to claim 1, wherein the proton attack substance is a quinone compound or an azo compound. It is difficult to dissolve and easy to fix to the electrode.
[0026]
The invention described in claim 4 is the ulcerative colorectal disease measuring electrode according to claim 3, wherein the quinone compound is a mercaptoquinone compound.
[0027]
The invention described in claim 5 is the ulcerative colorectal disease measuring electrode according to any one of claims 1 to 4, wherein the base is a substrate, and is therefore compact and easy to carry. is there.
[0028]
The invention described in claim 6 is characterized in that all of the first working electrode, the first counter electrode, the comparison electrode, the second working electrode, and the second counter electrode are formed in a thin film shape. Since it is an electrode for measuring ulcerative colorectal disease according to any one of Items 1 to 5, each electrode can be planarized to be thin and compact, and production is also easy.
[0029]
The invention described in claim 7 comprises the ulcerative colorectal disease measurement electrode according to any one of claims 1 to 6, and the first action when the sample liquid to be measured is dropped onto the ulcerative colorectal disease measurement electrode. A first power source that applies a voltage between an electrode and the first counter electrode; and a second power source that applies a voltage between the second working electrode and the counter electrode;
A controller that sweeps the potential between the first working electrode and the comparison electrode and controls the potential between the second working electrode and the counter electrode to a predetermined potential; and between the first working electrode and the first counter electrode A first detector that detects a flowing current; and a second detector that detects a current flowing between the second working electrode and the second counter electrode, and is detected by the first detector and the second detector. Since the ulcerative colorectal disease measuring device is provided with a calculation unit that calculates the ratio between the lactic acid concentration and the total fatty acid concentration from the measured current value, the total fatty acid detection unit measures the total fatty acid concentration by voltammetry, After measuring the total fatty acid concentration, the concentration of lactic acid produced in the reaction layer can be measured by the lactic acid detector, and the measurement of the total amount concentration of total fatty acids is upset due to pyruvic acid generated during lactic acid measurement. Without measurement Can, ulcerative diseases can correctly determine because calculating the ratio of the lactic acid concentration and total fatty acid concentration.
[0030]
The invention described in claim 8 comprises the ulcerative colorectal disease measurement electrode according to any one of claims 1 to 6, and the first action when the sample liquid to be measured is dropped onto the ulcerative colorectal disease measurement electrode. A first power source that applies a voltage between an electrode and the first counter electrode; and a second power source that applies a voltage between the second working electrode and the counter electrode;
The potential between the first working electrode and the first counter electrode is controlled to a predetermined first potential with reference to the comparison electrode, and the potential between the second working electrode and the second counter electrode is set to a predetermined second potential. A control unit for controlling, a first detection unit for detecting a current flowing between the first working electrode and the first counter electrode, and a second detection unit for detecting a current flowing between the second working electrode and the second counter electrode. An ulcerative colorectal disease measuring apparatus comprising: an arithmetic unit that calculates a ratio between a lactic acid concentration and a total fatty acid concentration based on a current value detected by the first detection unit and the second detection unit Therefore, the total fatty acid concentration can be measured at the total fatty acid detector by chronoamperometry, and the lactic acid produced in the reaction layer can be measured at the lactic acid detector after measuring the total fatty acid concentration. Because of the pyruvic acid that occurs in It can be measured without upset the measurement of the total amount concentration of fatty acids, ulcerative diseases can correctly determine because calculating the ratio of the lactic acid concentration and total fatty acid concentration.
[0031]
The invention described in claim 9 is: The ulcerative colorectal disease measuring electrode according to claim 1. A sample solution to be measured is dropped, and the potential of the first working electrode on which the proton attack layer is immobilized is swept with reference to the potential of the reference electrode, and the reduction current flowing between the working electrode and the first counter electrode is measured. Then, the sample liquid to be measured is permeated into a reaction layer containing an enzyme that oxidizes lactic acid, a predetermined voltage is applied between the second working electrode and the second counter electrode, and an oxidation current is measured. And the oxidation current From the ratio of total fatty acid concentration to lactic acid concentration (lactic acid concentration / short chain fatty acid concentration) of short chain fatty acids calculated from Since it is a method for judging ulcerative colorectal disease characterized by determining it as ulcerative colorectal disease, after measuring the total fatty acid concentration by voltammetry, it is selectively measured by enzymatic reaction of lactic acid. The concentration of total fatty acids does not go wrong with pyruvic acid.
[0032]
The invention described in claim 10 is: The ulcerative colorectal disease measuring electrode according to claim 1. A sample solution to be measured is dropped, and the potential of the first working electrode on which the proton attack layer is immobilized is swept with reference to the potential of the reference electrode, and the reduction current flowing between the working electrode and the first counter electrode is measured. Then, the sample liquid to be measured is permeated into a reaction layer containing an enzyme that oxidizes lactic acid, a predetermined voltage is applied between the second working electrode and the second counter electrode, and an oxidation current is measured. And the oxidation current From the ratio of total fatty acid concentration to lactic acid concentration (lactic acid concentration / short chain fatty acid concentration) of short chain fatty acids calculated from Since it is a method for judging ulcerative colorectal disease characterized by deciding as ulcerative colorectal disease, after measuring total fatty acid by chronoamperometry, it is selectively measured by enzymatic reaction of lactic acid. The concentration of total fatty acids does not go wrong with the pyruvic acid generated.
[0033]
Hereinafter, an embodiment of the present invention will be described with reference to the drawings.
[0034]
(Embodiment 1)
FIG. 1A is an overall configuration diagram of an ulcerative colon disease measuring electrode in Embodiment 1 of the present invention, and FIG. 1B is a cross-sectional view of the ulcerative colon disease measuring electrode in Embodiment 1 of the present invention. 2 (a) is a front view of the cover obtained by disassembling the ulcerative colorectal disease measurement electrode according to Embodiment 1 of the present invention, and FIG. 2 (b) is a spacer obtained by disassembling the ulcerative colorectal disease measurement electrode according to Embodiment 1 of the present invention. FIG. 2 (c) is a front view of the substrate in which the ulcerative colorectal disease measuring electrode in Embodiment 1 of the present invention is disassembled.
[0035]
1 and 2, 1 is an ulcerative colorectal disease measurement electrode (hereinafter referred to as measurement electrode) for electrochemically measuring total fatty acid and lactic acid by dropping a sample solution and 1a is made of an insulating material such as polyethylene terephthalate. The measurement electrode 1 substrate 2 is a perforated flat cover that covers the substrate 1 a and can inject the sample solution dropped from above into the measurement electrode 1. 3 is a liquid sandwiched between the substrate 1 a and the cover 2. It is a flat spacer in which an opening to be a reservoir is formed. The cover 2 and the spacer 3 are preferably formed of polyethylene terephthalate. In the first embodiment, the substrate 1a is employed as a base on which the electrode is placed. However, any material may be used as long as it is an insulating material that can hold the electrode. Reference numeral 4 denotes a sample injection hole formed in the cover 2 and distributes the sample to both the total fatty acid detection part A and the lactic acid detection part B. Reference numeral 5 denotes a communication groove located immediately below the sample injection hole 5 of the cover 2 and the communication groove. It is an opening for forming a sample holding chamber composed of two liquid reservoir openings to be connected. Here, the sample solution is prepared by mixing an organic solvent and an electrolyte from stool collected from defecation. The organic solvent is preferably ethanol, isopropyl alcohol, etc., and the electrolyte is preferably sodium chloride, potassium chloride, lithium chloride or the like. It is appropriate to prepare a sample solution by dissolving in an organic solvent so that stool serving as a sample is 20 g / L to 100 g / L, and mixing the electrolyte with 50 mg / L to 150 mg / L.
[0036]
First, the total fatty acid detection part A of the measurement electrode 1 will be described. 6 is a first counter electrode for constituting the total fatty acid detection part A, 7 is a first working electrode, and 8 is a reference electrode. In the first embodiment, the first working electrode 7 and the comparison electrode 8 are formed in a rectangular shape, and the first counter electrode 9 accommodates the two in a recess with a predetermined gap in a mosaic shape, and is substantially rectangular as a whole. Combined to form a shape.
[0037]
Reference numeral 9 denotes a first counter electrode pattern obtained by thinly printing a conductive carbon paste containing a resin binder on the substrate 1a by screen printing in order to form the total fatty acid detection part A in a thin film. 10 is a first working electrode pattern formed by thinly printing a conductive carbon paste to form the total fatty acid detection portion A, and 11 is a reference electrode pattern. Reference numeral 12 denotes a proton attack layer for extracting protons from the fatty acid of the sample and reducing it with the first working electrode 7 to form a pre-peak of fatty acid.
[0038]
The first counter electrode 6 is made of a conductive material such as platinum, graphite, gold, stainless steel, aluminum or the like, and is connected to the first counter electrode pattern 9 described above. The first counter electrode pattern 9 may be integrally formed with the conductive carbon paste. Next, the first working electrode 7 is composed of a carbon electrode called glassy carbon, a carbon material obtained by sintering a plastic foam called PFC at 1000 ° C. to 2000 ° C., or a thin film formed by vapor deposition or sputtering. A proton attack layer 12 is formed on the surface. The proton attack layer 12 is obtained by immobilizing a pre-proton attack substance by mercapto or drying a solvent after coating, and is not limited to bonding with protons (H +) dissociated from an acid, and does not dissociate to constitute an acid. It has the action of attacking and pulling out protons in the state. This pre-proton attack material includes a quinone compound, an azo compound, and the like, but these do not function to extract protons when they are in a natural potential. In other words, when the sample solution is dropped, the proton attack layer 12 becomes an anionized state of the pre-proton attack substance constituting the proton attack layer 12, and the acid is polarized by the applied voltage and the proton side becomes δ + and has a positive potential. This is because the protons in the acid are extracted, but this does not occur at the natural potential. Although it is connected to the first working electrode pattern 10, the manufacturing process can be simplified even if it is integrally formed with the conductive carbon paste together with the first working electrode pattern 10 instead of being made of a different material as described above. preferable. Examples of the quinone compound include p-benzoquinone, o-benzoquinone, diphenoquinone, naphthoquinone, anthraquinone, benzeneazohydroquinone, and derivatives of these quinones. Mercapto-mercaptoquinone compounds are preferred. Examples of the azo compound include azobenzene, azophenol, benzeneazomethane, benzeneazoethane, azonaphthalene, azotoluene, azobenzoic acid, azoaniline, azoanisolemethylazobenzene, 1-benzeneazonaphthalene, benzeneazonaphthol, oxyazobenzene, 2 4-dioxyazobenzene. An o-benzoquinone derivative is used for the pre-proton attack material of the first embodiment, and the side chain portion is mercaptoylated and bonded to the first working electrode 7.
[0039]
The comparison electrode 8 generates a reference potential for applying a predetermined reduction potential for reducing fatty acid to the first working electrode 7, and is a carbon electrode called gold, carbon or glassy carbon, or a plastic foam called PFC. Is made of a carbon material sintered at 1000 ° C. to 2000 ° C. The comparison electrode 8 is connected to the comparison electrode pattern 11. As with the first working electrode pattern 10, it is preferable to integrally form the conductive carbon paste.
[0040]
The reference electrode pattern 11, the first counter electrode pattern 9, and the first working electrode pattern 10 are formed along three lines in this order parallel to the longitudinal direction of the measurement electrode 1, and are respectively formed near the sample injection hole 4. The counter electrode 6, the first working electrode 7, and the comparison electrode 8 are connected. Parts other than the part where each electrode is formed and the part connected to the connector, that is, the surface of each pattern is covered with an insulating material.
[0041]
Next, the lactic acid detection part B of the measurement electrode 1 will be described.
[0042]
Reference numeral 13 denotes a second counter electrode for constituting the lactic acid detector B, and 14 denotes a second working electrode. In the first embodiment, the second working electrode 14 is formed in a rectangular shape, and the second counter electrode 13 has a circular shape with a rectangular opening formed therein. The second working electrode 14 is accommodated in the opening of the second counter electrode 13 with a predetermined gap in a mosaic shape, and is combined so as to be circular as a whole.
[0043]
Reference numeral 15 denotes a second counter electrode pattern obtained by thinly printing a conductive carbon paste containing a resin binder on the substrate 1a by screen printing in order to form the lactic acid detector B in a thin film. Similarly, reference numeral 16 denotes a second working electrode pattern formed by thinly printing a conductive carbon paste to form the lactic acid detection portion B. 17 is a buffer that cuts off the reaction that occurs in the total fatty acid detection part A and the lactic acid detection part B for measurement when the sample is dropped, and measures the lactic acid after detecting the total fatty acid. Is a layer. The buffer layer 17 is selected such that the sample solution penetrates after a predetermined time has elapsed. The material is a gel substance such as methyl cellulose acetate or agar. The thickness and material are determined depending on how long the penetration time is. Reference numeral 18 denotes a reaction layer made of an enzyme such as lactate oxidase necessary for oxidizing L-lactic acid to pyruvic acid. A hydrophilic polymer and an electron transfer described below are formed on the thin film of the second working electrode 14 and the second counter electrode 13. Formed through the body. Lactic acid includes D-lactic acid, but any lactic acid can be converted to pyruvic acid by mixing D-lactic acid with a lactate racemase that racemates to L-lactic acid. In addition, phosphate is mixed in the reaction layer 18. As the hydrophilic polymer, an alkylene oxide polymer is suitable, and as the electron carrier, ferrocene, potassium ferricyanide, and benzoquinone are suitable. In the first embodiment, sodium sulfonate-p-benzoquinone is used. Other water-soluble benzoquinones and further water-soluble quinone compounds may be used.
[0044]
When an oxidation potential of lactic acid is applied to the lactic acid detection part B, the lactic acid is deprived of protons by the enzyme and electron carrier as described above and oxidized to pyruvic acid, and the enzyme changes its active center from an oxidized form to a reduced form. . This reduced enzyme reacts with the oxidized electron carrier to return to the oxidized enzyme again, and the electron carrier further releases electrons from the oxidized form to return to the reduced form. As in the first embodiment, when lactate oxidase and lactate racemase are used as enzymes and sodium sulfonate-p-benzoquinone is used as an electron carrier, sodium sulfonate-p-benzoquinone to sodium sulfonate-p -Changes to hydroxybenzoquinone. Since the oxidation current that flows when oxidizing from hydroxybenzoquinone to benzoquinone is proportional to the amount of lactic acid contained in the sample solution, lactic acid in the sample solution can be measured by measuring this oxidation current.
[0045]
The second counter electrode 13 is made of a conductive material such as platinum, graphite, gold, stainless steel or the like, and is connected to the above-described second counter electrode pattern 15. It is preferable to integrally form with the conductive carbon paste together with the second counter electrode pattern 15. The second working electrode 14 is also made of a conductive material such as platinum, graphite, gold, stainless steel or the like, and is connected to the second working electrode 14. The conductive carbon paste together with the second working electrode pattern 16 is also used. It is good to form integrally.
[0046]
The second counter electrode pattern 15 and the second working electrode pattern 16 include a first counter electrode pattern 9, a first working electrode pattern 10, a second counter electrode pattern 15, a second working electrode pattern 16, and a reference electrode pattern. 11 are formed in parallel with the longitudinal direction of the measurement electrode 1 and are connected to the second counter electrode 13 and the second working electrode 14 in the vicinity of the sample injection hole 4, respectively. Since one end of the measurement electrode 1 is inserted into an electrode insertion connector (not shown) of the ulcerative colorectal disease measuring apparatus described later, the end of each electrode pattern may be bent near the end.
[0047]
As described above, when the measurement electrode of the first embodiment is inserted into the electrode insertion connector of the ulcerative colorectal disease measuring apparatus and the sample liquid is dropped from above the material injection hole 4, the sample liquid is measured through the communication groove. The total fatty acid detection part A and the lactic acid detection part B in the electrode 1 are each divided into two sample holding chambers and guided. Since the buffer layer 17 is provided in the lactic acid detection part B, the penetration of the sample liquid into the lactic acid detection part B is delayed, both are separated, and the total fatty acid detection by the total fatty acid detection part A is completed. Lactic acid detection can be performed.
[0048]
Detection of total fatty acids is performed as follows. When a voltage is applied between the first counter electrode 6 and the first working electrode 7 so that the potential of the first working electrode 7 becomes the reduction potential of the fatty acid as seen from the reference potential generated by the comparison electrode 8, the sample solution is dropped. Then, the pre-proton attack material becomes a proton attack material, and a reduction current flows by depriving the fatty acid of the proton. Short chain fatty acids produced in the large intestine due to ulcerative colorectal diseases are carboxylic acids having up to 6 carbon atoms such as acetic acid, propionic acid, butyric acid, valeric acid, and lactic acid, but the reduction potential of these carboxylic acids is relatively close. By applying a reduction potential in the vicinity, the concentration of total fatty acids produced in the body can be measured.
[0049]
There are two typical different methods for applying a voltage applied between the first counter electrode 6 and the first working electrode 7. One is a method in which the potential of the first working electrode 7 is swept with respect to the comparison electrode 8 in a range of +800 mV to −1000 mV. This method is called voltammetry. FIG. 5 (a) is an explanatory diagram of a pre-peak that appears when voltammetry is performed, and FIG. 5 (b) is a relationship diagram between the reduction current value and the fatty acid concentration. As shown in FIG. 5, the pre-peak value of the reduction current appearing in the potential-reduction current curve (voltagram) when swept is measured, and the pre-peak value is proportional to the total concentration of fatty acids to make use of the fatty acid concentration. The concentration is measured. The sweep rate is suitably 10 mV / s to 200 mV / s so that the electrode reaction is controlled by electron transfer.
[0050]
The second measurement method is a method in which the potential of the first working electrode 7 is applied to the comparison electrode 8 with a reduction potential of short chain fatty acids in the range of +100 mV to −600 mV in a pulsed or stepwise manner. However, when the first working electrode 7 is made of a material different from the above-exemplified material, there is a slight variation. This method is called chronoamperometry. FIG. 6 is an explanatory diagram of the Faraday current that appears when chronoamperometry is performed. As shown in FIG. 6, when the electric double layer is formed on the first working electrode 7, the pre-proton attack substance is anionized to become a proton attack substance and take away protons from the short-chain fatty acid. In Embodiment 1, since an o-benzoquinone derivative is used, it is reduced by electron transfer and hydrolyzed to become an o-hydroxybenzoquinone derivative. The reduction current that flows suddenly at this time is called the Faraday current, and the total concentration of short chain fatty acids is measured by measuring the Faraday current value.
[0051]
Next, lactic acid detection is performed as follows. Since the buffer layer 17 exists, the lactic acid detection is delayed until the total fatty acid detection unit A finishes the measurement. When an oxidation potential of lactic acid is applied between the second counter electrode 13 and the second working electrode 14 after a predetermined time has elapsed by the control unit of the ulcerative colorectal disease measuring apparatus, which will be described later as a third embodiment, lactic acid is converted into an enzyme and an electron. Proton is taken away by sodium sulfonate-p-benzoquinone, which is a transmitter, and oxidized to pyruvic acid, and the active center of the enzyme changes from oxidized to reduced. This reduced enzyme reacts with sodium sulfonate-p-benzoquinone to return to the oxidized enzyme again, and the sodium sulfonate-p-benzoquinone accepts electrons to become sodium sulfonate-p-hydroxybenzoquinone. To return to the quinone form. Since the oxidation current flowing at this time is proportional to the amount of lactic acid contained in the sample solution, the lactic acid in the sample solution can be measured by measuring this oxidation current.
[0052]
As described above, the measurement electrode 1 of Embodiment 1 can easily detect the total fatty acid concentration and the lactic acid concentration of the short-chain fatty acid by dropping the sample solution into the sample injection hole 4. This makes it possible to see the balance between the two, and when calculating the ratio of (lactic acid concentration / short chain fatty acid concentration) between patients with ulcerative colorectal disease and healthy individuals, it will be a big difference and to detect ulcerative colorectal disease with high accuracy Can do. Moreover, since it is electrochemical measurement, even if there are few samples, it can measure and it can detect rapidly. In the case of chronic diseases such as fistula, if there is bleeding (lactic acid concentration / short chain fatty acid concentration) is low, it is merely fistula and not ulcerative colon disease.
[0053]
(Embodiment 2)
FIG. 3A is a front view of the substrate A obtained by disassembling the ulcerative colorectal disease measurement electrode according to Embodiment 2 of the present invention, and FIG. 3B is an exploded view of the ulcerative colorectal disease measurement electrode according to Embodiment 2 of the present invention. FIG. 3C is a front view of the substrate B obtained by disassembling the ulcerative colorectal disease measurement electrode in Embodiment 2 of the present invention, and FIG. 4 is an ulcerative colorectal disease measurement electrode in Embodiment 2 of the present invention. FIG. The reaction and action are basically the same as those in the first embodiment, and the detailed description will be omitted and omitted here.
[0054]
3 and 4, 1′a is a substrate A constituting the total fatty acid detector A of the measurement electrode 1 made of an insulating material such as polyethylene terephthalate, and 1′b is a lactic acid detector of the measurement electrode 1 also made of an insulating material. Substrates B and 3 constituting B are flat spacers in which a communication groove capable of injecting a sample into the measurement electrode 1 and an opening serving as a liquid reservoir are formed. Reference numeral 19 denotes a sample injection hole formed in the spacer 3 for distributing the sample to both the total fatty acid detection part A and the lactic acid detection part B, and 20 denotes a sample holding chamber forming a liquid reservoir connected to the sample injection hole 19. It is an opening. The sample is made in the same manner as in the first embodiment.
[0055]
Here, the total fatty acid detection part A of the measurement electrode 1 of Embodiment 2 is demonstrated. 21 is a first counter electrode for constituting the total fatty acid detection part A, 22 is a first working electrode, and 23 is a reference electrode. The first working electrode 22 and the comparison electrode 23 are also formed in a rectangular shape in the second embodiment, and the second counter electrode 21 accommodates the two in a recess with a predetermined gap in a mosaic shape, and is substantially rectangular as a whole. Combined to form a shape.
[0056]
Reference numeral 24 denotes a first counter electrode pattern obtained by thinly printing a conductive carbon paste containing a resin binder by screen printing on the substrate 1′a in order to form the total fatty acid detection part A in a thin film. 25 is a first working electrode pattern formed by thinly printing a conductive carbon paste for forming the total fatty acid detection portion A, and 26 is a reference electrode pattern. Reference numeral 12 denotes a proton attack layer for extracting protons from the fatty acid of the sample and reducing it with the first working electrode 22 to form a pre-peak of fatty acid.
[0057]
The first counter electrode 21, the first working electrode 22, and the comparison electrode 23 are the same as those described in the first embodiment.
[0058]
The reference electrode pattern 26, the first counter electrode pattern 24, and the first working electrode pattern 25 are formed along the three in this order parallel to the longitudinal direction of the measurement electrode 1, and are respectively formed in the vicinity of the sample injection hole 19. The counter electrode 21, the first working electrode 23 and the comparison electrode 23 are connected.
[0059]
Next, the lactic acid detection part B of the measurement electrode 1 of Embodiment 2 is demonstrated.
[0060]
Reference numeral 27 denotes a second counter electrode for constituting the lactic acid detector B, and 28 denotes a second working electrode.
[0061]
The second working electrode 28 is also formed in a rectangular shape in the second embodiment, and the second counter electrode 27 has a circular shape with a rectangular opening formed therein. The second working electrode 28 is accommodated in the opening of the second counter electrode 27 with a predetermined gap in a mosaic shape, and is combined so as to be circular as a whole.
[0062]
Reference numeral 29 denotes a second counter electrode pattern obtained by thinly printing a conductive carbon paste containing a resin binder by screen printing on the substrate 1′b in order to form the lactic acid detector B in a thin film. Reference numeral 30 denotes a second working electrode pattern formed by thinly printing a conductive carbon paste for forming the lactic acid detection part B. Reference numeral 17 denotes a buffer layer similar to that of the first embodiment. Reference numeral 18 denotes a reaction layer made of the same enzyme as in the first embodiment.
[0063]
The second counter electrode pattern 29 and the second working electrode pattern 25 are formed along the longitudinal direction of the measurement electrode 1. The lactic acid detection unit B is stacked with the spacer 3 interposed therebetween at the position of the sample holding chamber forming opening 20 formed in the spacer 3 so as to face the total fatty acid detection unit A of the substrate A1′a.
[0064]
Thus, since the measurement electrode according to the second embodiment is configured, in addition to the operational effects exhibited by the measurement electrode 1 according to the first embodiment, the total fatty acid detection unit A and the lactic acid detection unit B are provided on each of the two substrates. The electrode and the wiring pattern are simply arranged, and the number of the sample holding chamber forming openings is one, so that the configuration of the spacer is simplified.
[0065]
(Embodiment 3)
FIG. 7 is a perspective view of the ulcerative colon disease measuring apparatus according to Embodiment 3 of the present invention, and FIG. 8 is a control circuit diagram of the ulcerative colon disease measuring apparatus according to Embodiment 3 of the present invention.
[0066]
In FIG. 7, 31 is an ulcerative colorectal disease measuring device main body, 32 is a start / stop button for starting measurement disposed on the upper surface of the ulcerative colorectal disease measuring device 31, and 33 is an ulcerative colorectal disease measuring device 31. A power button for turning the power on and off, 34 is a connector for inserting the measurement electrode 1 for detecting ulcerative disease and connecting it to the internal control circuit, and 35 is a ratio of (lactic acid concentration / short chain fatty acid concentration) of the sample When the ratio exceeds a predetermined threshold value, the LCD (display means) displays that there is a possibility of an ulcer disease.
[0067]
In FIG. 8, reference numeral 36 denotes a control unit which includes a timer and a memory. From the current values corresponding to the lactic acid concentration and the short-chain fatty acid concentration together with the control for applying a predetermined potential to each electrode of the measurement electrode 1. The lactic acid concentration and the short chain fatty acid concentration are calculated, the ratio of (lactic acid concentration / short chain fatty acid concentration) is calculated, and the LCD is further controlled. When the start / stop button 32 and the power button 33 are pressed, the control unit 36 turns on the corresponding switch and becomes operable. When the control unit 36 commands the start of detection of the total fatty acid concentration, the timer starts counting, and commands to detect lactic acid at the time of counting out. 37 is a D / A converter that converts data for applying a predetermined potential to the comparison electrode, the first counter electrode, and the second counter electrode into an analog signal, and 38 is for applying a predetermined potential to the first counter electrode or the second counter electrode. The operational amplifier 39 is a relay for switching output to the comparison electrode side and the counter electrode side, and 40 is a relay for switching output to the first counter electrode and the second counter electrode. 41 is a D / A converter that converts data for applying a predetermined potential to the first working electrode and the second working electrode into an analog signal, and 43 is a predetermined potential applied to the first working electrode or the second working electrode. An operational amplifier 44 for applying, a resistor 44 for measuring a current flowing between the first working electrode and the first counter electrode, a current flowing between the second working electrode and the second counter electrode, and 45 a first working electrode and a second working electrode. It is a relay for switching. A voltage amplifying unit 46 amplifies and outputs the voltage obtained by dividing the voltage across the resistor 44 by dividing the voltage drop by the resistance value, and 47 A converts the voltage dropped by the voltage amplifying unit 46 into data and inputs it to the control unit A. / D converter. The controller 36 stores this data in an internal memory as a current value flowing between the first working electrode and the first counter electrode and between the second working electrode and the second counter electrode.
[0068]
Next, how the control circuit of the third embodiment operates will be described. When the measurement electrode 1 is inserted into the connector 34, the sample solution is dropped into the sample injection hole 4, and the start / stop button 32 and the power button 33 are pressed, the control unit 36 incorporating the microcomputer turns on the corresponding switch. Become operational. In response to this, the control unit 36 starts timing by the counter, and in order to detect the fatty acid, the relay 39 is connected to the B contact side to energize the first counter electrode, the first working electrode, and the comparison electrode constituting the total fatty acid detection unit A. Then, the relay 40 is connected to the A ′ contact side and the relay 45 is connected to the A ″ side. Thereafter, the control unit 36 reads the data of the comparison electrode from the memory, converts it to analog by the D / A converter 37 and inputs it to the operational amplifier 38. The operational amplifier 38 uses an imaginary short to control the potential of the first counter electrode so that the reference electrode becomes the reference voltage according to the data, and the control unit 36 reads the data of the first working electrode from the memory, and D / The analog signal is converted into an analog signal by the A converter and input to the operational amplifier 43. The operational amplifier 43 has a voltage drop caused by the resistor 44 for detecting the current, and the first working electrode on the output side The first working electrode is controlled to a predetermined potential in accordance with the data, so as to prevent the potential from changing.
[0069]
When sweeping by voltammetry to detect short-chain fatty acids, the control unit 36 sweeps the potential of the first working electrode in the range of 10 mV / s to 200 mV / s, and +800 mV to −1000 mV. A potential that is a predetermined potential to be swept when the first working electrode is based on the potential of the comparison electrode is applied to the first counter electrode by an imaginary short circuit of the operational amplifier 38. At this time, the reduction current value flowing through the first working electrode is detected by a voltage drop by the resistor 44, amplified by the voltage amplifier 46, converted into data via the A / D converter 47, and input to the controller 36. The control unit 36 selects and memorizes data to be a pre-peak value from among the data constituting the voltammogram among the current value data. This data is proportional to the total short chain fatty acid concentration.
[0070]
By the way, short chain fatty acids can be measured by chronoamperometry in which a predetermined voltage is applied and a current flowing transiently is detected and quantified, instead of being swept by voltammetry to obtain a pre-peak. When measuring short chain fatty acids by this chronoamperometry, the control unit 36 applies a pulsed or stepped voltage to the first working electrode. At this time, the reduction current value flowing through the first working electrode is detected by the resistor 44, converted into data via the voltage amplification unit 46 and the A / D converter 47, and input to the control unit 36. The control unit 36 selects and memorizes data that is regarded as a Faraday current from the current value data. This data is proportional to the total short chain fatty acid concentration.
[0071]
After the fatty acid amount is measured by the total fatty acid detection unit A, the control unit 36 switches the relay 39 to the A contact, the relay 40 to the B ′ contact, and the relay 45 to the B ″ contact when the timer counts out. Since it is appropriately selected depending on the material and thickness of the buffer layer 17 of the electrode 1, the sample solution has already permeated into the lactic acid detection part B at the time of counting out and has caused a chemical reaction by the action of the enzyme in the reaction layer. Next, the control unit 36 reads the data of the second counter electrode from the memory, converts it to analog by the D / A converter 37, and inputs it to the operational amplifier 38. The potential of the first counter electrode is equal to the data via the follower formed by the operational amplifier 38. The control unit 36 reads out data for applying an oxidation potential of lactic acid to the second working electrode in order to detect lactic acid, and passes through a follower formed by the operational amplifier 43. Then, an oxidation potential of lactic acid is applied to the second working electrode, and the oxidation current value is detected by the resistor 44, and the control unit 36 stores this as data.
[0072]
When the current value detection of the short chain fatty acid and lactic acid is completed, the control unit 36 converts this into a short chain fatty acid concentration and a lactic acid concentration, and calculates and stores a ratio of (lactic acid concentration / short chain fatty acid concentration). Thereafter, the control unit 36 sends these data to the LCD for display.
[0073]
As described above, the ulcerative colorectal disease measuring apparatus according to the third embodiment measures total fatty acids by the total fatty acid detection unit A by voltammetry, and converts lactic acid produced in the reaction layer after measuring short chain fatty acids into the lactic acid detection unit. Because it is pyruvic acid that can be measured by B and is generated during the measurement of lactic acid, the measurement of the total amount concentration of short-chain fatty acids is not upset, and the ratio of lactic acid concentration to total fatty acid concentration is calculated. Can be determined correctly. Since the total fatty acid is measured by the total fatty acid detector by chronoamperometry, the total fatty acid can be accurately measured in a short time without sweeping.
[0074]
Moreover, since the short chain fatty acid is measured by voltammetry and then selectively measured by enzymatic reaction of lactic acid, the concentration of total fatty acid does not go wrong with the puruvic acid generated during the lactic acid measurement. The same applies when short chain fatty acids are measured by chronoamperometry.
[0075]
【The invention's effect】
As described above, according to the present invention, the following advantageous effects can be obtained.
[0076]
According to the ulcerative colorectal disease measuring electrode according to claim 1, by applying a voltage to each of the total fatty acid detection unit and the lactic acid detection unit, the proton attack substance deprotonates the total fatty acid concentration electrochemically. After the measurement, the lactic acid concentration generated by the enzyme reaction can be measured in the reaction layer that arrives later than the buffer layer.
[0077]
According to the electrode for measuring ulcerative colorectal disease described in claim 2, even if L-lactic acid is the only enzyme capable of specifically oxidizing lactic acid, D-lactic acid is racemized to accurately measure the total concentration of lactic acid. it can.
[0078]
According to the electrode for measuring ulcerative colorectal disease described in claim 3, it is difficult to dissolve blood, and it is easy to immobilize the electrode.
[0079]
According to the electrode for measuring ulcerative colorectal disease described in claim 4, the immobilization force becomes strong.
[0080]
According to the measurement electrode for ulcerative colorectal disease described in claim 5, it is compact and easy to carry.
[0081]
According to the electrode for measuring ulcerative colorectal disease described in claim 6, each electrode can be flattened to be thin and compact, and production is also easy.
[0082]
According to the ulcerative colorectal disease measuring device described in claim 7, the total fatty acid is measured by the total fatty acid detection unit by voltammetry, and the lactic acid produced in the reaction layer after measuring the total fatty acid is measured by the lactic acid detection unit. In addition, the measurement of the total amount of total fatty acids is not disturbed due to pyruvic acid generated during the measurement of lactic acid, and the ratio between the lactic acid concentration and the total fatty acid concentration is calculated, so that ulcerative colorectal disease can be correctly determined.
[0083]
According to the ulcerative colorectal disease measuring apparatus described in claim 8, the total fatty acid is measured by the total fatty acid detection unit by chronoamperometry, and the lactic acid generated in the reaction layer after measuring the total fatty acid is converted into the lactic acid detection unit. Thus, because of the pyruvic acid generated during the measurement of lactic acid, the measurement of the total amount of total fatty acids is not upset, and the ratio between the lactic acid concentration and the total fatty acid concentration is calculated, so that ulcerative colon disease can be correctly determined.
[0084]
According to the method for determining ulcerative colorectal disease described in claim 9, since the total fatty acid is measured by voltammetry and then selectively measured by enzymatic reaction of lactic acid, the total amount of pyruvic acid generated during the measurement of lactic acid is reduced. Fatty acid concentration never goes wrong.
[0085]
According to the method for determining ulcerative colorectal disease described in claim 10, since the total fatty acid is measured by chronoamperometry and then selectively measured by enzymatic reaction of lactic acid, The total fatty acid concentration never goes out of order.
[Brief description of the drawings]
FIG. 1A is an overall configuration diagram of an ulcerative colorectal disease measuring electrode according to Embodiment 1 of the present invention.
(B) Sectional view of the ulcerative colorectal disease measurement electrode in Embodiment 1 of the present invention
FIG. 2 (a) is a front view of the cover in which the ulcerative colorectal disease measurement electrode according to Embodiment 1 of the present invention is disassembled.
(B) Front view of a spacer in which the ulcerative colorectal disease measurement electrode in Embodiment 1 of the present invention is disassembled
(C) Front view of substrate obtained by disassembling ulcerative colorectal disease measurement electrode in Embodiment 1 of the present invention
FIG. 3 (a) is a front view of a substrate A in which an ulcerative colorectal disease measurement electrode according to Embodiment 2 of the present invention is disassembled.
(B) Front view of spacer in which ulcerative colorectal disease measurement electrode in Embodiment 2 of the present invention is disassembled
(C) Front view of substrate B in which ulcerative colorectal disease measurement electrode according to Embodiment 2 of the present invention is disassembled
FIG. 4 is a cross-sectional view of an ulcerative colorectal disease measuring electrode according to Embodiment 2 of the present invention.
FIG. 5A is an explanatory diagram of a pre-peak that appears when voltammetry is performed.
(B) Relationship between reduction current value and fatty acid concentration
FIG. 6 is an explanatory diagram of Faraday current that appears when chronoamperometry is performed.
FIG. 7 is a perspective view of an ulcerative colorectal disease measuring apparatus according to Embodiment 3 of the present invention.
FIG. 8 is a control circuit diagram of the ulcerative colon disease measuring apparatus according to the third embodiment of the present invention.
[Explanation of symbols]
1 Ulcerative colon disease measurement electrode (measurement electrode)
1a substrate
1'a Substrate A
1'b Substrate B
2 Cover
3 Spacer
4, 19 Sample injection hole
5, 20 Opening for sample holding chamber
6, 21 First counter electrode
7, 22 First working electrode
8, 23 Reference electrode
9, 24 First counter electrode pattern
10, 25 First working electrode pattern
11, 26 Reference electrode pattern
12 Proton attack layer
13, 27 Second counter electrode
14, 28 Second working electrode
15, 29 Second counter electrode pattern
16, 30 Pattern for second working electrode
17 Buffer layer
18 Reaction layer
31 Ulcerative colon disease measuring device
32 Start / Stop button
33 Power button
34 Conneco
35 LCD
36 Control unit
37, 41 D / A converter
38, 43 operational amplifier
39, 40, 45 Relay
44 Resistance
46 Voltage amplifier
47 A / D converter

Claims

基台上に形成された総脂肪酸検出部と乳酸検出部を備えた潰瘍性大腸疾患測定電極であって、
前記総脂肪酸検出部が、表面にプロトンアタック層が固定化された第１作用電極と、前記第１作用電極と対をなす第１対極と、基準の電位を示す比較電極を有し、
前記乳酸検出部が、第２作用電極と、前記第２作用電極と対をなす第２対極と、前記第２作用電極と前記第２対極の表面に設けられ、乳酸を酸化することができる酵素を含んだ反応層とを有し、
前記反応層の表面には乳酸検出を総脂肪酸検出から切り離すバッファ層が設けられたことを特徴とする潰瘍性大腸疾患測定電極。An electrode for measuring ulcerative colorectal disease comprising a total fatty acid detection part and a lactic acid detection part formed on a base,
The total fatty acid detection unit includes a first working electrode having a proton attack layer immobilized on a surface thereof, a first counter electrode paired with the first working electrode, and a reference electrode indicating a reference potential;
The lactic acid detector is provided on the surface of the second working electrode, the second counter electrode paired with the second working electrode, and the second working electrode and the second counter electrode, and is capable of oxidizing lactic acid And a reaction layer containing
An ulcerative colorectal disease measuring electrode, wherein a buffer layer for separating lactic acid detection from total fatty acid detection is provided on the surface of the reaction layer.

前記反応層に乳酸をラセミ化することができる酵素が含まれている請求項１記載の潰瘍性大腸疾患測定電極。The ulcerative colon disease measuring electrode according to claim 1, wherein the reaction layer contains an enzyme capable of racemizing lactic acid.

前記プロトンアタック物質がキノン化合物またはアゾ化合物であることを特徴とする請求項１〜２のいずれかに記載の潰瘍性大腸疾患測定電極。3. The ulcerative colorectal disease measuring electrode according to claim 1, wherein the proton attack substance is a quinone compound or an azo compound.

前記キノン化合物がメルカプトキノン化合物であることを特徴とする請求項３記載の潰瘍性大腸疾患測定電極。4. The ulcerative colorectal disease measuring electrode according to claim 3, wherein the quinone compound is a mercaptoquinone compound.

前記基台が基板であることを特徴とする請求項１〜４のいずれかに記載の潰瘍性大腸疾患測定電極。The ulcerative colorectal disease measuring electrode according to claim 1, wherein the base is a substrate.

前記第１作用電極、前記第１対極、比較電極、前記第２作用電極、前記第２対極のいずれもが薄膜状に形成されたことを特徴とする請求項１〜５のいずれかに記載の潰瘍性大腸疾患測定電極。6. The first working electrode, the first counter electrode, the comparison electrode, the second working electrode, and the second counter electrode are all formed in a thin film shape. 6. Ulcerative colon disease measurement electrode.

請求項１〜６のいずれかに記載の潰瘍性大腸疾患測定電極を備え、前記潰瘍性大腸疾患測定電極に被測定試料液を滴下したとき前記第１作用電極と前記第１対極間に電圧を印加する第１電源と、前記第２作用電極と前記対極間に電圧を印加する第２電源とを有し、
前記第１作用電極と前記比較電極間の電位を掃引するとともに前記第２作用電極と前記対極間の電位を所定の電位に制御する制御部と、前記第１作用電極と前記第１対極間に流れる電流を検知する第１検知部と、前記第２作用電極と前記第２対極間に流れる電流を検知する第２検知部を有し、前記第１検知部と前記第２検知部によって検知された電流値により乳酸濃度と総脂肪酸濃度の比を算出する演算部を備えたことを特徴とする潰瘍性大腸疾患測定装置。The ulcerative colorectal disease measurement electrode according to any one of claims 1 to 6, wherein when a sample liquid to be measured is dropped onto the ulcerative colorectal disease measurement electrode, a voltage is applied between the first working electrode and the first counter electrode. A first power source to apply, and a second power source to apply a voltage between the second working electrode and the counter electrode,
A controller that sweeps the potential between the first working electrode and the comparison electrode and controls the potential between the second working electrode and the counter electrode to a predetermined potential; and between the first working electrode and the first counter electrode A first detector that detects a flowing current; and a second detector that detects a current flowing between the second working electrode and the second counter electrode, and is detected by the first detector and the second detector. An apparatus for measuring ulcerative colorectal disease, comprising an arithmetic unit for calculating a ratio between a lactic acid concentration and a total fatty acid concentration based on a current value.

請求項１〜６のいずれかに記載の潰瘍性大腸疾患測定電極を備え、前記潰瘍性大腸疾患測定電極に被測定試料液を滴下したとき前記第１作用電極と前記第１対極間に電圧を印加する第１電源と、前記第２作用電極と前記対極間に電圧を印加する第２電源とを有し、
前記第１作用電極と前記第１対極間を前記比較電極を基準にして所定の第１の電位に制御するとともに前記第２作用電極と前記第２対極間の電位を所定の第２の電位に制御する制御部と、前記第１作用電極と前記第１対極間に流れる電流を検知する第１検知部と、前記第２作用電極と前記第２対極間に流れる電流を検知する第２検知部を有し、前記第１検知部と前記第２検知部によって検知された電流値により乳酸濃度と総脂肪酸濃度の比を算出する演算部を備えたことを特徴とする潰瘍性大腸疾患測定装置。The ulcerative colorectal disease measurement electrode according to any one of claims 1 to 6, wherein when a sample liquid to be measured is dropped onto the ulcerative colorectal disease measurement electrode, a voltage is applied between the first working electrode and the first counter electrode. A first power source to apply, and a second power source to apply a voltage between the second working electrode and the counter electrode,
The potential between the first working electrode and the first counter electrode is controlled to a predetermined first potential with reference to the comparison electrode, and the potential between the second working electrode and the second counter electrode is set to a predetermined second potential. A control unit for controlling, a first detection unit for detecting a current flowing between the first working electrode and the first counter electrode, and a second detection unit for detecting a current flowing between the second working electrode and the second counter electrode. An ulcerative colorectal disease measuring apparatus comprising: an arithmetic unit that calculates a ratio between a lactic acid concentration and a total fatty acid concentration based on current values detected by the first detection unit and the second detection unit.

請求項１〜６のいずれかに記載の潰瘍性大腸疾患測定電極に被測定試料液を滴下し、比較電極の電位を基準にしてプロトンアタック層が固定化された第１作用電極の電位を掃引するとともに、前記作用電極と第１対極間に流れる還元電流を測定し、次いで前記被測定試料液を乳酸が酸化される酵素を含んだ反応層に浸透させ、第２作用電極と第２対極間に所定の電圧を印加して酸化電流を測定して、前記還元電流と前記酸化電流から算出される短鎖脂肪酸の総脂肪酸濃度と乳酸濃度の比（乳酸濃度／短鎖脂肪酸濃度）から潰瘍性大腸疾患と判定することを特徴とする潰瘍性大腸疾患判定方法。 A sample liquid to be measured is dropped on the ulcerative colorectal disease measurement electrode according to any one of claims 1 to 6, and the potential of the first working electrode on which the proton attack layer is immobilized is swept based on the potential of the comparison electrode In addition, the reduction current flowing between the working electrode and the first counter electrode is measured, and then the sample liquid to be measured is permeated into the reaction layer containing an enzyme that oxidizes lactic acid, and between the second working electrode and the second counter electrode. A predetermined voltage is applied to the electrode to measure the oxidation current, and from the ratio of the total fatty acid concentration of the short chain fatty acid and the lactic acid concentration calculated from the reduction current and the oxidation current (lactic acid concentration / short chain fatty acid concentration), ulcerability A method for determining ulcerative colorectal disease, characterized by determining colorectal disease.

請求項１〜６のいずれかに記載の潰瘍性大腸疾患測定電極に被測定試料液を滴下し、比較電極の電位を基準にしてプロトンアタック層が固定化された第１作用電極と第１対極間に所定の第１の電圧を印加して還元電流を測定し、次いで前記被測定試料液を乳酸が酸化される酵素を含んだ反応層に浸透させ、第２作用電極と第２対極間に所定の第２の電圧を印加して酸化電流を測定して、前記還元電流と前記酸化電流から算出される短鎖脂肪酸の総脂肪酸濃度と乳酸濃度の比（乳酸濃度／短鎖脂肪酸濃度）から潰瘍性大腸疾患と判定することを特徴とする潰瘍性大腸疾患判定方法。 A first working electrode and a first counter electrode in which a sample solution to be measured is dropped on the ulcerative colorectal disease measuring electrode according to claim 1 and the proton attack layer is fixed with reference to the potential of the comparative electrode. A reduction current is measured by applying a predetermined first voltage between them, and then the sample liquid to be measured is permeated into a reaction layer containing an enzyme that oxidizes lactic acid, between the second working electrode and the second counter electrode. An oxidation current is measured by applying a predetermined second voltage, and the ratio of the total fatty acid concentration of the short chain fatty acid and the lactic acid concentration calculated from the reduction current and the oxidation current (lactic acid concentration / short chain fatty acid concentration) is calculated. A method for determining ulcerative colorectal disease, characterized in that it is determined as ulcerative colorectal disease.