JP3679121B2 - 皮下グルコース電極 - Google Patents
皮下グルコース電極 Download PDFInfo
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Description
この発明は、国立予防衛生研究所(DK42015)の部分的支援を受けている。従って、合衆国政府もこの発明に何らかの権利を有すると考える。
発明の分野
本発明は、生体内(in vivo)酵素バイオセンサに関し、特に、ワンポイントキャリブレーション(one-point calibration)、即ち1点較正でグルコースの皮下測定を行うミニチュアグルコースセンサに関する。
背景
糖尿病、特に不安定型糖尿病において頻繁或は継続的にグルコースを観測若しくは監視する必要性に応えるべく、広範にわたる生体内グルコース電極の研究がなされてきた。これらの電極に求められる特性には、安全性、診断精度及び信頼性、生体内再較正(in vivo recalibration)可能であること、少なくとも8時間の病院シフトの間の安定性、小サイズであること、挿脱容易性及び臨機中断を許容する迅速な応答性が含まれる。生体内再較正は、体液、例えば血液の単一サンプルを抽出してそのグルコース濃度を測定することに基づいて行わなければならない。これを“ワンポイントキャリブレーション(1点較正)”と呼ぶ。
安全性に対する鍵は、浸出性成分が存在しないこと、生体適合性及び潜在的に有害な外部物の体内への進入を最悪の場合でも大事に至らない量に制限することである。診断精度は、読み値の正確さが最悪であっても、これに基づく診断が正しいものでなければならない。医師がセンサの読み値に基づいて患者を診断する場合、センサが正しく機能しているかどうかの迅速な確認及び周期的な生体内再較正が可能であることが必要不可欠である。ゼログルコース濃度でゼロの信号に基づくとともに、時間における1点で血中グルコース濃度を測定するこのワンポイントキャリブレーション(1点較正)は、その信号ともども、必要不可欠であるが、これまでよく分らないものであった。感度は、必要とされる生体内再較正の頻度に対して過剰にならないように十分に安定したものでなければならない。センサは、患者の不快感を最小にし、且つ、組織の損傷を最小にして挿脱できるように十分に小さくしなければならない。センサは、皮下式で、診察室において患者或は職員によって挿脱できるのが望ましい。最後に、応答時間は、必要に応じて適時に矯正手段をとれるよう十分に速くなければならない。
これらの要望の幾つかに応えて、針型及び他の皮下電流滴定(amperometric)センサが検討された。これらのセンサの多くは、グルコースと酸素とのグルコースオキシダーゼ(GOX)触媒反応により発生するH2O2を電子酸化させるため白金イリジウム又はプラチナブラック(白金黒)を使用した。これらのセンサにおいて、GOXは、しばしばアルブミン及びグルタルアルデヒドと架橋結合することにより、過剰となり、固定化した。電子酸化性干渉物を除去するため、酢酸セルロースの膜及びNafion(商標)を含有するスルフォン化ポリマーが使用された。最も一般的な電子酸化性干渉物であるアスコルバート、尿酸塩及びアセトアミノフェンの除去に特別な注意が払われた。また、該干渉物に対処するため、二電極示差測定が使用され、一方の電極はグルコースと電子酸化性干渉物に対して感応し、他方の電極は干渉物のみに感応するものである。本発明にも適用できるもう1つの干渉物問題克服法は、干渉物の前酸化(preoxidation)を伴うものである。別の方法は、化学変化を介して検出層中のポリマーのレドックス(酸化還元)電位(redox potentials)をより低い電位に変換することを伴うものである。ポリマーのレドックス電位が標準カロメル電極(standard calomel electrode=SCE)に対して約-0.15Vと+0.15Vの間の範囲にあり、電極が生体内動作時に約-0.10Vと+0.25Vの間で均衡するとき、アスコルバート、尿酸塩及びアセトアミノフェンなどの干渉物の電子酸化の速度は、生理学的濃度範囲を通してグルコースの速度に比べて非常に遅い。従って、干渉物の電子酸化による電流もグルコースの場合と比べて小さい。
電極の生体適合性を高めるため、親水性ポリウレタン、ポリ(ビニルアルコール)及びポリHEMA膜が使用されてきた。
幾人かの研究者は、GOXを基盤とするグルコースセンサを生体内試験し、ラット、ウサギ、犬、豚、羊及び人間について満足できる結果を得た。これらの研究は、皮下組織が受容できるグルコース検知場所であることを立証した。血管内グルコース濃度と皮下グルコース濃度間に良好な相関関係が観測された。また、研究者らは、生体内センサ較正(検定)の必要性を示した。生体内グルコース観測にたいするもう1つのアプローチは、皮下ミクロ透析(microdialysis)と電気化学的検知とを組合わせて行われた。線形応答範囲を制御及び調整するため、電極は、通常グルコース移動制限膜を介して、グルコースの拡散を制限するようなされた。
信号のO2分圧依存性を減少させる拡散媒介物質がセンサから浸出する。このような浸出は、不要化学物質を体内に進入させ、特に小さなセンサにおける感度を低下させる。マイクロセンサ(microsensor)においては、媒介物質の外方拡散が放射状になり、感度の低下は急速である。この問題は“導通接続された(wired)”酵素電極、つまり電子伝導レドックスヒドロゲル(electron-conducting redox hydrogels)(wires=導線)を介して酵素を電極に接続することによって形成された電極において克服された。ブドウ糖酸化酵素(グルコースオキシダーゼ)は、母格に電子リレー可能[Os(bpy)2C1]+/2+レドックス(酸化還元)中心(redox centers)を有する高分子電解質で“導通接続”された。ヒドロゲルは、電極上で酵素とワイヤ(導線)を架橋結合することによって形成された。これらの電極は高い電流密度を有し、SCEに対し0.3Vの電位で作動された。電子酸化性干渉物は“導通接続された”酵素電極の第2の非導通接続過酸化水素発生層でペルオキシダーゼ触媒作用の前酸化(preoxidation)を介して除去された。
発明の概要
生体内一点較正を含む、上記の全要求に応える得る生体内グルコース観測用の小寸法(例えば0.29mm)で、凹部を備える非腐食金属(例えば金、白金、パラジウム)若しくはカーボン線電極が製造された。該電極は、絶縁された金製ワイヤから金をエッチングで取り除くことにより形成された凹部にアクティブ(活性)ポリマー(active polymer)層を付着若しくは堆積させることによって構成された。
アクティブポリマー層は、検知(検出)層、グルコースフラックス制限層、生体適合性層及び任意のペルオキシダーゼ系干渉物除去層を含み、凹部内で機械的損傷から保護される。(前述したように、低レドックス電位ポリマーを使用する際はペルオキシダーゼ系干渉除去層は必要とされない。)該凹部とそのポリマー層も、検知層に接触する該ワイヤ電極へのグルコースの移動を減少させた。
グルコースフラックスを制限することにより、診断に関係するグルコース濃度範囲に跨がる所望の線形応答範囲が得られた。本発明のバイオセンサは、例えば生体内の約2〜30μmのグルコース及び約0.5〜10μmのラクタートを正確に測定することができる。該センサは、浸出性成分を有さず、その4つの架橋結合ポリマー層はわずか約5μgの固定化材と、わずか数ナノグラムのポリマー結合のオスミウムを含有する。4つの層のうちの1層における干渉物の前酸化はセンサの生体内一点較正を可能とする。
【図面の簡単な説明】
図1は、本発明による電極の概略構成図である。
図2は、PVI5-Os(白抜き三角形)を使用して形成した電極でのグルコース電子酸化の電流密度とPVI3-Os(黒塗り三角形)を使用したときを比較して得られたデータを示すグラフ図である。
図3は、凹部の底で発生した電流の従属性を比較して得られたデータを示すグラフ図である。
図4は、検知層に発生した電流とポリマーレドックス中心を電子還元或は酸化するのに要する電荷との比の検知層の厚みに対する従属性を比較して得られたデータを示すグラフ図である。
図5は、厚さの異なる検知層と組成及び厚さの異なる拡散制限層とを有する電極によって発生する電流の変化を比較して得られたデータを示すグラフ図である。黒塗り円:厚さ4μmのPAL/PAZ(比は1:1)の拡散制限層でコーティングされたPVI5−Os(52%)、rGOX(35%)、PEGDGE(13%)製の厚さ7.5μmの検知層。白抜き円:厚さ5.0μmの検知層。黒塗り三角形:厚さ12.5μmの検知層、7μmのPAL/PAZ(比は1:2)の拡散制限層。白抜き三角形:厚さ7.5μmの検知層で、厚さ4.5μm、PAL/PAZ(比は1:2)の拡散制限層。
図6は、ラクタートとグルコースの不在及び存在時におけるアスコルバートの存在時に発生する電流の従属性を比較して得られたデータを示すグラフ図である。アスコルバート(A)、ラクタート(L)とグルコース(G)の濃度を示す。アルコルバートは電子酸化性干渉物である。ラクタート付加時に、ラクタートの電子酸化電流は抑制される一方、グルコースの電子酸化電流は抑制されない。
図7は、ラットを動物モデルに、電流密度とそれに対応する皮下に差込まれた本発明の電極を用いて測定した皮下グルコース濃度を示す。大きな黒塗り円はYSIアナライザーを使用して採集した血液サンプルで測定した血中グルコース濃度を示す。
図8は、図7の血中グルコース測定値の臨床上の関連性を分析したクラーク式臨床図表(Clarke-type clinical grid)である。
図9は、図7の24のグルコース分析結果が、どちらか一方の埋め込まれた電極の一点較正に使用した際に得られた有効な相関関係の全てを示すクラーク式臨床図表である。
図10は、冗長電極を介して1点較正の向上を試験したクラーク式臨床図表であり、その読み値は、埋め込まれた一対の電極による同時読み値間の全ての差に対して算出された標準偏差内であった。
発明の詳細な説明
本発明は、絶縁された非腐食電導金属(例えば金、白金、パラジウム)或はカーボン製のワイヤ(導線)を基調とする小径(例えば290μm)で、生体内1点較正を許容するO.D.皮下グルコースセンサを含む。図1に示すように、その構成は、小径(例えば250μm)の非腐食金属或はカーボン製のワイヤ2を電気的絶縁体4(例えばポリイミド)でコーティングし、エッチング又は金属或はカーボンの部分除去によって形成される凹部6に次のようなアクティブ(活性)ポリマー層を形成することを含む。アクティブポリマー層は、固定(不動)化され、“導通接続された(wired)”ブドウ糖酸化酵素(グルコースオキシダーゼ)層8と、例えばポリアリルアミン(PAL)をポリエチレンイミン(PAZ)と架橋結合させることにより形成された電気絶縁性グルコース拡散制限層10と、(例えば架橋結合したホースラディッシュペルオキシターゼ及びラクタートオキシダーゼ製の)任意なものとしての干渉除去層12と、(例えば光架橋結合(photo crosslinking)を許容するため被覆されたポリエチレン酸化物(PEO)製の)生体適合膜14とから成る。ワイヤ(導線)2の外径aは、好ましくは約0.25mm或はそれ以下であり、絶縁ワイヤの外径bは、好ましくは約0.3mm或はそれ以下である。絶縁された電極における凹部6は、周囲環境に露出する電極の先端部16から絶縁シース(sheath)におけるワイヤ2上の上端部18まで、約0.150mm以下、好ましくは約0.125mmの長さcだけ延びている。
電極は浸出性成分を有していない。ポリマーと酵素の合計量は好ましくは約5μgである。生理学的に関連する2〜20mMの濃度レンジを通してグルコース応答は線形に近いものである。電極は少なくとも約36時間はアスコルバート、尿酸塩及びアセトアミノフェノールに反応しない。これらの10〜90%応答時間は2mMグルコースで約90秒、20mMのグルコースで約30秒である。約30秒間の平衡状態の後、電極の感度は10mMグルコース中において37℃で72時間安定し、電流は平均から±5%以内で偏移する。測定される分析物の濃度がゼロのとき、電極は信号、例えば電流、電荷或は電位を実質的に全く出力しない。
血糖値を監視するラットに皮下埋設された2つの電極及びこれらの絶対非補正電流出力は血中グルコース濃度と比例していた。細静脈の血液グルコース濃度と胸部皮下領域のセンサの電流出力との間の相関関係及び同一ラットの肩甲骨間の相関関係の分析は、探査した箇所と器官とが極めて異なる場合であっても、生体内一点較正が有効であることを示した。この分析は、埋め込み冗長センサが価値あることをも示した。1点で較正(検定)したときの個々のセンサの読み値に基づいて臨床判定を行ったところ、94%が臨床的に正確である。冗長センサを使用するとともに、1つの標準偏差内にあった読み値対だけ採用することにより、臨床的に正しい決定の確率が99%に上昇した。
本書で略述した原理を用いて変更したバイオセンサを得るため、前述したバイオセンサの各種構成部分或は部材の代りに周知の材料を使用してもよいことは当業者の理解するところである。例えば、以下の代替が予想される。
ベース電極:
本発明によるセンサのベース電極は、例えば硝子質カーボン、グラファイト、白金、パラジウム或は金の非腐食金属若しくはカーボンワイヤで形成してもよい。金が好ましく、以下の本発明の実施例にも金が使用される。
絶縁体:
電導性金属或はカーボン製ワイヤは、アクティブポリマー部材を収容する凹部の周りに壁を形成する電気絶縁材料でコーティングされる。絶縁材料は、例えばポリウレタン、テフロン(フッソ化ポリマー)、ポリエチレンテレフタラート(PET、Dacron)或はポリイミドでもよい。絶縁材は、好ましくは正常な体液、例えば皮下組織と平衡状態のとき、5%以下の水を含有する生体適合ポリマーである。
凹部:
一般に、電極の先端の凹部は、長さcがおよそ20〜150μm、好ましくは約50〜125μmである。
エッチング方法:
本書で説明する電極の先端から金属をエッチングする方法は、シアン化合物の代りに、塩化物、臭化物或はヨウ化物を浴で使用してもよい。臭化物は、毒性が少なく、Au(CN)2 -、AuBr4 -のように水溶性陰イオンであるので、好ましい。従って、水性HBrにおいて、金属、例えば金は、以下のように電気分解で溶解する際、十分な酸化電圧を加えることによってエッチングできる。
Au+4HBr−→HAuBr4+3/2H2
導通接続酵素層:
検知酵素含有層において、グルコースオキシダーゼ(ブドウ糖酸化酵素)を他のレドックス酵素(redox enzymes)に代えて、他の臨床上関連する化合物を測定してもよい。例えば、ラクタートオキシダーゼを生体内のラクタート検出に使用してもよい。ラクタートの検出は器官が血液を介して酸素を十分に受けているか否かの判定に重要である。
実用的なレドックスポリマー(酸化還元樹脂)及び検知層の製造方法は、例えば米国特許第5,264,104号、第5,356,786号、第5,262,035号及び第5,320,725号(項目3、4、5、7、8)に記載されている。他のレドックスポリマーは、例えばポリ(1−ビニルイミダゾール)、ポリ(4−ビニルピリジン)、或は[Os(bpy)2Cl]+/2+、[Os(4、4′−ジメチルビピリジン)2Cl]+/2+、[Os(4、4′−ジメチルフェナントロリン)2Cl]+/2+、[Os(4、4′−ジメトキシフェナントロリン)2Cl]+/2+、[Os(4、4′−ジメトキシビピリジン)2Cl]+/2+と錯体を作るイミダゾールやピリジンからイミタゾール環までの錯体のポリ(アクリルアミド 共1−ビニル(co-1-vinyl)イミダゾール)等の共重合体を含む。イミダゾール環化合物が好ましい。これは、これらの錯体が比較的低いレドックス電位、例えばSCE電位のレドックス電位に近いからである。これらの低い電位において、干渉物の電子酸化速度と電流がこれにより発生する。
バリヤ層:
ポリマー製バリヤ層は電気的に絶縁しており、検知層へのグルコースの拡散を制限する。バリヤ層は、例えばポリアリルアミン(PAL)とポリエチレンイミン(PAZ)を架橋結合させることにより形成してもよい。これに代えて、PALの全体または一部を両性イオンポリマーに置き換えてもよい。両性イオンポリマーは、ポリ(ビニルピリジン)をブロモ酢酸で4級化合物化し、0.15MNaClを透析することにより、或はポリスルホン酸(polysulfonic acid)等のポリ陰イオン(polyanion)により得られる。
バリヤ層は、ポリ陰イオンポリマーを含有してもよい。この場合、アスコルバートと尿酸塩のような陰イオン干渉物の浸透の速度が低下する。この層もまた静電気の結合によってポリ陰イオンの保持力を強め、生体適合性層によって湿潤を改善するポリ陽イオン(polycation)を含有してもよい。
干渉物除去層:
上記したように、この層は、任意のものであり、PVI15−dmeOsのように低い電位を有するレドックスポリマーが使用されるときは不要である(オハラ他Analytical Chemistry、1994,64:2451-2457)。グルコースバイオセンサの操作電位をおよそ−0.10〜0.25Vとしたとき、アスコルバート、尿酸塩及びアセトアミノフェノンのような干渉物の電子酸化の速度は通常の濃度範囲でのグルコースの場合と比べて非常に遅い。
別個の干渉物除去層を使用するとき、事前に活性化できる、或はできない、ペルオキシダーゼ酵素を含有することが好ましい。そのような干渉物除去層は、例えば米国特許第5,356,786号(項目4)及び米国特許出願No.08/161,682に開示されており、後者は干渉物除去層バイオセンサの構造と機能を開示している。グルコースバイオセンサは、好ましくは、干渉物除去層でペルオキシダーゼと結合するラクタートオキシダーゼ(LOX)を含有している。しかし、ラクタート検知に使用するバイオセンサでは、グルコースオキシダーゼはペルオキシダーゼと共に使用される。同様に、干渉物除去層の酵素組成を特定の機能のために変えてもよい。
生体適合性層:
一般に、生体適合性層は、例えば生きている組織や血液のような生理学的環境と平衡するときに20重量%以上の水を含有するポリマー組成物などのヒドロゲルから成る。ポリ(エチレンオキシド)テトラアクリル酸塩のような架橋結合したポリ(エチレンオキシド)が1例である。ポリマー組成物は、毒性がなく、生体系に適合するものでなければならない。
多重層凹部付きバイオセンサの形成方法:
絶縁された非腐食金属又はカーボン製ワイヤは、上記のごとくエッチングされており、先端に凹部を具有してX−Y位置決め部材として作用するブロックに配置される。ワイヤはブロックを鉛直に横断し、例えば圧力をかけて所定位置に保持される。ワイヤ付きブロックは、それぞれ半円筒状の鉛直に走る多数の溝を有する部材で形成することができる。ワイヤは、これらの溝に配置され、ネジを用いて部材を組立ててブロックにする。例えば、ブロックは(30×30ワイヤ列に対して900)の等間隔に離隔する穴を有するアルミニウムで形成してもよく、各穴に1本のワイヤを設ける。ブロックは、絶縁ワイヤの凹部に液体を注入する固定マイクロノズルの下に配置する。
ブロック及びマイクロノズルの配置における精度の必要条件を緩和するため、ノズルを電気的に帯電させ、ワイヤをノズルとは反対の極性に帯電させるか、或は接地又は少なくともノズルとワイヤとの間に電位差が生じるように電位をもたせる。ノズルが帯電しているため、放出される微小落滴もノズルと同じ極性(正又は負)に帯電される。ノズル(例えば接地電位に対する)の電位が高くなればなるほど、放出される液滴の電荷(電位)も高くなる。コーティングされるワイヤの先端が接地電位とされる場合或は反対極性の電荷を有する場合には、液滴の吹出し口が鉛直になっていなくとも、つまりマイクロノズルがワイヤの先端上に正確に整列していなくとも、帯電した液滴は凹部へ誘導されて電極に付着する。
更に、ノズルの電位が(接地に対して)高くなればなるほど、放出される液滴の電気量も大きくなる。帯電が十分に高いとき、液滴は、その電荷による静電反発力(electrostatic repulsion)のため、2つ或はそれ以上の微細液滴に分裂する。従って、微細液滴は、電極と正確に整列したノズルから発していなくても、すべて凹部付き電極面に“ドリフト”(ドリフトとは、電界の支援によって移ることを意味する)し、該面上に集まる。
このコーティング方法は、凹部内のものに限らず、いかなる小型バイオセンサの形成にも有効である。
凹部付きバイオセンサの臨床上の用途:
本発明の凹付きバイオセンサは、十分な感度と安定度を有し、グルコース及びラクタートのような臨床上の関連化合物の測定用の極小皮下センサとして使用できる。電極は、約2〜30μMの範囲でグルコースを、約0.5〜10mMの範囲でラクタートを正確に測定する。埋め込まれたバイオセンサは、1つの機能として、例えば患者のグルコース濃度が過度に近い或は高いときにアラーム音を発する。埋め込まれた電極対を使用する際、アラームが誘発される次の3つのケースがある。低グルコース濃度の場合と、高グルコース濃度の場合と、2つのセンサの読み値対の間の差若しくはズレによって決定されるセンサの故障の場合である。アラームの誘発に十分な差異は、例えば10分以下でない規定期間にわたって持続する標準偏差の2又は3倍以上とすればよい。このようなシステムは、睡眠中の患者に有効であり、致命的な機能が継続監視される病院の緊急若しくは集中治療室でも有効であろう。
本発明のバイオセンサの他の機能は、糖尿病患者の血中グルコースレベルを正常に近い状態に維持することで患者を支援することである。多くの糖尿病患者は、低血糖による昏睡状態及び死の危険のため、通常より高い血中グルコース濃度を保持している。しかし、例えば約40%或はそれ以上の通常より高い血中グルコース濃度を保つと、腎臓障害は勿論、網膜症や失明を招く。連続的でないにせよ、頻繁にグルコース濃度を観測してグルコース濃度を最適レベルに近い状態に維持するため、皮下バイオセンサを使用することが望ましい。
皮下バイオセンサは、食後或はグルコース投与(例えばグルコース耐性試験)後の、グルコース濃度の上昇及び低下率の測定に使用できる。また、該センサは、グルコース濃度を規定範囲内に維持するため自動或は手動制御される帰還ループにも有効である。例えばインシュリンポンプと共に使用する際、センサのグルコース読み値が設定値を上回わると、ポンプから一定量のインシュリンが供給される。
これらの全適用例において、埋め込まれたセンサの読み値が正確であることを素速く確認できることが必要不可欠である。一点較正が有効であるときに限り、迅速確認及び素速い再較正が可能となる。一般に、センサの応答が関連濃度レンジを通して線形であっても、較正は、グルコース濃度が違うときに患者から採取した少なくとも2つの血液或は流体サンプルを必要とする。グルコース濃度が十分変化して2点較正による適正機能を実証するには通常数時間かかる。1点だけを用いて確認及び再較正する能力は、本発明の極めて望ましい特徴である。
皮下バイオセンサの臨床利用においては、冗長センサ(例えば少なくとも2個)が好ましい。このような冗長は、1時点での複数センサの読み値の差の増加、例えば2つの標準偏差を超える開きを認識することによって一方のセンサの故障を知らせることができる。冗長センサは、互いに近接して或は離れた場所に埋め込んでもよい。
バイオセンサは、比較的邪魔にならないように皮下組織に埋め込み、例えば回転や移動によって外れないような位置に埋め込むのが望ましい。また、温度に合せて読み値が補正されない(通常は補正される)場合、センサは体温、例えば37℃程度になる位置に差込むのが好ましく、好ましくは布で被覆される。手頃な場所としては、腹、内腿、腕が含まれる。
ここでは、グルコースを分析するため連続的な電流測定を行う場合を説明しているが、グルコース濃度を観測する電気的測定は、連続的でも間欠的でもよい。電流測定でも、電位測定でも、電荷測定であってもよい。測定中に実質的に変化しない電流或は電位が観測される安定状態測定であっても、所定時間内の電流若しくは電位の変化量が観測される動的(ダイナミック)測定であってもよい。これらの測定は、検知電極に加え、少なくとも1個の電極を必要とする。この第2電極は、皮膚上に配置しても、例えば皮下に埋設してもよい。電流を測定する場合、差込まれた検知電極と第2電極とを接続する回路にポテンシオスタットを設け、これをAg/AgCl電極のような基準電極とすれば有益である。電流を測定する場合、基準電極はカウンタ(計数)電極としても働く。計数電極は、白金、カーボン、パラジウム或は金製の別個の第3電極であってもよい。
検知電極を体内に差込むことに加え、体内からの流体、特に皮下領域からの流体を外部センサへ送ることもできる。この場合、その皮下領域にミクロ濾過(microfiltration)ファイバを設けて検知電極を含有するセル(電解槽)を横断する流体を排出容器に引き込むこむことが好ましい。この電解槽は、好ましくは、例えば計数電極として作用する基準電極などの第2電極を含む。この代わりに、基準及び計数電極を別個の電極としてもよい。電量測定は、2つの電極、検知電極及び計数電極のみを必要とする。体液流は間欠的又は連続的とされる。差込みミクロ濾過ファイバ以外に、ミクロ透析(microdialysis)ファイバを使用してもよく、好ましくはポンプと併用される。
バイオセンサの向上した安定性:
本発明によるバイオセンサの安定性及び寿命を向上させるため、生来的に比較的安定の酵素及びレドックスポリマーを使用すると有利である。しかし、酵素及びレドックスポリマーが、信号(電流)を発生させるグルコース電子酸化過程で劣化しても、埋設した電極の耐用年数を大きく延ばし、挿入後に要する再較正の頻度を大幅に下げることができる。
埋め込み電極の有効年数を延ばし、必要とされる再較正の頻度を減少させる単純な手段は、測定期間中だけバイアス、つまり電位をかけることによって電極を“オン”にし、次いで、付加した電圧をオフにする或は下げることを含み、もってより少ない電流が流れるようにする。グルコース濃度は急峻に変化しないので、5分おき或は10分おきに1回の測定を実行することで概ね十分である。
もう1つの手段は、十分な感度及び検知性の維持に相応して、可能な限り検知層へのグルコースフラックスを減少させることである。検知層へのグルコースフラックスの減少は電流を減少させる。従って、これによって電極が安定する、つまり感度の喪失が遅くなっても、フラックス依存電流は、過度に減少してはならない。通常、2mMのグルコース濃度で3〜5nAの電流で十分である。PAL/PAZ層のような1つ或はそれ以上のグルコースフラックス減少ポリマー層を用いてグルコースフラックスを引下げると、センサの寿命が延びる。
実験例
例1
電極作製
電極は、290μmの外径(O.D.)を有するポリアミド絶縁された250μm径の金ワイヤ(カリフォルニア州グローバー市のカリフォルニア・ファイン・ワイヤ社/California Fine Wire Co.製)で形成した。熱収縮可能チューブ(カリフォルニア州メンロパークのレイチェム/RaychemのThermofit(登録商標)、RNF 100 3/64”BKと1/16”BK)と2成分銀エポキシ(エポテックH2OE;マサチューセッツ州ビレリカのエポキシテック社/Epoxy Tech,Inc.製)を電極作製に用いた。
グルコース検知層は、遺伝子学処理されたグルコースオキシダーゼ(rGOX)(純度35%、カリフォルニア州エメリーヴィルのチロン社/Chiron Corp.製)を、イミダゾール(imidazoles)の一部を[Os(bpy)2Cl]+/2+に錯体化することによって形成したポリ(ビニルイミダゾール)(PVI)の誘導ポリマーと架橋結合することによって形成した。PVI−Osと呼ばれるレドックスポリマーは、先に公開されたプロトコル(Ohara et al.1993 Anal. Chem. 65:24)に従って合成された。ポリ(エチレングリコール)ジグリシジルエーテル(poly(ethylene glycol)diglycidyl ether)400(PEDGE、ペンシルバニア州ワリントンのポリサイエンス/Polysciences製)は架橋結合剤として使用した。
検知層と干渉物除去層との間のバリア層は、多官能アジリジン(Polyfunctional aziridine=PAZ)(XAMA−7、バージニア州ポーツマスのバージニア・ケミカルズ/Virginia Chemicals製)と架橋結合したポリアリルアミン(PAL、Polysciences製)で形成した。
干渉物除去層は、ホースラディッシュペルオキシダーゼ(HRP)タイプVI(カタログNo.P−8375、310U/mg、本書でHRP−VIと表示、ミズーリ州セントルイスのSigma製)及び免疫学検定用のHRP(No.814407、min1000U/mg、本書ではHRP−BMと表示、インディアナ州インディアナポリスのブーリンガー・マンハイム/Boehringer-Mannheim製)を、ペディオコッカス社(Pediococcus sp.)製ラクタートオキシダーゼ(カタログNo.1361、40U/mg、LOXと表示、マサチューセッツ州ケンブリッジのゲンザイム/Genzyme製)及び組み替え型微生物源(recombinant microbial source)(カタログNo.1381、rLOXと表示、ゲンザイム製)と相互固定して作製した。相互固定は、1992年、Anal. Chem. 64:2889〜2896のMaidan and Hellerに記載された方法に従い、過ヨウ素酸ナトリウム(sodium periodate)(カタログNo.S−1147、シグマ社製)を用いて行なわれた。
生体適合性層は、10%水性ポリ(エチレンオキシド)テトラアクリレート(PEO−TA)で形成した。光架橋結合可能ポリマーを形成するため、塩化アクリロイルとの反応によりPEOをアクリル化した。該18,500g/molのPEO(ポリサイエンス製)は、2つのα、ωヒドロキシ終止9000g/molPEO単位を結合したビスフェノールAビスエポキシド(bisphenol A bisepoxide)上の2つの水酸基によるテトラヒドロキシル化した化合物である。2〜5モル超過の塩化アクリロイル(ウィスコンシン州ミルウォーキーのアルドリッチ/Aldrich製)はポリマー(ベンゼンにおいて10%w/vPEO)をアクリル化するために使用された。トリエチルアミン(triethylamine)(ニューヨク州パリのマリンクロット/Mallinkrodt製)は、塩化アクリロイルと等モルのプロトン(陽子)受容体(アクセプタ)として使用される。
使用される他の化学物質は、シグマ社より全て入手可能な牛の血清アルブミン(BSA)分画V(カタログNo.A−2153)、BSA、アスコルビン酸、尿酸、4−アセタミノフェノール、L(+)型の乳酸及び過酸化水素30%であった。全化学物質は入手したままの状態で使用した。溶液は(特に指定してない限り)蒸留された非イオン化水で作製した。グルコース観測は、抗生物質−抗真菌剤溶液(antibiotic-antimycotic solution)(シグマ社カタログNo.A−8909)を含有する緩衝液、牛血清中で37℃でラットにおいて行なった。
器具:凹付き金電極の製造において、ガルバノスタットモードで動作するポテンシオスタット/ガルバノスタット(PARモデル173、ニュージャージー州プリンストンのプリンストン・アプライド・リサーチ/Princeton Applied Research製)及びソニケータ(sonicator)(ペンシルバニア州ピッツバーグのフィッシャー・サイエンティフィック/Fisher Scientific製)が使用された。サイクリックボルタモグラム(Cyclic voltammograms)は、ポテンシオスタット(PARモデル273A)と、プラチナワイヤカウンタ及びSCE基準電極を備える従来の電気化学セルとで記録され、PAR270ソフトウェアで評価された。グルコース信号は、バイポテンシオスタット(bipotentiostat)(Biometra EP30)及び2チャネルストリップチャートレコーダ(2 channel strip-chart recorder)で観測した。凹部付き電極は、マイクロマニピュレータ(micromanipulator)(Narishige、ニューヨーク州シークリフ)を用いて顕微鏡(Bausch & Lomb製)下でコーティングされた。マイクロピペットは、マイクロピペット引き抜き器(Narishige)で引き抜かれた。温度は、等温サーキュレーター(isothermal circulater)(フィッシャー・サイエンティフィック/Fisher Scientific製)で制御された。
電極作製:
長さ5cmのポリアミド絶縁金製ワイヤは、鋭いカミソリ刃で切断した。絶縁したステンレス鋼ワイヤに金属ワイヤの一端を銀エポキシを用いて電気的に接触させ、接合部を絶縁熱収縮チューブで被覆した。凹部形成電気化学エッチング法は、10mlの3M青酸カリ中で、金製ワイヤを作用電極とし、白金或は金製ワイヤを計数電極として行なわれた。ワイヤはビーカーの底に接触するように配置され、全ての電極は計数電極から等距離とされた。ビーカーはエッチング処理時に超音波処理された。金製ワイヤの端部は上方へ曲げられ、もって超音波処理機(ソニケータ)による撹拌はエッチング工程中に発生した酸素の気泡を上昇させて、消失させる。その後、電極をよく洗い、30分間水中に浸した。
ポリアミド絶縁ワイヤ2において、例えば溝のような凹部6は、ガルバノスタットの制御下で金の電気化学エッチングによって形成される。電気量を制御することによってAu(CN)2 -として電子酸化され、溶解した金の総量が定まる。溝へのCN−の移動及び溝から出るAu(CN)2 -の移動の速度に制限がないように条件(例えば、少なくとも約0.2M、好ましくは3Mの高濃度の青酸カリ及び超音波浴槽)を設定したとき、平らな金のワイヤ表面は、0.5乃至2.0のアスペクト比で溝の底に生成される。従って、CN濃度が十分高く、ワイヤが超音波振動したとき、金製ワイヤの先端は平らとなる。8mAの電流における電極当たり1.5クーロンの通過により、約125μmの深さのキャビティ或は溝が形成された。1電子の酸化に対する理論上の効率では、3.08mgの金がエッチングされることになる。実際にエッチングされた金の量はわずか0.076mgであり、十分なCN-或は水の酸化を示す。いずれにせよ、該工程は1回5分以下で処理加工される20個の電極の複製ができ、正確で速い。凹部形成工程は1回分の量である30個の凹部付き電極に対して(対物マイクロメータ/objective micrometerを用いて)認められた偏差は±10μmであり、高度な複製ができる。コーティングの前に、顕微鏡下で電極の金表面の平坦度と正確な深さを調べた。
図1は、本発明の電極の側面視断面図であり、金製ワイヤ2、絶縁コーティング4及び凹部或は溝6を示す。凹部の金表面は、キャビティ或は溝6を架橋結合可能な異なる層の成分及びそれらの架橋結合剤を含有する水溶性溶液で満たすことでコーティングされた。溶液は、マイクロマニプレータを用いて、(ポリエチレンチューブ及び収縮チューブによってマイクロシリンジに連結された)マイクロピペットとともに顕微鏡下に導入される。個々の層を形成した後、電極を室温で一晩空気中で養生した。
電極の構造:
電極は、4つの層を凹部或は溝6内に順次堆積させることによって作製された。これらの層は、検知層8、絶縁層10、干渉除去層12及び生体適合層14である。“導通接続された(wired)”レドックス酵素を含有する検知層は、金製ワイヤの近傍に、接触配置される。絶縁層10は、検知層8及びペルオキシダーゼ系干渉物除去層12間に配置される。生体適合層14は、凹部6内の残り空間を満たし、電極の外側の環境と接触する。これらの薄いポリマー層は、ポリイミドスリーブ4内に封じ込めることによって適切に保護されている。
検知層8は、酵素が共有結合するレドックスヒドロゲルを介してrGOXと金製電極を“導通接続する”ことによって形成された。電極は以下のごとく作製された。10mg/ml溶液を、
1.水中におけるPVI-Osレドックスポリマー、
2.水中における架橋結合剤、PEDGE、及び
3.pH8.15に調節された10mMのHEPES溶液中の酵素、rGOXから形成された。
レドックスヒドロゲルは、3種の溶液を混合することによって形成され、もって最終成分は、(重量で)レドックスポリマーが52%、酵素が35%、架橋結合剤が13%であった。
絶縁層10は、レドックスヒドロゲルと干渉除去酵素(HRPとLOX)との間の電気接触を防止した。PAL:PAZを絶縁材料として使用した。膜は、PAL溶液(pH7.0の100mM HEPES緩衝剤中で4.5mg)と新たに準備したPAZ溶液(30mg/ml)を1/1、1/2或は1/3の体積比で混合することによって得た溶液から堆積させた。PAZ溶液は、準備してから15分以内に使用した。
干渉除去層12は、先に開示したプロトコル、1992年Maidan & Heller、Anal.Chem.、64:2889−2896、に従って作製した。12mg/ml新規作製過ヨウ素酸ナトリウム溶液50μlを、0.1M重炭酸ナトリウム中に20mg/mlのHRP(HRP−VI或はHRP−BM)及び100mg/mlのLOX(LOX或はrLOX)を含有する100μlの溶液に添加し、混合物を2時間暗闇に温置した。代替的に、HRPの酸化をLOX添加及び架橋結合の前に実施してもよい。
生体適合層14の薄膜は、UV光線(カリフォルニア州サンガブリエルのUVP社、Blak-Ray、360nMのスペクトルピーク、200mW/cm2サンプルのUV放射照度)に1分間露出することによって光架橋結合させた。使用した開始剤は、2,2−ジメトキシ−2−フェニルアセトフェノン(Aldrich)である。1−ビニル−2−ピロリジンワン(Aldrich)中の開始剤300mg/mlの溶液をプレポリマー混合物に添加えた。開始剤溶液約30μlを、テトラアクリル酸塩化PEOの10%w/w水溶性溶液のミリリットル当たりで添加した。プレポリマーは、電極の内側にその場で架橋結合した。該薄膜は、凹部をプレポリマー溶液で2度に渡って満たし、電極をキャビティが満たされる毎にUV光線源に露出することによって作製された。
電極の生体外試験:
生体外実験は、作用電極としての酵素改質形成ワイヤと、計数電極及び飽和カロメル基準電極(SCE)としての白金線とを備える従来の3電極電子化学セルを用いて25℃及び37℃でバッチ方式で行なわれた。電解液は、pH7.15で0.15mM NaClを含有する20mMのリン酸塩緩衝塩類泉(phosphate buffered-saline)であった。血清中での実験は、100μLの抗生物質−抗真菌剤(antibiotic-antimycotic)溶液を10mlの血清に添加し、37℃で行なった。実験中、リン酸塩緩衝塩類泉及び血清は撹拌された。作動電位は、PVI−Osポリマーを用いての実験のためSCEに対して0.3Vとした。
構造及び性能:
溝6の深さc及びその中のポリマー層の厚さは、検知層へのマス(質量)の移動、つまりグルコースフラックスの移動を制御する。これらのパラメータを制御することにより、見掛けミハエリス定数(Km)は、約20〜30mMのグルコースに調節される。溝6のポリイミド壁4も、4つのポリマー及びポリマー/酵素層8、10、12、14を機械的損傷から保護し、体内のこれらの層の損失の危険性を減少させる。グルコース酸化電流は組織に露出される端部16への質量移動によるというよりも、凹部6及びそのポリマー薄膜8、10、12、14を介するグルコースの質量移動により制限されるので、実際上、電流は運動に対して無感応である。明らかに、凹部検知層8におけるグルコースの電子酸化速度は、グルコースが溝の外側の流体と接触する界面へ拡散する速度より遅い。
PVI5−Osは、HRP及びLOXの干渉除去層を使用する場合の検知層の“導線”として好ましいが、干渉除去層が存在しいないとき、つまり、より低いレドックス電位を有するレドックスポリマーが好ましいときはそうではない。下付き文字(5)は、平均して、5つ目毎のビニルイミダゾールマー(vinylimidazole mer)が電子中継オスミウムセンター(electron-relaying osmium center)を有することを示す。PVI5−Os及び(3つ目毎の1−ビニルイミダゾールマー/1-vinylimidazole merがオスミウムセンターを有する)PVI3−Osが設けられた電極の使用を図2で比較しており、PVI5−Osを備える電極におけるグルコース電子酸化の電流密度(白抜き三角形)が、PVI3−Osの場合(黒塗り三角形)より高いことを示している。
凹部の深さ及び検知層:
溝の深さに対する電流の依存度を見るため、PVI5−Os及びRGOXの総量を一定に保持して、深さ125、250及び500μmの溝について調べた(図3)。より深いキャビティにおける多くの電流損は、減ったグルコース質量移動から発生したのではなく、成分溶液のミクロ液滴が電極形成過程で溝に導入された際にポリイミド壁に酵素及びポリマーの一部が吸収保持されることに起因している。水で繰返し濯ぐことにより、吸収された壁上のポリマー及び酵素の一部は、電極表面に流されて電流を増加させる。最も高い電流は5回の洗浄後に確認された。コーティング回数を増加させることによって検知層の厚さが増大した際(図4)、検知層におけるレドックスポリマーを電子還元或は電子酸化するのに要する電荷に対する電流の比は最大に達し、その後下降した。好適な125μmの凹部に対し、約13μmの厚さの導通接続されたrGOX検知層を生成する10回のコーティングによって生体内使用のための所望の特徴を備えるセンサが形成された。
絶縁層:
この層は、“導通接続”rGOX層から干渉除去層(HRP及びLOX)のレドックス酵素を電気的に絶縁し、検知層へのグルコースフラックスを制限し、もって電極の耐用年数を延ばす。pH7.09でポリ陽イオン網(polycationic network)を形成するPAZ及び架橋結合PALが好ましい。約7μm厚の架橋結合膜を形成するため3回のコーティングを行い、PAL:PAZ=1:2(図5)を用いて最良の結果、つまり最良の電流出力安定性が得られた。
干渉除去層:
干渉物、特にアスコルバート、尿酸塩及びアセトアミノフェノールは、LOX及びHRPを含有する第3層で酸化される。この層において、血中典型濃度が1mMのラクタートはO2と反応してH2O2及びビルビン酸を形成する。HRPの存在下、H2O2はアスコルバート、炭酸塩及びアセトアミノフェノールを酸化させて水になる。好適な相互固定化プロセスは、次の2つのステップを包含する。即ち、HRPのオリゴ糖官能基をアルデヒドへ過ヨウ素酸塩酸化し、次いでLOXと混合して、HRP−アルデヒドとLOXアミン(例えばリシン)との間及びHRP−アルデヒドとアミンと間に多数のシッフ塩基(Schiff bases)を形成することである。干渉除去層の厚さは約85μmであり、連続する、例えば6回のコーティングによって生成された。図6は電子酸化可能干渉物が通常のレベルでラクタートの存在時に除去されたことを示す。LOXは架橋結合HRP−LOX層における活性をゆっくりと失った。こうして該層の干渉物除去能力が低下した。37℃で36時間作動した後、十分なアスコルバートを加えて0.1mMの濃度を達成したとき、測定可能電流の増加が記録された。
生体適合性層:
好適な生体適合性層は、例えば光架橋結合テトラアクリル化された18,500Daポリ(エチレンオキシド)(パタック他/Pathak et al.,1993,J.Am.Chem.Soc.,114:8311-8312)から成っている。2コーティングの連続光架橋結合によって形成されたこの層の厚さは約20μmである。光架橋結合過程に要する1分間のUV露出は感度を16%±2%低下させた。
例2 センサの生体内使用
この実験の目的は、生体内一点較正の有効性を確立することであった。2つのセンサはラットの皮下に1つは胸部に、2つめは肩甲骨の間に同時に埋設した。採取血液とセンサで立証した皮下流体との間の差をできるだけ極端にするため、つまりサンプリングした器官が異なり、サンプリング場所が離れていたとしても、一点較正が有効であるかどうかを調べるため、血液は末端血管(tail vein)或は細静脈から採取した。絶対未補正センサ電流を連続的に観測しながら、採取サンプルの血中グルコースレベルを定期的に測定した。
生体内実験(6〜10時間)は、300gの雄のスプレーグ−ドーリー(Sprague-Dawley)ラットで行なわれた。ラットに一晩絶食させ、実験に先立ってペントバービタルナトリウム(sodium pentobarbital)の腹腔内(i.p.)注射を用いて麻酔した。その後、硫酸アトロピンをi.p.投与し、呼吸の低下を抑制した。ラットに麻酔をかけたら、ラットの腹部の一部を剃り、導体ゲルを塗り、Ag/AgCl表皮基準電極を取付けた。この電極は計数(カウンタ)電極としても働いた。次いで、センサをラットの胸部に22ゲージPer−Q−Cathイントロデューサ(テキサス州サンアントニオのジェスコ・インターナショナル/Gesco International製)を用いて皮下に埋め込むか、小規模な外科切開を介して皮下の細胞内領域に埋設した。センサの移動を避けるため、センサを皮膚にテープで固定した。センサは、基準電極とともに、内蔵バイポテンシオスタットに接続された。センサの作動電位は、0.3V vs. Ag/AgClとし、Ag/AgCl電極は基準計数電極としても作用する。センサ読み値は、データロガー(data logger)(ラストラック・レンジャー/Rustrak Ranger製、ロードアイランド州イーストグリニッジ)を用いて集められ、実験の最後にコンピュータに転送された。実験中、ラットの体温を恒常性毛布(homeostatic blanket)によって37℃に保った。血液サンプリングを開始する前に、センサが基礎信号レベルに到達するまで少なくとも1時間待った。血液のサンプルは末端血管(細静脈)から得ており、すべての血液サンプルはグルコースアナライザ(ワイエスアイ/YSI社製、オハイオ州イエロースプリング;型式23A)を用いて分析した。
血液サンプルの採取開始から約30分後、シリンジポンプ(ハーバード・アパラタス/Harvard Appratus、マサチューセッツ州サウスナティック)を用いてi.p.グルコース注入を120mgグルコース/minラット体重kgの割合で開始した。グルコースの注入はおよそ1時間続けられた。
図7に分るように、410分で電流が急降下した。このような降下は他の皮下に差込まれた電極を用いた測定において400分と600分との間で観測されたが、37℃の緩衝剤中で操作された電極では観測されなかった。故障した電極を引き抜いて緩衝剤中で再試験をおこなったとき、電極の原感度の殆どが無事であることが分った。この見かけの不作動の原因は、良好な電解質接触を達成するラットの皮膚上の計数/基準Ag/AgCl電極の故障であり、埋め込まれたセンサの故障ではなかった。各電極の較正曲線を算出するため任意に選んだ点、つまりスケールを作製するための1つの血中グルコースレベルの決定及び1つの電流測定値を用い、図7からの全データを更なる補正なしで、クラーク式(Clark-type/Clark et al.,1987,Diabetes Care 5:622〜627)臨床図表(図8)にプロットした。この分析において、図表のA範囲に入る点は臨床上は正確なものと考えられ、B範囲に入る点は臨床上正しいと考えられる。C範囲に入る点は正しくはないが、不適切な治療に至らないと考えられる。D範囲及びE範囲に入る点は正確なものではなく、治療をこれらに頼るとすると、不適切なものになると考えられる。
両電極から得た全ての点がA範囲及びB範囲にあり、48の内の43の点がA範囲にある。肩甲骨間に埋設された電極に対する100mg/dlグルコース付近のB範囲にある3点は410分の実験の最後の3点であった。計数/基準電極の皮膚との不十分な電解質接触による400−600分における故障モード及びラクタートオキシダーゼの不活性による36時間後の故障によって干渉除去が失われるが、一点較正(ワンポイントキャリブレーション)が実用的であることが分る。このような較正の後、皮下センサの読み値は、補正なしで臨床上有用な血中グルコースレベルの評価を提供する。
図9は、どちらかの埋め込み電極の一点較正に24個のグルコース分析の各々を用いたときに得られた全ての有り得る相関関係の分布を示すものである。この分布には、2×24×24=1152の点が存在する。これらのうち78%はA範囲に、15%がB範囲に、1%がC範囲に、6%がD範囲にあり、E範囲には点が存在しない。
図10においては、冗長電極を用いて一点較正の向上を試験した。まず、電極Aの読み値を電極Bのものに対し、電極Aの平均出力で割った電極Bの平均出力を各読み値に乗ずることによって標準化した。次いで、24組の埋設電極B読み値と埋設電極Aの補正読み値との間の差について標準偏差を求めた。その後で、標準偏差以上に異なる読み値の全組を除いた。組数が24から11に減らされた。全点の82%がA範囲にあり、17%はB範囲に、1%はD範囲に、C範囲とE範囲には点が全く存在しなかった。この分布は、採取血液サンプル中のグルコース濃度の1回の測定によって較正が可能であることを物語っている。また、これらは、冗長皮下センサの使用によって診断精度が向上することをも示す。全セットから標準偏差以上に異なるデータポイントを選び出すことにより、埋め込みセンサの読み値に基づく診断で誤診する確率が6倍も減少した。
安定度とその他の特性:
安定性を向上させるため、GOXではなくて、熱安定性のよい組替型GOX(rGOX;Heller,1992,J.Phys.Chem.96:3579-3587)をセンサに使用し、酵素の代謝(turnover)ではなく、センサの電流拡散を制限するため、グルコースの移動を減らした。グルコースフラックスは、3つの外側層及び検知層自体によって弱められた。検知層が過剰なグルコースオキシダーゼを含有しているので、その作用は、弱められたグルコースフラックスを電子酸化するのに要する以上の活性を大きく越え、センサの安定度が向上する。
安定性は、周知の方法、例えば37℃の10mMグルコースの0.1mMアスコルバート存在下で試験すればよい。典型的な最適化電極の電流出力は約35nAで、イーディーホフスティー(Eadie-Hofstee)プロットから得た見掛けKmは、約20mMであった(表1)。10〜90%応答時間は約1分であった。
予期したように、また図5に見られるように、膜がより薄くて、グルコースの質量移動が増大した。つまり、電流は高かったが、薄い膜については安定性が向上した。薄い(約1μmの)検知膜電極(10-2A cm-2 M-1以下)が高感度であるため、感度のオーダーの減少を安定度と引き換えにしてもよく、このとき電流は測定し易いよう十分に高く維持された。
図5にみられるように、安定したセンサの感度は、37℃で72時間の操作に対して±5%以上は変化しない。最初の小さな感度減少の後、40時間後に最高値まで上昇し、最後の72時間の感度は最初のものと殆ど同じであった。
本発明の電極の特性は、表1に要約される。各記載は5つの試験した電極の平均値を表わしたものである。基準線電流は、典型的に0.5nA以下であり、ノイズは10pAである。生理学的グルコース濃度範囲(2〜20mM)を通して観察された電流は、少なくとも指数100だけノイズ等価電流(noise equivalent current)を上回っている。見掛けKmは約20mMであり、10%乃至90%応答時間は、経年電極の場合、最低の生理学関連グルコース濃度(2mM)で約90秒、最高(20mM)で20秒である。
0mMグルコースでのゼロ基準線は、0.1mMのアスコルバートの存在下で36時間安定している。観測された安定度及び干渉物があるときの有効ゼロポイントの存在は、センサを72時間生体内で使用し、1点較正を介して、つまり、個々の分析に対して単一の血液サンプルを採取することにより、生体内試験/再較正が可能であることを示唆する。
Claims (12)
- 分析物の濃度を測定する電気化学バイオセンサであって、
非腐食金属又はカーボン製の電極と、
前記電極と隣接し、かつ、電気的に接触し、架橋結合されたレドックスポリマーとレドックス酵素とから成る検知層と、
前記バイオセンサの外側面をコートし、前記検知層と接触し、生体適合性を有し、分析物の拡散を制限する少なくとも1つの層と、
から成ることを特徴とする電気化学バイオセンサ。 - 前記少なくとも1つの層は、前記検知層に接触している分析物拡散制限層と、前記バイオセンサの前記外側面と接触している生体適合性層と、から成ることを特徴とする請求項1の電気化学バイオセンサ。
- 前記少なくとも1つの層は、前記検知層に接触している分析物拡散制限層と、前記分析物拡散制限層と接触している干渉除去層と、前記干渉除去層及び前記バイオセンサの前記外側面と接触している生体適合性層と、から成ることを特徴とする請求項1の電気化学バイオセンサ。
- 前記干渉除去層は、ペルオキシダーゼ酵素から成ることを特徴とする請求項3の電気化学バイオセンサ。
- 前記干渉除去層は、更に、過酸化水素発生反応を触媒する酵素を含むことを特徴とする請求項4の電気化学バイオセンサ。
- 前記生体適合性層は、生理学的体液と平衡のときに、20重量%以上の水を含有する生体適合性ポリマーから成ることを特徴とする請求項2乃至5のいずれかの電気化学バイオセンサ。
- 前記検知層は、架橋結合されたレドックスポリマーと、レドックス酵素と、架橋結合剤と、から成ることを特徴とする請求項1乃至6のいずれかの電気化学バイオセンサ。
- 前記架橋結合剤は、ポリ(エチレングリコール)ジグリシジルエーテルであることを特徴とする請求項7の電気化学バイオセンサ。
- 前記レドックスポリマーは、ポリ(4−ビニルピリジン)又はOs3+/2+とRu3+/2+とFe3+/2+とで構成されるグループから選択された金属イオンと結合する(4−ビニルピリジン)の共重合体から誘導されることを特徴とする請求項1乃至8のいずれかに記載の電気化学バイオセンサ。
- 前記レドックス酵素は、グルコースオキシダーゼであることを特徴とする請求項1乃至9のいずれかの電気化学バイオセンサ。
- 前記電極は、導電性ワイヤの一部であることを特徴とする請求項1乃至10のいずれかの電気化学バイオセンサ。
- 前記レドックスポリマーのレドックス電位は、pH7.4の水溶液中で標準カロメロ電極に対して酸化が+0.15V以下であり、還元が−0.15V以下であることを特徴とする請求項1乃至11のいずれかの電気化学バイオセンサ。
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US08/299,526 US5593852A (en) | 1993-12-02 | 1994-09-01 | Subcutaneous glucose electrode |
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