JP3703787B2

JP3703787B2 - Method for measuring analyte concentration using conductive diamond electrode and apparatus therefor

Info

Publication number: JP3703787B2
Application number: JP2002265886A
Authority: JP
Inventors: 嶋昭藤; ▲煕▼ 一堀; 多謙介本; ブルス・ベンカタ・サラダ; 波裕之斯
Original assignee: Japan Science and Technology Agency; National Institute of Japan Science and Technology Agency
Current assignee: Japan Science and Technology Agency; National Institute of Japan Science and Technology Agency
Priority date: 2002-09-11
Filing date: 2002-09-11
Publication date: 2005-10-05
Anticipated expiration: 2022-09-11
Also published as: JP2004101437A

Description

【０００１】
【発明の背景】
発明の分野
本発明は、導電性ダイヤモンド電極を用いた被検物質濃度を測定するための方法および装置に関する。具体的には、測定妨害物質を含む被検試料液中における被検物質濃度、特に尿酸およびアスコルビン酸を含む被検試料液中における尿酸濃度、を測定するための方法および装置に関する。
【０００２】
背景技術
尿酸は、人体内ではプリン体の最終分解産物として生成されるものである。人間の血清中における尿酸濃度の正常値は、男性では４〜７ｍｇ／ｄｌ、女性では２．５〜６ｍｇ／ｄｌである。また、人間の尿中に排せつされる尿酸量の正常値は、０．２〜１ｇ／２４時間であり、これは尿量を１Ｌと仮定した場合には約１．２〜６ｍＭの濃度に換算される。しかしながら、体内における尿酸の過剰な生成、あるいは腎機能の低下による尿酸の体外排出量の減少により、血液中の尿酸値が高くなることがある。これは、高尿酸血症や関節などに尿酸塩が析出する痛風の原因となる。したがって、体内尿酸値を測定することは、これらの疾患を予防し、あるいは痛風患者の尿酸値を制御する上で極めて重要である。
【０００３】
従来の尿酸測定法として、酵素を用いた方法、および高速液体クロマトグラフィー（ＨＰＬＣ）を用いた分析法が知られている。これらの方法にあっては、操作が複雑であるため専門的知識を必要とし、分析機器も高価となる。
【０００４】
一方、ダイヤモンドは本来抵抗率が１０^１３Ωｃｍ程度の絶縁材料であるが、微量不純物のドープにより導電性を獲得する。この導電性ダイヤモンドには、種々の用途が期待されている。その一つに電気化学用の電極としての利用がある。
導電性とされたダイヤモンドを電気化学用の電極として見ると、広い電位窓を有し、かつバックグラウンド電流が極めて小さいという優れた特長を有する。さらに、物理的、化学的に安定であり、耐久性に優れるとの特長もまた有する。導電性ダイヤモンド（好ましくはその薄膜）を有する電極は、ダイヤモンド電極と一般的に呼ばれるに至っている。
【０００５】
ダイヤモンド電極に関する先駆的研究はIwakiらによって行われた（Iwaki et al., Nuclear Instruments and Methods, 209-210, 1129(1983)）。彼らは、アルゴンや窒素イオンを打ち込んで表面導電性を付与した単結晶ダイヤモンドの電気伝導材料としての性質を研究したものである。同時に、電解質溶液中におけるサイクリックボルタモグラムも示した。その後、ホットフィラメントを用いて気相合成した多結晶ダイヤモンド電極の特性が報告されている（Pleskov et al., J. Electroanal. Chem., 228, 19(1993)）。
【０００６】
本発明者らの一部は、気相合成したダイヤモンド電極を用いて、窒素酸化物の還元について先に報告した（Tenne et al., J. Electroanal. Chem., 347, 409(1993)）。この研究では、ドーパントとしてホウ素を導入したｐ型半導体ダイヤモンドを電極として使用した。その後、ダイヤモンド電極としては、ホウ素をドーパントとするｐ型半導体またはより導電性の高い金属様導電性ダイヤモンドの利用が主流となるに至る。１９９０年代に入って、ダイヤモンド電極の研究が複数のグループにより行われ、１９９５年以降は、より大面積のダイヤモンド薄膜が得られるプラズマＣＶＤ（ＰＣＶＤ）装置を用いて得られたダイヤモンド電極の研究が、電気化学分野にも散見されるに至っている。
【０００７】
陽極酸化処理されたダイヤモンド薄膜電極を用いて尿酸を測定する方法が知られている（例えば、特許文献１参照）。この方法では、被測定液を好ましくはｐＨ１という極めて強い酸性域に制御することにより、測定妨害物質であるアスコルビン酸のピーク電圧と尿酸のピーク電圧とを分離して、尿酸濃度の測定を可能としている。
【０００８】
【特許文献１】
特開２００１−１４７２１１号公報
【０００９】
【発明の概要】
本発明者らは、今般、ある種の被検試料液において、導電性ダイヤモンド電極により二種以上の被検物質が検出され、しかもその二種以上の被検物質のそれぞれの濃度を簡便かつ高精度に測定できるとの知見を得た。また、本発明者らは、二種以上の被検物質のうち一種のみが濃度測定の対象とされる測定対象物質であり、残りの被検物質が測定対象物質の濃度測定において排除されるべき物質である場合において、測定妨害物質の影響を排除するための煩雑な操作を行うことなく、導電性ダイヤモンド電極により測定対象物質の濃度を簡便かつ高精度に測定できるとの知見を得た。より具体的には、尿酸およびアスコルビン酸を含む被検試料液において、弱酸性〜中性（例えばｐＨ５〜８）という穏やかな液性下で、導電性ダイヤモンド電極により尿酸濃度を簡便かつ高精度に測定できるとの知見を得た。
【００１０】
したがって、本発明は、二種以上の被検物質のそれぞれの濃度、特に測定対象物質および測定妨害物質を含む被検試料液中の測定対象物質濃度、を簡便かつ高精度に測定することをその目的としている。
【００１１】
そして、本発明による、導電性ダイヤモンド電極を用いた被検物質濃度の測定方法は、
共に導電性ダイヤモンド電極上で同一電圧の印加により酸化または還元反応を生じうる、第一の被検物質および第二の被検物質を含む被検試料液を用意し、
該被検試料液に、導電性ダイヤモンド電極からなる第一作用電極と、導電性ダイヤモンド電極からなる第二作用電極と、対電極とを接触させ、
前記第一作用電極と前記対電極との間に、第一作用電極上で前記第二の被検物質の酸化または還元反応が生じるが、前記第一の被検物質の酸化または還元反応が実質的に生じない電圧を印加し、該電圧下における電流値を測定し、得られた電流値から前記被検試料液中の第二の被検物質の濃度を算出し、
該第二の被検物質濃度から、前記第二作用電極と前記対電極との間に第二作用電極上で前記第一の被検物質および前記第二の被検物質の両方の酸化または還元反応が生じる電圧を印加した場合における、得られる電流値への前記第二の被検物質の寄与分を算出し、
前記第二作用電極と前記対電極との間に、第二作用電極上で前記第一の被検物質および第二の被検物質の両方の酸化または還元反応が生じる電圧を印加し、該電圧下における電流値を測定し、該電流値から前記第二の被検物質の寄与分を差し引いて、前記第一の被検物質単独に由来する電流値を算出し、該電流値から前記被検試料中の第一の被検物質濃度を算出すること
を含んでなる。
【００１２】
また、本発明による、導電性ダイヤモンド電極を用いた被検物質濃度の測定装置は、
導電性ダイヤモンド電極からなる第一作用電極と、
導電性ダイヤモンド電極からなる第二作用電極と、
対電極と、
前記第一作用電極、第二作用電極、および対電極を被検試料液に接触させる手段と、
前記第一作用電極と前記対電極との間に、第一作用電極上で第二の被検物質の酸化または還元反応が生じるが、第一の被検物質の酸化または還元反応が実質的に生じない電圧を印加して、該電圧下における電流値を測定するとともに、前記第二作用電極と前記対電極との間に、前記第一の被検物質および第二の被検物質の両方の酸化または還元反応が生じる電圧を印加して、該電圧下における電流値を測定する測定手段と、
前記第一作用電極で得られた電流値から前記被検試料液中の第二の被検物質濃度を算出して、該第二の被検物質濃度から第二作用電極における電流値への前記第二の被検物質の寄与分を算出し、さらに、前記第二作用電極における電流値から前記第二の被検物質の寄与分を差し引いて、第一の被検物質単独に由来する電流値を算出し、該電流値から前記被検試料液中の第一の被検物質濃度を算出する算出手段と
を備えてなる。
【００１３】
【発明の具体的説明】
被検試料液
本発明による方法の被検対象となるものは、共に導電性ダイヤモンド電極上で同一電圧の印加により酸化または還元反応を生じうる、第一の被検物質および第二の被検物質を含む被検試料液である。第一の被検物質は、導電性ダイヤモンド電極上において、ある特定の電圧の印加により酸化または還元反応を生じる物質であり、その反応により生じる電流値を利用して濃度測定を行う対象となるものである。一方、第二の被検物質は、導電性ダイヤモンド電極上において第一の被検物質と同一の電圧の印加により酸化または還元反応を生じるとともに、第一の被検物質の酸化または還元反応を実質的に生じない別の電圧の印加によっても酸化または還元反応を生じるものである。第二の被検物質は、互いに異なる電圧の印加によっても酸化または還元反応を生じることができるものであれば２種以上であってもよい。このように、第一および第二の被検物質には、共に導電性ダイヤモンド電極上で酸化または還元反応を生じる電圧が存在する。したがって、第一の被検物質の濃度測定に際して、第二の被検物質は、第一の被検物質の濃度測定を妨害する結果となる。これは、第一の披検物質の酸化または還元反応の際、第二の被検物質も酸化または還元反応による余分な電流を生じるため、電流値に基づく第一の被検物質の正確な定量を行うことができなくなるからである。
【００１４】
上述したように、第二の被検物質には、第一の被検物質と共に酸化または還元反応を生じる電圧以外に、第二の被検物質の酸化または還元反応が生じるが、第一の被検物質の酸化または還元反応が実質的に生じない電圧が存在する。そして、この電圧を導電性ダイヤモンド電極に印加することにより第二の被検物質の濃度を測定することができる。本発明においては、そのようにして得られた第二の被検物質の濃度に基づいて、第一の被検物質の酸化または還元反応の際に生じる電流値の補正を行うことにより、第一の被検物質の濃度測定を可能とする。
【００１５】
本発明によれば第一の被検物質および第二の被検物質の両方の濃度を測定できるので、第一および第二の被検物質の両方が測定対象物質とされる場合が、本発明の一つの典型的な態様である。一方、本発明による方法は、第一の被検物質が濃度測定の対象とされる測定対象物質であり、第二の被検物質が測定対象物質の濃度測定において排除されるべき測定妨害物質であるときに、好ましく用いられる。いずれの態様も、いずれかまたは一方の被検物質量を知るとの目的において相違するのみで、その本質においては同じであるから、以下、後者の態様に基づいて本発明の内容を説明する。
【００１６】
本発明の好ましい態様によれば、測定対象物質が尿酸であり、かつ測定妨害物質がアスコルビン酸であるのが好ましい。尿酸は、尿または血清中にアスコルビン酸と共に含まれる物質であり、その尿酸濃度を知ることは医学上極めて有益である。一方、アスコルビン酸は尿酸測定を妨害する測定妨害物質となる。これは、尿酸およびアスコルビン酸が、共に、陽極酸化ダイヤモンド電極上において０．８Ｖ付近で酸化反応によるピーク電流を生じるためである。すなわち、尿中の尿酸濃度を導電性ダイヤモンド電極により測定しようと試みても、そのままではアスコルビン酸に起因する電流に妨害されて、尿酸濃度の正確な測定を行うことは出来ない。ところが、アスコルビン酸には、上記０．８Ｖにも、０．２Ｖ付近でアスコルビン酸単独のピーク電流を生じる。そこで、本発明によれば、このアスコルビン酸の０．２Ｖ付近における電流値を利用して、アスコルビン酸の濃度をまず算出する。次いで、この濃度に基づく所定の補正を行うことにより、尿酸濃度を測定することができる。したがって、本発明の測定方法によれば、アスコルビン酸を分離する操作を行ったり、あるいは、被検試料液を極めて強い酸性域に制御して尿酸とアスコルビン酸の各ピーク電位を分離することなく、簡便かつ高精度に尿酸を測定することができる。
【００１７】
上記好ましい態様における被検試料液は、尿酸およびアスコルビン酸が含まれていると考えられる、血清、尿、その他の生体由来の液などであることができる。尿または血清を被験試料とし、本発明の方法により尿中の尿酸濃度を知ることにより、高尿酸血症、通風、尿路結石症などの即時診断に有効となる。
【００１８】
本発明の好ましい態様によれば、被検試料液のｐＨが５〜８であるのが好ましい。本発明の測定方法は、測定対象物質と測定妨害物質の酸化または還元反応が同一電位となることを許容するため、例えば、前述した特開２００１−１４７２１１号公報のように、好ましくはｐＨ１という極めて強い酸性下において、測定妨害物質であるアスコルビン酸のピーク電位を、尿酸のピーク電位と分離する必要がない。したがって、尿酸およびアスコルビン酸を含む被検試料液において、弱酸性〜中性（例えばｐＨ５〜８）という穏やかな液性下で、尿酸濃度を測定できるので、披検試料液の取り扱いがし易く、安全性が高くなる。また、本発明の別の好ましい態様によれば、測定に先立ち、前記被検試料液のｐＨを５〜８に調整する工程をさらに行ってもよい。
【００１９】
本発明の好ましい態様によれば、被検試料液がリン酸緩衝液をさらに含んでなるのが好ましく、リン酸緩衝液によりｐＨの調整を行うことができる。この場合、尿酸によるピーク電位と、アスコルビン酸による尿酸と重ならないピーク電位との差が大きくなり、より精度の高い測定が可能となる。
【００２０】
測定系および測定方法
本発明による測定方法において、被検物質測定のための電気化学的系は、作用電極としてのダイヤモンド電極を２個以上設けたこと以外は、一般的な電気化学的な系であることが出来る。すなわち、本発明にあっては、作用電極としての２個以上のダイヤモンド電極を、対電極とともに被検試料液に接触させ、作用電極の各々について対電極との間にそれぞれ異なる電圧を印加する。
【００２１】
この系を模式的に表せば図１の通りとなる。図１に示される系は、被検試料液１を収納した容器２に、被検試料液１と接触するように、第一作用電極３、第二作用電極４、および対電極５を備えてなる。第一作用電極３および第二作用電極４は共に導電性ダイヤモンド電極である。本発明の好ましい態様によれば、参照電極６を系に更に備え、各作用電極３，４と、対電極５とに印加される電圧の絶対値をそれぞれ知ることができるように構成されてもよい。なお、以下の説明において、被検試料液１は、第一の被検物質としての測定対象物質および第二の被検対象物質としての測定妨害物質を含むものとする。
【００２２】
本発明において、第一作用電極と対電極との間に印加される電圧は、第一作用電極上で測定妨害物質の酸化または還元反応が生じるが、測定妨害物質の酸化または還元反応が実質的に生じない電圧とする。すなわち、この印加電圧下では測定妨害物質の酸化または還元反応のみが生じる。本発明の好ましい態様によれば、測定効率および精度の観点から、この印加電圧は、測定妨害物質の酸化または還元のピーク電流を与える電圧であることが好ましい。ここで、ピーク電流を与える電圧とは、例えばサイクリックボルタンメトリーにより、最大電流値を与える電圧として求めることが出来る。本発明者らの得た知見によれば、測定対象物質が尿酸であり、かつ測定妨害物質がアスコルビン酸である弱酸性〜中性付近の被検試料液の場合、陽極酸化導電性ダイヤモンド電極上で測定妨害物質のピーク電流のみを与える電圧は、０．１〜０．３Ｖ（対Ａｇ／ＡｇＣｌ電極）範囲内であり、より具体的には、約０．２Ｖ（対Ａｇ／ＡｇＣｌ電極）である。
【００２３】
上記印加電圧下、第一作用電極と対電極との間に生じる電流値を測定し、得られた電流値から被検試料液中の測定妨害物質濃度を算出する。アスコルビン酸などの測定妨害物質の酸化または還元により第一作用電極と対電極との間に生じる電流値は、系内の測定妨害物質の濃度に正比例する。従って、一旦ある電圧値における電流値と測定妨害物質濃度との関係を求めておけば、その関係から、得られた電流値に対応する被験試料液中の測定妨害物質の濃度を容易に知ることが出来る。すなわち、本発明の好ましい態様にあっては、測定妨害物質の濃度と電流値との検量線を予め作成しておき、この検量線と、得られた電流値とを対比することにより、測定妨害物質の濃度を知ることが出来る。
【００２４】
測定妨害物質は第二作用電極においても測定対象物質と共に検出されるが、上記のように得られた測定妨害物質の濃度から、第二作用電極で測定される電流値への測定妨害物質の寄与分を算出する。すなわち、第二作用電極の場合においても、第一作用電極の場合と同様に、予め、印加しようとする電圧値における電流値と測定妨害物質濃度との関係を求めておけば、その関係から、得られた被験試料液中の測定妨害物質の濃度に対応する電流値を容易に知ることが出来る。本発明の好ましい態様にあっては、測定妨害物質の濃度と電流値との検量線を予め作成しておき、この検量線と、第一作用電極で測定された測定妨害物質濃度とを対比することにより、第二作用電極で測定される電流値への測定妨害物質の寄与分を知ることが出来る。
【００２５】
本発明において、第二作用電極と前記対電極との間に印加される電圧は、第二作用電極上で測定対象物質および測定妨害物質の酸化または還元反応が生じる電圧とする。すなわち、この印加電圧下では測定対象物質および測定妨害物質の両方の酸化または還元反応が生じている。本発明の好ましい態様によれば、測定効率および精度の観点から、この印加電圧は、測定対象物質および測定妨害物質の両方の酸化または還元のピーク電流を与える電圧であることが好ましい。ここで、ピーク電流を与える電圧とは、例えばサイクリックボルタンメトリーにより、最大電流値を与える電圧として求めることが出来る。本発明者らの得た知見によれば、測定対象物質が尿酸であり、かつ測定妨害物質がアスコルビン酸である弱酸性〜中性付近の被検試料液の場合、陽極酸化導電性ダイヤモンド電極上で測定対象物質および測定妨害物質の両方のピーク電流を与える電圧は、０．７〜０．９Ｖ（対Ａｇ／ＡｇＣｌ電極）の範囲内であり、より具体的には、約０．８Ｖ（対Ａｇ／ＡｇＣｌ電極）である。
【００２６】
上記電圧下、第二作用電極と対電極との間に生じる電流値を測定する。そして、得られた電流値から先に算出した測定妨害物質の寄与分を差し引いて、測定対象物質単独に由来する電流値を算出し、この電流値から被検試料中の測定対象物質濃度を算出する。尿酸などの測定対象物質の酸化または還元により第一作用電極と対電極との間に生じる電流値は、系内の測定対象物質の濃度に正比例する。従って、一旦ある電圧値における電流値と測定対象物質濃度との関係を求めておけば、その関係から、得られた電流値に対応する被験試料液中の測定対象物質の濃度を容易に知ることが出来る。すなわち、本発明の好ましい態様にあっては、測定対象物質の濃度と電流値との検量線を予め作成しておき、この検量線と、得られた電流値とを対比することにより、測定対象物質の濃度を知ることが出来る。
【００２７】
本発明の別の好ましい態様によれば、ピーク電流付近でバックグランド電流が著しく増大してしまう場合においては、バックグランド電流が測定に悪影響を及ぼさない程度に低い範囲内で最もピーク電流に近い電圧、例えばバックグラウンド電流と酸化反応により生じる電流との差が最も大きくなる電圧、を選択するのが、安定かつ高感度の測定を行える点で好ましい。さらに、本発明の別の好ましい態様によれば、最大電流値を与える電圧は、回転電極法またはマイクロ電極法により求めることが出来る。回転電極法またはマイクロ電極法によれば、測定条件等による測定誤差の可能性をより排除出来る点で有利である。
【００２８】
本発明において第一作用電極および第二作用電極の形状等は特に限定されないが、後記する図２に示されるような板状のものであっても、またディスク上の電極を回転させながら用いるいわゆる回転電極であってもよい。
【００２９】
本発明において対電極としては、白金、炭素、ステンレス、金、ダイヤモンド、ＳｎＯ_２等の利用が好ましい。
【００３０】
本発明の好ましい態様によれば、参照電極を被験試料中に接触させ、ダイヤモンド電極と、対電極との間に、ダイヤモンド電極上で酸化または還元反応の生じる電圧の絶対値を制御することが、測定精度の観点から好ましい。参照電極は公知のものを利用することが出来、飽和カロメル電極（ＳＣＥ）、標準水素電極、銀塩化銀電極、水銀塩化水銀電極、水素パラジウム電極等が利用可能である。
【００３１】
ダイヤモンド電極およびその製造
ダイヤモンドは本来優れた絶縁体である。しかしながら、３族や５族の不純物を添加することによって、半導体〜金属様の導電性を示すようになる。本発明にあっては、半導体〜金属様の導電性を示すダイヤモンドを電極として使用する。
【００３２】
このような導電性を付与するために添加される物質としては、上記の通り３族および５族の元素が挙げられ、さらに好ましくはホウ素、窒素、リンが挙げられ、最も好ましくはホウ素または窒素である。この導電性を付与するために添加される物質の添加量は、ダイヤモンドに導電性を付与できる範囲で適宜決定されてよいが、例えば１×１０^−２〜１０^−６Ωｃｍ程度の導電性を与える量添加されることが好ましい。この導電性を付与するために添加される物質の添加量は、導電性ダイヤモンドの製造工程における添加量により制御されることが一般的である。
【００３３】
本発明の好ましい態様によれば、基材上に導電性ダイヤモンドの薄膜を形成させるとともに、導線を接続させ、電極とすることが好ましい。基材としては、Ｓｉ（例えば、単結晶シリコン）、Ｍｏ、Ｗ、Ｎｂ、Ｔｉ、Ｆｅ、Ａｕ、Ｎｉ、Ｃｏ、Ａｌ_２Ｏ_３、ＳｉＣ、Ｓｉ_３Ｎ_４、ＺｒＯ_２、ＭｇＯ、黒鉛、単結晶ダイヤモンド、ｃＢＮ、石英ガラス等が挙げられ、特に単結晶シリコン、Ｍｏ、Ｗ、Ｎｂ、Ｔｉ、ＳｉＣ、単結晶ダイヤモンドの利用が好ましい。
【００３４】
この態様の電極を図２を用いて更に説明する。図２（ａ）は、ダイヤモンド電極１１の断面図であり、この電極は、基材１２の上に形成された導電性ダイヤモンド薄膜１３とからなり、さらにこの導電性ダイヤモンド薄膜１３には導線１５が例えば金コーティング１４を介して接続される。図２（ｂ）は、ダイヤモンド電極１１の斜視図であり、基材１２の上に形成された導電性ダイヤモンド薄膜１３からなり、さらにこの導電性ダイヤモンド薄膜１３を電極とするための金コーティング１４を介して導線１５が接続される。
【００３５】
導電性ダイヤモンド薄膜の厚さは、特に限定されないが、１〜１００μｍ程度の厚さが好ましく、より好ましくは５〜５０μｍ程度である。
【００３６】
さらに本発明の好ましい態様によれば、本発明によるダイヤモンド電極は、マイクロ電極の形態をとることが出来る。マイクロ電極の概念は既に公知であり、本発明においてマイクロ電極形態のダイヤモンド電極とは、Ｐｔ、Ｗ、Ｍｏ等の細線の末端を鋭利に切断し、電解研磨により末端をさらに鋭利にした後、その末端表面に導電性ダイヤモンドの薄膜を形成した構成のものを意味する。
【００３７】
本発明の好ましい態様によれば、導電性ダイヤモンド薄膜は、化学気相成長法により好ましく製造される。化学気相成長法とは、気相中で気体原料を化学反応させて物質を合成する方法であり、ＣＶＤ（Chemical Vapor Deposition)法と一般に呼ばれる。この方法は、半導体製造プロセスにおいて広く利用されており、本発明における導電性ダイヤモンド薄膜の製造にも合目的的な改変のもと利用可能である。
【００３８】
ダイヤモンドの化学気相合成は、メタンなどの含炭素気体と水素を混合したものを原料気体として、それを励起源により励起させ、基板上に供給して堆積させることにより行われる。
【００３９】
励起源としては、熱フィラメント、マイクロ波、高周波、直流グロー放電、直流アーク放電、燃焼炎などが挙げられる。また、これらを複数組み合わせて核生成密度を調整したり、大面積化や均一化を図ることも可能である。
【００４０】
原料としては、炭素の含まれている多くの種類の、励起源により分解、励起されて、Ｃ、Ｃ_２などの活性な炭素、およびＣＨ、ＣＨ_２、ＣＨ_３、Ｃ_２Ｈ_２などの炭化水素ラジカルを生じさせる化合物を利用可能である。好ましい具体例としては、気体としてＣＨ_４、Ｃ_２Ｈ_２、Ｃ_２Ｈ_４、Ｃ_１０Ｈ_１６、ＣＯ、ＣＦ_４、液体としてＣＨ_３ＯＨ、Ｃ_２Ｈ_５ＯＨ、（ＣＨ_３）_２ＣＯ、固体として黒鉛、フラーレンなどが挙げられる。
【００４１】
気相合成法にあって、ダイヤモンドに導電性を付与する物質の添加は、例えば添加物質のディスクを系内に置き、炭素源原料と同様に励起させ、炭素気相に添加物質を導入する方法、炭素源に予め添加物質を添加し、系内に炭素源と共に導入し、励起源により励起し、炭素気相に添加物質を導入する方法等により行うことが出来る。本発明の好ましい態様によれば、後者の方法が好ましい。とりわけ、炭素源としてアセトン、メタノールなどの液体を用いる場合、これに酸化ボロン（Ｂ_２Ｏ_３）を溶解してボロン源とする方法が、ボロンの濃度の制御が容易で、かつ簡便であることから好ましい。例えば、気相合成法にあって、炭素源にホウ素を添加する場合、１０〜１２，０００ｐｐｍ程度が一般的であり、また１，０００〜１０，０００ｐｐｍ程度が好ましい。
【００４２】
本発明の好ましい態様によれば、導電性ダイヤモンド薄膜の製造はプラズマ化学気相合成法により行われることが好ましい。このプラズマ化学気相合成法は、化学反応を引き起こす活性化エネルギーが大きく、反応が速いとの利点を有する。さらに、この方法によれば、熱力学的に高温でなければ存在しない化学種を生成して、低い温度での反応が可能となる。プラズマ化学気相合成法による導電性ダイヤモンド薄膜の製造は、本発明者らの一部を含めいくつかの報告が既にあり（例えば、Yano et al., J. Electrochem. Soc., 145(1998) 1870）、これら報告に記載の方法に従って行うことが好ましい。
【００４３】
本発明の好ましい態様によれば、導電性ダイヤモンド電極が陽極酸化されたものであるのが好ましい。何ら処理が施されていない、作製されたままの（as grown）導電性ダイヤモンド電極は、表面が水素終端化されているが、陽極酸化を行うことにより、導電性ダイヤモンド電極の表面は酸素終端化される。そして、この陽極酸化ダイヤモンド電極、すなわち酸素終端化されたダイヤモンド電極を用いると、アスコルビン酸の酸化反応における２つのピーク電位間の差が、水素終端化された導電性ダイヤモンド電極の場合よりも大きくなる。本発明者らの知見によれば、アスコルビン酸の酸化反応における２つのピーク電位間の差は、酸素終端化ダイヤモンド電極の場合には０．５Ｖと大きいのに対して、水素終端化ダイヤモンド電極の場合には０．３Ｖと小さい。したがって、陽極酸化ダイヤモンド電極、すなわち酸素終端化されたダイヤモンド電極を用いることにより、０．２Ｖ（対Ａｇ／ＡｇＣｌ電極）付近に観察されるアスコルビン酸単独のピーク電流を高精度に測定することができる。その結果、尿酸濃度をより高い精度で測定することができる。
【００４４】
導電性ダイヤモンド電極の陽極酸化は、水酸化ナトリウム溶液中で導電性ダイヤモンドに正の電圧を印加して、ダイヤモンドを酸化させることにより行うことができる。
【００４５】
測定装置
本発明による測定方法に用いる測定装置は、図１に示されるように、作用電極として２個のダイヤモンド電極を用いること以外は、一般的な電気化学的な系であることが出来るが、本発明の好ましい態様によれば、所定の溶液を一定流速でキャリアとして系内を流し、そのキャリア溶液中に被験試料を注入して測定を行う、フローセルを用いたフローインジェクション法による装置を用いるのが好ましい。
【００４６】
フローセルを用いたフローインジェクション法を用いる測定装置の概略は図３に示される通りである。フローセル３１には、キャリア溶液溜２１から、ポンプ２２によりキャリア溶液が注入される。ポンプ２２とフローセル３１との間には、被験試料注入口２３および披検試料導入部２４が設けられ、キャリア溶液に被験試料が注入可能とされる。フローセル３１を通過したキャリア溶液は、廃液溜２６に集められる。フローセル３１の基本構成は、前述した図１に示される測定系と同様であり、被検試料液を収納する容器３２と、被検試料液と接触するように、第一作用電極３３、第二作用電極３４、対電極３５、および参照電極３６を備えてなる。
【００４７】
電源・電流計４１には、フローセル３１より、第一作用電極３３に接続される導線ＷＥ１、第二作用電極３４に接続される導線ＷＥ２、対電極３５に接続される導線ＣＥ、および参照電極３６に接続される導線ＲＥが接続される。この電源・電流計４１は、第一作用電極と対電極との間、および第二作用電極と対電極との間に、それぞれ所定の電圧を印加する手段と、この印加電圧下で第一作用電極および第二作用電極における各電流値を測定する手段とを兼ねるものである。すなわち、この電源・電流計４１によって、第一作用電極と対電極との間に、第一作用電極上で測定妨害物質の酸化反応が生じるが、測定対象物質の酸化反応が実質的に生じない電圧を印加し、該電圧下で第一作用電極における酸化または還元反応に伴う電流値を測定する。また、第二作用電極と対電極との間に、測定対象物質および測定妨害物質の酸化反応が生じる電圧を印加し、該電圧下で第二作用電極における酸化または還元反応に伴う電流値を測定する。これらの電流値は、電流値比較・濃度算出装置４２に送られる。
【００４８】
この装置４２には、第一作用電極に印加される電圧における測定妨害物質濃度対電流値の検量線データ４３ａ、第二作用電極に印加される電圧における測定妨害物質濃度対電流値の検量線データ４３ｂ、および第二作用電極に印加される披検物質対電流値の検量線データ４３ｃが送られ、この装置内において、電源・電流計４１から送られた第一作用電極における電流値と、検量線データ４３ａを比較し、測定妨害物質の濃度を算出する。得られた測定妨害物質濃度と、検量線データ４３ｂを比較し、第二作用電極における電流値への測定妨害物質の寄与分を算出する。そして、電源・電流計４１から送られた第二作用電極における電流値から測定妨害物質の寄与分を差し引く。こうして得られた測定対象物質単独に由来する電流値と、検量線データ４３ｃを比較し、測定対象物質の濃度を算出する。このように、この装置４２は、得られた各電流値から被験試料液中の測定対象物質の濃度を算出する手段を備える。得られた測定対象物質濃度は、表示装置４４により表示される。
【００４９】
測定は以下のように行われる。まず、被験試料を注入しないキャリア溶液のみを流し、いわゆるバックグラウンド電流を出来るだけ小さくし、かつ安定させる。ダイヤモンド電極はバックグラウンド電流が小さいことを特長とする。次に、被験試料を被験試料注入口２３より注入する。この注入は継続して行われてもよいが、ピーク電流を測定可能な程度の量を一時期に注入してもよい。
【００５０】
【実施例】
以下の実施例によって本発明をさらに詳細に説明するが、本発明はこれら実施例に限定されるものではない。
【００５１】
例１：陽極酸化ダイヤモンド電極の作製
公知の方法に従い、導電性ｎ型シリコン（１１１）面上に、ホウ素を高濃度（１０，０００ppm、Ｂ／Ｃ比）で導入したダイヤモンド薄膜を、マイクロ波プラズマＣＶＤ装置を用いて形成した。得られたダイヤモンド薄膜付シリコン基板を０．１Ｍの水酸化ナトリウム溶液に浸漬し、２．６Ｖ（対Ａｇ／ＡｇＣｌ電極）の電圧を７５分間印加して、ダイヤモンド薄膜の表面を酸化した。こうして、陽極酸化ダイヤモンド電極を得た。
【００５２】
例２：ディファレンシャルパルスボルタンメトリーの測定
尿酸濃度１００μＭの溶液、アスコルビン酸濃度５００μＭの溶液、尿酸濃度１００μＭおよびアスコルビン酸濃度５００μＭの混合溶液をそれぞれ用意した。これらの各溶液について、ディファレンシャルパルスボルタンメトリーを測定した。ディファレンシャルパルスボルタンメトリーの測定条件は、以下の条件で行った。
測定温度：室温
作用電極：例１で作製した陽極酸化ダイヤモンド電極
対電極：白金電極
参照電極：Ａｇ／ＡｇＣｌ電極
支持電解質：０．１Ｍリン酸緩衝液（ｐＨ６．８）
パルス幅：５０ｍ秒
パルス周期：５００ｍ秒
パルス高さ：５０ｍＶ
【００５３】
その結果は、図４に示される通りであった。図４において、「ＵＡ」とは尿酸濃度１００μＭの溶液に、「ＡＡ」とはアスコルビン酸濃度５００μＭの溶液に、「ＵＡ＋ＡＡ」とは尿酸濃度１００μＭおよびアスコルビン酸濃度５００μＭの混合溶液に、「ＢＧ」とはバックグラウンド電流にそれぞれ対応する。図４に示されるように、アスコルビン酸のピーク電流が０．２Ｖおよび０．８Ｖ付近に観察され、尿酸のピーク電流が０．８Ｖ付近に観察された。
【００５４】
例３：アスコルビン酸の検量線の作成
０．１Ｍのリン酸緩衝溶液（ｐＨ６．８）にアスコルビン酸を加え、アスコルビン酸濃度０、２５、５０、１００、および５００μＭの溶液を用意した。この溶液に、作用電極として例１で調製したダイヤモンド電極を浸け、また対電極として白金電極を浸け、さらに参照電極としてＡｇ／ＡｇＣｌ電極を浸けて、電気化学測定用フローセルを構成した。
作用電極に０．２Ｖ（対参照電極）の電圧を印加し、各尿酸濃度におけるピーク電流を測定した。得られたピーク電流値とアスコルビン酸濃度から、図５に示される検量線ＡＡ１が得られた。
また、印加電圧を０．８Ｖ（対参照電極）としたこと以外は上記同様にして、各尿酸濃度におけるピーク電流を測定した。得られたピーク電流値とアスコルビン酸濃度から、図５に示される検量線ＡＡ２が得られた。
図５に示されるように、０．２Ｖおよび０．８Ｖ（対参照電極）のいずれの電位においても、アスコルビン酸濃度に比例して検出電流も推移した。
【００５５】
例４：尿酸の検量線の作成
０．１Ｍのリン酸緩衝溶液（ｐＨ６．８）に尿酸を加え、尿酸酸濃度０、５、１０、２０、５０、および１００μＭの溶液を用意した。この溶液に、作用電極として例１で調製したダイヤモンド電極を浸け、また対電極として白金電極を浸け、さらに参照電極としてＡｇ／ＡｇＣｌ電極を浸けて、電気化学測定用フローセルを構成した。
作用電極に０．８Ｖ（対参照電極）の電圧を印加し、各尿酸濃度におけるピーク電流を測定した。得られたピーク電流値と尿酸濃度から、図６に示される検量線ＵＡが得られた。図６に示されるように、尿酸濃度に比例して検出電流も推移した。
【００５６】
このように、例３および４において各成分の濃度に比例する検量線が得られた。したがって、これらの検量線を用いることにより、尿酸およびアスコルビン酸を含む被検試料液における尿酸濃度を、ダイヤモンド電極により簡便かつ高精度に測定できる。
【図面の簡単な説明】
【図１】本発明による測定法に用いられる測定系の概略を示す図である。
【図２】ダイヤモンド電極の構造を示す図であり、（ａ）はダイヤモンド電極の断面図であり、（ｂ）はダイヤモンド電極の斜視図である。
【図３】本発明による測定装置の基本構成を示す図である。
【図４】尿酸濃度１００μＭの溶液、アスコルビン酸濃度５００μＭの溶液、尿酸濃度１００μＭおよびアスコルビン酸濃度５００μＭの混合溶液の、ディファレンシャルパルスボルタンメトリーの測定結果である。
【図５】０．２Ｖおよび０．８Ｖ（対参照電極）の印加電圧において測定された、アスコルビン酸濃度とピーク電流の値との検量線である。
【図６】０．８Ｖ（対参照電極）の印加電圧において測定された、尿酸濃度とピーク電流の値との検量線である。[0001]
BACKGROUND OF THE INVENTION
Field of Invention
The present invention relates to a method and an apparatus for measuring a test substance concentration using a conductive diamond electrode. Specifically, the present invention relates to a method and apparatus for measuring a test substance concentration in a test sample solution containing a measurement interfering substance, particularly a uric acid concentration in a test sample solution containing uric acid and ascorbic acid.
[0002]
Background art
Uric acid is produced as a final degradation product of purines in the human body. The normal value of uric acid concentration in human serum is 4-7 mg / dl for men and 2.5-6 mg / dl for women. Moreover, the normal value of the amount of uric acid excreted in human urine is 0.2 to 1 g / 24 hours, which is converted to a concentration of about 1.2 to 6 mM when the amount of urine is assumed to be 1 L. Is done. However, uric acid levels in the blood may increase due to excessive production of uric acid in the body or a decrease in the amount of uric acid excreted due to a decrease in renal function. This causes hyperuricemia and gout in which urate is deposited on joints and the like. Therefore, measuring the uric acid level in the body is extremely important in preventing these diseases or controlling the uric acid level in gout patients.
[0003]
As a conventional method for measuring uric acid, a method using an enzyme and an analysis method using high performance liquid chromatography (HPLC) are known. In these methods, since the operation is complicated, specialized knowledge is required, and the analysis equipment is also expensive.
[0004]
On the other hand, diamond inherently has a resistivity of 10¹³Although it is an insulating material of about Ωcm, conductivity is obtained by doping with a small amount of impurities. Various uses are expected for this conductive diamond. One of them is the use as an electrode for electrochemical use.
When the conductive diamond is viewed as an electrode for electrochemical use, it has excellent features of having a wide potential window and extremely low background current. Furthermore, it has the characteristics that it is physically and chemically stable and has excellent durability. Electrodes having conductive diamond (preferably a thin film thereof) have been commonly referred to as diamond electrodes.
[0005]
Pioneering work on diamond electrodes was performed by Iwaki et al. (Iwaki et al., Nuclear Instruments and Methods, 209-210, 1129 (1983)). They studied the properties of single-crystal diamond that was given surface conductivity by implanting argon or nitrogen ions as an electrically conductive material. At the same time, a cyclic voltammogram in the electrolyte solution is also shown. Subsequently, the properties of polycrystalline diamond electrodes synthesized by vapor phase using hot filaments have been reported (Pleskov et al., J. Electroanal. Chem., 228, 19 (1993)).
[0006]
Some of the inventors have previously reported the reduction of nitrogen oxides using a diamond electrode synthesized in a gas phase (Tenne et al., J. Electroanal. Chem., 347, 409 (1993)). In this study, p-type semiconductor diamond introduced with boron as a dopant was used as an electrode. After that, as diamond electrodes, the use of p-type semiconductors with boron as a dopant or metal-like conductive diamond having higher conductivity has become mainstream. In the 1990s, research on diamond electrodes was conducted by a plurality of groups. Since 1995, research on diamond electrodes obtained using a plasma CVD (PCVD) apparatus capable of obtaining a diamond thin film with a larger area has been conducted. It has also been found in the electrochemical field.
[0007]
A method of measuring uric acid using an anodized diamond thin film electrode is known (for example, see Patent Document 1). In this method, it is possible to measure the uric acid concentration by separating the peak voltage of ascorbic acid, which is a measurement interfering substance, and the peak voltage of uric acid by controlling the liquid to be measured in an extremely strong acidic range of preferably pH 1. Yes.
[0008]
[Patent Document 1]
JP 2001-147 211 A
[0009]
SUMMARY OF THE INVENTION
The present inventors have recently detected two or more kinds of test substances with a conductive diamond electrode in a certain kind of test sample solution, and moreover, the concentration of each of the two or more kinds of test substances is simply and high. The knowledge that it can measure with accuracy was obtained. In addition, the inventors of the present invention have only one of two or more kinds of test substances to be measured for concentration measurement, and the remaining test substances should be excluded in the measurement of the concentration of the measurement target substance. In the case of a substance, the inventors have obtained knowledge that the concentration of a substance to be measured can be easily and accurately measured with a conductive diamond electrode without performing a complicated operation for eliminating the influence of a measurement interfering substance. More specifically, in a test sample solution containing uric acid and ascorbic acid, the uric acid concentration can be easily and accurately controlled by a conductive diamond electrode under a mild liquidity of weakly acidic to neutral (for example, pH 5 to 8). The knowledge that it can measure was obtained.
[0010]
Therefore, the present invention is to measure each concentration of two or more kinds of test substances, in particular, a measurement target substance concentration in a test sample solution containing a measurement target substance and a measurement interfering substance simply and with high accuracy. It is aimed.
[0011]
And, according to the present invention, a method for measuring the concentration of a test substance using a conductive diamond electrode is as follows:
Prepare a test sample solution containing a first test substance and a second test substance, both of which can cause an oxidation or reduction reaction by applying the same voltage on a conductive diamond electrode,
A first working electrode made of a conductive diamond electrode, a second working electrode made of a conductive diamond electrode, and a counter electrode in contact with the test sample solution;
An oxidation or reduction reaction of the second test substance occurs on the first working electrode between the first working electrode and the counter electrode, but the oxidation or reduction reaction of the first test substance is substantially Applying a voltage that does not occur automatically, measuring the current value under the voltage, calculating the concentration of the second test substance in the test sample liquid from the obtained current value,
From the concentration of the second test substance, oxidation or reduction of both the first test substance and the second test substance on the second working electrode between the second working electrode and the counter electrode. Calculate the contribution of the second test substance to the current value obtained when a voltage at which reaction occurs is applied,
A voltage that causes oxidation or reduction reaction of both the first test substance and the second test substance on the second working electrode is applied between the second working electrode and the counter electrode, and the voltage The current value at the bottom is measured, and the current value derived from the first test substance alone is calculated by subtracting the contribution of the second test substance from the current value, and the test value is calculated from the current value. Calculate the first analyte concentration in the sample
Comprising.
[0012]
In addition, the measurement device for the concentration of a test substance using a conductive diamond electrode according to the present invention includes:
A first working electrode comprising a conductive diamond electrode;
A second working electrode comprising a conductive diamond electrode;
A counter electrode;
Means for bringing the first working electrode, the second working electrode, and the counter electrode into contact with a test sample solution;
An oxidation or reduction reaction of the second test substance occurs on the first working electrode between the first working electrode and the counter electrode, but the oxidation or reduction reaction of the first test substance is substantially A voltage that does not occur is applied, and the current value under the voltage is measured. Between the second working electrode and the counter electrode, both of the first test substance and the second test substance are measured. Measuring means for applying a voltage at which oxidation or reduction reaction occurs and measuring a current value under the voltage;
The second test substance concentration in the test sample liquid is calculated from the current value obtained at the first working electrode, and the second test substance concentration to the current value at the second working electrode is calculated. Calculate the contribution of the second test substance, and further subtract the contribution of the second test substance from the current value at the second working electrode to obtain a current value derived from the first test substance alone Calculating means for calculating a first test substance concentration in the test sample liquid from the current value;
It is equipped with.
[0013]
DETAILED DESCRIPTION OF THE INVENTION
Test sample solution
An object to be tested by the method according to the present invention is a test including a first test substance and a second test substance, both of which can cause an oxidation or reduction reaction by applying the same voltage on a conductive diamond electrode. Sample liquid. The first test substance is a substance that undergoes an oxidation or reduction reaction upon application of a specific voltage on a conductive diamond electrode, and is a target for concentration measurement using the current value generated by the reaction. It is. On the other hand, the second test substance causes an oxidation or reduction reaction on the conductive diamond electrode by applying the same voltage as that of the first test substance, and substantially causes the oxidation or reduction reaction of the first test substance. An oxidation or reduction reaction is caused by the application of another voltage that is not generated. Two or more kinds of second test substances may be used as long as they can cause an oxidation or reduction reaction even when different voltages are applied. Thus, both the first and second test substances have a voltage that causes an oxidation or reduction reaction on the conductive diamond electrode. Therefore, when measuring the concentration of the first test substance, the second test substance results in interfering with the concentration measurement of the first test substance. This is because, during the oxidation or reduction reaction of the first test substance, the second test substance also generates an extra current due to the oxidation or reduction reaction, so that accurate quantification of the first test substance based on the current value is performed. It is because it becomes impossible to perform.
[0014]
As described above, the second test substance undergoes oxidation or reduction reaction of the second test substance in addition to the voltage causing the oxidation or reduction reaction with the first test substance. There is a voltage at which the oxidation or reduction reaction of the test substance does not substantially occur. Then, the concentration of the second test substance can be measured by applying this voltage to the conductive diamond electrode. In the present invention, based on the concentration of the second test substance thus obtained, the current value generated during the oxidation or reduction reaction of the first test substance is corrected, whereby the first test substance is corrected. This makes it possible to measure the concentration of the test substance.
[0015]
According to the present invention, since the concentrations of both the first test substance and the second test substance can be measured, the present invention can be applied to the case where both the first and second test substances are measured substances. This is one typical embodiment. On the other hand, in the method according to the present invention, the first test substance is a measurement target substance to be subjected to concentration measurement, and the second test substance is a measurement interfering substance to be excluded in the concentration measurement of the measurement target substance. It is preferably used at certain times. Each embodiment is different only in the purpose of knowing the amount of one or one of the test substances, and is essentially the same in nature, so the contents of the present invention will be described below based on the latter embodiment.
[0016]
According to a preferred embodiment of the present invention, it is preferable that the measurement target substance is uric acid and the measurement interfering substance is ascorbic acid. Uric acid is a substance contained in urine or serum together with ascorbic acid, and it is extremely useful medically to know its uric acid concentration. On the other hand, ascorbic acid is a measurement interfering substance that interferes with uric acid measurement. This is because uric acid and ascorbic acid both generate a peak current due to an oxidation reaction at around 0.8 V on the anodized diamond electrode. That is, even if an attempt is made to measure the uric acid concentration in urine with a conductive diamond electrode, the uric acid concentration cannot be measured accurately because it is hindered by the current caused by ascorbic acid. However, ascorbic acid produces a peak current of ascorbic acid alone in the vicinity of 0.2 V as well as the above 0.8V. Therefore, according to the present invention, the concentration of ascorbic acid is first calculated using the current value of 0.2 V of ascorbic acid. Subsequently, the uric acid concentration can be measured by performing a predetermined correction based on this concentration. Therefore, according to the measurement method of the present invention, without performing the operation of separating ascorbic acid, or without separating each peak potential of uric acid and ascorbic acid by controlling the test sample solution in a very strong acidic range, Uric acid can be measured easily and with high accuracy.
[0017]
The test sample solution in the above preferred embodiment can be serum, urine, other biological fluids, etc. that are considered to contain uric acid and ascorbic acid. Using urine or serum as a test sample, knowing the uric acid concentration in urine by the method of the present invention is effective for immediate diagnosis of hyperuricemia, ventilation, urolithiasis, and the like.
[0018]
According to a preferred embodiment of the present invention, the pH of the test sample solution is preferably 5-8. Since the measurement method of the present invention allows the oxidation or reduction reaction of the measurement target substance and the measurement interfering substance to have the same potential, for example, as described in Japanese Patent Application Laid-Open No. 2001-147511 described above, the pH is preferably extremely high. Under strong acidity, it is not necessary to separate the peak potential of ascorbic acid, which is a measurement interfering substance, from the peak potential of uric acid. Therefore, in the test sample liquid containing uric acid and ascorbic acid, since the uric acid concentration can be measured under a mild liquidity of weak acidity to neutrality (for example, pH 5 to 8), it is easy to handle the test sample liquid. Increases safety. Moreover, according to another preferable aspect of this invention, you may further perform the process of adjusting the pH of the said test sample liquid to 5-8 prior to a measurement.
[0019]
According to a preferred embodiment of the present invention, the test sample solution preferably further comprises a phosphate buffer, and the pH can be adjusted with the phosphate buffer. In this case, the difference between the peak potential due to uric acid and the peak potential that does not overlap with uric acid due to ascorbic acid increases, and measurement with higher accuracy becomes possible.
[0020]
Measuring system and measuring method
In the measurement method according to the present invention, the electrochemical system for measuring the test substance can be a general electrochemical system except that two or more diamond electrodes as working electrodes are provided. That is, in the present invention, two or more diamond electrodes as working electrodes are brought into contact with the test sample solution together with the counter electrodes, and different voltages are applied to the working electrodes between the counter electrodes.
[0021]
A schematic representation of this system is shown in FIG. The system shown in FIG. 1 includes a first working electrode 3, a second working electrode 4, and a counter electrode 5 so that a container 2 containing a test sample solution 1 is in contact with the test sample solution 1. Become. Both the first working electrode 3 and the second working electrode 4 are conductive diamond electrodes. According to a preferred aspect of the present invention, the system may further include a reference electrode 6 so that the absolute values of the voltages applied to the working electrodes 3 and 4 and the counter electrode 5 can be known. Good. In the following description, the test sample solution 1 includes a measurement target substance as a first test substance and a measurement interfering substance as a second test target substance.
[0022]
In the present invention, the voltage applied between the first working electrode and the counter electrode causes an oxidation or reduction reaction of the measurement interfering substance on the first working electrode. Voltage that does not occur in That is, only the oxidation or reduction reaction of the measurement interfering substance occurs under this applied voltage. According to a preferred embodiment of the present invention, from the viewpoint of measurement efficiency and accuracy, the applied voltage is preferably a voltage that gives a peak current for oxidation or reduction of the measurement interfering substance. Here, the voltage giving the peak current can be obtained as a voltage giving the maximum current value by, for example, cyclic voltammetry. According to the knowledge obtained by the present inventors, in the case of a weakly acidic to neutral test sample liquid in which the substance to be measured is uric acid and the measurement interfering substance is ascorbic acid, on the anodized conductive diamond electrode The voltage that gives only the peak current of the measurement interfering substance is in the range of 0.1 to 0.3 V (vs. Ag / AgCl electrode), more specifically about 0.2 V (vs. Ag / AgCl electrode). is there.
[0023]
Under the applied voltage, a current value generated between the first working electrode and the counter electrode is measured, and a measured interfering substance concentration in the test sample liquid is calculated from the obtained current value. The current value generated between the first working electrode and the counter electrode due to oxidation or reduction of a measurement interfering substance such as ascorbic acid is directly proportional to the concentration of the measurement interfering substance in the system. Therefore, once the relationship between the current value and the measurement interfering substance concentration at a certain voltage value is obtained, the concentration of the measurement interfering substance in the test sample solution corresponding to the obtained current value can be easily known from the relationship. I can do it. That is, in a preferred embodiment of the present invention, a calibration curve between the concentration of the measurement interfering substance and the current value is prepared in advance, and the measurement interference is compared with the obtained current value. You can know the concentration of the substance.
[0024]
The measurement interfering substance is detected at the second working electrode together with the measurement target substance, but the contribution of the measurement interfering substance to the current value measured at the second working electrode from the concentration of the measurement interfering substance obtained as described above. Calculate minutes. That is, also in the case of the second working electrode, as in the case of the first working electrode, if the relationship between the current value and the concentration of the measurement interfering substance in the voltage value to be applied is obtained in advance, from that relationship, The current value corresponding to the concentration of the measurement interfering substance in the obtained test sample solution can be easily known. In a preferred embodiment of the present invention, a calibration curve between the concentration of the measurement interfering substance and the current value is prepared in advance, and the calibration curve is compared with the measurement interfering substance concentration measured at the first working electrode. Thus, the contribution of the measurement interfering substance to the current value measured at the second working electrode can be known.
[0025]
In the present invention, the voltage applied between the second working electrode and the counter electrode is a voltage at which the measurement target substance and the measurement interfering substance are oxidized or reduced on the second working electrode. That is, under this applied voltage, the oxidation or reduction reaction of both the measurement target substance and the measurement interfering substance occurs. According to a preferred embodiment of the present invention, from the viewpoint of measurement efficiency and accuracy, the applied voltage is preferably a voltage that gives a peak current for oxidation or reduction of both the measurement target substance and the measurement interfering substance. Here, the voltage giving the peak current can be obtained as a voltage giving the maximum current value by, for example, cyclic voltammetry. According to the knowledge obtained by the present inventors, in the case of a weakly acidic to neutral test sample liquid in which the substance to be measured is uric acid and the measurement interfering substance is ascorbic acid, on the anodized conductive diamond electrode The voltage that gives the peak currents of both the measurement target substance and the measurement interfering substance in the range of 0.7 to 0.9 V (vs. Ag / AgCl electrode), more specifically, about 0.8 V (vs. Ag / AgCl electrode).
[0026]
Under the above voltage, a current value generated between the second working electrode and the counter electrode is measured. Then, the contribution of the measurement interfering substance calculated earlier is subtracted from the obtained current value to calculate the current value derived from the measurement target substance alone, and the concentration of the measurement target substance in the test sample is calculated from this current value. To do. A current value generated between the first working electrode and the counter electrode due to oxidation or reduction of a measurement target substance such as uric acid is directly proportional to the concentration of the measurement target substance in the system. Therefore, once the relationship between the current value at a certain voltage value and the concentration of the measurement target substance is obtained, the concentration of the measurement target substance in the test sample solution corresponding to the obtained current value can be easily known from the relationship. I can do it. That is, in a preferred embodiment of the present invention, a calibration curve between the concentration of the substance to be measured and the current value is prepared in advance, and the calibration target is compared with the obtained current value to obtain the measurement target. You can know the concentration of the substance.
[0027]
According to another preferred embodiment of the present invention, in the case where the background current increases remarkably in the vicinity of the peak current, the voltage closest to the peak current within a range that is low enough that the background current does not adversely affect the measurement. For example, it is preferable to select a voltage at which the difference between the background current and the current generated by the oxidation reaction is the largest because stable and highly sensitive measurement can be performed. Furthermore, according to another preferred embodiment of the present invention, the voltage that gives the maximum current value can be determined by the rotating electrode method or the microelectrode method. The rotating electrode method or the microelectrode method is advantageous in that the possibility of measurement errors due to measurement conditions and the like can be further eliminated.
[0028]
In the present invention, the shape and the like of the first working electrode and the second working electrode are not particularly limited, but even a plate-like one as shown in FIG. 2 to be described later may be used while rotating the electrode on the disk. It may be a rotating electrode.
[0029]
In the present invention, as the counter electrode, platinum, carbon, stainless steel, gold, diamond, SnO₂Etc. are preferable.
[0030]
According to a preferred embodiment of the present invention, the reference electrode is brought into contact with the test sample, and the absolute value of the voltage at which oxidation or reduction reaction occurs on the diamond electrode between the diamond electrode and the counter electrode is controlled. This is preferable from the viewpoint of measurement accuracy. A known electrode can be used as the reference electrode, and a saturated calomel electrode (SCE), a standard hydrogen electrode, a silver-silver chloride electrode, a mercury mercury chloride electrode, a hydrogen palladium electrode, and the like can be used.
[0031]
Diamond electrode and its manufacture
Diamond is inherently an excellent insulator. However, by adding Group 3 or Group 5 impurities, semiconductor-to-metal conductivity is exhibited. In the present invention, diamond having a semiconductor to metal-like conductivity is used as an electrode.
[0032]
Examples of the substance added to impart such conductivity include elements of Group 3 and Group 5 as described above, more preferably boron, nitrogen, and phosphorus, and most preferably boron or nitrogen. is there. The amount of the substance added for imparting conductivity may be appropriately determined within a range in which conductivity can be imparted to diamond. For example, 1 × 10^-2-10^-6It is preferable to add an amount that gives conductivity of about Ωcm. In general, the amount of a substance added for imparting conductivity is controlled by the amount added in the manufacturing process of conductive diamond.
[0033]
According to a preferable aspect of the present invention, it is preferable to form a conductive diamond thin film on a substrate and connect a conductive wire to form an electrode. As the base material, Si (for example, single crystal silicon), Mo, W, Nb, Ti, Fe, Au, Ni, Co, Al₂O₃, SiC, Si₃N₄, ZrO₂MgO, graphite, single crystal diamond, cBN, quartz glass and the like, and single crystal silicon, Mo, W, Nb, Ti, SiC, and single crystal diamond are particularly preferable.
[0034]
The electrode of this aspect will be further described with reference to FIG. FIG. 2A is a cross-sectional view of the diamond electrode 11, which is composed of a conductive diamond thin film 13 formed on the substrate 12, and a conductive wire 15 is provided on the conductive diamond thin film 13. For example, it is connected via a gold coating 14. FIG. 2B is a perspective view of the diamond electrode 11, which is composed of a conductive diamond thin film 13 formed on the substrate 12, and a gold coating 14 for using the conductive diamond thin film 13 as an electrode. The conducting wire 15 is connected through the via.
[0035]
Although the thickness of a conductive diamond thin film is not specifically limited, The thickness of about 1-100 micrometers is preferable, More preferably, it is about 5-50 micrometers.
[0036]
Further in accordance with a preferred embodiment of the present invention, the diamond electrode according to the present invention can take the form of a microelectrode. The concept of a microelectrode is already known, and in the present invention, a diamond electrode in the form of a microelectrode is obtained by sharply cutting the end of a fine wire such as Pt, W, Mo, etc. It means a structure in which a conductive diamond thin film is formed on the end surface.
[0037]
According to a preferred embodiment of the present invention, the conductive diamond thin film is preferably produced by a chemical vapor deposition method. The chemical vapor deposition method is a method of synthesizing a substance by chemically reacting gaseous raw materials in a gas phase, and is generally called a CVD (Chemical Vapor Deposition) method. This method is widely used in the semiconductor manufacturing process, and can be used for manufacturing the conductive diamond thin film in the present invention under a purposeful modification.
[0038]
The chemical vapor synthesis of diamond is performed by using a mixture of a carbon-containing gas such as methane and hydrogen as a source gas, exciting it with an excitation source, supplying it on a substrate, and depositing it.
[0039]
Examples of the excitation source include a hot filament, microwave, high frequency, direct current glow discharge, direct current arc discharge, and combustion flame. It is also possible to adjust the nucleation density by combining a plurality of these, and to increase the area and make it uniform.
[0040]
As a raw material, many kinds of carbon containing carbon are decomposed and excited by an excitation source, and C, C₂Active carbon such as CH, CH₂, CH₃, C₂H₂Compounds that generate hydrocarbon radicals such as can be used. As a preferable specific example, as gas, CH₄, C₂H₂, C₂H₄, C₁₀H₁₆, CO, CF₄, CH as liquid₃OH, C₂H₅OH, (CH₃)₂Examples of CO and solid include graphite and fullerene.
[0041]
In the gas phase synthesis method, for example, a method for adding a substance that imparts conductivity to diamond is a method of introducing an additive substance into a carbon gas phase by, for example, placing a disk of the additive substance in the system and exciting the same as the carbon source material. Alternatively, an additive substance may be added in advance to the carbon source, introduced into the system together with the carbon source, excited by the excitation source, and introduced into the carbon gas phase. According to a preferred embodiment of the present invention, the latter method is preferred. In particular, when a liquid such as acetone or methanol is used as the carbon source, boron oxide (B₂O₃) Is preferred because it is easy to control the concentration of boron and is simple. For example, in the gas phase synthesis method, when boron is added to the carbon source, it is generally about 10 to 12,000 ppm, and preferably about 1,000 to 10,000 ppm.
[0042]
According to a preferred embodiment of the present invention, the conductive diamond thin film is preferably produced by a plasma chemical vapor synthesis method. This plasma chemical vapor synthesis method has the advantage that the activation energy causing a chemical reaction is large and the reaction is fast. Furthermore, according to this method, a chemical species that does not exist unless it is thermodynamically high is generated, and a reaction at a low temperature is possible. There have already been several reports on the production of conductive diamond thin films by the plasma chemical vapor synthesis method, including some of the present inventors (eg, Yano et al., J. Electrochem. Soc., 145 (1998)). 1870), preferably according to the methods described in these reports.
[0043]
According to a preferred embodiment of the present invention, the conductive diamond electrode is preferably anodized. The as-grown conductive diamond electrode, which has not been subjected to any treatment, has a hydrogen-terminated surface, but by anodizing, the surface of the conductive diamond electrode is oxygen-terminated. Is done. When this anodized diamond electrode, that is, an oxygen-terminated diamond electrode is used, the difference between the two peak potentials in the ascorbic acid oxidation reaction is larger than that of a hydrogen-terminated conductive diamond electrode. . According to the knowledge of the present inventors, the difference between the two peak potentials in the oxidation reaction of ascorbic acid is as large as 0.5 V in the case of the oxygen-terminated diamond electrode, whereas in the hydrogen-terminated diamond electrode, In this case, it is as small as 0.3V. Therefore, by using an anodized diamond electrode, that is, an oxygen-terminated diamond electrode, the peak current of ascorbic acid alone observed in the vicinity of 0.2 V (vs. Ag / AgCl electrode) can be measured with high accuracy. . As a result, the uric acid concentration can be measured with higher accuracy.
[0044]
Anodization of the conductive diamond electrode can be performed by oxidizing the diamond by applying a positive voltage to the conductive diamond in a sodium hydroxide solution.
[0045]
measuring device
As shown in FIG. 1, the measuring apparatus used in the measuring method according to the present invention can be a general electrochemical system except that two diamond electrodes are used as the working electrode. According to a preferred embodiment of the present invention, it is preferable to use an apparatus based on a flow injection method using a flow cell, in which a predetermined solution is flowed through the system as a carrier at a constant flow rate, and a test sample is injected into the carrier solution for measurement. .
[0046]
An outline of a measuring apparatus using a flow injection method using a flow cell is as shown in FIG. A carrier solution is injected into the flow cell 31 from a carrier solution reservoir 21 by a pump 22. A test sample inlet 23 and a test sample introduction unit 24 are provided between the pump 22 and the flow cell 31, so that the test sample can be injected into the carrier solution. The carrier solution that has passed through the flow cell 31 is collected in the waste liquid reservoir 26. The basic configuration of the flow cell 31 is the same as that of the measurement system shown in FIG. 1 described above, and the first working electrode 33 and the second working electrode 33 are in contact with the container 32 for storing the sample liquid to be tested and the test sample liquid. A working electrode 34, a counter electrode 35, and a reference electrode 36 are provided.
[0047]
The power source / ammeter 41 includes, from the flow cell 31, a conductive wire WE1 connected to the first working electrode 33, a conductive wire WE2 connected to the second working electrode 34, a conductive wire CE connected to the counter electrode 35, and a reference electrode 36. A conducting wire RE connected to is connected. The power source / ammeter 41 includes means for applying a predetermined voltage between the first working electrode and the counter electrode, and between the second working electrode and the counter electrode, and the first action under the applied voltage. It also serves as a means for measuring each current value in the electrode and the second working electrode. That is, the power source / ammeter 41 causes an oxidation reaction of the measurement interfering substance on the first working electrode between the first working electrode and the counter electrode, but does not substantially cause an oxidation reaction of the measurement target substance. A voltage is applied, and the current value associated with the oxidation or reduction reaction at the first working electrode is measured under the voltage. In addition, a voltage is generated between the second working electrode and the counter electrode that causes an oxidation reaction of the measurement target substance and the measurement interfering substance, and the current value accompanying the oxidation or reduction reaction at the second working electrode is measured under the voltage. To do. These current values are sent to the current value comparison / concentration calculation device 42.
[0048]
The device 42 includes calibration curve data 43a for measured interfering substance concentration versus current value at the voltage applied to the first working electrode, and calibration curve data for measured interfering substance concentration versus current value at the voltage applied to the second working electrode. 43b and calibration substance data 43c of the test substance versus current value applied to the second working electrode are sent, and in this apparatus, the current value at the first working electrode sent from the power source / ammeter 41, and the calibration The line data 43a is compared, and the concentration of the measurement interfering substance is calculated. The obtained measurement interfering substance concentration is compared with the calibration curve data 43b, and the contribution of the measurement interfering substance to the current value at the second working electrode is calculated. Then, the contribution of the measurement interfering substance is subtracted from the current value at the second working electrode sent from the power source / ammeter 41. The current value derived from the measurement target substance alone thus obtained is compared with the calibration curve data 43c to calculate the concentration of the measurement target substance. As described above, the device 42 includes means for calculating the concentration of the measurement target substance in the test sample solution from the obtained current values. The obtained measurement target substance concentration is displayed by the display device 44.
[0049]
The measurement is performed as follows. First, only the carrier solution into which the test sample is not injected is allowed to flow, so that the so-called background current is minimized and stabilized. Diamond electrodes are characterized by a low background current. Next, the test sample is injected from the test sample injection port 23. Although this injection may be performed continuously, an amount that can measure the peak current may be injected at a time.
[0050]
【Example】
The present invention will be described in more detail with reference to the following examples, but the present invention is not limited to these examples.
[0051]
Example 1: Preparation of anodized diamond electrode
According to a known method, a diamond thin film into which boron was introduced at a high concentration (10,000 ppm, B / C ratio) was formed on a conductive n-type silicon (111) surface using a microwave plasma CVD apparatus. The obtained silicon substrate with a diamond thin film was immersed in a 0.1 M sodium hydroxide solution, and a voltage of 2.6 V (vs. Ag / AgCl electrode) was applied for 75 minutes to oxidize the surface of the diamond thin film. Thus, an anodized diamond electrode was obtained.
[0052]
Example 2: Differential pulse voltammetry measurement
A solution having a uric acid concentration of 100 μM, a solution having an ascorbic acid concentration of 500 μM, and a mixed solution having a uric acid concentration of 100 μM and an ascorbic acid concentration of 500 μM were prepared. For each of these solutions, differential pulse voltammetry was measured. The measurement conditions for differential pulse voltammetry were as follows.
Measurement temperature: room temperature
Working electrode: Anodized diamond electrode prepared in Example 1
Counter electrode: Platinum electrode
Reference electrode: Ag / AgCl electrode
Supporting electrolyte: 0.1 M phosphate buffer (pH 6.8)
Pulse width: 50 ms
Pulse period: 500 ms
Pulse height: 50 mV
[0053]
The result was as shown in FIG. In FIG. 4, “UA” is a solution with a uric acid concentration of 100 μM, “AA” is a solution with an ascorbic acid concentration of 500 μM, “UA + AA” is a mixed solution with a uric acid concentration of 100 μM and an ascorbic acid concentration of 500 μM, and “BG” Corresponds to the background current. As shown in FIG. 4, the peak current of ascorbic acid was observed around 0.2V and 0.8V, and the peak current of uric acid was observed around 0.8V.
[0054]
Example 3: Creating a calibration curve for ascorbic acid
Ascorbic acid was added to a 0.1 M phosphate buffer solution (pH 6.8) to prepare solutions having ascorbic acid concentrations of 0, 25, 50, 100, and 500 μM. In this solution, the diamond electrode prepared in Example 1 was immersed as a working electrode, a platinum electrode was immersed as a counter electrode, and an Ag / AgCl electrode was immersed as a reference electrode to constitute a flow cell for electrochemical measurement.
A voltage of 0.2 V (vs. reference electrode) was applied to the working electrode, and the peak current at each uric acid concentration was measured. A calibration curve AA1 shown in FIG. 5 was obtained from the obtained peak current value and ascorbic acid concentration.
Further, the peak current at each uric acid concentration was measured in the same manner as described above except that the applied voltage was 0.8 V (counter reference electrode). A calibration curve AA2 shown in FIG. 5 was obtained from the obtained peak current value and ascorbic acid concentration.
As shown in FIG. 5, the detected current also changed in proportion to the ascorbic acid concentration at any potential of 0.2 V and 0.8 V (counter reference electrode).
[0055]
Example 4: Creating a calibration curve for uric acid
Uric acid was added to a 0.1 M phosphate buffer solution (pH 6.8) to prepare solutions having uric acid concentrations of 0, 5, 10, 20, 50, and 100 μM. In this solution, the diamond electrode prepared in Example 1 was immersed as a working electrode, a platinum electrode was immersed as a counter electrode, and an Ag / AgCl electrode was immersed as a reference electrode to constitute a flow cell for electrochemical measurement.
A voltage of 0.8 V (vs. reference electrode) was applied to the working electrode, and the peak current at each uric acid concentration was measured. A calibration curve UA shown in FIG. 6 was obtained from the obtained peak current value and uric acid concentration. As shown in FIG. 6, the detected current also changed in proportion to the uric acid concentration.
[0056]
Thus, a calibration curve proportional to the concentration of each component was obtained in Examples 3 and 4. Therefore, by using these calibration curves, the uric acid concentration in the test sample solution containing uric acid and ascorbic acid can be measured easily and with high precision using the diamond electrode.
[Brief description of the drawings]
FIG. 1 is a diagram showing an outline of a measurement system used in a measurement method according to the present invention.
2A and 2B are diagrams showing a structure of a diamond electrode, wherein FIG. 2A is a cross-sectional view of the diamond electrode, and FIG. 2B is a perspective view of the diamond electrode.
FIG. 3 is a diagram showing a basic configuration of a measuring apparatus according to the present invention.
FIG. 4 shows differential pulse voltammetry measurement results of a solution having a uric acid concentration of 100 μM, a solution having an ascorbic acid concentration of 500 μM, and a mixed solution having a uric acid concentration of 100 μM and an ascorbic acid concentration of 500 μM.
FIG. 5 is a calibration curve of ascorbic acid concentration and peak current value measured at applied voltages of 0.2 V and 0.8 V (vs. reference electrode).
FIG. 6 is a calibration curve of uric acid concentration and peak current value measured at an applied voltage of 0.8 V (vs. reference electrode).

Claims

A method for measuring a test substance concentration using a conductive diamond electrode,
Prepare a test sample solution containing a first test substance and a second test substance, both of which can cause an oxidation or reduction reaction by applying the same voltage on a conductive diamond electrode,
A first working electrode made of a conductive diamond electrode, a second working electrode made of a conductive diamond electrode, and a counter electrode in contact with the test sample solution;
An oxidation or reduction reaction of the second test substance occurs on the first working electrode between the first working electrode and the counter electrode, but the oxidation or reduction reaction of the first test substance is substantially Applying a voltage that does not occur automatically, measuring the current value under the voltage, calculating the concentration of the second test substance in the test sample liquid from the obtained current value,
From the concentration of the second test substance, oxidation or reduction of both the first test substance and the second test substance on the second working electrode between the second working electrode and the counter electrode. Calculate the contribution of the second test substance to the current value obtained when a voltage at which reaction occurs is applied,
A voltage that causes oxidation or reduction reaction of both the first test substance and the second test substance on the second working electrode is applied between the second working electrode and the counter electrode, and the voltage The current value is measured below, the current value derived from the first test substance alone is calculated by subtracting the contribution of the second test substance from the current value, and the test value is calculated from the current value. Calculating a first analyte concentration in the sample.

The first test substance is a measurement target substance to be subjected to concentration measurement, and the second test substance is a measurement interfering substance to be excluded in the concentration measurement of the measurement target substance. The method described.

The method according to claim 2 , wherein the measurement target substance is uric acid and the measurement interfering substance is ascorbic acid.

The method according to any one of claims 1 to 3, wherein the pH of the test sample solution is 5 to 8.

The method according to any one of claims 1 to 3, further comprising a step of adjusting the pH of the test sample solution to 5 to 8 prior to measurement.

The voltage applied between the first working electrode and the counter electrode is 0.1 to 0.3 V (vs. Ag / AgCl electrode), and the voltage applied between the second working electrode and the counter electrode The method according to any one of claims 3 to 5, wherein is 0.7 to 0.9 V (vs. Ag / AgCl electrode).

The method according to claim 1, wherein the conductive diamond electrode is anodized.

The method according to claim 1, wherein the surface of the conductive diamond electrode is oxygen-terminated.

The method according to any one of claims 1 to 8, wherein the test sample solution comprises urine or serum.

The method according to claim 1, wherein the test sample solution further comprises a phosphate buffer.

The supply of the test sample solution is performed by injecting the test sample solution into the carrier solution while flowing the solution as a carrier through the system at a constant flow rate. The method described in 1.

The step of calculating the concentration of the second test substance from the current value due to the oxidation or reduction reaction at the first working electrode was obtained with the calibration curve of the second test substance concentration and the current value prepared in advance. The method according to claim 1, wherein the method is performed by comparing the current value.

The step of calculating the contribution of the second test substance to the current value due to the oxidation or reduction reaction at the second working electrode includes a calibration curve of the second test substance concentration and the current value prepared in advance, The method as described in any one of Claims 1-12 performed by contrasting with the 2nd test substance density | concentration measured with the working electrode.

The step of calculating the first test substance concentration in the test sample from the current value derived from the first test substance alone is a calibration of the first test substance concentration and the current value created in advance. The method according to claim 1, wherein the method is performed by comparing the line and the obtained current value.

Three or more kinds of test substances exist in the test sample solution, and two or more first working electrodes exist independently, and different test substances are respectively provided on these first working electrodes. The method according to any one of claims 1 to 15, wherein a different voltage is applied to each electrode so that an oxidation or reduction reaction of the test substance occurs.

Two or more kinds of measurement interfering substances exist in the test sample solution, and two or more first working electrodes exist independently, and different measurements are made on each of these first working electrodes. 16. A method according to any one of claims 2 to 15, wherein a different voltage is applied to each electrode so that an interfering substance oxidation or reduction reaction occurs.

An apparatus for measuring a test substance concentration using a conductive diamond electrode,
A first working electrode comprising a conductive diamond electrode;
A second working electrode comprising a conductive diamond electrode;
A counter electrode;
Means for contacting the first working electrode, the second working electrode, and the counter electrode with a test sample solution;
An oxidation or reduction reaction of the second test substance occurs on the first working electrode between the first working electrode and the counter electrode, but the oxidation or reduction reaction of the first test substance is substantially A voltage that does not occur is applied, and the current value under the voltage is measured. Between the second working electrode and the counter electrode, both of the first test substance and the second test substance are measured. Measuring means for applying a voltage at which oxidation or reduction reaction occurs and measuring a current value under the voltage;
The second test substance concentration in the test sample liquid is calculated from the current value obtained at the first working electrode, and the second test substance concentration to the current value at the second working electrode is calculated. Calculate the contribution of the second test substance, and further subtract the contribution of the second test substance from the current value at the second working electrode to obtain a current value derived from the first test substance alone. And a calculating means for calculating a first test substance concentration in the test sample liquid from the current value.

The first test substance is a measurement target substance to be subjected to concentration measurement, and the second test substance is a measurement interfering substance to be excluded in the concentration measurement of the measurement target substance. The device described.

The apparatus according to claim 18, wherein the measurement target substance is uric acid and the measurement interfering substance is ascorbic acid.