JPWO2001098766A1 - Method for measuring the concentration of a xanthine compound and a sensor used therefor - Google Patents

Abstract

Provided is a method capable of easily and specifically measuring the concentrations of xanthine compounds such as caffeine, theophylline, theobromine, and xanthine which are clinically or foodically important. The property that the xanthine-based compound represented by the following formula (I) is specifically oxidized electrochemically in a diamond electrode is used. That is, a diamond electrode having conductive diamond and a counter electrode are prepared, and a voltage at which an oxidation reaction occurs on the diamond electrode is applied between the diamond electrode and the counter electrode. The value is measured, and the concentration of the compound represented by the formula (I) in the test sample is calculated from the obtained current value using a calibration curve prepared in advance. (Wherein, R1, R2 and R3 independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)

Description

（式中、Ｒ^１、Ｒ^２およびＲ^３は、独立して、水素原子または炭素数１〜６のアルキル基を表す）
導電性ダイヤモンドを有するダイヤモンド電極と、対電極とを用意し、
前記ダイヤモンド電極と、前記対電極とを被験試料に接触させ、
前記ダイヤモンド電極と、前記対電極との間に、前記ダイヤモンド電極上で酸化反応の生じる電圧を印加し、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験試料中の前記式（Ｉ）で表される化合物の濃度を算出すること
を含んでなるものである。
また本発明の別の態様によれば、上記測定法に用いられるセンサが提供され、そのセンサは、導電性ダイヤモンドを有するダイヤモンド電極からなるものである。
さらに、本発明の別の態様によれば、上記測定法を実施するための装置が提供され、その装置は、
導電性ダイヤモンドを有するダイヤモンド電極と、
対電極と、
前記ダイヤモンド電極と、対電極とを被験試料に接触させる手段と、
前記ダイヤモンド電極と、前記対電極との間に、前記ダイヤモンド電極上で酸化反応の生じる電圧を印加する手段と、
該印加電圧下における電流値を測定する手段と、
得られた電流値から前記被験試料中の前記式（Ｉ）で表される化合物の濃度を算出する手段と
を少なくとも備えてなるものである。
また、本発明の別の態様によれば、被験試料中の下記の式（Ｉ）で表される化合物：

（式中、Ｒ^１、Ｒ^２およびＲ^３は、独立して、水素原子または炭素数１〜６のアルキル基を表す）
の濃度を測定するための、導電性ダイヤモンドを有するダイヤモンド電極の使用、が提供される。
［発明の具体的説明］
被検化合物
本発明による方法によれば、試料溶液中における式（Ｉ）の化合物の濃度を選択的に知ることが出来る。本発明者らは、前記したように、導電性ダイヤモンド電極上において、式（Ｉ）の化合物が電気化学的に特異的に酸化されること、さらには、導電性ダイヤモンドを作用電極とし、対電極との間に生じる電流値が系内の式（Ｉ）の化合物の濃度に正比例するとの関係も確認出来た。その結果、被験試料中の式（Ｉ）の化合物を定量的に検出することが可能となった。
被験試料は、式（Ｉ）の化合物が含まれていると考えられる生体由来の血液、体液等、または食品もしくは食品の希釈溶液または懸濁液であってよい。
式（Ｉ）において、Ｒ^１、Ｒ^２およびＲ^３が表す炭素数１〜６のアルキル基は、直鎖状であっても分岐鎖状であってもよく、また置換基を有していてもよい。
本発明において、特に生化学的に重要と思われる式（Ｉ）の化合物は以下の通りである。

キサンチンは、例えばキサンチン尿症患者において大量に尿中に排出される。キサンチン結晶の析出は、いわゆる膀胱結石につながるおそれがある。従って、キサンチンの尿中濃度を短時間に、かつ簡便に測定可能とする本発明による測定法は、その診断および治療に有用である。また、テオフィリンは、気管支拡張剤、強心利尿剤等として臨床において利用されており、その血中中濃度等を知ることは有用である。さらに、テオブロミンは、テオフィリンと同様に強心作用および利用作用を有しており、その血中濃度等を知ることが出来ることは意義がある。さらにまた、カフェインは、医薬用途以外にも多くの食品に含まれ、食品の製造および栄養学上、その濃度を簡便に知ることが出来ることの意義は大きいと言える。
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〜金属様の導線性を示すダイヤモンドを電極として使用する。
このような導線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、最も好ましくはホウ素または窒素であれる。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。
本発明によるダイヤモンド電極は、この導線性ダイヤモンドを電極として用いたものである。このダイヤモンド電極は、電気化学反応を水中で行うと、酸化反応として水の電気分解により酸素が発生せず、上記した式（Ｉ）の化合物の酸化反応を特異的に生じさせるとの極めて興味深い特性を有していた。この特性は、導電性ダイヤモンドの表面状態にかかわらず発揮されるものと考えられる。すなわち、後記するような化学気相成長法により、水素ガスをキャリアとして用いて製造された何の処理もされていない（ａｓ ｇｒｏｗｎ）ダイヤモンド薄膜の表面には、末端炭素原子に水素原子が結合した状態に有ることが通常であるが、この終端水素原子を他の原子に置換した導電性ダイヤモンドによっても、上記式（Ｉ）の化合物の酸化反応を特異的に生じさせることが出来るものと考えられる。
導電性ダイヤモンドそれ自体を基材の支持によらず電極とすることも可能であるが、本発明の好ましい態様によれば、基材上に導線性ダイヤモンドの薄膜を形成し、この薄膜に導線を接続させ、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。
この態様の電極を図１を用いて更に説明する。図１（ａ）は、ダイヤモンド電極１の断面図であり、この電極は、基材２の上に形成された導電性ダイヤモンド薄膜３とからなり、さらにこの導電性ダイヤモンド薄膜３には導線５が例えば金コーティング４を介して接続される。図１（ｂ）は、ダイヤモンド電極１の斜視図であり、基材２の上に形成された導電性ダイヤモンド薄膜３とからなり、さらにこの導電性ダイヤモンド薄膜３を電極とするための金コーティング４を介して導線５が接続される。
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。
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。
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。
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。
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はかることも可能である。
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。
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程度が一般的にであり、また１，０００〜１０，０００ｐｐｍ程度が好ましい。
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。
測定法およびそのための装置
本発明にあっては、導電性ダイヤモンドが上記式（Ｉ）の化合物を電気化学的に特異的に酸化し、導電性ダイヤモンドを作用電極とし、対電極との間に生じる電流値が系内の式（Ｉ）の化合物の濃度に正比例するとの性質を利用して、被験試料中の式（Ｉ）の化合物を定量的に検出する。その測定は電流値を知ることにより行われるため、測定時間は短くかつ簡便であり、本発明による方法は短時間の内に容易に式（Ｉ）の化合物濃度を知ることが出来る点で極めて有利である。
また、ダイヤモンド電極は、式（Ｉ）の化合物以外の化合物を酸化する可能性を有するが、式（Ｉ）の化合物に極めて特異的に感応する。本発明者らの知る限り、上記したカフェイン、テオフィリン、テオブロミン、およびキサンチンの存在が予想される生体または食品由来の試料において、式（Ｉ）の化合物以外の物質が存在していても、それらには反応せず、式（Ｉ）の化合物のみをダイヤモンド電極は酸化する。従って、本発明による測定法は、式（Ｉ）の化合物のみを特異的に測定することを可能にする。式（Ｉ）の化合物の混合物、さらにはダイヤモンド電極に反応しうる物質が被験試料中に存在してるまたはその可能性がある場合には、液体クロマトグラフィーなどの前処理を行い、式（Ｉ）の化合物をダイヤモンド電極に応答性を有する他の物質と峻別して、本発明による測定法に付することが好ましいことは言うまでもない。
さらに、化合物ごとに酸化される電圧が異なる、すなわち後記するピーク電圧が異なることから、式（Ｉ）の化合物の複数を峻別が、印可電圧の制御により可能となる。
また、本発明による測定法によれば、式（Ｉ）の化合物を約１μｍｏｌ／ｌ〜飽和濃度まで広い濃度範囲で測定することが出来る。
本発明による式（Ｉ）の化合物の濃度測定法において、式（Ｉ）の化合物の定量のための電気化学的系は、導電性ダイヤモンドを作用電極とした以外は、一般的な電気化学的な系であることが出来る。
すなわち、本発明による式（Ｉ）の化合物の濃度測定法にあっては、導電性ダイヤモンドを作用電極とし、対電極とともに被験試料に接触させ、この二つの電極間に、ダイヤモンド電極上で酸化反応の生じる電圧を印加し、この電圧下における電流値を測定する。そして、この得られた電流値から被験試料中の式（Ｉ）で表される化合物の濃度を算出する。上記したように、式（Ｉ）の化合物の酸化により、導電性ダイヤモンドと対電極との間に生じる電流値は、系内の式（Ｉ）の化合物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式（Ｉ）の化合物の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中の式（Ｉ）の化合物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、式（Ｉ）で表される化合物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、式（Ｉ）の化合物の濃度を知ることが出来る。
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。
本発明において、作用電極であるダイヤモンド電極と、対電極との間に印加される電圧は、ダイヤモンド電極上で式（Ｉ）の化合物の酸化反応が生じるものであれば限定されないが、測定効率および精度の観点から、この印加電圧は、式（Ｉ）の化合物の酸化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。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。
さらに、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、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。
本発明による測定方法において、電気化学的系は、導電性ダイヤモンドを作用電極とした以外は、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測定が好ましい。
フローセルを用いたフローインジェクション法の概略は図２（ａ）に示される通りである。フローセル２１には、キャリア溶液溜６から、ポンプ７によりキャリア溶液が注入される。ポンプ７とフローセル２１との間には、被験試料注入口８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２１を通過したキャリア溶液は、廃液溜９に集められる。
フローセル２１の基本構造は図２（ｂ）に示されるとおりである。フローセル２１は、ダイヤモンド電極１、対電極２２、および参照電極２３が、キャリア溶液および被験試料が流れる流路２４内に露出され、被験試料と接触出来るよう構成されている。ダイヤモンド電極２１は、基本的に図１に示される構造を有し、図１におけるダイヤモンド薄膜３が、流路２４内に露出され、キャリア溶液および被験試料と接触する。キャリア溶液は、流路２４の流入口２５から入り、図中の矢印のように流れ、流出口２６に至る。ダイヤモンド電極１の導線Ａおよび対電極２２の導線Ｂの間に、式（Ｉ）の化合物をダイヤモンド電極１上で酸化を生じさせる電圧、好ましくはピーク電流を生じさせる電圧を印加する。
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８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。式（Ｉ）の化合物が被験試料中に含まれている場合、式（Ｉ）の化合物がダイヤモンド電極１上で酸化され、酸化反応に伴う電流値が測定できる。この電流値より、式（Ｉ）の化合物の濃度を知る。
本発明による測定法にあっては、被験試料のｐＨは任意の特定の値に固定して測定に付されることが好ましい。式（Ｉ）の化合物の一部については、ピーク電流を与える電圧のｐＨ依存性は大きくないが、測定対象物と好ましい被験試料のｐＨの範囲は以下の通りである。

本発明の別の好ましい態様によれば、フローセル２１におけるダイヤモンド電極１をマイクロ電極の形態とすることも好ましい。また、本発明の別の好ましい態様によれば、ダイヤモンド電極１を回転電極としてもよい。
以上の通り、本発明の別の態様によれば式（Ｉ）の化合物の濃度測定法が提供されるが、本発明の別の態様によれば、導電性ダイヤモンドを有するダイヤモンド電極からなる、式（Ｉ）の化合物の濃度測定のためのセンサが提供される。
さらに、本発明の別の態様によれば、被験試料中の式（Ｉ）の化合物の濃度を測定する装置が提供される。この装置の基本構成は図３に示される通りである。図３の装置において、電源・電流計３１には、図２に記載のフローセルより、ダイヤモンド電極１に接続される導線Ａ、対電極２２に接続される導線Ｂ、および参照電極２３に接続される導線Ｃが接続される。この電源・電流計３１は、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反応の生じる電圧を印加する手段と、この印加電圧下における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３１によって、ダイヤモンド電極１および対電極２２の間に、式（Ｉ）の化合物をダイヤモンド電極１上で酸化を生じさせる電圧、好ましくはピーク電流を生じさせる電圧を印加し、さらに式（Ｉ）の化合物のダイヤモンド電極１上での酸化反応に伴う電流値を測定する。この電流値は、電柱値比較・濃度算出装置３２に送られる。この装置３２には、検量線データ３３が送られ、この装置内において、電源・電流計３１から送られた電流値と、検量線データを比較し、式（Ｉ）の化合物の濃度を算出する。すなわち、得られた電流値から被験試料中の前記式（Ｉ）で表される化合物の濃度を算出する手段を備える。得られた式（Ｉ）の化合物の濃度は、表示装置３４により表示される。
［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。
ダイヤモンド電極の調製
ダイヤモンド薄膜を、ＡＳＴｅＸ社製、マイクロ波ＣＶＤ成膜装置を用い、マイクロ波プラズマアシストＣＶＤ法により調製した。具体的には以下の通りである。
まず、シリコン基板（Ｓｉ（１００））の表面を、０．５μｍのダイヤモンド粉で研磨した後、ＣＶＤ成膜装置の基板ホルダにセットした。炭素源としてアセトンとメタノールとの混合物（混合比９：１（体積比））を用いた。さらに、この混合物に酸化ホウ素（Ｂ_２Ｏ_３）を、ホウ素／炭素比で１０^４ｐｐｍとなる量溶解した。炭素源の混合物に純水素ガスを通し、溶存気体と置換した。一方、チャンバー内に純水素ガスを５３２ｃｃ／分の流速で流し、さらにその圧力を１１５×ｔｏｒｒ（１１５×１３３．３２２Ｐａ）とした。次に、２．４５ＧＨｚのマイクロ波をチャンバー内に注入し、放電させ、出力が５ＫＷとなるよう調製した。装置が安定したことを確認した後、炭素源混合物にキャリアガスとして純水素ガスを１５ｃｃ／分の流速で流し、チャンバー内に導入し、成膜を行った。成膜速度は１〜４μｍ／時であった。約３０μｍの厚さとなるまで成膜を行った。基板の加熱は行わなかったが、定常状態において約８５０〜９５０℃の温度となっていることが観察された。
得られたダイヤモンド薄膜のラマンスペクトルをとると、１３３３ｃｍ^−１に単一ピークのみが観察された。また、電気伝導度は約１０^−３Ωｃｍ程度であり、０．５Ｍ硫酸中においてサイクリックボルタモグラムを測定した結果、−１．２５〜＋２．３Ｖ（対ＳＨＥ）と、広い電位窓を有することが確認出来た。
ボルタンメトリー測定
ボルタンメトリー測定を、ガラスセル中において、Ｈｏｋｕｔｏ Ｄｅｎｋｏ ＨＡ−５０２ポテンショスタット、Ｈｏｋｕｔｏ Ｄｅｎｋｏ ＨＢ−１１ファンクションジェネレーター、およびＲｉｋｅｎ Ｄｅｎｓｈｉ ｘ−ｙレコーダーを用いて、室温にて行った。上記のようにして得られたダイアモンド薄膜を有するダイヤモンド電極を作用電極（露出面積０．０７ｃｍ^２）として用い、また白金箔を対電極として用いた。参照電極として、飽和カロメル電極（ＳＣＥ）を用いた。
実験例１：ピーク電流値と濃度との関係
（ａ） キサンチン、テオフィリン（１級）、テオブロミン、およびカフェイン（特級）をＷａｋｏ社から求め、Ｍｉｌｌｉ−Ｑ水（Ｍｉｌｌｉｐｏｒｅ）を用いて１〜４００μｍｏｌ／ｌの濃度範囲で水溶液を調製した。なお、０．１ＭＮａＣｌＯ_４を含む空気飽和Ｂｒｉｔｔｏｎ−Ｒｏｂｉｎｓｏｎ緩衝液（０．０４Ｍ）を電解質として用い、ｐＨは通常のガラス電極を用いて測定した。
（ｂ） 全ての水溶液のｐＨを１．８とし、電位掃引速度は２０ｍＶｓ^−１のボルタンメトリー測定を行い、ピーク電流の値を求めた。さらに、得られたピーク電流値と、濃度との関係を調べると、原点を通る線形の関係が得られた。最小二乗法により得られた検量線：濃度Ｉ＝α×化合物濃度Ｍは、以下の通りである。

なお、キサンチンについては、濃度約１５０μｍｏｌ／ｌを超えるとピーク電流値は濃度依存性を失った。これは、キサンチンの緩衝液への貧溶解性が原因と思われる。
（ｃ） いくつかの濃度の溶液に対して、５回測定を行い、ピーク電流値の再現性を、ピーク電流値の偏差（％）として確認した。
その結果は、以下の通りであった。

実験例２：ピーク電流値とｐＨのとの関係
（ａ） ５０μＭテオフィリン溶液について、ｐＨ１、２、３、４、および５の条件の下、２０ｍＶｓ^−１の掃引速度で陽極ボルタモグラフを記録した。その結果は、図４に示されるとおりであった。この結果から、ｐＨが増加すると、テオフィリンについてのピーク電圧が負のシフトを生じることが観察された。
（ｂ） キサンチン、テオフィリン、テオブロミン、およびカフェインそれぞれについて、５０μＭの溶液を用意し、ピーク電圧と、ｐＨとの関係を調べた。その結果は、以下の通りであった。

上記の通り、キサンチンおよびテオフィリンでは、広汎なｐＨ範囲（１．８〜１２．０）に亘って再現性あるピークが示されたが、テオブロミンおよびカフェインの場合には、ｐＨ値が約５．５では、酸化ピークは明確で無くなる傾向を示した。
実験例３：カフェインの定量
（ａ）検量線の作成
ｐＨ１の０．１ＭＨＣｌＯ_４を溶媒とし、種々のカフェイン濃度の溶液を用意した。この溶液について、電位掃引速度を２０ｍＶｓ^−１とするボルタンメトリー測定を行い、ピーク電流の値とカフェイン濃度との関係を得て、検量線を作成した。得られた検量線は、図５（ａ）および（ｂ）に示される通りであった。
（ｂ）カフェインの定量
市販のコーラ飲料をｐＨ１の０．１ＭＨＣｌＯ_４を溶媒とし、１２５倍希釈した。この希釈溶液について、図３に示されるフローセルを用いた、フローインジェクション法により、ピーク電流値を求めた。印加電圧は、対塩化銀電極で１．５Ｖとした。得られたピーク電流値は、０．５１μＡであり、上記検量線から、１２５倍希釈溶液中のカフェイン濃度は８．１ｍｍｏｌ／ｌと算出された。市販のコーラ飲料中のカフェイン濃度は１９４．２１ｍｇ／ｌとされた。
実験例４：テオフィリンの定量
（ａ）検量線の作成
実施例３（ａ）と同様にして、テオフィリンにつき、ピーク電流の値とカフェイン濃度との関係を得て、検量線を作成した。得られた検量線は、図６に示される通りであった。
（ｂ）テオフィリンの定量
市販のテオフィリン１００ｍｇ錠をｐＨ１の０．１ＭＨＣｌＯ_４を溶媒とし、まずその５０ｍｌに溶解し、更にその溶液０．０４ｍｌを前記溶媒２０ｍｌに溶解した。この溶液に付き、印加電圧を１．４Ｖとした以外は、実施例３（ｂ）と同様にしてピーク電流値を求めた。ピーク電流値は１７００ｎＡであり、上記検量線から、被測定溶液中のテオフィリン濃度は１９．９６ｍｍｏｌ／ｌと算出された。この濃度からテオフィリン１００ｍｇ錠中のテオフィリン量は９９．８ｍｇと算出された。
【図面の簡単な説明】
図１は、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１の断面図であり、この電極は、基材２の上に形成された導電性ダイヤモンド薄膜３とからなる。（ｂ）は、ダイヤモンド電極１の斜視図であり、基材２の上に形成された導電性ダイヤモンド薄膜３とからなる。
図２は、本発明による測定法に用いられるフローセルの基本構造を示す図である。
図３は、本発明による測定装置の基本構成を示す図である。
図４は、５０μＭテオフィリン溶液について、ｐＨ１、２、３、４、および５の条件の下、２０ｍＶｓ^−１の掃引速度で陽極ポルタモグラフの結果を示す図である。
図５は、カフェイン濃度と、ピーク電流の値との検量線である。
図６は、テオフィリン濃度と、ピーク電流の値との検量線である。[Background of the Invention]
The present invention relates to a method for measuring the concentration of xanthine-based compounds such as caffeine, theophylline, theobromine, and xanthine, which are clinically or foodically important, using a diamond electrode, and used in the method. The present invention relates to a sensor and a device therefor.
BACKGROUND ART Although diamond is an insulating material having a resistivity of about 10 < ¹³ > [Omega] cm, conductivity is obtained by doping a trace amount of impurities. Various uses are expected for this conductive diamond. One of them is utilization as an electrode for electrochemical use. When diamond made conductive is viewed as an electrode for electrochemical use, it has the excellent features of having a wide potential window and extremely low background current. Furthermore, it also has features 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.
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, which was implanted with argon or nitrogen ions to impart surface conductivity, as an electrically conductive material. At the same time, a cyclic voltammogram in the electrolyte solution was also shown. Thereafter, the properties of polycrystalline diamond synthesized by gas phase using a hot filament have been reported (Pleskov et al., J. Electroanal. Chem., 228, 19 (1993)).
Some of the present 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, a p-type semiconductor diamond into which boron was introduced as a dopant was used as an electrode. After that, as a diamond electrode, the use of a p-type semiconductor using boron as a dopant or a metal-like conductive diamond having higher conductivity has become mainstream. In the 1990s, diamond electrode research was conducted by several groups. Since 1995, diamond obtained by using a plasma CVD (PCVD) apparatus capable of obtaining a diamond thin film having a larger area than vapor phase synthesis. Electrode research has also been seen in the electrochemical field.
On the other hand, various xanthine compounds having a xanthine structure have various physiological functions in animals and plants.
For example, xanthine (3,7-dihydro-1H-purine-2,6-dione) belongs to the purines and plays an important role as an intermediate in the degradation of adenine and guanine to uric acid, and thus has a biological role. Is considered extremely important. In addition, xanthine is poorly soluble in water and xanthine, although very rare, may precipitate as aggregates due to metabolic abnormalities.
Theophylline (3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione), theobromine (3,7-dihydro-3,7-dimethyl-1H-purine-2,6-dione) and N-methyl derivatives of xanthine, such as caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione), are alkaloids widely distributed in plant products and beverages. And are known to have many physiological effects such as gastric acid secretion, diuresis, and central nervous system stimulation. In particular, theophylline is useful as a bronchodilator and as a therapeutic agent for bronchial asthma.
Therefore, if the abundance of these xanthine compounds in a living body or a sample derived from a living body or in a food can be easily known, it is extremely significant in medical pharmacy or food production.
[Summary of the Invention]
The present inventors have now found that a diamond electrode is specifically sensitive to a xanthine-based compound, and that its abundance can be quantified from the current value at its oxidation potential. The present invention is based on such findings.
Accordingly, an object of the present invention is to provide a method capable of easily and specifically measuring the concentration of a xanthine compound in a short time.
Another object of the present invention is to provide a sensor used in the above method and a device therefor.
According to a first aspect of the present invention, there is provided a method for measuring the concentration of a compound represented by the following formula (I) in a test sample, the method comprising:

(Wherein, R ¹ , R ² and R ³ independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)
Prepare a diamond electrode having a conductive diamond, and a counter electrode,
The diamond electrode and the counter electrode are brought into contact with a test sample,
A voltage at which an oxidation reaction occurs on the diamond electrode is applied between the diamond electrode and the counter electrode, and a current value under the voltage is measured,
Calculating the concentration of the compound represented by the formula (I) in the test sample from the obtained current value.
According to another aspect of the present invention, there is provided a sensor used in the above-mentioned measuring method, the sensor comprising a diamond electrode having conductive diamond.
Further, according to another aspect of the present invention, there is provided an apparatus for performing the above-described measurement method, the apparatus comprising:
A diamond electrode having conductive diamond,
A counter electrode,
The diamond electrode, means for contacting the counter electrode with the test sample,
Means for applying a voltage at which an oxidation reaction occurs on the diamond electrode, between the diamond electrode and the counter electrode,
Means for measuring a current value under the applied voltage;
Means for calculating the concentration of the compound represented by the formula (I) in the test sample from the obtained current value.
According to another aspect of the present invention, a compound represented by the following formula (I) in a test sample:

(Wherein, R ¹ , R ² and R ³ independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)
Use of a diamond electrode with conductive diamond for measuring the concentration of
[Specific description of the invention]
Test compound According to the method of the present invention, the concentration of the compound of the formula (I) in the sample solution can be selectively known. As described above, the present inventors have found that the compound of formula (I) is electrochemically specifically oxidized on a conductive diamond electrode, and further, the conductive diamond is used as a working electrode, Was confirmed to be directly proportional to the concentration of the compound of formula (I) in the system. As a result, it has become possible to quantitatively detect the compound of the formula (I) in the test sample.
The test sample may be blood, a body fluid, or the like, or a food or a diluted solution or suspension of the food, which is thought to contain the compound of formula (I).
In the formula (I), the alkyl group having 1 to 6 carbon atoms represented by R ¹ , R ² and R ³ may be linear or branched, and may have a substituent. Is also good.
In the present invention, the compounds of the formula (I) which are considered particularly biochemically important are as follows.

Xanthine is excreted in large amounts in the urine, for example in patients with xanthineuria. Precipitation of xanthine crystals can lead to so-called bladder stones. Therefore, the measuring method according to the present invention, which enables the urinary concentration of xanthine to be measured in a short time and simply, is useful for the diagnosis and treatment. Theophylline is used clinically as a bronchodilator, a cardiotonic diuretic and the like, and it is useful to know its blood concentration and the like. Furthermore, theobromine has a cardiotonic effect and a utilization effect similarly to theophylline, and it is significant that its blood concentration and the like can be known. Furthermore, caffeine is contained in many foods in addition to pharmaceutical uses, and it can be said that the significance of easily knowing its concentration in food production and nutrition is significant.
Diamond electrodes and their manufacture Diamonds are inherently excellent insulators. However, by adding a Group 3 or Group 5 impurity, the semiconductor or metal becomes conductive. In the present invention, diamond exhibiting semiconductor-metal-like conductive properties is used as an electrode.
Examples of the substance added to impart such a conductive property include Group 3 and Group 5 elements as described above, more preferably include boron, nitrogen, and phosphorus, and most preferably include boron or nitrogen. There. The amount of the substance added for imparting this conductivity may be appropriately determined within a range in which the conductivity can be imparted to diamond, and for example, gives a conductivity of about 1 × 10 ⁻² to 10 ⁻⁶ Ωcm. It is preferable to add in an amount. Generally, the amount of the substance added for imparting the conductivity is controlled by the amount added in the process of producing the conductive diamond.
The diamond electrode according to the present invention uses the conductive diamond as an electrode. This diamond electrode has an extremely interesting property that when an electrochemical reaction is performed in water, oxygen is not generated by water electrolysis as an oxidation reaction, and specifically causes an oxidation reaction of the compound of the above formula (I). Had. It is considered that this property is exhibited regardless of the surface state of the conductive diamond. That is, a hydrogen atom was bonded to a terminal carbon atom on the surface of an untreated (as grown) diamond thin film manufactured using a hydrogen gas as a carrier by a chemical vapor deposition method described later. It is usually in the state, but it is considered that the oxidation reaction of the compound of the above formula (I) can be specifically caused by the conductive diamond in which this terminal hydrogen atom is replaced by another atom. .
Although it is possible to use the conductive diamond itself as an electrode instead of supporting the base material, according to a preferred embodiment of the present invention, a conductive diamond thin film is formed on the base material, and a conductive wire is formed on the thin film. Preferably, they are connected to form electrodes. As the substrate, Si (e.g., single crystal _{silicon), Mo, W, Nb,} Ti, Fe, Au, Ni, Co, Al 2 O 3, SiC, Si 3 N 4, ZrO 2, MgO, graphite, single Examples thereof include crystalline diamond, cBN, and quartz glass. In particular, single crystal silicon, Mo, W, Nb, Ti, SiC, and single crystal diamond are preferably used.
The electrode of this embodiment will be further described with reference to FIG. FIG. 1A is a cross-sectional view of a diamond electrode 1, which comprises a conductive diamond thin film 3 formed on a base material 2, and furthermore, a conductive wire 5 is formed on the conductive diamond thin film 3. For example, they are connected via a gold coating 4. FIG. 1B is a perspective view of the diamond electrode 1, comprising a conductive diamond thin film 3 formed on a substrate 2, and a gold coating 4 for using the conductive diamond thin film 3 as an electrode. Is connected to the conductive wire 5.
The thickness of the conductive diamond thin film is not particularly limited, but is preferably about 1 to 100 μm, more preferably about 5 to 50 μm.
According to a further 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. In the present invention, a diamond electrode in the form of a microelectrode is formed by sharply cutting the end of a fine wire such as Pt, making the end more sharp by electrolytic polishing, and then forming a conductive surface on the end surface. Means a structure in which a thin film of conductive diamond is formed.
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 a gaseous raw material in a gas phase, and is generally called a CVD (Chemical Vapor Deposition) method. This method is widely used in a semiconductor manufacturing process, and can be used for manufacturing the conductive diamond thin film of the present invention with appropriate modification.
Chemical vapor synthesis of diamond is performed by using a mixture of hydrogen and a carbon-containing gas such as methane as a raw material gas, exciting it with an excitation source, and supplying and depositing it on a substrate.
Examples of the excitation source include a hot filament, a microwave, a high frequency, a DC glow discharge, a DC arc discharge, and a combustion flame. It is also possible to adjust the nucleation density by combining a plurality of them, or to increase the area and uniformity.
As a raw material, activated carbon such as C and C ₂ and carbonized such as CH, CH ₂ , CH ₃ and C ₂ H ₂ are decomposed and excited by many kinds of excitation sources containing carbon. Compounds that generate hydrogen radicals are available. Preferred specific examples include CH ₄ , C ₂ H ₂ , C ₂ H ₄ , C ₁₀ H ₁₆ , CO, CF ₄ as a gas, CH ₃ OH, C ₂ H ₅ OH, (CH ₃ ) ₂ CO as a liquid, Examples of the solid include graphite, fullerene, and the like.
In the gas phase synthesis method, the addition of a substance that imparts conductivity to diamond is, for example, a method in which a disc of the added substance is placed in the system, excited in the same manner as the carbon source material, and the added substance is introduced into the carbon gas phase. The method can be carried out by, for example, adding an additive to the carbon source in advance, introducing the additive into the system together with the carbon source, exciting with an excitation source, and introducing the additive 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 a carbon source, a method of dissolving boron oxide (B ₂ O ₃ ) in the liquid to obtain a boron source is such that the control of the boron concentration is easy and simple. Is preferred. For example, in the case of adding boron to a carbon source in a gas phase synthesis method, the amount is generally about 10 to 12,000 ppm, and preferably about 1,000 to 10,000 ppm.
According to a preferred embodiment of the present invention, the production of the conductive diamond thin film is preferably performed by a plasma enhanced chemical vapor deposition method. This plasma-enhanced chemical vapor synthesis method has the advantage that activation energy for causing a chemical reaction is large and the reaction is fast. Further, according to this method, a chemical species which does not exist thermodynamically unless the temperature is high is generated, and the reaction can be performed at a low temperature. Some reports have already been made on the production of conductive diamond thin films by the plasma chemical vapor deposition method, including some of the present inventors (for example, Yano et al., J. Electrochem. Soc., 145 (1998)). 1870), preferably according to the methods described in these reports.
Measurement method and apparatus therefor In the present invention, the conductive diamond oxidizes the compound of the above formula (I) electrochemically specifically, the conductive diamond is used as a working electrode, and the counter electrode is Using the property that the current value generated during the period is directly proportional to the concentration of the compound of the formula (I) in the system, the compound of the formula (I) in the test sample is quantitatively detected. Since the measurement is performed by knowing the current value, the measuring time is short and simple, and the method according to the present invention is extremely advantageous in that the concentration of the compound of the formula (I) can be easily known in a short time. It is.
Also, diamond electrodes have the potential to oxidize compounds other than the compound of formula (I), but are very specifically sensitive to compounds of formula (I). As far as the present inventors know, even if substances other than the compound of the formula (I) are present in a sample derived from a living body or a food in which the above-mentioned caffeine, theophylline, theobromine and xanthine are expected to be present, And the diamond electrode oxidizes only the compound of formula (I). Thus, the assay according to the invention makes it possible to specifically measure only compounds of formula (I). When a mixture of the compound of the formula (I) or a substance capable of reacting with the diamond electrode is present in the test sample or is possibly present, a pretreatment such as liquid chromatography is carried out, and the compound of the formula (I) It is needless to say that it is preferable that the compound of formula (1) is distinguished from other substances having a responsiveness to the diamond electrode and subjected to the measuring method according to the present invention.
Furthermore, since the oxidized voltage is different for each compound, that is, the peak voltage described later is different, a plurality of compounds of the formula (I) can be distinguished by controlling the applied voltage.
Further, according to the measurement method of the present invention, the compound of the formula (I) can be measured in a wide concentration range from about 1 μmol / l to a saturation concentration.
In the method for measuring the concentration of the compound of the formula (I) according to the present invention, the electrochemical system for quantifying the compound of the formula (I) is a general electrochemical system except that conductive diamond is used as a working electrode. Can be a system.
That is, in the method for measuring the concentration of the compound of the formula (I) according to the present invention, a conductive diamond is used as a working electrode, brought into contact with a test sample together with a counter electrode, and an oxidation reaction between the two electrodes is performed on the diamond electrode. Is applied, and the current value under this voltage is measured. Then, the concentration of the compound represented by the formula (I) in the test sample is calculated from the obtained current value. As described above, the current value generated between the conductive diamond and the counter electrode due to the oxidation of the compound of formula (I) is directly proportional to the concentration of the compound of formula (I) in the system. Therefore, once the relationship between the current value at a certain voltage value and the concentration of the compound of the formula (I) is determined, the relationship of the compound of the formula (I) in the test sample corresponding to the obtained current value is obtained from the relationship. The concentration can be easily known. That is, in a preferred embodiment of the present invention, a calibration curve between the concentration of the compound represented by the formula (I) and the current value is prepared in advance, and the calibration curve is compared with the obtained current value. By doing so, the concentration of the compound of formula (I) can be known.
In the present invention, it is preferable to use platinum, carbon, stainless steel, gold, diamond, SnO ₂ or the like as the counter electrode.
In the present invention, the voltage applied between the working electrode, the diamond electrode, and the counter electrode is not limited as long as an oxidation reaction of the compound of the formula (I) occurs on the diamond electrode. From the viewpoint of accuracy, the applied voltage is preferably a voltage that gives a peak current of oxidation of the compound of the formula (I). Here, the peak current can be determined as a voltage that gives a maximum current value by, for example, cyclic voltammetry. Further, according to another preferred embodiment of the present invention, the voltage giving the maximum current value can be obtained by a rotating electrode method or a micro electrode method. The rotating electrode method or the micro electrode method is advantageous in that the possibility of a measurement error due to measurement conditions and the like can be further eliminated.
Further, according to a preferred embodiment of the present invention, the reference electrode is brought into contact with the test sample, and between the diamond electrode and the counter electrode, controlling the absolute value of the voltage at which an oxidation reaction occurs on the diamond electrode, It is preferable from the viewpoint of measurement accuracy. As the reference electrode, a known electrode can be used, and a standard hydrogen electrode, a silver-silver chloride electrode, a mercury-mercury chloride electrode, a hydrogen-palladium electrode and the like can be used.
In the measurement method according to the present invention, the electrochemical system may be a general electrochemical system, except that the conductive diamond is used as a working electrode. It is preferable to use a flow injection method using a flow cell, in which a solution is allowed to flow through a system as a carrier at a constant flow rate, and a test sample is injected into the carrier solution for measurement.
An outline of a flow injection method using a flow cell is as shown in FIG. The carrier solution is injected into the flow cell 21 from the carrier solution reservoir 6 by the pump 7. A test sample inlet 8 is provided between the pump 7 and the flow cell 21 so that the test sample can be injected into the carrier solution. The carrier solution that has passed through the flow cell 21 is collected in the waste liquid reservoir 9.
The basic structure of the flow cell 21 is as shown in FIG. The flow cell 21 is configured such that the diamond electrode 1, the counter electrode 22, and the reference electrode 23 are exposed in a flow path 24 through which the carrier solution and the test sample flow, and can be brought into contact with the test sample. The diamond electrode 21 basically has the structure shown in FIG. 1, and the diamond thin film 3 in FIG. 1 is exposed in the channel 24 and comes into contact with the carrier solution and the test sample. The carrier solution enters through the inlet 25 of the flow channel 24, flows as indicated by the arrow in the drawing, and reaches the outlet 26. Between the conductive wire A of the diamond electrode 1 and the conductive wire B of the counter electrode 22, a voltage for causing the compound of formula (I) to oxidize on the diamond electrode 1, preferably a voltage for generating a peak current, is applied.
The measurement is performed as follows. First, only a carrier solution into which a test sample is not injected is caused to flow, so-called background current is reduced as much as possible and stabilized. Diamond electrodes are characterized by low background current. Next, a test sample is injected from the test sample inlet 8. This injection may be performed continuously, but may be performed at a time in such an amount that the peak current can be measured. When the compound of the formula (I) is contained in the test sample, the compound of the formula (I) is oxidized on the diamond electrode 1, and the current value accompanying the oxidation reaction can be measured. From this current value, the concentration of the compound of formula (I) is known.
In the measurement method according to the present invention, it is preferable that the pH of the test sample is fixed to an arbitrary specific value before the measurement. For some of the compounds of the formula (I), the pH at which the peak current is applied is not highly pH-dependent, but the ranges of the pH of the analyte and the preferred test sample are as follows.

According to another preferred embodiment of the present invention, it is preferable that the diamond electrode 1 in the flow cell 21 is in the form of a micro electrode. According to another preferred embodiment of the present invention, the diamond electrode 1 may be a rotating electrode.
As described above, according to another aspect of the present invention, there is provided a method for measuring the concentration of the compound of the formula (I). According to another aspect of the present invention, there is provided a method comprising the steps of: A sensor is provided for measuring the concentration of the compound of (I).
Further, according to another aspect of the present invention, there is provided an apparatus for measuring the concentration of a compound of formula (I) in a test sample. The basic configuration of this device is as shown in FIG. In the apparatus shown in FIG. 3, the power supply / ammeter 31 is connected to the conductor A connected to the diamond electrode 1, the conductor B connected to the counter electrode 22, and the reference electrode 23 from the flow cell shown in FIG. The conductor C is connected. The power supply / ammeter 31 serves as a means for applying a voltage at which an oxidation reaction occurs on the diamond electrode between the diamond electrode and the counter electrode, and a means for measuring a current value under the applied voltage. That is, a voltage that causes the compound of formula (I) to oxidize the compound of formula (I) on the diamond electrode 1, preferably a voltage that causes a peak current, is applied between the diamond electrode 1 and the counter electrode 22 by the power supply / ammeter 31. Further, a current value accompanying the oxidation reaction of the compound of the formula (I) on the diamond electrode 1 is measured. This current value is sent to the utility pole value comparison / concentration calculation device 32. Calibration curve data 33 is sent to the device 32. In this device, the current value sent from the power / ammeter 31 is compared with the calibration curve data to calculate the concentration of the compound of the formula (I). . That is, a means for calculating the concentration of the compound represented by the formula (I) in the test sample from the obtained current value is provided. The obtained concentration of the compound of the formula (I) is displayed on the display device.
[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.
Preparation of diamond electrode A diamond thin film was prepared by a microwave plasma assisted CVD method using a microwave CVD film forming apparatus manufactured by ASTeX. Specifically, it is as follows.
First, the surface of a silicon substrate (Si (100)) was polished with 0.5 μm diamond powder, and then set on a substrate holder of a CVD film forming apparatus. A mixture of acetone and methanol (mixing ratio 9: 1 (volume ratio)) was used as a carbon source. Further, in this mixture, boron oxide (B ₂ O ₃ ) was dissolved in an amount to give a boron / carbon ratio of 10 ⁴ ppm. Pure hydrogen gas was passed through the mixture of carbon sources to replace the dissolved gas. On the other hand, pure hydrogen gas was flowed into the chamber at a flow rate of 532 cc / min, and the pressure was set to 115 × torr (115 × 133.322 Pa). Next, a microwave of 2.45 GHz was injected into the chamber and discharged, so that the output was adjusted to 5 KW. After confirming that the apparatus was stable, a pure hydrogen gas as a carrier gas was flowed through the carbon source mixture at a flow rate of 15 cc / min, and introduced into the chamber to form a film. The deposition rate was 1-4 μm / hour. Film formation was performed until the thickness became about 30 μm. Although the substrate was not heated, it was observed that the temperature was about 850-950 ° C. in a steady state.
When the Raman spectrum of the obtained diamond thin film was taken, only a single peak was observed at 1333 cm ⁻¹ . Further, the electric conductivity is about 10 ⁻³ Ωcm, and as a result of measuring the cyclic voltammogram in 0.5 M sulfuric acid, it has a wide potential window of −1.25 to +2.3 V (vs. SHE). I was able to confirm.
Voltammetric measurements Voltammetric measurements were performed in a glass cell at room temperature using a Hokuto Denko HA-502 potentiostat, Hokuto Denko HB-11 function generator, and a Riken Denshi xy recorder. A diamond electrode having a diamond thin film obtained as described above was used as a working electrode (exposed area 0.07 cm ² ), and a platinum foil was used as a counter electrode. A saturated calomel electrode (SCE) was used as a reference electrode.
Experimental Example 1 : Relationship between peak current value and concentration (a) Xanthine, theophylline (primary), theobromine, and caffeine (special grade) were obtained from Wako, and 1 to 400 μmol using Milli-Q water (Millipore). An aqueous solution was prepared in a concentration range of / l. Note that an air-saturated Britton-Robinson buffer (0.04 M) containing 0.1 M NaClO ₄ was used as an electrolyte, and the pH was measured using a normal glass electrode.
(B) The pH of all aqueous solutions was set to 1.8, and the potential sweep rate was measured by voltammetry at 20 mVs- ¹ to determine the value of the peak current. Further, when the relationship between the obtained peak current value and the concentration was examined, a linear relationship passing through the origin was obtained. Calibration curve obtained by the least squares method: concentration I = α × compound concentration M is as follows.

In addition, as for xanthine, when the concentration exceeded about 150 μmol / l, the peak current value lost concentration dependency. This may be due to poor solubility of xanthine in the buffer.
(C) Measurements were performed five times on solutions of several concentrations, and the reproducibility of the peak current value was confirmed as a deviation (%) of the peak current value.
The results were as follows.

Experimental Example 2 : Relationship between peak current value and pH (a) With respect to a 50 μM theophylline solution, an anodic voltammograph was recorded under conditions of pH ¹ , 2, 3, 4, and 5 at a sweep rate of 20 mVs ⁻¹ . The result was as shown in FIG. From this result, it was observed that as the pH increased, the peak voltage for theophylline caused a negative shift.
(B) A 50 μM solution was prepared for each of xanthine, theophylline, theobromine, and caffeine, and the relationship between peak voltage and pH was examined. The results were as follows.

As noted above, xanthine and theophylline exhibited reproducible peaks over a broad pH range (1.8-12.0), whereas theobromine and caffeine had pH values of about 5. In No. 5, the oxidation peak tended to be unclear.
Experimental Example 3 Quantification of Caffeine (a) Preparation of Calibration Curve Solutions of various concentrations of caffeine were prepared using 0.1 M HClO ₄ at pH 1 as a solvent. This solution was subjected to voltammetric measurement at a potential sweep rate of 20 mVs ^-1 to obtain a relationship between the peak current value and the caffeine concentration, and to prepare a calibration curve. The calibration curve obtained was as shown in FIGS. 5 (a) and 5 (b).
(B) Quantification of Caffeine A commercially available cola drink was diluted 125-fold with 0.1 M HClO ₄ at pH 1 as a solvent. The peak current value of this diluted solution was determined by a flow injection method using the flow cell shown in FIG. The applied voltage was 1.5 V at the silver chloride electrode. The obtained peak current value was 0.51 μA, and the caffeine concentration in the 125-fold diluted solution was calculated to be 8.1 mmol / l from the above calibration curve. The caffeine concentration in the commercially available cola drink was 194.21 mg / l.
Experimental Example 4 : Quantification of theophylline (a) Preparation of calibration curve In the same manner as in Example 3 (a), a calibration curve was prepared for theophylline by obtaining the relationship between the peak current value and the caffeine concentration. The obtained calibration curve was as shown in FIG.
(B) Quantification of Theophylline A commercially available theophylline 100 mg tablet was dissolved in 50 ml of 0.1 M HClO ₄ having a pH of 1 as a solvent, and 0.04 ml of the solution was dissolved in 20 ml of the solvent. The peak current value was determined in the same manner as in Example 3 (b) except that the applied voltage was set to 1.4 V for this solution. The peak current value was 1700 nA, and the theophylline concentration in the solution to be measured was calculated to be 19.96 mmol / l from the above calibration curve. From this concentration, the amount of theophylline in the theophylline 100 mg tablet was calculated to be 99.8 mg.
[Brief description of the drawings]
FIG. 1 is a view showing the structure of a diamond electrode. FIG. 1A is a cross-sectional view of a diamond electrode 1, which is composed of a conductive diamond thin film 3 formed on a substrate 2. (B) is a perspective view of the diamond electrode 1, which comprises a conductive diamond thin film 3 formed on a substrate 2.
FIG. 2 is a diagram showing a basic structure of a flow cell used in the measuring method according to the present invention.
FIG. 3 is a diagram showing a basic configuration of the measuring device according to the present invention.
FIG. 4 is a diagram showing the results of anodic porthamography for a 50 μM theophylline solution under the conditions of pH ¹ , 2, 3, 4, and 5 at a sweep rate of 20 mVs ⁻¹ .
FIG. 5 is a calibration curve of the caffeine concentration and the value of the peak current.
FIG. 6 is a calibration curve of theophylline concentration and the value of the peak current.

[0002]
The use of a p-type semiconductor or a highly conductive metal-like conductive diamond as a punt has become mainstream. In the 1990's, research on diamond electrodes was conducted by several groups. Since 1995, diamond electrodes obtained using a plasma CVD (PCVD) apparatus capable of obtaining a diamond thin film having a larger area than vapor phase synthesis. Has been found in the electrochemical field.
On the other hand, various xanthine compounds having a xanthine structure have various physiological functions in animals and plants.
For example, xanthine (3,7-dihydro-1H-purine-2,6-dione) belongs to the purines and plays an important role as an intermediate in the degradation of adenine and guanine to uric acid, and thus has a biological role. Is considered extremely important. In addition, xanthine is poorly soluble in water and xanthine, although very rare, may precipitate as aggregates due to metabolic abnormalities.
Theophylline (3,7-dihydro-1,3-dimethyl-1H-purine-2,6-dione), theobromine (3,7-dihydro-3,7-dimethyl-1H-purine-2,6-dione) and N-methyl derivatives of xanthine, such as caffeine (3,7-dihydro-1,3,7-trimethyl-1H-purine-2,6-dione), are alkaloids widely distributed in plant products and beverages. And are known to have many physiological effects such as gastric acid secretion, diuresis, and central nervous system stimulation. In particular, theophylline is useful as a bronchodilator and as a therapeutic agent for bronchial asthma.
Therefore, if the abundance of these xanthine compounds in a living body or a sample derived from a living body or in a food can be easily known, it is extremely significant in medical pharmacy or food production.
[Summary of the Invention]
The present inventors have now found that a diamond electrode is specifically sensitive to a xanthine-based compound, and that its abundance can be quantified from the current value at its oxidation potential. The present invention is based on such findings.
Accordingly, an object of the present invention is to provide a method capable of easily and specifically measuring the concentration of a xanthine compound in a short time.

キサンチンは、例えばキサンチン尿症患者において大量に尿中に排出される。キサンチン結晶の析出は、いわゆる膀胱結石につながるおそれがある。従って、キサンチンの尿中濃度を短時間に、かつ簡便に測定可能とする本発明による測定法は、その診断および治療に有用である。また、テオフィリンは、気管支拡張剤、強心利尿剤等として臨床において利用されており、その血中濃度等を知ることは有用である。さらに、テオブロミンは、テオフィリンと同様に強心作用および利用作用を有しており、その血中濃度等を知ることが出来ることは意義がある。さらにまた、カフェインは、医薬用途以外にも多くの食品に含まれ、食品の製造[0005]
FIG. 6 is a calibration curve of theophylline concentration and the value of the peak current.
[Specific description of the invention]
Test compound According to the method of the present invention, the concentration of the compound of the formula (I) in the sample solution can be selectively known. As described above, the present inventors have found that the compound of formula (I) is electrochemically specifically oxidized on a conductive diamond electrode, and further, the conductive diamond is used as a working electrode, Was confirmed to be directly proportional to the concentration of the compound of formula (I) in the system. As a result, it has become possible to quantitatively detect the compound of the formula (I) in the test sample.
The test sample may be blood, a body fluid, or the like, or a food or a diluted solution or suspension of the food, which is thought to contain the compound of formula (I).
In the formula (I), the alkyl group having 1 to 6 carbon atoms represented by R ¹ , R ² and R ³ may be linear or branched, and may have a substituent. Is also good.
In the present invention, the compounds of the formula (I) which are considered particularly biochemically important are as follows.

Xanthine is excreted in large amounts in the urine, for example in patients with xanthineuria. Precipitation of xanthine crystals can lead to so-called bladder stones. Therefore, the measuring method according to the present invention, which enables the urinary concentration of xanthine to be measured in a short time and simply, is useful for the diagnosis and treatment. In addition, theophylline is used clinically as a bronchodilator, a cardiotonic diuretic and the like, and it is useful to know its blood concentration and the like. Furthermore, theobromine has a cardiotonic effect and a utilization effect similarly to theophylline, and it is significant that its blood concentration and the like can be known. Furthermore, caffeine is included in many foods besides pharmaceutical use,

[0012]
However, according to another aspect of the present invention, there is provided a sensor for measuring the concentration of a compound of formula (I), comprising a diamond electrode having conductive diamond.
Further, according to another aspect of the present invention, there is provided an apparatus for measuring the concentration of a compound of formula (I) in a test sample. The basic configuration of this device is as shown in FIG. In the apparatus shown in FIG. 3, the power supply / ammeter 31 is connected to the conductor A connected to the diamond electrode 1, the conductor B connected to the counter electrode 22, and the reference electrode 23 from the flow cell shown in FIG. The conductor C is connected. The power supply / ammeter 31 serves as a means for applying a voltage at which an oxidation reaction occurs on the diamond electrode between the diamond electrode and the counter electrode, and a means for measuring a current value under the applied voltage. That is, a voltage that causes the compound of formula (I) to oxidize the compound of formula (I) on the diamond electrode 1, preferably a voltage that causes a peak current, is applied between the diamond electrode 1 and the counter electrode 22 by the power supply / ammeter 31. Further, a current value accompanying the oxidation reaction of the compound of the formula (I) on the diamond electrode 1 is measured. This current value is sent to the current value comparison / concentration calculation device 32. Calibration curve data 33 is sent to the device 32. In this device, the current value sent from the power / ammeter 31 is compared with the calibration curve data to calculate the concentration of the compound of the formula (I). . That is, a means for calculating the concentration of the compound represented by the formula (I) in the test sample from the obtained current value is provided. The obtained concentration of the compound of the formula (I) is displayed on the display device.
[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.
Preparation of diamond electrode A diamond thin film was prepared by a microwave plasma assisted CVD method using a microwave CVD film forming apparatus manufactured by ASTeX. Specifically, it is as follows.
First, the surface of a silicon substrate (Si (100)) was polished with 0.5 μm diamond powder, and then set on a substrate holder of a CVD film forming apparatus. A mixture of acetone and methanol (mixing ratio 9: 1 (volume ratio)) was used as a carbon source. In addition,

上記の通り、キサンチンおよびテオフィリンでは、広汎なｐＨ範囲（１．８〜１２．０）に亘って再現性あるピークが示されたが、テオブロミンおよびカフェインの場合には、ｐＨ値が約５．５では、酸化ピークは明確で無くなる傾向を示した。
実験例３：カフェインの定量
（ａ）検量線の作成
ｐＨ１の０．１ＭＨＣｌＯ_４を溶媒とし、種々のカフェイン濃度の溶液を用意した。この溶液について、電位掃引速度を２０ｍＶｓ^−１とするボルタンメトリー測定を行い、ピーク電流の値とカフェイン濃度との関係を得て、検量線を作成した。得られた検量線は、図５（ａ）および（ｂ）に示される通りであった。
（ｂ）カフェインの定量
市販のコーラ飲料をｐＨ１の０．１ＭＨＣｌＯ_４を溶媒とし、１２５倍希釈した。この希釈溶液について、図３に示されるフローセルを用いた、フローインジェクション法により、ピーク電流値を求めた。印加電圧は、対銀塩化銀電極で１．５Ｖとした。得られたピーク電流値は、０．５１μＡであり、上記検量線から、[0015]
Under conditions, anodic voltammography was recorded at a sweep rate of 20 mVs- ¹ . The result was as shown in FIG. From this result, it was observed that as the pH increased, the peak voltage for theophylline caused a negative shift.
(B) A 50 μM solution was prepared for each of xanthine, theophylline, theobromine, and caffeine, and the relationship between peak voltage and pH was examined. The results were as follows.

As noted above, xanthine and theophylline exhibited reproducible peaks over a broad pH range (1.8-12.0), whereas theobromine and caffeine had pH values of about 5. In No. 5, the oxidation peak tended to be unclear.
Experimental Example 3 Quantification of Caffeine (a) Preparation of Calibration Curve Solutions of various concentrations of caffeine were prepared using 0.1 M HClO ₄ at pH 1 as a solvent. This solution was subjected to voltammetric measurement at a potential sweep rate of 20 mVs ^-1 to obtain a relationship between the peak current value and the caffeine concentration, and to prepare a calibration curve. The calibration curve obtained was as shown in FIGS. 5 (a) and 5 (b).
(B) Quantification of Caffeine A commercially available cola drink was diluted 125-fold with 0.1 M HClO ₄ at pH 1 as a solvent. The peak current value of this diluted solution was determined by a flow injection method using the flow cell shown in FIG. The applied voltage was 1.5 V at the silver-silver chloride electrode. The obtained peak current value was 0.51 μA. From the above calibration curve,

Claims (14)

A method for measuring the concentration of a compound represented by the following formula (I) in a test sample:

(Wherein, R ¹ , R ² and R ³ independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)
Prepare a diamond electrode having a conductive diamond, and a counter electrode,
The diamond electrode and the counter electrode are brought into contact with a test sample,
A voltage at which an oxidation reaction occurs on the diamond electrode is applied between the diamond electrode and the counter electrode, and a current value under the voltage is measured,
A method comprising calculating the concentration of the compound represented by the formula (I) in the test sample from the obtained current value. 2. The method according to claim 1, wherein the diamond electrode has a diamond thin film made conductive by the incorporation of a Group 3 or 5 element. The method according to claim 1, wherein the diamond electrode has a diamond thin film that has been made conductive by the incorporation of one or more elements selected from the group consisting of boron, nitrogen, and phosphorus. The method according to any one of claims 1 to 3, wherein the compound represented by the formula (I) is one or more selected from the group consisting of caffeine, theophylline, theobromine, and xanthine. The step of calculating the concentration of the compound represented by the formula (I) in the test sample from the obtained current value includes the step of calculating the concentration of the compound represented by the formula (I) prepared in advance and the current value. The method according to claim 1, wherein the method is performed by comparing a calibration curve with an obtained current value. The method according to any one of claims 1 to 5, wherein when the measurement object is theobromine or xanthine, the measurement is performed with the pH of the test sample being 5.5 or less. The method according to claim 1, wherein a voltage at which an oxidation reaction occurs on the diamond electrode between the diamond electrode and the counter electrode is a voltage that gives a peak current. A reference electrode is brought into contact with a test sample, and between the diamond electrode and the counter electrode, further comprising controlling an absolute value of a voltage at which an oxidation reaction occurs on the diamond electrode. The method according to any one of claims 7 to 10. A sensor for measuring the concentration of a compound of formula (I) as defined in claim 1, comprising a diamond electrode having conductive diamond. The sensor according to claim 1, wherein the diamond electrode has a diamond thin film made conductive by mixing boron. The sensor according to claim 9 or 10, wherein the compound represented by the formula (I) is at least one selected from the group consisting of caffeine, theophylline, theobromine, and xanthine. An apparatus for measuring the concentration of a compound of formula (I) as defined in claim 1 in a test sample,
A diamond electrode having conductive diamond,
A counter electrode,
The diamond electrode, means for contacting the counter electrode with the test sample,
Means for applying a voltage at which an oxidation reaction occurs on the diamond electrode, between the diamond electrode and the counter electrode,
Means for measuring a current value under the applied voltage;
Means for calculating the concentration of the compound represented by the formula (I) in the test sample from the obtained current value. Further comprising a reference electrode,
Means for bringing the reference electrode into contact with the test sample;
The apparatus according to claim 12, further comprising means for controlling an absolute value of a voltage at which an oxidation reaction occurs on the diamond electrode, between the diamond electrode and the counter electrode. A compound represented by the following formula (I) in a test sample:

(Wherein, R ¹ , R ² and R ³ independently represent a hydrogen atom or an alkyl group having 1 to 6 carbon atoms)
Use of a diamond electrode having conductive diamond for measuring the concentration of methane.

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