JP2009069085A - Phenylalanine sensor and measurement method - Google Patents

Phenylalanine sensor and measurement method Download PDF

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JP2009069085A
JP2009069085A JP2007240318A JP2007240318A JP2009069085A JP 2009069085 A JP2009069085 A JP 2009069085A JP 2007240318 A JP2007240318 A JP 2007240318A JP 2007240318 A JP2007240318 A JP 2007240318A JP 2009069085 A JP2009069085 A JP 2009069085A
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phenylalanine
electrode
dehydrogenase
diaphorase
electron mediator
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JP4702341B2 (en
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Hiroaki Shinohara
寛明 篠原
Yasuhisa Asano
泰久 浅野
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Toyama University
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Abstract

<P>PROBLEM TO BE SOLVED: To provide a phenylalanine sensor with which a limitation concentration of phenylalanine to be detected is low and measurement for analysis can be performed in a short time and repeated usage is possible, and to provide a phenylalanine measurement method using the same. <P>SOLUTION: The phenylalanine sensor includes a working electrode with phenylalanine dehydrogenase and diaphorase being fixed, an electrode system consisting of counter-electrodes, and a reagent solution containing coenzyme and an electronic mediator. <P>COPYRIGHT: (C)2009,JPO&INPIT

Description

本発明は、血液、尿、唾液等の生体試料中のフェニルアラニン濃度、あるいは食品試料中のフェニルアラニン濃度を測定するバイオセンサ及びバイオセンサを用いたフェニルアラニンの測定方法に関する。   The present invention relates to a biosensor for measuring phenylalanine concentration in a biological sample such as blood, urine, saliva or the like, or a phenylalanine concentration in a food sample, and a method for measuring phenylalanine using the biosensor.

各種基質に対する脱水素酵素反応を用いて、その酵素反応により生じる補酵素の還元型補酵素（ＮＡＤＨ，ＮＡＤＰＨ）を直接的に分光学的あるいは電気化学的に定量すること、さらには、この還元型補酵素により生成する還元型電子メディエーターや還元型色素を電気化学的あるいは比色法や蛍光法といった分光学的方法により検出するバイオセンサが開発されている。   Using a dehydrogenase reaction on various substrates, the reduced coenzyme (NADH, NADPH) of the coenzyme produced by the enzyme reaction is directly spectroscopically or electrochemically quantified. Biosensors have been developed that detect reduced electron mediators and reduced dyes produced by coenzymes by electrochemical or spectroscopic methods such as colorimetric methods and fluorescent methods.

フェニルアラニンは必須アミノ酸の１つであり、生体試料中のフェニルアラニンの濃度測定はフェニルケトン尿症（ＰＫＵ）の診断に有用であることから、フェニルアラニンを基質とした酵素反応を用いたバイオセンサも開発されている。
例えば、非特許文献１には、フェニルアラニンデヒドロゲナーゼ（フェニルアラニン脱水素酵素）、ＮＡＤ＋、ジアホラーゼ、さらにヨードニトロテトラゾリウムの混合液中に、フェニルアラニンを加えると生じたＮＡＤＨによりテトラゾリウムがホルマザンとなり、その４９２ｎｍの３０分間の吸光度増加から、血漿中のフェニルアラニンを３０〜１２００μＭの範囲で定量検出することを記載する。
非特許文献２には、スライドグラス上に組み換えフェニルアラニンデヒドロゲナーゼを固定化し、その上に形成した微小ウェル中に、ジアホラーゼ、ＮＡＤ＋、レサズリン、血液サンプルから抽出したフェニルアラニンの混合液を添加し、１時間後、酵素反応で生じた還元型レサズリンの蛍光を蛍光スキャナーで計測することにより、２４〜７８０μＭの濃度範囲でフェニルアラニンの検出ができることを記載する。
また、非特許文献３には、フェニルアラニンデヒドロゲナーゼ、ＮＡＤ＋、電子メディエーターとしての３，４−ＤＨＢをカーボンペーストの中に同時に包括固定した電極を用い、試薬を一切添加せず、ヒト尿中のフェニルアラニンの検出を酸化電流の増加より行い、０．５〜８０ｍＭの検出を実現できることを示している。
非特許文献４には、フェニルアラニンデヒドロゲナーゼを架橋法で共有結合固定化したアセチルセルロース膜をカーボン電極の先端に装着して、ＮＡＤ＋を含む緩衝液中で＋７００ｍＶに電位設定して、フェニルアラニン添加時に酵素反応で生じるＮＡＤＨの電気化学酸化電流を測定することにより、２５μＭ〜９ｍＭのフェニルアラニンの検出方法を記載する。
特許文献５には、試薬としてＬ−フェニルアラニン脱水素酵素と補酵素である酸化型ニコチンアミドアデニンジヌクレオチド（ＮＡＤ＋）もしくは酸化型ニコチンアミドアデニンヌクレオチドリン酸（ＮＡＤＰ＋）および電子メディエーターを、少なくとも作用極と対極からなる電極系と一体化することによって構成される旨が開示されている。 Since phenylalanine is one of the essential amino acids and the measurement of the concentration of phenylalanine in biological samples is useful for the diagnosis of phenylketonuria (PKU), biosensors using enzyme reactions using phenylalanine as a substrate have also been developed. ing.
For example, Non-Patent Document 1 discloses that tetrazolium is converted to formazan by NADH generated when phenylalanine is added to a mixed solution of phenylalanine dehydrogenase (phenylalanine dehydrogenase), NAD + , diaphorase, and iodonitrotetrazolium, and 492 nm of 30 It is described that the phenylalanine in plasma is quantitatively detected in the range of 30 to 1200 μM from the increase in absorbance per minute.
In Non-Patent Document 2, recombinant phenylalanine dehydrogenase is immobilized on a slide glass, and a mixture of diaphorase, NAD + , resazurin, and phenylalanine extracted from a blood sample is added to a microwell formed thereon, and 1 hour Subsequently, it is described that phenylalanine can be detected in a concentration range of 24 to 780 μM by measuring the fluorescence of reduced resazurin produced by the enzyme reaction with a fluorescence scanner.
Non-Patent Document 3 discloses that phenylalanine dehydrogenase, NAD + , and an electrode in which 3,4-DHB as an electron mediator is comprehensively immobilized in a carbon paste at the same time, without adding any reagent, phenylalanine in human urine. It is shown that the detection of 0.5 to 80 mM can be realized by performing the detection of from the increase of the oxidation current.
In Non-Patent Document 4, an acetylcellulose membrane in which phenylalanine dehydrogenase is covalently immobilized by a crosslinking method is attached to the tip of a carbon electrode, and the potential is set to +700 mV in a buffer solution containing NAD +. A method for detecting 25 μM to 9 mM phenylalanine is described by measuring the electrochemical oxidation current of NADH produced in the reaction.
In Patent Document 5, L-phenylalanine dehydrogenase and a coenzyme oxidized nicotinamide adenine dinucleotide (NAD + ) or oxidized nicotinamide adenine nucleotide phosphate (NADP + ) and an electron mediator are used as reagents. It is disclosed that it is configured by integrating with an electrode system composed of a pole and a counter electrode.

しかし、これらの発明の多くが、デヒドロゲナーゼ、補酵素（ＮＡＤ＋あるいはＮＡＤＰ＋）、電子メディエーターや発色色素をウェル中で均一な混合溶液として測定する、もしくは高分子膜担体や導電性微粉末材料中に吸着や包括固定化して測定するものであり、測定の後、酵素は使い捨てにするものがほとんどであった。
また、分光学的測定法では試料の処理方法が煩雑で且つ分析時間に３０分以上も要する問題があり、電流計測方法においてはフェニルアラニンの検出限界が１００μＭレベルと高かった。 However, many of these inventions measure dehydrogenases, coenzymes (NAD + or NADP + ), electron mediators and coloring dyes as uniform mixed solutions in wells, or in polymer membrane carriers and conductive fine powder materials. Most of the enzymes were disposable after the measurement.
In addition, the spectroscopic measurement method has a problem that the sample processing method is complicated and the analysis time takes 30 minutes or more. In the current measurement method, the phenylalanine detection limit is as high as 100 μM.

U.Wendel, W.Hummel, and U. Langenbeck, Monitoring of phenylketonuria: A colorimetric method for the determination of plasma phenylalanine using -phenylalanine dehydrogenase, Analytical Biochemistry, 180(1), 91-94(1989).U. Wendel, W. Hummel, and U. Langenbeck, Monitoring of phenylketonuria: A colorimetric method for the determination of plasma phenylalanine using -phenylalanine dehydrogenase, Analytical Biochemistry, 180 (1), 91-94 (1989). S.Tachibana, M.Suzuki and Y.Asano, Application of an enzyme chip to the microquantification of l-phenylalanine, Analytical Biochemistry, 359( 1), 72-78 (2006).S. Tachibana, M. Suzuki and Y. Asano, Application of an enzyme chip to the microquantification of l-phenylalanine, Analytical Biochemistry, 359 (1), 72-78 (2006). D.J.Weiss, M.Dorris, A Loh and L.Peterson, Dehydrogenase based reagentless biosensor for monitoring phenylketonuria, Biosensors and Bioelectronics, 22(11), 2436-2441(2007).D.J. Weiss, M. Dorris, A Loh and L. Peterson, Dehydrogenase based reagentless biosensor for monitoring phenylketonuria, Biosensors and Bioelectronics, 22 (11), 2436-2441 (2007). R.Villalonga, A.Fujii, H.Shinohara, S.Tachibana and Y.Asano, Covalent immobilization of phenylalanine dehydrogenase on cellulose membrane for biosensor construction, Sensors and Actuators B: Chemical, In Press, Accepted Manuscript, Available online 27 July 2007.R. Villalonga, A. Fujii, H. Shinohara, S. Tachibana and Y. Asano, Covalent immobilization of phenylalanine dehydrogenase on cellulose membrane for biosensor construction, Sensors and Actuators B: Chemical, In Press, Accepted Manuscript, Available online 27 July 2007 . 国際公開ＷＯ００／０４３７８パンフレットInternational Publication WO00 / 04378 Pamphlet

本発明は、フェニルアラニンの検出限界濃度が低く、短時間にて分析測定できるとともに繰り返し使用も可能なフェニルアラニンセンサ及びこれを用いたフェニルアラニンの測定方法の提供を目的とする。   An object of the present invention is to provide a phenylalanine sensor that has a low detection limit concentration of phenylalanine, can be analyzed and measured in a short time, and can be repeatedly used, and a method for measuring phenylalanine using the same.

本発明に係るフェニルアラニンセンサは、フェニルアラニン脱水素酵素とジアホラーゼとを固定化した作用極及び、対極からなる電極系と、補酵素及び電子メディエーターを含有する試薬液とを備えたことを特徴とする。
作用極となる電極上に、フェニルアラニン脱水素酵素とジアホラーゼとを固定化する方法はメルカプトカルボン酸の自己集積化膜等を用いた共有結合法が簡便で同時固定化できるので好ましい。 The phenylalanine sensor according to the present invention is characterized by comprising a working electrode on which phenylalanine dehydrogenase and diaphorase are immobilized, an electrode system comprising a counter electrode, and a reagent solution containing a coenzyme and an electron mediator.
The method of immobilizing phenylalanine dehydrogenase and diaphorase on the electrode serving as the working electrode is preferable because a covalent bond method using a self-assembled film of mercaptocarboxylic acid or the like is simple and can be simultaneously immobilized.

本発明においては、脱水素酵素とジアホラーゼの二種類の異なる酵素を同一電極上に共有結合法にて同時固定化した点に特徴があり、第１段階の脱水素酵素反応により生成するＮＡＤＨ又はＮＡＤＰＨが第２段階のジアホラーゼ等による酵素反応で即座に電子メディエーターの還元に使われることから、基質の検出性能が高く、脱水素酵素及び電子メディエーターの選定により各種基質のバイオセンサとして活用できるものであり、第１段階酵素反応として、グルタミン酸脱水素酵素、ロイシン脱水素酵素等をフェニルアラニン脱水素酵素と組み合せることでマルチバイオセンサも可能になる。   The present invention is characterized in that two different enzymes, dehydrogenase and diaphorase, are simultaneously immobilized on the same electrode by a covalent bond method, and NADH or NADPH produced by the first-stage dehydrogenase reaction. Is used for the reduction of electron mediators immediately in the second stage of enzyme reaction such as diaphorase, etc., so it has high substrate detection performance and can be used as a biosensor for various substrates by selecting dehydrogenase and electron mediator. As a first stage enzyme reaction, a multi-biosensor can be realized by combining glutamate dehydrogenase, leucine dehydrogenase and the like with phenylalanine dehydrogenase.

補酵素としては、酸化型ニコチンアミドアデニンジヌクレオチド（ＮＡＤ＋）又は酸化型ニコチンアミドアデニンヌクレオチドリン酸（ＮＡＤＰ＋）が好ましい。
フェニルアラニン基質とフェニルアラニン脱水素酵素との酵素反応により生じた還元型ニコチンアミドアデニンジヌクレオチド（ＮＡＤＨ）又は還元型ニコチンアミドアデニンヌクレオチドリン酸から電子メディエーターに電子移動し、このメディエーターが還元型の電子メディエーターになる。
還元型の電子メディエーターの存在下において電極系に所定の電圧を印加すると基質濃度に応じて酸化電流が応答電流として出力される。
従って、本発明において電子メディエーターはＮＡＤＨ又はＮＡＤＰＨにより電気化学的に還元され、電極において酸化される可逆的反応物質であれば特に限定されない。
電子メディエーターとしてはフェロセン誘導体、キノン類、フェリシアン化カリウム、オスミウム錯体、ルテニウム錯体、フェノチアジン誘導体、フェナジンメトサルフェート誘導体、ｐ−アミノフェノール、メルドーラブルー、２，６−ジクロロフェノールインドフェノールのいずれかが良く、フェロセン誘導体の代表例にはフェロセンメタノールがあり、キノン類の代表例にはベンゾキノンがある。
例えば、フェロセンメタノールは下記化学式のように酸化、還元反応を示す。
The coenzyme is preferably oxidized nicotinamide adenine dinucleotide (NAD + ) or oxidized nicotinamide adenine nucleotide phosphate (NADP + ).
Electron transfer from reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine nucleotide phosphate generated by an enzymatic reaction between a phenylalanine substrate and phenylalanine dehydrogenase to an electron mediator, which mediator becomes a reduced electron mediator Become.
When a predetermined voltage is applied to the electrode system in the presence of a reduced electron mediator, an oxidation current is output as a response current according to the substrate concentration.
Therefore, in the present invention, the electron mediator is not particularly limited as long as it is a reversible reactant that is electrochemically reduced by NADH or NADPH and oxidized at the electrode.
As an electron mediator, any one of ferrocene derivatives, quinones, potassium ferricyanide, osmium complexes, ruthenium complexes, phenothiazine derivatives, phenazine methosulfate derivatives, p-aminophenol, meldola blue, 2,6-dichlorophenol indophenol, A typical example of a ferrocene derivative is ferrocenemethanol, and a typical example of quinones is benzoquinone.
For example, ferrocenemethanol exhibits oxidation and reduction reactions as shown in the following chemical formula.

このようなバイオセンサを用いた生体中又は食品中に含まれるフェニルアラニンの測定方法であって、フェニルアラニン脱水素酵素とジアホラーゼとを固定化した作用極及び、対極からなる電極系を用いて、フェニルアラニン脱水素酵素を触媒にしてフェニルアラニン基質からＮＡＤ＋又はＮＡＤＰ＋に電子移動反応をさせ、生じたＮＡＤＨ又はＮＡＤＰＨからジアホラーゼを触媒として電子メディエーターに電子移動反応させて、所定の印加電圧下における還元型電子メディエーターの酸化応答電流値を測定することを特徴とする。 A method for measuring phenylalanine contained in living organisms or foods using such a biosensor, wherein phenylalanine dehydration is performed using an electrode system comprising a working electrode and a counter electrode in which phenylalanine dehydrogenase and diaphorase are immobilized. An electron transfer reaction from a phenylalanine substrate to NAD + or NADP + using an elementary enzyme as a catalyst, and an electron transfer reaction from the resulting NADH or NADPH to an electron mediator using diaphorase as a catalyst, a reduced electron mediator under a predetermined applied voltage The oxidation response current value of is measured.

本発明は、電極上においてフェニルアラニン脱水素酵素とジアホラーゼの２種類の酵素を相互に近傍に固定化したことにより、第１段階のフェニルアラニン脱水素酵素の酵素反応で生じたＮＡＤＨ又はＮＡＤＰＨが第２段階のジアホラーゼの酵素反応により電子メディエーターを還元するのでフェニルアラニン脱水素酵素の見かけの平衡定数Ｋｍ値が７５μＭから４０μＭに減少し、より低濃度でのフェニルアラニンの検出が可能になる。
また、これにより容易に血中のフェニルアラニンを測定できる。
なお、正常人では血中フェニルアラニンは２０〜１１０μＭであるがフェニルケトン尿症患者は約１２０μＭ以上といわれている。 In the present invention, two types of enzymes, phenylalanine dehydrogenase and diaphorase, are immobilized in the vicinity of each other on the electrode, so that NADH or NADPH generated by the enzyme reaction of the first stage phenylalanine dehydrogenase is the second stage. Since the electron mediator is reduced by the enzyme reaction of diaphorase, the apparent equilibrium constant Km value of phenylalanine dehydrogenase is reduced from 75 μM to 40 μM, and phenylalanine can be detected at a lower concentration.
This also makes it possible to easily measure phenylalanine in the blood.
In normal individuals, blood phenylalanine is 20 to 110 μM, but phenylketonuria patients are said to be about 120 μM or more.

本発明に係るフェニルアラニンセンサの構造及び測定方法を以下説明するが、２種類以上の酵素を同一電極上に固定化するものである限りにおいて広く展開でき、本実施例に限定されるものではない。   The structure and measurement method of the phenylalanine sensor according to the present invention will be described below, but it can be widely developed as long as two or more kinds of enzymes are immobilized on the same electrode, and is not limited to this example.

（電極への酵素の固定）
直径１．６ｍｍの金ディスク電極を、２〜２０ｍＭになるようメルカプトカルボン酸のスクシンイミドエステルを溶かしたジメチルスルホキシド（ＤＭＳＯ）溶液に１〜２時間浸漬し、その自己組織化膜を形成した。
次いで約５％のポリアリルアミン（ＰＡＡ）を溶かしたリン酸緩衝液（ｐＨ７．０）に３０分〜１晩浸漬してＰＡＡをこの自己組織化膜に結合させた。
さらに５％グルタルアルデヒド水溶液で３０分から２時間処理した後、２ｍｇ／ｍＬのフェニルアラニン脱水素酵素（ＰｈｅＤＨ）と２ｍｇ／ｍＬのジアホラーゼ（ＤＩ）の等量混合液に３０分〜一晩浸けて電極にこれらの酵素を同時に修飾した。
その構造を図１に模式的に示す。
なお、自己組織化膜を形成する際のメルカプトカルボン酸スクシンイミドエステルとしては、メルカプトプロピオン酸やメルカプトウンデカン酸等のスクシンイミドエステル体が利用可能であるが、鎖長が適当に長いメルカプトカルボン酸を用いて自己組織化膜を作製したほうが、その疎水性分子薄膜のバリヤ効果により、後のフェニルアラニンセンサの出力測定の際に血中などに含まれるアスコルビン酸、尿酸などの負電荷を持ち電気化学反応性の夾雑物質による妨害を受けにくくできることを新たに明らかにしており、現状ではメルカプトウンデカン酸スクシンイミドエステルの利用が好ましい。
また、さらに血中や食品サンプル中に共存するアスコルビン酸などの電気化学反応性物質の妨害を抑制したいときには、市販の陰イオン性ポリマーである商品名：Ｎａｆｉｏｎ（ナフィオン）（登録商標）の溶液を酵素固定化電極表面に滴下乾燥して、修飾電極を仕上げると有効である。
こうして作製した酵素固定化電極は、洗浄後使用時までは、酵素の活性を最適に保つグリシン‐ＫＯＨ緩衝液（ｐＨ９．５）中に浸漬して、冷蔵保存した。 (Immobilization of enzyme to electrode)
A gold disk electrode having a diameter of 1.6 mm was immersed in a dimethyl sulfoxide (DMSO) solution in which a succinimide ester of mercaptocarboxylic acid was dissolved to 2 to 20 mM for 1 to 2 hours to form a self-assembled film.
Subsequently, it was immersed in a phosphate buffer solution (pH 7.0) containing about 5% polyallylamine (PAA) for 30 minutes to overnight to bind PAA to the self-assembled film.
Further, after treatment with 5% glutaraldehyde aqueous solution for 30 minutes to 2 hours, immerse in an equal volume mixture of 2 mg / mL phenylalanine dehydrogenase (PheDH) and 2 mg / mL diaphorase (DI) for 30 minutes to overnight. These enzymes were modified simultaneously.
The structure is schematically shown in FIG.
In addition, as the mercaptocarboxylic acid succinimide ester for forming the self-assembled film, succinimide ester bodies such as mercaptopropionic acid and mercaptoundecanoic acid can be used, but a mercaptocarboxylic acid having an appropriately long chain length is used. When the self-assembled film is produced, due to the barrier effect of the hydrophobic molecular thin film, the negative reaction such as ascorbic acid and uric acid contained in the blood, etc. in the subsequent measurement of the output of the phenylalanine sensor, has electrochemical reactivity. It has been newly clarified that it can be hardly disturbed by contaminants, and at present, the use of mercaptoundecanoic acid succinimide ester is preferable.
In addition, when it is desired to suppress interference with electrochemically reactive substances such as ascorbic acid coexisting in blood or food samples, a solution of a commercial name anionic polymer: Nafion (registered trademark) is used. It is effective to finish the modified electrode by dripping and drying on the surface of the enzyme-immobilized electrode.
The enzyme-immobilized electrode thus prepared was immersed in a glycine-KOH buffer (pH 9.5) that keeps the enzyme activity optimally and stored refrigerated until use after washing.

（フェニルアラニンセンサの応答測定）
フェニルアラニンセンサの応答は、Ｇｌｙ−ＫＯＨ緩衝液（ｐＨ９．５）に、ＮＡＤ＋を１．５ｍＭ、フェロセンメタノールを０．１ ｍＭ溶かした測定液中に酵素固定化電極をセットして測定した。
そこにフェニルアラニン溶液を順次添加し、０〜５００ｍＶ（ｖｓ．Ａｇ／ＡｇＣｌ）の電位範囲でサイクリックボルタンメトリー（ＣＶ）を用いて触媒電流の増加を調べた。
ＣＶの走査速度は５ｍＶ／ｓで行った（１サイクルの測定で２００秒）。
対極にはＰｔ電極を、参照電極はＡｇ／ＡｇＣｌ電極を使用した。
その構造を図２に模式的に示す。 (Measurement of phenylalanine sensor response)
The response of the phenylalanine sensor was measured by setting the enzyme-immobilized electrode in a measurement solution in which 1.5 mM of NAD + and 0.1 mM of ferrocenemethanol were dissolved in Gly-KOH buffer (pH 9.5).
A phenylalanine solution was sequentially added thereto, and an increase in the catalyst current was examined using cyclic voltammetry (CV) in a potential range of 0 to 500 mV (vs. Ag / AgCl).
The CV scanning speed was 5 mV / s (200 seconds for one cycle measurement).
A Pt electrode was used as the counter electrode, and an Ag / AgCl electrode was used as the reference electrode.
The structure is schematically shown in FIG.

本発明に係るバイオセンサの計測原理を図３に示す。
作製したフェニルアラニンセンサは、図４に示すように、フェニルアラニン濃度の増加とともにＣＶにおける触媒電流の増加が観測された。
また、２９０ｍＶでの触媒電流値とフェニルアラニン濃度の関係を図５に示した。
この結果から本センサのフェニルアラニン定量可能範囲は、２０μＭ〜１６０μＭであった。
また、比色法（均一系）で測定したＰｈｅＤＨフェニルアラニンに対するＫｍ値は７５μＭであったが、一方、本センサのフェニルアラニンに対する見かけのＫｍ値は４２μＭであり、高感度検出に有利であった。 The measurement principle of the biosensor according to the present invention is shown in FIG.
In the produced phenylalanine sensor, as shown in FIG. 4, an increase in the catalyst current at CV was observed with an increase in the phenylalanine concentration.
FIG. 5 shows the relationship between the catalyst current value at 290 mV and the phenylalanine concentration.
From this result, the phenylalanine quantifiable range of this sensor was 20 μM to 160 μM.
Further, the Km value for PheDH phenylalanine measured by a colorimetric method (homogeneous system) was 75 μM, whereas the apparent Km value for phenylalanine of this sensor was 42 μM, which was advantageous for high-sensitivity detection.

図６にフェニルアラニンセンサとフェニルアラニン脱水素酵素の基質特異性の比較を示す。
本センサは比色法で測定したフェニルアラニン脱水素酵素自体とほぼ同様の基質特異性を示した。
フェニルアラニンに対する選択性が高いことが明らかになった。
本発明は血液中や食品中のフェニルアラニンの定量によりフェニルケトン尿症等の先天性代謝異常症早期診断や食品中の有用成分の検出に適用できる。
また、フェニルアラニン脱水素酵素とジアホラーゼを電極表面に直接共有結合固定化してあるため、電極を洗浄し、補酵素と電子メディエーターを加えた緩衝液中に浸漬すれば繰り返し使用できるので経済的である。 FIG. 6 shows a comparison of the substrate specificities of the phenylalanine sensor and phenylalanine dehydrogenase.
This sensor showed almost the same substrate specificity as the phenylalanine dehydrogenase itself measured by the colorimetric method.
It was revealed that the selectivity to phenylalanine is high.
The present invention can be applied to early diagnosis of inborn errors of metabolism such as phenylketonuria and detection of useful components in foods by quantifying phenylalanine in blood and foods.
Moreover, since phenylalanine dehydrogenase and diaphorase are directly covalently immobilized on the electrode surface, it can be used repeatedly if the electrode is washed and immersed in a buffer solution containing a coenzyme and an electron mediator.

電極構造を示す。An electrode structure is shown. 計測システムを示す。The measurement system is shown. センサの計測原理を示す。The measurement principle of the sensor is shown. フェニルアラニン濃度に依存したセンサのサイクリックボルタモグラムの変化を示す。The change of the cyclic voltammogram of the sensor depending on the phenylalanine concentration is shown. ２９０ｍＶにおける触媒電流のフェニルアラニン濃度依存性を示す。The dependence of the catalyst current at 290 mV on the phenylalanine concentration is shown. フェニルアラニンセンサとフェニルアラニン脱水素酵素の基質特異性の比較を示す。The comparison of the substrate specificity of a phenylalanine sensor and phenylalanine dehydrogenase is shown.

Claims (5)

フェニルアラニン脱水素酵素とジアホラーゼとを固定化した作用極及び、対極からなる電極系と、補酵素及び電子メディエーターを含有する試薬液とを備えたことを特徴とするフェニルアラニンセンサ。   A phenylalanine sensor comprising an electrode system composed of a working electrode and a counter electrode on which phenylalanine dehydrogenase and diaphorase are immobilized, and a reagent solution containing a coenzyme and an electron mediator. 作用極は、電極上にフェニルアラニン脱水素酵素とジアホラーゼとを共有結合法により同時固定化したものであることを特徴とする請求項１記載のフェニルアラニンセンサ。   2. The phenylalanine sensor according to claim 1, wherein the working electrode is obtained by simultaneously immobilizing phenylalanine dehydrogenase and diaphorase on the electrode by a covalent bond method. 補酵素は、ＮＡＤＨ又はＮＡＤＰＨであることを特徴とする請求項１又は２記載のフェニルアラニンセンサ。   The phenylalanine sensor according to claim 1 or 2, wherein the coenzyme is NADH or NADPH. 電子メディエーターは、フェロセン誘導体、キノン類、フェリシアン化カリウム、オスミウム錯体、ルテニウム錯体、フェノチアジン誘導体、フェナジンメトサルフェート誘導体、ｐ−アミノフェノール、メルドーラブルー、２，６−ジクロロフェノールインドフェノールのいずれかであることを特徴とする請求項１〜３のいずれかに記載のフェニルアラニンセンサ。   The electron mediator is one of ferrocene derivatives, quinones, potassium ferricyanide, osmium complexes, ruthenium complexes, phenothiazine derivatives, phenazine methosulfate derivatives, p-aminophenol, meldora blue, and 2,6-dichlorophenol indophenol. The phenylalanine sensor according to any one of claims 1 to 3. 生体中又は食品中に含まれるフェニルアラニンの測定方法であって、
フェニルアラニン脱水素酵素とジアホラーゼとを固定化した作用極及び、対極からなる電極系を用いて、
フェニルアラニン脱水素酵素を触媒にしてフェニルアラニン基質からＮＡＤ＋又はＮＡＤＰ＋に電子移動反応をさせ、
生じたＮＡＤＨ又はＮＡＤＰＨからジアホラーゼを触媒として電子メディエーターに電子移動反応させて、所定の印加電圧下における還元型電子メディエーターの酸化応答電流値を測定することを特徴とするフェニルアラニンの測定方法。 A method for measuring phenylalanine contained in a living body or food,
Using an electrode system consisting of a working electrode and a counter electrode in which phenylalanine dehydrogenase and diaphorase are immobilized,
Electron transfer reaction from phenylalanine substrate to NAD + or NADP + using phenylalanine dehydrogenase as a catalyst,
A method for measuring phenylalanine, which comprises subjecting the generated NADH or NADPH to an electron transfer reaction to an electron mediator using diaphorase as a catalyst and measuring an oxidation response current value of a reduced electron mediator under a predetermined applied voltage.
JP2007240318A 2007-09-17 2007-09-17 Phenylalanine sensor and phenylalanine measuring method Expired - Fee Related JP4702341B2 (en)

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