JP3869686B2 - Microorganism measuring apparatus, microbe measuring electrode chip used therefor, and microorganism measuring method - Google Patents

Description

ここで、複素誘電率Ｋは（数２）で表され、
【００１４】
【数２】

また、懸濁液の複素誘電率ε^＊ _ｍは（数３）で表され、
【００１５】
【数３】

ε_ｍ ：懸濁液の誘電率
ε^＊ _ｍ ：懸濁液の複素誘電率
ε^＊ _ｉ ：微生物の複素誘電率
σ 懸濁液の導電率
ω ：電界の角周波数
また、
ａ ：球形近似したときの微生物の半径
Ｒｅ［Ｋ］ ：微生物と懸濁液の複素誘電率に依存するパラメータ
Ｅ ：電界強度
である。この（数１）（数２）（数３）から明らかなように、懸濁液と微生物の大きさが一定であれば、誘電泳動力Ｆ_ＤＥＰはパラメータＲｅ［Ｋ］に比例することがわかる。
【００１６】
図８は細胞質導電率をパラメータとするＲｅ［Ｋ］の周波数特性図である。図８において、誘電泳動に用いる電界の周波数fをパラメータとして、誘電泳動力を細胞質導電率σ_iの関数として表している。図８によると、周波数１０ｋＨｚ〜１ＭＨｚで正の誘電泳動力が働き、それ以外では負の誘電泳動力が働くのがわかる。細胞質導電率σ_iと周波数ｆによっては負の誘電泳動力が働き、誘電泳動力Ｆ_ＤＥＰが微生物に作用しても、捕集できない場合があることが分かる。
【００１７】
従って、微生物（細胞質導電率σ_i）の種類に応じて周波数ｆを選択すると、正の誘電泳動力を作用させて捕集したり、負の誘電泳動力を作用させて排除することも可能である。但し、実際の誘電泳動には懸濁液導電率等の影響も考慮しなければならない。
【００１８】
しかし、微生物の種類によって、パラメータＲｅ［Ｋ］が正で、他の菌種のそれがすべて負となるようなことはなく、誘電泳動力の正負の振り分けは一般化できることではない。すなわち、微生物固有の誘電特性（誘電率、導電率）を利用して、多種の微生物の混合懸濁液中から特定の微生物のみに選択的に正の誘電泳動力を作用させ、ＤＥＰＩＭ法によりその微生物のみを検出することは困難である。
【００１９】
また、ＤＥＰＩＭ法を実際に応用する場合は、多種且つ未知の種類の微生物が含まれている懸濁液から特定の微生物のみを検出することが要求されることが多いと予想されるが、従来のＤＥＰＩＭ法ではこのような選択的微生物検出は不可能である。
【００２０】
【発明が解決しようとする課題】
以上説明したように、従来のコロニーカウント法は、微生物を含有した懸濁液を培地上に散布し、この微生物の増殖に伴って形成されるコロニーの数から生菌数を定量するが、コロニーが形成されるまでに1日から数日という驚くほどの長時間を必要とするものであった。そして、複数の細菌が含有された試料液の場合には、補助処理なしには特定の細菌を選んで定量することはできない。また、専門家による煩雑な手作業が必要であり、検査に必要なランニングコストの上昇や、人為的ミスを招来する可能性すらあった。
【００２１】
また、ＡＴＰ法は、ＡＴＰ抽出試薬によるＡＴＰの抽出や、酵素を作用させたり、発光量の測定、ＡＴＰ量の推定等は専門家による高度の知識が必要で、測定に時間が要求され、測定に当ってはバックグラウンドが多く、検査が不確実になるものであった。微生物として細菌、酵母、真菌等の区別ができない方法であり、選択的な定量が難しいものであった。
【００２２】
そして、ＥＬＩＳＡ法は、他の方法と同様に複雑な実験手順を踏まなくてはならず、高度の専門性が要求され、吸光度の測定に適した検査対象濃度を得るために濃縮を行う必要があり、検出には長時間をかけなければならないという欠点がある。
【００２３】
そして、ＰＣＲ法は、死菌のＤＮＡも増幅させるため、事前に培養の手順を踏み、検査対象の活性を調べる必要がある。また、ＤＮＡが爆発的に増加するため、細菌の初期数を推定するのは困難で、定量性に欠けるという問題を有すものであった。専門性が要求されるのは他の方法以上である。
【００２４】
本発明者が他の研究者らとともに提案したＤＥＰＩＭ法は、微生物の濃縮と菌濃度検出のいずれのプロセスも電気的に同時に行うため、専門家が行わなくとも短時間で微生物を定量することができるが、複数種類の微生物を混合した懸濁液の特定の微生物だけに誘電泳動力を作用させて検出することはできないものであった。他の微生物の定量方法が専門家によって長時間の測定を行わざるを得ない中で、ＤＥＰＩＭ法だけがこれらを解決できるものであるが、複数種類の微生物が含有された試料液の中で特定の微生物を高感度に定量できるものではなかった。従って、複数種類の微生物が含有された試料液であっても、特定の微生物を迅速、高感度で選択的に定量できる新しい微生物測定装置が切望される。
【００２５】
そこで、本発明は、複数種類の微生物が含有された試料液の中で特定の微生物を高感度で迅速に、専門家によらなくとも簡易に定量できる微生物測定装置を提供することを目的とする。
【００２６】
また、本発明は、複数種類の微生物が含有された試料液の中で特定の微生物を高感度で迅速に、専門家によらなくとも簡易に定量でき安価な微生物測定用電極チップを提供することを目的とする。
【００２７】
そして、本発明は、複数種類の微生物が含有された試料液の中で特定の微生物を高感度で迅速に、専門家によらなくとも簡易に定量できる微生物測定方法を提供することを目的とする。
【００２８】
【課題を解決するための手段】
上記の問題点を解決するため本発明の微生物測定装置は、電極間で濃縮された微生物に対して抗原抗体反応させるための抗体液を前記測定チャンバーに供給する抗体液供給部とを備え、演算制御部が、誘電泳動用電源部によって第１の交流電圧を印加して複数種類の微生物を濃縮し、抗体供給部から抗体液を供給して特定の微生物と特異的に反応させ、該特定の微生物には流動作用に伴う流体力より大きな誘電泳動力を作用させるとともに、他の微生物には流動作用に伴う流体力より小さな誘電泳動力を作用させる第２の交流電圧を印加し、特定の微生物を凝集するとともに他の微生物を分離し、測定部によって測定した電極間のコンダクタンス変化から特定の微生物数を算出して出力することを特徴とする。
【００２９】
これにより、複数種類の微生物が含有された試料液の中で特定の微生物を高感度で迅速に、専門家によらなくとも簡易に定量することができる。
【００３０】
本発明の微生物測定用電極チップは、電極間の基板上には特定の微生物と特異的に反応する抗体が固相化され、反応した特定の微生物以外の微生物は流体力で分離され、電極間のコンダクタンス変化から特定の微生物数を算出して出力することを特徴とする。
【００３１】
これにより、複数種類の微生物が含有された試料液の中で特定の微生物を高感度で迅速に、専門家によらなくとも簡易に定量でき安価な微生物測定用電極チップを提供することができる。
【００３２】
本発明の微生物測定方法は、一対の電極間に交流電圧を印加して複数種類の微生物を第１の誘電泳動で濃縮し、濃縮された微生物に抗体液を供給し、該抗体液と特定の微生物と特異的に反応させ、さらにこの微生物に第２の誘電泳動と流動作用を加え、特定の微生物には流体力より大きな誘電泳動力、他の微生物には流体力より小さな誘電泳動力を作用させることによって特定の微生物を凝集するとともに他の未反応の微生物を分離し、その後前記電極間のインピーダンスを測定して特定の微生物のみの微生物数を算出することを特徴とする。
【００３３】
これにより、複数種類の微生物が含有された試料液の中で特定の微生物を高感度で迅速に、専門家によらなくとも簡易に定量することができる。
【００３４】
【発明の実施の形態】
本発明の請求項１に記載の発明は、試料液を収容することができる測定チャンバーと、測定チャンバー内の試料液に浸漬され、不平等電界を発生して第１の誘電泳動により該試料液に含有される複数種類の微生物を濃縮し、この濃縮された微生物と抗体とを反応させたときに第２の誘電泳動と流動作用を加えることにより抗体と特異的に反応した特定の微生物だけを残して他の微生物を分離し、このときのインピーダンスを測定することができる一対の電極と、電極間に交流電圧を印加する誘電泳動用電源部と、電極間のインピーダンスを測定することができる測定部と、誘電泳動用電源部と測定部の制御を行う演算制御部と、測定対象の微生物毎に測定制御データと検量線データを格納した測定用テーブルが格納されたメモリ部と、電極間で濃縮された微生物に対して抗原抗体反応させるための抗体液を測定チャンバーに供給する抗体液供給部とを備え、演算制御部が、誘電泳動用電源部によって第１の交流電圧を印加して複数種類の微生物を濃縮し、抗体供給部から抗体液を供給して特定の微生物と特異的に反応させ、該特定の微生物には流動作用に伴う流体力より大きな誘電泳動力を作用させるとともに、他の微生物には流動作用に伴う流体力より小さな誘電泳動力を作用させる第２の交流電圧を印加し、特定の微生物を凝集するとともに他の微生物を分離し、測定部によって測定した電極間のコンダクタンス変化から特定の微生物数を算出して出力することを特徴とする微生物測定装置であるから、交流電圧を電極間に印加して試料液に含有される複数種類の微生物を第１の誘電泳動により濃縮するため、濃縮のために長時間をかける必要がなく、この濃縮した微生物に特定の微生物と特異的に反応する抗体液を供給し、第２の誘電泳動と流動作用を加えることにより特定の微生物だけ凝集して他の微生物から分離するから、複数種類の微生物の中から測定対象の特定の微生物だけを選択することができ、選択した後の特定の微生物をインピーダンス測定することにより、コンダクタンス変化から微生物数を測定するため、混合懸濁液の中で特定の微生物を高感度で迅速に、専門家によらなくとも簡易に定量することができる。
【００３５】
本発明の請求項２に記載の発明は、抗体液供給部が、抗体液を収容した抗体液タンクと、測定チャンバーに抗体液を導入する抗体液供給管と、該抗体供給管に設けられた抗体液バルブを備えて、複数種類の微生物を濃縮した後に抗体液を測定チャンバーに供給することを特徴とする請求項１に記載の微生物活性測定装置であるから、抗体液バルブを操作することにより、濃縮した複数種類の微生物に抗体液を自動的に供給することができる。
【００３６】
本発明の請求項３に記載の発明は、測定チャンバーには攪拌装置が設けられ、試料液の攪拌を行うことを特徴とする請求項１または２に記載の微生物活性測定装置であるから、微生物濃度を均一化し、電極近傍に補修することができる。さらに、抗原抗体反応後に、抗体と特異的に反応した特定の微生物以外の微生物を攪拌による流動によって分離することができる。
【００３７】
本発明の請求項４に記載の発明は、測定チャンバーには洗浄液の洗浄管が設けられるとともに、洗浄管に洗浄バルブが設けられ、演算制御部が時計手段の行う計時に基づいて、洗浄バルブを開いて洗浄液を測定チャンバーに供給し、流体力で特定の微生物を分離して洗い流すことを特徴とする請求項１〜３のいずれかに記載の微生物活性測定装置であるから、抗体と特異的に反応した特定の微生物以外の微生物を洗浄液の流体力で分離することができ、分離した未反応の微生物を洗い流してインピーダンス測定を可能にすることができる。
【００３８】
本発明の請求項５に記載の発明は、試料液を収容することができる測定チャンバーと、測定チャンバー内の試料液に浸漬され、不平等電界を発生して第１の誘電泳動により該試料液に含有される複数種類の微生物を濃縮し、この濃縮された微生物と抗体とを反応させたときに第２の誘電泳動と流動作用を加えることにより抗体と特異的に反応した特定の微生物だけを残して他の微生物を分離し、このときのインピーダンスを測定することができる一対の電極を備えた微生物測定用電極チップと、電極間に交流電圧を印加する誘電泳動用電源部と、電極間のインピーダンスを測定することができる測定部と、誘電泳動用電源部と測定部の制御を行う演算制御部と、測定対象の微生物毎に測定制御データと検量線データを格納した測定用テーブルが格納されたメモリ部とを備え、微生物測定用電極チップには、一対の電極の基板上に特定の微生物と特異的に反応する抗体が固相化され、演算制御部が、誘電泳動用電源部によって交流電圧を印加して複数種類の微生物を濃縮し、固相化された抗体と特定の微生物とを特異的に反応させて第２の誘電泳動力と流動作用に伴う流体力によって分離し、測定部によって測定した電極間のコンダクタンス変化から特定の微生物数を算出して出力することを特徴とする微生物測定装置であるから、交流電圧を電極間に印加して試料液に含有される複数種類の微生物を第１の誘電泳動により濃縮するため濃縮のために長時間をかける必要がなく、特定の微生物と特異的に反応する抗体を固相化し、特定の微生物だけ微生物測定用電極チップに固定し第２の誘電泳動と流動作用を加えることにより他の微生物を分離するから、複数種類の微生物の中から測定対象の特定の微生物だけを選択することができ、選択した後の特定の微生物をインピーダンス測定することにより、コンダクタンス変化から微生物数を測定するため、混合懸濁液の中で特定の微生物を高感度で迅速に、専門家によらなくとも簡易に定量することができる。
【００３９】
本発明の請求項６に記載の発明は、抗体と特異的に反応した特定の微生物だけを残して他の微生物を分離するとき、該特定の微生物と他の微生物に交流電圧を印加してそれぞれに誘電泳動力を作用させ、該誘電泳動力と流動作用に伴う流体力の差を利用して分離することを特徴とする請求項５記載の微生物測定装置であるから、反応した特定の微生物の集合体と他の微生物の大きさの違いによる差が誘電泳動力と流体力の差に変換され、この差を利用することで分離が簡単且つ迅速に行える。
【００４０】
本発明の請求項７に記載の発明は、請求項６記載の微生物測定装置で使用される微生物測定用電極チップであって、電極間の基板上には特定の微生物と特異的に反応する抗体が固相化され、反応した特定の微生物以外の微生物は流体力で分離され、電極間のコンダクタンス変化から特定の微生物数を算出して出力することを特徴とする微生物測定用電極チップであるから、特定の微生物と特異的に反応する抗体を固相化し、特定の微生物だけ微生物測定用電極チップに固定し、他の微生物を流体力で分離することができ、複数種類の微生物の中から測定対象の特定の微生物だけを選択することが安価に実現できる。
【００４１】
本発明の請求項８に記載の発明は、一対の電極間に交流電圧を印加して複数種類の微生物を第１の誘電泳動で濃縮し、濃縮された微生物に抗体液を供給し、該抗体液と特定の微生物と特異的に反応させ、さらにこの微生物に第２の誘電泳動と流動作用を加え、特定の微生物には流体力より大きな誘電泳動力、他の微生物には流体力より小さな誘電泳動力を作用させることによって特定の微生物を凝集するとともに他の未反応の微生物を分離し、その後電極間のインピーダンスを測定して特定の微生物のみの微生物数を算出することを特徴とする微生物測定方法であるから、交流電圧を電極間に印加して試料液に含有される複数種類の微生物を第１の誘電泳動により濃縮するため濃縮に長時間をかける必要がなく、この濃縮した微生物に特定の微生物と特異的に反応する抗体液を供給し、第２の誘電泳動と流動作用を加えて特定の微生物だけ凝集して他の微生物を分離するから、複数種類の微生物の中から測定対象の特定の微生物だけを選択することができ、選択した後の特定の微生物をインピーダンス測定することにより、コンダクタンス変化から微生物数を測定するため、混合懸濁液の中で特定の微生物を高感度で迅速に、専門家によらなくとも簡易に定量することができる。
【００４２】
本発明の請求項９に記載の発明は、一対の電極間に交流電圧を印加して複数種類の微生物を第１の誘電泳動で濃縮し、電極間に固相化された抗体を特定の微生物と特異的に反応させ、さらにこの微生物に第２の誘電泳動と流動作用を加え、該第２の誘電泳動力と流体力によって抗体と特異的に反応した特定の微生物だけを残して他の微生物を分離し、その後電極間のインピーダンスを測定して特定の微生物のみの微生物数を算出することを特徴とする微生物測定方法であるから、交流電圧を電極間に印加して試料液に含有される複数種類の微生物を第１の誘電泳動により濃縮するため濃縮に長時間をかける必要がなく、特定の微生物と特異的に反応する抗体を固相化し、特定の微生物だけ微生物測定用電極チップに固定し第２の誘電泳動と流動作用を加えることにより他の微生物を分離するから、複数種類の微生物の中から測定対象の特定の微生物だけを選択することができ、選択した後の特定の微生物をインピーダンス測定することにより、コンダクタンス変化から微生物数を測定するため、混合懸濁液の中で特定の微生物を高感度で迅速に、専門家によらなくとも簡易に定量することができる。
（実施の形態１）
以下、本発明における実施の形態１における微生物検出装置及び微生物検出方法について説明する。図１は本発明の実施の形態１における微生物測定装置の構成図、図２（ａ）は本発明の実施の形態１における微生物測定装置の電極部が特定の微生物を選択的に測定するための濃縮プロセス図、図２（ｂ）は本発明の実施の形態１における微生物測定装置の電極部が特定の微生物を選択的に測定するための抗原抗体反応プロセス図、図２（ｃ）は本発明の実施の形態１における微生物測定装置の電極部が特定の微生物を選択的に測定するための洗浄プロセス図、図３は本発明の実施の形態１における微生物測定装置の微生物選択可能領域図、図４は本発明の実施の形態１における測微生物定装置の特定の微生物の測定結果図である。
【００４３】
図１において、１はＤＥＰＩＭ法 (Dielectrophoretic Impedance Measurement Method)で特定の微生物を測定するために複数の微生物が懸濁液に混合された試料液を収容する測定チャンバー、２は微生物数を測定するための電極部、２ａ，２ｂは電極部２に設けられた一対の電極である。本実施の形態１は、この電極2ａ，２ｂ間で行う後述する誘電泳動によって、微生物の濃縮作用、抗原抗体反応によって凝集した特定の微生物と他の微生物を流体力で分離するための篩い分け作用を行う。
【００４４】
すなわち、本実施の形態１の特徴は、誘電泳動によって複数の微生物を含有した試料液を電極２ａ，２ｂ間に前処理として濃縮し、この前処理で濃縮を終えた微生物に対して特定の微生物と特異的に反応する抗体液を導入する。抗原抗体反応によってこの特定の微生物を凝集させた後、電極２ａ，２ｂ間に弱い誘電泳動力を作用させながら、後述するように洗浄液の導入と攪拌のいずれかもしくは双方で電極２ａ，２ｂ上の試料液を流動させ、この流体力と誘電泳動力の関係によって未反応の他の微生物だけを分離除去し、最終的に洗浄液で洗い流す。その後電極２ａ，２ｂ間で特定微生物数をカウントすることを特徴としている。これらの処理がすべて電極部２の表面で行われる。なお、実施の形態１では、凝集による集合体の大きさの差を利用するものであるが、この大きさの違いは流体抵抗や誘電泳動力等に反映される。
【００４５】
ここで、上記した複数種類の微生物の中で特定の種類の微生物を分離する前提となる抗原抗体反応処理について説明を行う。特定の微生物に含まれる固有のタンパク質のみに特異的に反応する抗体の液を加えると、抗原抗体反応を起こす。この性質を利用すれば、特定の微生物（抗原）を選択的に検出することが可能となる。本実施の形態１の場合、誘電泳動によって微生物全体を濃縮後、抗原抗体反応により特定の微生物同士を凝集させ、未反応の微生物は凝集しないから、この大きさの違いによって生じる誘電泳動力の差を利用し、洗浄液導入や攪拌の流体力によって特定の微生物だけを残して測定する。従来のように培養することなく、抗原抗体反応を行うのに必要な微生物濃度を誘電泳動力で得るため、短時間で検出することができる。微生物数は電気的なインピーダンス信号として検出するため、定量的測定が可能である。
【００４６】
電極２ａ，２ｂの形状は、誘電泳動を行うために不平等電界を発生して、電極２ａ，２ｂ間にパールチェインと呼ばれる微生物の鎖を多数橋絡できる形状、例えばキャッスルウォール型電極、櫛歯型電極等の形状が好ましい。キャッスルウォール型電極は、両側に１ピッチ（例えば５０μｍ〜１００μｍ）おきに突出する多数の矩形電極片が形成された電極２ａ，２ｂを１ピッチずらして５μｍ〜１０μｍ離して配設したもので、電極２ａ，２ｂの各矩形電極片のエッジ間に電界が集中するものである。また、櫛歯型電極は、櫛のように歯（例えば３０μｍ〜１００μｍ幅）を形成された一対の電極が溝に入れ子状に挿入、組み合わされ、狭いギャップ（例えば５μｍ〜１０μｍ幅）で対向した電極であり、電界は厚さ方向のエッジ間に不平等電界が形成され、パールチェインが多数形成されるものである。微生物の大きさでギャップやピッチは最も適当な値を選択するのが望ましい。
【００４７】
そして、電極２ａ，２ｂはクロムや白金等の薄膜電極として構成し、ガラス基板やプラスチック基板にスパッタリングや蒸着、メッキ等で成膜し、フォトリソグラフィー等でエッチングして形成するのがよい。薄膜の厚さは５０ｎｍ〜２００ｎｍ程度のものが多数のパールチェインを形成する上で望ましい。なお、電極２ａ，２ｂの材質はクロムや白金に限らず、交流電圧を印加したとき電気分解が生じないイオン化傾向の小さい金属であればよい。
【００４８】
測定チャンバー１は、図１に示すように電極部２を別体として内部に収容するほかに、電極部２の基板を覆った構造とすることもできる。例えば、電極２ａ，２ｂの周囲にスペーサを配置して、このスペーサ上にガラス板やプラスチック板等を載せて接着すれば微小な測定チャンバー１をコンパクトに作ることができる。試料液が少量である場合に有効であり、攪拌に代えて閉じた流れで流動させて微生物を電極２ａ，２ｂ付近に導き、微生物濃度を均一化することができる。
【００４９】
次に、３は電極２ａ，２ｂ間に誘電泳動を発生させるために交流電圧を印加する誘電泳動用電源部、４は電極２ａ，２ｂ間のインピーダンスを測定することができる測定部、５はマイクロプロセッサ等から構成され、作業域に制御用のプログラムや各種データをロードして機能し、少なくとも誘電泳動用電源部３及び測定部４を制御するとともに、演算を行う演算制御部、６は時計手段である。誘電泳動用電源部３は、電極２ａ，２ｂ間に印加する交流電圧の電圧と周波数を演算制御部５によって制御される。本実施の形態１においては、１ｋＨｚ〜１０ＭＨｚの間で周波数を変えることができ、ピーク間電圧（以下、ｐｐと表す）１Ｖｐｐ〜２０Ｖｐｐを印加できるファンクションジェネレータを採用されている。なお、本明細書で交流電圧というのは、正弦波のほか、ほぼ一定の周期で流れの向きを変える三角波、方形波等の電圧を意味し、正負両サイドの電流の平均値が等しいものである。周波数は微生物の種類や懸濁液導電率によって変化し、電圧は電極２ａ，２ｂ間で電気分解が発生せず、誘電泳動力が大きくなるような値が選択される。
【００５０】
測定部４には５００Ω程度の電流検出用の抵抗が設けられ、図１に示す電圧印加回路に直列に挿入されており、これを流れる電流を測定して、電極２ａ，２ｂ間のコンダクタンス（抵抗成分の逆数）を算出している。すなわち、演算制御部５は、時計手段６の計時する、例えば１０ｓｅｃ間隔というタイミングで、誘電泳動用電源部３の周波数と電圧を制御し、電極２ａ，２ｂ間に最適な周波数の交流電圧を印加すると同時に、このとき測定部４に電圧印加回路を流れる電流の測定を行わせ、試料液中の微生物が濃縮されたことによる抵抗成分の測定を行い、逆数をとってコンダクタンスの算出をしている。時計手段６はこのサンプリングのタイミングだけでなく、実施の形態１における微生物測定装置の自動運転のためのスケジューリングのための時間管理を行う。
【００５１】
さて、７は演算制御部５にロードするプログラムや各種データを格納したメモリ部、７ａはメモリ部７の中に設けられ、特定の微生物を捕集して濃縮するための測定用制御データと、コンダクタンスを示す測定部４の出力（以下、ＤＥＰＩＭ出力）から微生物数を換算するための検量線データ等を格納した測定用テーブルである。測定用制御データは、微生物毎に、微生物を懸濁した試料液の懸濁液導電率と、微生物に正の誘電泳動力を作用させることが可能な最適周波数がデータ化されている。同様に、（表１）に示すように、測定用テーブル７ａには、微生物毎に抗原抗体反応を起こさせる抗体の種類、捕集のため印加する電圧Ｖ_C1、流体抵抗を利用して分離するとき印加しておく電圧Ｖ_{C ２}、抗原抗体反応で凝集させるための設定時間ｔ_ｃ１、洗浄を行う洗浄時間ｔ_ｃ２が格納されている。
【００５２】
【表１】

続いて、図１において、８は微生物名や抗体の種類等を入力するためのデータ入力部、９はデータ入力のための表示を行ったり、演算制御部５が検量線データに基づいて算出した微生物数を表示するためのＬＣＤ等の表示部である。また、１０は微生物を含有した試料液を貯めた試料液槽、１１は試料液槽１０と測定チャンバー１を接続し、試料液槽１０内の試料液を測定チャンバー１に導く供給管である。１２は測定チャンバー１から測定後の試料液を排出するための排出管、１３は供給管１１に設けられた電磁弁等の供給バルブ、１４は排出管１２に設けられた排出バルブである。
【００５３】
１５はスターラ等の撹拌装置であって、時計手段６の行う計時に基づいて演算制御部５が誘電泳動を行う前と間に試料液の攪拌を行うものである。これにより、試料液を供給管１１から導入して誘電泳動するとき、微生物濃度を均一化するとともに、多くの微生物を電極２ａ，２ｂ近傍に導くことができる。さらに、必要に応じ、攪拌装置１５は抗原抗体反応が終了した後、反応で凝集化した特定の微生物を残して他の微生物を分離するために流れを起こし、大きさに対する誘電泳動力の差を利用して不要な微生物を洗い流す作用を有している。
【００５４】
また、測定チャンバー１を、電極２ａ，２ｂの周囲にスペーサを配置して、このスペーサ上にガラス板等を載せて接着した微小な測定チャンバー１の場合等には、攪拌装置１５に代えて排出管１２を試料液層１０に接続して閉回路として流動させることで微生物を電極２ａ，２ｂ付近に導くとともに、微生物濃度を均一化する方法と組み合わせで用いるのが適当である。
【００５５】
１６は洗浄液タンク、１７は洗浄管、１８は洗浄バルブである。抗原抗体反応によってこの特定の微生物を凝集させ、攪拌装置１５で流れを発生して未反応の微生物を流体力（後述する誘電泳動力に抗して加える流体の力であって、粘性力に基づく流体抵抗に基づく力）で分離するとともにこれを洗い流す。試料液槽１０や洗浄液タンク１６はヘッド（水頭）が確保できない場合や調節が難しい場合には、ポンプを設けるのも好適である。時計手段６の時間管理で、演算制御部５が所定の時間ｔｃ２が経過すると、洗浄バルブ１８を開いて洗浄液を測定チャンバー１に供給し、電極２ａ，２ｂ間を洗浄する。なお、上述のように未反応の微生物を分離して洗い流すために洗浄液を用いるが、未反応の微生物を分離するプロセスにおいては、洗浄液による分離に代えて、攪拌装置１５の攪拌作用を併用することもできるし、場合によっては攪拌装置１５単独の攪拌作用で分離することも可能である。
【００５６】
１９は特定の微生物と特異的に反応する抗体を含有した抗体液を収容した抗体液タンク、２０は抗体液を測定チャンバー１に導入する抗体導入管、２１は抗体供給バルブである。抗体液タンク１９と抗体導入管２０、抗体供給バルブ２１が本実施の形態１の抗体供給部を構成する。本実施の形態１においては、誘電泳動によって複数の微生物を含有した試料液を電極２ａ，２ｂ間に抗原抗体反応前の前処理として濃縮し、この前処理で濃縮した微生物に特定の微生物とだけ特異的に反応する抗体液を導入する。抗原抗体反応によってこの特定の微生物を凝集させ、攪拌装置１５や洗浄液によって凝集しなかった微生物を流体力で分離し、最後は洗浄液で洗い流し、その後電極２ａ，２ｂ間で特定微生物数をカウントする。これらの処理はすべて電極部２の表面で行われる。
【００５７】
さらに、本実施の形態１の微生物測定装置は、測定セルフチェック機構を備えてそれほど知識がなくとも正しく測定できるようになっている。すなわち、本微生物測定装置が正しく準備されていなければ、測定を行っても無駄になってしまう。そこで、本微生物測定装置は測定開始直後にセルフチェックを行う。測定開始したばかりの状態では微生物が捕集されていない。この状態で電極２ａ，２ｂ間のインピーダンス測定を行ってＤＥＰＩＭ信号を検出すると、懸濁液の導電率と誘電率だけに依存した初期値を示す。もし、演算制御部５が、測定開始直後にＤＥＰＩＭ信号を検出して、懸濁液が示す初期値が通常値の範囲と大きく異なっていると判断すると、電極部２が破損して短絡されていたり汚染されているなどの可能性があり、アラームを鳴らし、または表示部９によって警告を表示して電極や装置の確認や交換を促す。懸濁液が示す初期値の通常値は、測定用テーブル７ａに懸濁液毎に用意しておくか、格納されている検量線データを利用して判定すればよい。このようなセルフチェックを行うことによって、専門家でなくとも簡単に微生物数の測定が確実に行える。
【００５８】
続いて、図２（ａ）（ｂ）（ｃ），図３，図４に基づき、本実施の形態１の微生物数測定装置及び微生物数測定方法により特定の微生物だけを選択的に測定する手順について説明する。図２（ａ）（ｂ）（ｃ）では簡単のため、懸濁されている微生物は微生物Ａ，Ｂの２種類のみであり、微生物濃度はほぼ等しい場合を想定している。図２（ａ）に示すように、ステップ１において誘電泳動による前捕集を行う。試料液中に設置した電極２ａ，２ｂ間に電圧Ｖ_ｃ１を印加し、誘電泳動力によりすべての微生物Ａ，Ｂを捕集する。ＤＥＰＩＭ信号が飽和傾向を示したら前捕集を終了する。これら微生物Ａ，Ｂの大きさや誘電特性に顕著な違いがない場合、（数１）から分かるように両者にはほぼ等しい誘電泳動力が作用し、共に電極に捕集される。この時、ＤＥＰＩＭ法によるインピーダンス計測を行うと、微生物Ａ，Ｂどちらの捕集もほぼ同じインピーダンス変化をもたらし、このような場合両者を区別することはできない。
【００５９】
ステップ２において、複数種類の微生物Ａ，Ｂから抗原抗体反応で特定の微生物Ａのみを選択する。すなわち、微生物Ａ，Ｂの前捕集が完了した状態で、微生物Ａと特異的に反応する抗体を含有した抗体液を添加する。このとき誘電泳動により微生物Ａは密集して互いに接触した状態となっており、このような状態の微生物に抗体を作用させると抗体が架橋となって微生物Ａのみが集合体を形成し、いわゆる凝集反応が起こる。時計手段６が所定の時間ｔ_ｃ１を計時すると、凝集反応が終了したと判断する。
【００６０】
続いて、ステップ３において、時間ｔ_ｃ１が経過すると、演算制御部５は電圧を誘電泳動力による微生物捕集がそれ以上起こらない程度の電圧Ｖ_ｃ２に低下させる。ただし、電極２ａ，２ｂ間では十分に高い電界が維持されるので既に捕集された微生物は拡散せずにそのまま保持される。そして、流体力で微生物の選別を行う。（数１）から明らかなように、誘電泳動力Ｆ_ＤＥＰは基本的に電界（電圧V）の二乗ならびに微生物の大きさ（半径ａ）の３乗に比例し（数４）が成立する。
【００６１】
【数４】

但し、Ａは比例定数である。誘電泳動を行う際、測定チャンバー１内の試料液を攪拌し、流れを生じさせると、試料液中の微生物には粘性力に基づく流体抵抗が発生する。攪拌や洗浄液の流れで生じる流速は低いためレイノルズ数との関係から、微生物と試料液の相対速度が一定の場合、通常粘性力Ｆ_Ｖは微生物の大きさ（半径a）に比例し（数５）が成立する。
【００６２】
【数５】

但し、Bは比例定数である。
（数４）（数５）より、粘性力Ｆ_Ｖに対する誘電泳動力Ｆ_ＤＥＰの比を相対誘電泳動力Ｆ*_ＤＥＰとして定義すると、Ｆ*_ＤＥＰは（数６）で表される。
【００６３】
【数６】

（数６）の関係を図示すると図３に示すようになる。抗体液を添加するより微生物が凝集反応を起こすとその見かけ上の半径が大きくなり、電圧の二乗Ｖ^２に対する増加割合は上昇する。すなわち、凝集前の微生物に作用する相対誘電泳動力Ｆ*_ＤＥＰは、図３中の直線ＯＡに沿って変化するが、凝集後は直線ＯＢに沿って変化する。
【００６４】
電極２ａ，２ｂ表面に流れがある場合、粘性力Ｆ_Ｖに打ち勝って誘電泳動力で微生物を電極に捕集・保持するためには、相対誘電泳動力Ｆ*_ＤＥＰが一定以上の大きさである必要がある。目安としては、誘電泳動力が粘性力よりも大きい場合、即ち相対誘電泳動力Ｆ*_ＤＥＰ＞１をその条件と考えることができる。この相対誘電泳動力Ｆ*_ＤＥＰの閾値を図中では直線ＣＤで表しており、この直線よりも上側の領域（図中ハッチング部分）では誘電泳動力が相対的に流れから受ける粘性力Ｆ_Ｖよりも勝っている。
【００６５】
図３中で電圧をＶ_C1に設定したときの相対誘電泳動力Ｆ*_ＤＥＰは、凝集した微生物（半径ａ₁）、凝集していない微生物（半径ａ₂）のどちらに対しても直線ＣＤより上側にあるので、粘性力に打ち勝って微生物を捕集することができる。一方、電圧をＶ_{C ２}に設定したときの相対誘電泳動力Ｆ*_ＤＥＰは、凝集した微生物に対しては点Ｅとなり直線ＣＤより上側にあるので微生物を捕集することができるが、凝集していない微生物では直線ＣＤより下側の点Ｆとなり、粘性力Ｆ_Ｖの方が優勢であるため誘電泳動力で微生物を捕集できない。
【００６６】
従って、ステップ１での前捕集を電圧Ｖ_C1で行えば、微生物Ａ，Ｂの両方が誘電泳動で捕集される。しかし、ステップ２に進み凝集反応を行った後、ステップ３で電圧をＶ_{C ２}まで低下させ、試料液を流動させると、凝集体を形成した微生物Ａは電極２ａ，２ｂに捕集されたままであるが、凝集していない微生物Ｂは粘性力Ｆ_Ｖによって電極２ａ，２ｂから離脱して洗い流されてしまう。即ち、最終的に検出対象である微生物Ａだけが電極に捕集された状態で残ることになり、この状態でインピーダンスを測定すればＤＥＰＩＭ法により微生物Ａだけを選択的に検出することができる。
【００６７】
以上説明したステップ１〜３に従って、具体的に、大腸菌と緑膿菌の等量混合した試料液から大腸菌を選択的に測定したときの測定結果を図４に示す。図４において、測定は懸濁液導電率０．１ｍＳ／ｍ、誘電泳動周波数１ＭＨｚの条件下で行ったものである。大腸菌と緑膿菌は大きさや誘電特性が類似しており、従来のＤＥＰＩＭ法では試料液からどちらかだけを選択的に検出することはできない。
【００６８】
図４の測定は、ステップ１で電圧１０Ｖで誘電泳動による前捕集を行っている。この時認められるコンダクタンスの過渡的増加は、図２（ａ）に示すように誘電泳動力により試料液中の大腸菌と緑膿菌が共に電極ギャップ間に捕集された結果生じたものである。次に、ステップ２で試料液に大腸菌の抗体を１０ｍｌ添加し、図２（ｂ）に示すように大腸菌の凝集体を電極２ａ，２ｂ間で形成した。この時コンダクタンスは一定に保たれたままである。凝集反応終了後、ステップ３で電圧を１Ｖに低下させ、図２（ｃ）に示すように試料液を攪拌し洗浄液を導入して粘性力によって凝集していない緑膿菌のみを電極から離脱させると、コンダクタンスが約５０％急激に低下している。ステップ３の後で観測されるコンダクタンス（残留コンダクタンス）は凝集して電極２ａ，２ｂ間に保持されている大腸菌の濃度に対応しており、この残留コンダクタンスによって試料液中の大腸菌濃度を定量されたことになる。
【００６９】
なお、以上の説明は二種類の微生物が混合された試料液を対象としているが、三種類以上の場合であっても同じ方法で特定の微生物のみを選択的に検出できるものである。
（実施の形態２）
以下、本発明における実施の形態２における微生物検出装置及び微生物検出方法について説明する。図５は本発明の実施の形態２における微生物測定装置の構成図、図６（ａ）は本発明の実施の形態２における微生物測定装置の測定実施前の電極チップ図、図６（ｂ）は本発明の実施の形態２における微生物測定装置の電極部が特定の微生物を選択的に測定するための凝集プロセス図、図６（ｃ）は本発明の実施の形態２における微生物測定装置の電極部が特定の微生物を選択的に測定するための洗浄プロセス図、図７は本発明の実施の形態２における測微生物定装置の特定の微生物の測定結果図である。実施の形態１の微生物測定装置と同一符号の部材は、実施の形態２の微生物測定装置においても同一の部材を示しており、詳細な説明は実施の形態１の微生物測定装置に譲って詳細な説明は省略する。
【００７０】
図５において、１は複数の微生物が混合された試料液を収容する測定チャンバー、２’は微生物数を測定するための微生物測定用電極チップ、２ａ，２ｂは微生物測定用電極チップ２’に設けられた一対の電極である。図６（ａ）（ｂ）（ｃ）において、２ｃは微生物測定用電極チップ２’上で電極２ａ，２ｂ間に固相化された抗体である。本明細書においては、抗体をガラス基板等の表面に吸着させ、抗原抗体反応に利用することを抗体の固相化という。本実施の形態２においては、誘電泳動によって複数の微生物を含有した試料液を電極２ａ，２ｂ間に抗原抗体反応の前処理として濃縮し、この前処理で濃縮した微生物に対して特定の微生物と特異的に反応する固相化された抗体２ｃを作用させる。抗原抗体反応によってこの特定の微生物は微生物測定用電極チップ２’上に抗体２ｃを介して固定される。後述するように攪拌装置１５によって試料液を流動させ、未反応の微生物を分離し、洗浄液を導入して未反応の微生物を洗い流す。その後電極２ａ，２ｂ間で特定微生物数をカウントする。これらの処理がすべて微生物測定用電極チップ２’の表面で行われる。
【００７１】
固相化の方法は、物理的方法や化学的方法など様々であるが、本実施の形態２においては、抗体２ｃは微生物測定用電極チップ２’の電極基板をアセトンで清浄化し、その後透析済みの抗体溶液を電極基板上に滴下し、そのままの状態で４℃に保ち、１５から１８時間放置して固相化する。なお、微生物測定用電極チップ２’は測定毎に交換するといった使い捨てタイプの電極とすることができる。
【００７２】
３は誘電泳動用電源部、４は電極２ａ，２ｂ間のインピーダンスを測定することができる測定部、５は演算制御部、６は時計手段である。また、７はメモリ部、７ａはメモリ部７の中に設けられ、特定の微生物を濃縮するための測定用制御データと、微生物数を換算するための検量線データ等を格納した測定用テーブルである。実施の形態２の測定用制御データは（表２）に示すようなもので、測定用テーブル７ａには、微生物毎に抗原抗体反応を起こさせる抗体の種類、捕集のため印加する電圧Ｖ_C1、流体力で分離するとき印加しておく電圧Ｖ_{C ２}、抗原抗体反応で凝集させるための設定時間ｔ_ｃｌ、洗浄を行う洗浄時間ｔ_ｃ２が少なくとも格納されている。
【００７３】
【表２】

８は微生物名や懸濁液導電率を入力するためのデータ入力部、９は表示部、１０は試料液槽、１１は試料液を測定チャンバー１に導く供給管である。１２は測定チャンバー１から測定後の試料液を排出するための排出管、１３は供給管１１に設けられた電磁弁等の供給バルブ、１４は排出管１２に設けられた排出バルブである。１５は撹拌装置である。１６は洗浄液タンク、１７は洗浄管、１８は洗浄バルブである。
【００７４】
続いて、図６（ａ）（ｂ）（ｃ），図７に基づき、本実施の形態２の微生物数測定装置及び微生物数測定方法により特定の微生物だけを選択的に測定する手順について説明する。図６（ａ）（ｂ）（ｃ）では簡単のため、懸濁されている微生物は微生物Ａ，Ｂの２種類のみであり、微生物濃度はほぼ等しい場合を想定している。図６（ａ）に示すように、ステップ１において、抗体２ｃの微生物測定用電極チップ２’への固相化を行う。ＤＥＰＩＭ法で用いる微生物測定用電極チップ２’は、ガラス基板の表面にクロムなどの金属薄膜を作製した後にフォトリソグラフィーによりパターン形成したものであり、電極２ａ，２ｂ間はガラス基板が露出した状態であり、このギャップ部分に抗体２ｃを固相化する。
【００７５】
次に、ステップ２において、微生物の誘電泳動による前捕集と、固相化した抗体２ｃによる微生物測定用電極チップ２’への特定の微生物の固定を行う。すなわち、試料液中に設置した電極に電圧Ｖ_ｃ１を印加し、誘電泳動力により先ずすべての微生物Ａ，Ｂを捕集する。ＤＥＰＩＭ信号が飽和傾向を示したら前捕集は終了である。微生物Ａ，Ｂの大きさや誘電特性に顕著な違いがない場合、（数１）から予想されるように両者にはほぼ等しい誘電泳動力が作用し、共に電極２ａ，２ｂに捕集される。この時、ＤＥＰＩＭ法によるインピーダンス測定を行うと、微生物Ａ，Ｂどちらの捕集もほぼ同じインピーダンス変化をもたらし、両者を区別することはできない。誘電泳動された微生物Ａ，Ｂの一部は、電極２ａ，２ｂ間のガラス基板表面に接触する状態で保持される。ステップ１で微生物Ａの抗体がガラス基板上に固相化してあるので、ガラス基板表面に接触した微生物Ａは抗体と結合し、従って結果的にガラス表面に固定される。
【００７６】
続いて、ステップ３において、洗浄液の流体抵抗（粘性力）による選別を行う。時計手段６が予め設定した時間ｔ_ｃ１が経過したら、抗原抗体反応は終了したと判断し、電圧Ｖ_ｃ２に低下させて誘電泳動力を減少させ、さらに攪拌か洗浄液の導入で試料液を流動させ微生物に流体力（粘性力）を作用させる。粘性力が適当な値であれば、ステップ２で固相化された抗体２ｃによりガラス表面に固定された微生物Ａは、電圧を下げても電極２ａ，２ｂ間に保持される。一方、固定化されていない微生物Ｂは流動する液の粘性力によって電極２ａ，２ｂから離脱して最終的には洗浄液で洗い流されてしまう。すなわち、最終的には検出対象である微生物Ａだけが電極に捕集された状態で残ることになる。この状態でインピーダンスを測定すれば本実施の形態２の微生物測定装置で微生物Ａだけが選択的に検出されたことになる。
【００７７】
以上説明したステップ１〜３に従って、具体的に、大腸菌と緑膿菌の等量混合した試料液から大腸菌を選択的に測定したときの測定結果を図７に示す。図７において、測定は懸濁液導電率０．１ｍＳ／ｍ、誘電泳動周波数１ＭＨｚの条件下で行ったものである。大腸菌と緑膿菌は大きさや誘電特性が類似しており、従来のＤＥＰＩＭ法では混合懸濁液からどちらかだけを選択的に検出することはできない。
【００７８】
まずステップ１において、電極のガラス基板表面に大腸菌の抗体を物理的吸着法によって固相化する。ステップ２では電圧１０Ｖで誘電泳動による前捕集を行っている。この時認められるコンダクタンスの過渡的増加は、誘電泳動力により懸濁液中の大腸菌と緑膿菌が共に電極ギャップ間に捕集された結果生じたものである。ＤＥＰＩＭ法ではこのコンダクタンスの初期増加率から菌濃度を定量的に推定することができるが、混合懸濁液では両方の細菌の混合濃度に対応した信号が得られる。また、この時捕集された大腸菌の一部は、ガラス基板表面に固相化された抗体２ｃと結合する。
【００７９】
前捕集終了後、ステップ３で電圧を１Ｖに低下させ、図６（ｃ）に示すように攪拌や洗浄液の導入で流動を起こし、粘性力により固相化した抗体２ｃと結合していない緑膿菌のみを電極から離脱させると、コンダクタンスが約５０％急激に低下した。ステップ３の後で観測されるコンダクタンス（残留コンダクタンス）は固相化された抗体２ｃによって電極２ａ，２ｂ間に保持されている大腸菌の濃度に対応しており、この残留コンダクタンスによって混合懸濁液中の大腸菌濃度を定量できる。
【００８０】
以上の説明は二種類の微生物の試料液を対象としているが、三種類以上の場合であっても同じ方法で特定の微生物のみを選択的に検出できる。
【００８１】
【発明の効果】
本発明の請求項１に記載の微生物測定装置は、複数種類の微生物を第１の誘電泳動により濃縮し、抗体供給部から抗体液を供給し、特定の微生物と特異的に反応させ、これに第２の誘電泳動と流動作用を加えることにより凝集して他の微生物を分離し、電極間のコンダクタンス変化から微生物数を算出して出力するから、濃縮のために長時間をかける必要がなく、この濃縮した微生物に特定の微生物と特異的に反応する抗体液を供給し、第２の誘電泳動と流動作用を加えることで複数種類の微生物の中から測定対象の特定の微生物だけを選択することができ、選択した後の特定の微生物をインピーダンス測定することにより、コンダクタンス変化から微生物数を測定するため、混合懸濁液の中で特定の微生物を高感度で迅速に、専門家によらなくとも簡易に定量することができる。
【００８２】
本発明の請求項２に記載の微生物測定装置は、抗体液供給部が抗体液タンクと、抗体液供給管と、抗体液バルブを備えたから、抗体液バルブを操作することにより、濃縮した複数種類の微生物に抗体液を自動的に供給することができる。
【００８３】
本発明の請求項３に記載の微生物測定装置は、測定チャンバーには攪拌装置が設けられ、演算制御部が誘電泳動を行った後に、特定の微生物以外の微生物を分離するための試料液の攪拌を行うから、微生物濃度を均一化し、電極近傍に補修することができる。さらに、抗原抗体反応後に、抗体と特異的に反応した特定の微生物以外の微生物を攪拌による流動によって分離することができる。
【００８４】
本発明の請求項４に記載の微生物測定装置は、演算制御部が時計手段の行う計時に基づいて、洗浄バルブを開いて洗浄液を測定チャンバーに供給し、試料液を排出した後チャンバー内を洗浄するから、抗体と特異的に反応した特定の微生物以外の微生物を洗浄液の流体力で分離することができ、分離した未反応の微生物を洗い流してインピーダンス測定を可能にすることができる。
【００８５】
本発明の請求項５に記載の微生物測定装置は、微生物測定用電極チップに、一対の電極の基板上に特定の微生物と特異的に反応する抗体が固相化され、交流電圧を印加して複数種類の微生物を濃縮し、固相化された抗体と特定の微生物とを特異的に反応させて分離し、電極間のコンダクタンス変化から特定の微生物数を算出して出力するから、交流電圧を電極間に印加して試料液に含有される複数種類の微生物を第１の誘電泳動により濃縮するため、濃縮のために長時間をかける必要がなく、特定の微生物と特異的に反応する抗体を固相化し、特定の微生物だけ微生物測定用電極チップに固定し第２の誘電泳動と流動作用を加えることにより他の微生物を分離するから、複数種類の微生物の中から測定対象の特定の微生物だけを選択することができ、選択した後の特定の微生物をインピーダンス測定することにより、コンダクタンス変化から微生物数を測定するため、混合懸濁液の中で特定の微生物を高感度で迅速に、専門家によらなくとも簡易に定量することができる。
【００８６】
本発明の請求項６に記載の微生物測定装置は、特定の微生物と他の微生物に交流電圧を印加してそれぞれに誘電泳動力を作用させ、該誘電泳動力と流動作用による流体力の差を利用して分離するから、反応した特定の微生物の集合体と他の微生物の大きさの違いによる差が誘電泳動力と流体力の差に変換され、この差を利用することで分離が簡単且つ迅速に行える。
【００８７】
本発明の請求項７に記載の微生物測定用電極チップは、電極間の基板上には特定の微生物と特異的に反応する抗体が固相化されたから、特定の微生物と特異的に反応する抗体を固相化し、特定の微生物だけ微生物測定用電極チップに固定し、他の微生物を洗浄することができ、複数種類の微生物の中から測定対象の特定の微生物だけを選択することが安価に実現できる。
【００８８】
本発明の請求項８に記載の微生物測定方法は、複数種類の微生物を第１の誘電泳動で濃縮し、濃縮された微生物に抗体液を供給し、該抗体液と特定の微生物と特異的に反応させ、さらにこの微生物に第２の誘電泳動と流動作用を加えて他の未反応の微生物を分離し、その後電極間のインピーダンスを測定して特定の微生物のみの微生物数を算出から、交流電圧を電極間に印加して試料液に含有される複数種類の微生物を第１の誘電泳動により濃縮するため濃縮に長時間をかける必要がなく、この濃縮した微生物に特定の微生物と特異的に反応する抗体液を供給し、第２の誘電泳動と流動作用を加えて特定の微生物だけ凝集して他の微生物を分離するから、複数種類の微生物の中から測定対象の特定の微生物だけを選択することができ、選択した後の特定の微生物をインピーダンス測定することにより、コンダクタンス変化から微生物数を測定するため、混合懸濁液の中で特定の微生物を高感度で迅速に、専門家によらなくとも簡易に定量することができる。
【００８９】
本発明の請求項９に記載の微生物測定方法は、複数種類の微生物を第１の電気泳動で濃縮し、電極間に固相化された抗体によって特定の微生物と特異的に反応させ、これを第２の誘電泳動と流動作用を加え、該第２の誘電泳動力と流体力によって分離し、その後電極間のインピーダンスを測定して特定の微生物のみの微生物数を算出するから、交流電圧を電極間に印加して試料液に含有される複数種類の微生物を第１の誘電泳動により濃縮するため濃縮のために長時間をかける必要がなく、特定の微生物と特異的に反応する抗体を固相化し、特定の微生物だけ微生物測定用電極チップに固定し第２の誘電泳動と流動作用を加えることにより他の微生物を分離するから、複数種類の微生物の中から測定対象の特定の微生物だけを選択することができ、選択した後の特定の微生物をインピーダンス測定することにより、コンダクタンス変化から微生物数を測定するため、混合懸濁液の中で特定の微生物を高感度で迅速に、専門家によらなくとも簡易に定量することができる。
【図面の簡単な説明】
【図１】本発明の実施の形態１における微生物測定装置の構成図
【図２】（ａ）本発明の実施の形態１における微生物測定装置の電極部が特定の微生物を選択的に測定するための濃縮プロセス図
（ｂ）本発明の実施の形態１における微生物測定装置の電極部が特定の微生物を選択的に測定するための抗原抗体反応プロセス図
（ｃ）は本発明の実施の形態１における微生物測定装置の電極部が特定の微生物を選択的に測定するための洗浄プロセス図
【図３】本発明の実施の形態１における測微生物定装置の微生物選択可能領域図
【図４】本発明の実施の形態１における測微生物定装置の特定の微生物の測定結果図
【図５】図５は本発明の実施の形態２における微生物測定装置の構成図
【図６】（ａ）本発明の実施の形態２における微生物測定装置の未処理時の電極チップ図
（ｂ）本発明の実施の形態２における微生物測定装置の電極部が特定の微生物を選択的に測定するための凝集プロセス図
（ｃ）本発明の実施の形態２における微生物測定装置の電極部が特定の微生物を選択的に測定するための洗浄プロセス図
【図７】本発明の実施の形態２における測微生物定装置の特定の微生物の測定結果図
【図８】細胞質導電率をパラメータとするＲｅ［Ｋ］の周波数特性図
【符号の説明】
１ 測定チャンバー
２ 電極部
２’ 微生物測定用電極チップ
２ａ，２ｂ 電極
２ｃ 抗体
３ 誘電泳動用電源部
４ 測定部
５ 演算制御部
６ 時計手段
７ メモリ部
７ａ 測定用テーブル
８ データ入力部
９ 表示部
１０ 試料液槽
１１ 供給管
１２ 排出管
１３ 供給バルブ
１４ 排出バルブ
１５ 撹拌装置
１６ 洗浄液タンク
１７ 洗浄管
１８ 洗浄バルブ
１９ 抗体液タンク
２０ 抗体導入管
２１ 抗体供給バルブ[0001]
BACKGROUND OF THE INVENTION
The present invention relates to a microorganism measuring apparatus in which anyone can select a specific microorganism and detect it simply and quantitatively, an electrode chip used therefor, and a microorganism detecting method.
[0002]
[Prior art]
In recent years, mass infection by microorganisms, particularly malignant pathogens such as pathogenic Escherichia coli O-157, and bacterial contamination in food production processes have become one of the social problems. It is difficult to immediately take the best measures without mistakes due to such mass infections and bacterial contamination cases, and the later the countermeasures are delayed, the more the damage will spread and the more damage will be caused. The biggest reason for the delay in countermeasures is that the investigation of the pathogen causing the infection cannot be performed quickly and with high sensitivity. Therefore, a new technique for detecting pathogenic bacteria at an early stage and with high sensitivity is eagerly desired.
[0003]
At present, the most commonly used method for detecting pathogenic bacteria is the colony counting method. That is, in this method, a suspension sample containing microorganisms is sprayed on a medium, and the number of viable bacteria is quantified from the number of colonies (colonies) formed as the microorganisms grow. However, this colony counting method requires a surprisingly long time of 1 to several days until colonies are formed, and although it can be quantified, it can increase the damage in urgent group infections and food poisoning. It couldn't be stopped. In many cases, the test sample solution contains a plurality of bacteria, and specific bacteria cannot be quantified without an auxiliary treatment for selective quantification. In addition, complicated manual operations such as sampling from the test object, concentration and dilution, planting in the medium, and counting the number of colonies (CFU) are necessary. For these reasons, it was impossible to deny the possibility of an increase in running costs necessary for the inspection and human error.
[0004]
In the present specification, microorganisms generally include all microorganisms that are subject to so-called microbiology, such as bacteria, fungi, actinomycetes, rickettsia, mycoplasma, and viruses.
[0005]
As another method for selectively quantifying microorganisms, there is an ATP (Adenosine Tri Phosphate) method using luminescence. Since cells such as animals, plants, and bacteria always contain ATP (adenosine triphosphate), the ATP contained in the bacteria in the test sample is extracted with the ATP extraction reagent, and the extracted ATP is then added to the firefly. The amount of ATP is detected by measuring the amount of luminescence with the enzyme (luciferase). Since the ATP content in the microorganism is a certain numerical value, the number of bacteria contained in the sample is estimated by detecting the amount of ATP estimated from the luminescence intensity.
[0006]
However, extraction of ATP with an ATP extraction reagent, action of an enzyme, measurement of luminescence, estimation of ATP, etc. require advanced and complicated work by specialists, and it is not possible for amateurs to make quick measurements. There wasn't. In addition, since all living cells have ATP, they become background ATP in the measurement, so that the separation technique is not perfect and the test becomes uncertain. This method cannot distinguish bacteria, yeasts, fungi and the like as microorganisms, and is difficult to selectively quantify.
[0007]
Furthermore, there is an ELISA (Enzyme-Linked Immunosorbent Assay) method. The ELISA method is a method for measuring a substance to be examined with high sensitivity by labeling an antibody or an antigen with an enzyme, and is mainly used in clinical examinations and the like. A test sample solution is brought into contact with a solid phase such as a plastic surface, and test bacteria in the solution are adsorbed via the proteins of the bacteria. Thereafter, except for the liquid phase, only the bacteria adsorbed on the solid phase are left, and a solution of an antibody that specifically reacts only with the protein contained in the target bacteria is added thereto. If the amount of antigen (bacteria) added first and the amount of antibody added later are within an appropriate range, the amount of antibody bound by the antigen-antibody reaction changes depending on the amount of antigen adsorbed on the solid phase. . If the antibody used is labeled with an enzyme, there will be a lot of enzyme in proportion to the amount of bound antibody, and the enzyme reaction will occur strongly. If a chemical substance that develops color by enzymatic reaction is used, the amount of bound antibody can be determined by measuring the intensity of color (absorbance), and the amount of antigen protein (test bacteria) originally present can also be determined.
[0008]
However, this test method requires complicated experimental procedures, requires a high degree of expertise, and requires concentration to obtain a test target concentration suitable for absorbance measurement. There is a disadvantage of having to spend a long time. In addition, the ELISA method described above measures the color development by modifying the antibody, but also uses the antigen-antibody reaction, and there are an aggregation method and a precipitation method as methods for easily confirming with the naked eye. However, high concentrations of microorganisms are required for measurement using this agglutination method and sedimentation method, and the number of microorganisms must be dramatically increased by culturing at the previous stage, and detection requires a very long time. It is.
[0009]
Next, PCR (Polymerase Chain Reaction) is a technique for replicating billions of genes in a short time based on the sequence of a specific gene using the polymerase chain reaction. First, DNA is extracted from the test object and heated to unwind the double helix structure of the DNA so that it is in a single-stranded state. A gene consists of four bases, and each base binds to a specific base. This base sequence is called a gene sequence, and a pair of gene fragments (called primers) is added thereto. This primer binds to the target gene sequence with extremely high accuracy. By adding a thermostable enzyme (Taq enzyme) to this primer, the original double-stranded DNA is removed from the site where the primer is bound to the single-stranded gene. Let it play. By repeating this process, the DNA increases exponentially, and only the DNA to be examined is detected by the fluorescence detector. However, since dead DNA is also amplified, it is necessary to follow the culture procedure in advance and examine the activity of the test object. And the method of measuring this microbial activity simply and quantitatively is not established. Moreover, since DNA increases explosively, it is difficult to estimate the initial number of bacteria, and there is a problem of lack of quantitativeness.
[0010]
Since the conventional technique has a fatal common defect that the quantification of microorganisms takes time, the present inventor, together with other researchers, measures the number of microorganisms by combining dielectrophoresis and electrical impedance. A method (DEPIM (Dielectrophoretic Impedance Measurement Method)) was proposed.
[0011]
This DEPIM method is characterized in that both the microbial concentration process and the microbial concentration detection process are electrically performed. The concentration process concentrates bacteria on the integrated microelectrode by a dielectrophoresis phenomenon (a phenomenon in which an electric force acts on dielectric particles polarized in an electric field and moves in a certain direction). Quantitative estimation of bacterial concentration from changes in interelectrode impedance during concentration (Yanami, et al .: “Measurement of suspension concentration of Escherichia coli by dielectrophoretic impedance measurement (1): Principle and outline of the device”), IEEJ Proceedings '99, pp.337-340 (1999); Tomita, et al .: “Measurement of Suspension Concentration of Escherichia coli by Dielectrophoretic Impedance Measurement (2) —Estimated Model of Suspension Concentration”, Collection '99, pp.341-344 (1999)).
[0012]
In order to detect microorganisms by this DEPIM method, it is necessary to collect the microorganisms on the microelectrode by dielectrophoretic force. In other words, the number of bacteria collected on the microelectrode is reduced under conditions where the dielectrophoretic force does not sufficiently act, so that the detection signal is reduced. Dielectrophoretic force F acting on microorganisms_DEPIn terms of complex numbers, is theoretically given by (Equation 1) below.
[0013]
[Expression 1]

Here, the complex dielectric constant K is expressed by (Equation 2),
[0014]
[Expression 2]

Also, the complex permittivity ε of the suspension^* _mIs represented by (Equation 3),
[0015]
[Equation 3]

ε_m  : Dielectric constant of suspension
ε^* _m  : Complex permittivity of suspension
ε^* _i  : Complex permittivity of microorganisms
σ Suspension conductivity
ω: Angular frequency of electric field
Also,
a: Radius of microorganism when approximated by a sphere
Re [K]: a parameter that depends on the complex permittivity of the microorganism and the suspension
E: Electric field strength
It is. As is clear from (Equation 1), (Equation 2), and (Equation 3), if the size of the suspension and the microorganism is constant, the dielectrophoretic force F_DEPIs proportional to the parameter Re [K].
[0016]
FIG. 8 is a frequency characteristic diagram of Re [K] using cytoplasmic conductivity as a parameter. In FIG. 8, with the frequency f of the electric field used for dielectrophoresis as a parameter, the dielectrophoretic force is expressed as cytoplasmic conductivity σ._iIt is expressed as a function of According to FIG. 8, it can be seen that a positive dielectrophoretic force works at a frequency of 10 kHz to 1 MHz, and a negative dielectrophoretic force works otherwise. Cytoplasmic conductivity σ_iDepending on the frequency f, the negative dielectrophoretic force works, and the dielectrophoretic force F_DEPIt can be seen that there are cases in which even if it acts on microorganisms, it cannot be collected.
[0017]
Therefore, microorganisms (cytoplasmic conductivity σ_iWhen the frequency f is selected according to the type of (), it is possible to collect by applying a positive dielectrophoretic force or to eliminate it by applying a negative dielectrophoretic force. However, in actual dielectrophoresis, the influence of suspension conductivity and the like must be taken into consideration.
[0018]
However, depending on the type of microorganism, the parameter Re [K] is positive and that of other bacterial species is not all negative, and the positive / negative distribution of the dielectrophoretic force cannot be generalized. In other words, by utilizing the dielectric properties (dielectric constant, conductivity) inherent to microorganisms, a positive dielectrophoretic force is selectively applied to only specific microorganisms from a mixed suspension of various microorganisms, and the DEPIM method is used to It is difficult to detect only microorganisms.
[0019]
Further, when the DEPIM method is actually applied, it is expected that it is often required to detect only specific microorganisms from a suspension containing various and unknown types of microorganisms. Such selective microorganism detection is impossible with the DEPIM method.
[0020]
[Problems to be solved by the invention]
As described above, in the conventional colony counting method, a suspension containing microorganisms is sprayed on a medium, and the number of viable bacteria is quantified from the number of colonies formed as the microorganisms grow. It took a surprisingly long time from one to several days to form. In the case of a sample solution containing a plurality of bacteria, it is not possible to select and quantify specific bacteria without auxiliary treatment. In addition, complicated manual work by specialists is required, and there is a possibility that the running cost required for the inspection increases and human error is caused.
[0021]
In addition, the ATP method requires advanced knowledge by experts to extract ATP with an ATP extraction reagent, allow enzyme to act, measure the amount of luminescence, and estimate the amount of ATP. At that time, there was a lot of background and the inspection was uncertain. This method cannot distinguish bacteria, yeast, fungi, etc. as microorganisms, and is difficult to selectively quantify.
[0022]
The ELISA method has to follow a complicated experimental procedure like the other methods, requires a high degree of expertise, and needs to be concentrated in order to obtain a test target concentration suitable for absorbance measurement. There is a disadvantage that detection requires a long time.
[0023]
Since the PCR method also amplifies DNA of dead bacteria, it is necessary to follow the culture procedure in advance and check the activity of the test object. Moreover, since DNA increases explosively, it is difficult to estimate the initial number of bacteria, and there is a problem of lack of quantitativeness. Expertise is required more than other methods.
[0024]
The DEPIM method proposed by the inventor together with other researchers performs both the microbial concentration and microbial concentration detection processes electrically at the same time. Although it was possible, the dielectrophoretic force was not allowed to be detected only for specific microorganisms in a suspension in which a plurality of types of microorganisms were mixed. While other methods for quantifying microorganisms must be measured by specialists for a long time, only the DEPIM method can solve these problems, but it is specified in sample liquids containing multiple types of microorganisms. These microorganisms could not be quantified with high sensitivity. Therefore, a new microorganism measuring apparatus capable of selectively quantifying specific microorganisms quickly, with high sensitivity, even for a sample solution containing a plurality of types of microorganisms is desired.
[0025]
Therefore, an object of the present invention is to provide a microbe measurement apparatus capable of quantitatively quantifying a specific microbe in a sample solution containing a plurality of types of microbes with high sensitivity and speed without using an expert. .
[0026]
In addition, the present invention provides an inexpensive microbe measurement electrode tip that can quantitate a specific microbe in a sample solution containing a plurality of types of microbes with high sensitivity and speed, easily and without using an expert. With the goal.
[0027]
And this invention aims at providing the microbe measurement method which can quantify a specific microbe in a sample liquid containing a plurality of kinds of microbes with high sensitivity, quickly and easily without using an expert. .
[0028]
[Means for Solving the Problems]
  In order to solve the above problems, the microorganism measuring apparatus of the present invention includes an antibody solution supply unit that supplies an antibody solution for antigen-antibody reaction to microorganisms concentrated between electrodes to the measurement chamber, and The control unit is controlled by the power supply unit for dielectrophoresis.FirstApplying AC voltage to concentrate multiple types of microorganisms, supplying antibody solution from the antibody supply unit to allow specific reactions with specific microorganismsThe second microorganism is applied with a dielectrophoretic force that is greater than the fluid force associated with the flow action and the other microorganism is subjected to a dielectrophoretic force that is less than the fluid force associated with the flow action. Specific microorganismsAgglomerateAs well asAnother microorganism is separated, and a specific number of microorganisms is calculated and output from a change in conductance between the electrodes measured by the measuring unit.
[0029]
Thereby, it is possible to quantify a specific microorganism in a sample solution containing a plurality of types of microorganisms with high sensitivity and quickly without using a specialist.
[0030]
In the electrode chip for measuring microorganisms of the present invention, an antibody that specifically reacts with a specific microorganism is immobilized on a substrate between the electrodes, and microorganisms other than the reacted specific microorganism are separated by a fluid force. The specific number of microorganisms is calculated from the change in conductance and output.
[0031]
Thereby, it is possible to provide an inexpensive microbe measurement electrode tip that allows specific microorganisms to be quantified in a sample solution containing a plurality of types of microorganisms with high sensitivity and speed, easily and without using specialists.
[0032]
  The microorganism measuring method of the present invention applies an alternating voltage between a pair of electrodes to remove a plurality of types of microorganisms.FirstConcentrate by dielectrophoresis, supply antibody solution to the concentrated microorganism, and react with the antibody solution specifically with a specific microorganism.In addition, the second dielectrophoresis and flow action are applied to the microorganism, and the specific microorganism is acted on by applying a dielectrophoretic force larger than the fluid force to the specific microorganism and a dielectrophoretic force smaller than the fluid force to the other microorganism.AgglomerateAs well asAnother unreacted microorganism is separated, and then the impedance between the electrodes is measured to calculate the number of microorganisms of only a specific microorganism.
[0033]
Thereby, it is possible to quantify a specific microorganism in a sample solution containing a plurality of types of microorganisms with high sensitivity and quickly without using a specialist.
[0034]
DETAILED DESCRIPTION OF THE INVENTION
  The invention according to claim 1 of the present invention is a measurement chamber capable of containing a sample solution, and is immersed in the sample solution in the measurement chamber to generate an unequal electric field.FirstConcentrate multiple types of microorganisms contained in the sample solution by dielectrophoresisThen, when this concentrated microorganism is reacted with the antibody, a second dielectrophoresis and a flow action are applied.Isolate other microorganisms, leaving only specific microorganisms that have reacted specifically with the antibody, and measure the impedance at this timecan doA pair of electrodes, a dielectrophoresis power supply unit that applies an alternating voltage between the electrodes, a measurement unit that can measure impedance between the electrodes, a dielectrophoresis power supply unit, and an arithmetic control unit that controls the measurement unit; , A memory part that stores measurement control data and calibration curve data for each microorganism to be measured, and an antibody solution for antigen-antibody reaction against microorganisms concentrated between the electrodes in the measurement chamber An antibody solution supply unit for supplying the operation control unit to the dielectrophoresis power supply unitFirstApply AC voltage to concentrate multiple types of microorganisms, and supply antibody solution from the antibody supply unitTheIt reacts specifically with specific microorganismsThe second microorganism is applied with a dielectrophoretic force that is greater than the fluid force associated with the flow action and the other microorganism is subjected to a dielectrophoretic force that is less than the fluid force associated with the flow action. Agglomerate certain microorganismsFrom the change in conductance between electrodes measured by the measuring part after separating other microorganismsspecificSince it is a microorganism measuring device characterized by calculating and outputting the number of microorganisms, an AC voltage is applied between the electrodes.A plurality of types of microorganisms contained in the sample solutionTo concentrate by dielectrophoresis,There is no need to spend a long time for concentration, and this concentrated microorganism is supplied with an antibody solution that reacts specifically with a specific microorganism.By adding a second dielectrophoresis and flow actionSince only specific microorganisms are aggregated and separated from other microorganisms, only specific microorganisms to be measured can be selected from a plurality of types of microorganisms, and impedance measurement is performed on specific microorganisms after selection. Since the number of microorganisms is measured from the change in conductance, specific microorganisms in the mixed suspension can be quantified with high sensitivity and speed, without using a specialist.
[0035]
In the invention according to claim 2 of the present invention, the antibody solution supply section is provided in the antibody solution tank containing the antibody solution, the antibody solution supply pipe for introducing the antibody solution into the measurement chamber, and the antibody supply pipe. 2. The microbial activity measuring apparatus according to claim 1, further comprising an antibody solution valve, wherein the antibody solution is supplied to the measurement chamber after concentrating a plurality of types of microorganisms. The antibody solution can be automatically supplied to a plurality of concentrated microorganisms.
[0036]
The invention according to claim 3 of the present invention is the microorganism activity measuring device according to claim 1 or 2, wherein the measuring chamber is provided with a stirring device, and the sample solution is stirred. The concentration can be made uniform and repaired in the vicinity of the electrode. Furthermore, after the antigen-antibody reaction, microorganisms other than specific microorganisms that have specifically reacted with the antibody can be separated by a flow by stirring.
[0037]
According to a fourth aspect of the present invention, the measurement chamber is provided with a cleaning pipe for cleaning liquid, a cleaning valve is provided in the cleaning pipe, and the cleaning valve is provided on the basis of the time measured by the clock control means. The microbial activity measuring device according to any one of claims 1 to 3, wherein the microbial activity measuring device according to any one of claims 1 to 3, wherein the microbial activity measuring device according to any one of claims 1 to 3 is opened and supplied to a measurement chamber to separate and wash away specific microorganisms by fluid force. Microorganisms other than the reacted specific microorganism can be separated by the fluid force of the washing liquid, and the separated unreacted microorganism can be washed away to enable impedance measurement.
[0038]
  The invention according to claim 5 of the present invention is a measurement chamber capable of containing a sample solution, and is immersed in the sample solution in the measurement chamber to generate an unequal electric field.FirstConcentrate multiple types of microorganisms contained in the sample solution by dielectrophoresisThen, when this concentrated microorganism is reacted with the antibody, a second dielectrophoresis and a flow action are applied.Leaving only certain microorganisms that have specifically reacted with the antibodyIsolate other microorganisms, A microbe measurement electrode chip having a pair of electrodes that can measure the impedance at this time, a dielectrophoresis power supply unit that applies an alternating voltage between the electrodes, and a measurement that can measure the impedance between the electrodes A control unit for controlling the power supply unit for dielectrophoresis and the measurement unit, and a memory unit for storing a measurement table storing measurement control data and calibration curve data for each microorganism to be measured. In the measurement electrode chip, an antibody that specifically reacts with a specific microorganism is immobilized on a pair of electrode substrates, and a calculation control unit applies an AC voltage by a dielectrophoresis power supply unit to apply a plurality of types of electrodes. By concentrating microorganisms and reacting the immobilized antibody with specific microorganisms specificallyDue to the second dielectrophoretic force and the fluid force associated with the flow actionSince it is a microorganism measuring apparatus characterized by calculating and outputting a specific number of microorganisms from a change in conductance between electrodes measured by a measuring unit, an alternating voltage is applied between the electrodes.A plurality of types of microorganisms contained in the sample solutionBecause it is concentrated by dielectrophoresis, there is no need to spend a long time for concentration. An antibody that specifically reacts with a specific microorganism is immobilized on a solid phase, and only a specific microorganism is immobilized on a microbe measurement electrode chip.By adding a second dielectrophoresis and flow actionSince other microorganisms are separated, only specific microorganisms to be measured can be selected from multiple types of microorganisms, and the number of microorganisms is measured from changes in conductance by impedance measurement of specific microorganisms after selection. Therefore, specific microorganisms in the mixed suspension can be quantified with high sensitivity and speed, without using an expert.
[0039]
  The invention according to claim 6 of the present invention separates other microorganisms while leaving only specific microorganisms that have specifically reacted with the antibody.The specific microorganismAnd applying an AC voltage to other microorganisms to cause the dielectrophoretic force to act on each,And fluid force associated with fluid actionSeparation using the difference ofDo6. The microorganism measuring apparatus according to claim 5, wherein the difference due to the difference in the size of the reacted specific microorganism aggregate and other microorganisms is dielectrophoretic force.And fluid forceBy using this difference, separation can be performed easily and quickly.
[0040]
The invention according to claim 7 of the present invention is a microbe measurement electrode chip used in the microbe measurement apparatus according to claim 6, wherein the antibody specifically reacts with a specific microorganism on the substrate between the electrodes. Since this is an electrode chip for measuring microorganisms characterized in that microorganisms other than specific microorganisms that have been solid-phased are separated by fluid force, and the specific number of microorganisms is calculated and output from the change in conductance between the electrodes. An antibody that specifically reacts with a specific microorganism can be solid-phased, and only a specific microorganism can be immobilized on a microbe measurement electrode chip, and other microorganisms can be separated by fluid force. Selecting only a specific microorganism of interest can be realized at low cost.
[0041]
  The invention according to claim 8 of the present invention applies a plurality of types of microorganisms by applying an alternating voltage between a pair of electrodes.First dielectrophoresisThe antibody solution is supplied to the concentrated microorganism, and the antibody solution reacts specifically with the specific microorganism.In addition, the second dielectrophoresis and flow action are applied to the microorganism, and the specific microorganism is acted on by applying a dielectrophoretic force larger than the fluid force to the specific microorganism and a dielectrophoretic force smaller than the fluid force to the other microorganism.AgglomerateAs well asIt is a microorganism measurement method characterized by separating other unreacted microorganisms and then calculating the number of microorganisms of only a specific microorganism by measuring the impedance between the electrodes.A plurality of types of microorganisms contained in the sample solutionSince it is concentrated by dielectrophoresis, it is not necessary to spend a long time for concentration, and an antibody solution that specifically reacts with a specific microorganism is supplied to this concentrated microorganism.Add the second dielectrophoresis and flow actionSince only specific microorganisms are aggregated and other microorganisms are separated, only a specific microorganism to be measured can be selected from a plurality of types of microorganisms, and by impedance measurement of the selected specific microorganism, Since the number of microorganisms is measured from the change in conductance, specific microorganisms in the mixed suspension can be quantified with high sensitivity and speed, without using a specialist.
[0042]
  The invention according to claim 9 of the present invention applies an AC voltage between a pair of electrodes to remove a plurality of types of microorganisms.First dielectrophoresisAntibody concentrated on the electrode and immobilized between electrodesTheReact specifically with specific microorganisms,Furthermore, the second dielectrophoresis and flow action are applied to the microorganism, and the second dielectrophoresis force and fluid forceLeaving only certain microorganisms that have specifically reacted with the antibodyIsolate other microorganisms,Then, the impedance between the electrodes is measured to calculate the number of microorganisms of only a specific microorganism, so that an alternating voltage is applied between the electrodes.A plurality of types of microorganisms contained in the sample solutionConcentration by dielectrophoresis eliminates the need for a long time for concentration. An antibody that specifically reacts with a specific microorganism is immobilized on a solid phase, and only specific microorganisms are immobilized on a microbe measurement electrode chip.By adding a second dielectrophoresis and flow actionSince other microorganisms are separated, only specific microorganisms to be measured can be selected from multiple types of microorganisms, and the number of microorganisms is measured from changes in conductance by impedance measurement of specific microorganisms after selection. Therefore, specific microorganisms in the mixed suspension can be quantified with high sensitivity and speed, without using an expert.
(Embodiment 1)
  Hereinafter, a microorganism detection apparatus and a microorganism detection method according to Embodiment 1 of the present invention will be described. FIG. 1 is a configuration diagram of a microorganism measuring device according to Embodiment 1 of the present invention, and FIG. 2A is a diagram for the electrode unit of the microorganism measuring device according to Embodiment 1 of the present invention to selectively measure a specific microorganism. Fig. 2 (b) is an enrichment process diagram, Fig. 2 (b) is an antigen-antibody reaction process diagram for the electrode part of the microorganism measuring apparatus according to Embodiment 1 of the present invention to selectively measure a specific microorganism, and Fig. 2 (c) is the present invention. FIG. 3 is a cleaning process diagram for selectively measuring a specific microorganism by the electrode section of the microorganism measuring device according to the first embodiment of the present invention. FIG. 3 is a diagram showing a region where microorganisms can be selected by the microorganism measuring device according to the first embodiment of the present invention. FIG. 4 is a measurement result diagram of a specific microorganism in the microbe measuring apparatus according to Embodiment 1 of the present invention.
[0043]
In FIG. 1, 1 is a measurement chamber for storing a sample solution in which a plurality of microorganisms are mixed in a suspension in order to measure a specific microorganism by the DEPIM method (Dielectrophoretic Impedance Measurement Method), and 2 is for measuring the number of microorganisms. The electrode portions 2a and 2b are a pair of electrodes provided on the electrode portion 2. In the first embodiment, by the later-described dielectrophoresis performed between the electrodes 2a and 2b, the microbial concentration action and the sieving action for separating the specific microorganisms aggregated by the antigen-antibody reaction and other microorganisms by fluid force I do.
[0044]
  That is, the feature of the first embodiment is that a sample solution containing a plurality of microorganisms is concentrated as a pretreatment between the electrodes 2a and 2b by dielectrophoresis, and a specific microorganism is selected from the microorganisms that have been concentrated in this pretreatment. An antibody solution that specifically reacts with is introduced. After aggregating this specific microorganism by the antigen-antibody reaction, a weak dielectrophoretic force is applied between the electrodes 2a and 2b, and the electrode 2a and 2b is introduced on the electrodes 2a and 2b by introducing a washing liquid and / or stirring as described later. The sample liquid is flowed, and only other unreacted microorganisms are separated and removed by the relationship between the fluid force and the dielectrophoretic force, and finally washed away with the washing liquid. Thereafter, the number of specific microorganisms is counted between the electrodes 2a and 2b. All of these processes are performed on the surface of the electrode part 2. In the first embodiment, by aggregationAggregateThe difference in size is used, but this difference in size is reflected in fluid resistance, dielectrophoretic force, etc..
[0045]
Here, the antigen-antibody reaction process which is a premise for separating a specific kind of microorganisms from the above-described plurality of kinds of microorganisms will be described. When an antibody solution that specifically reacts only with a specific protein contained in a specific microorganism is added, an antigen-antibody reaction occurs. By utilizing this property, it becomes possible to selectively detect a specific microorganism (antigen). In the case of the first embodiment, after concentrating the entire microorganisms by dielectrophoresis, specific microorganisms are aggregated by antigen-antibody reaction, and unreacted microorganisms do not aggregate. Therefore, the difference in dielectrophoretic force caused by this difference in size. , And measure by leaving only specific microorganisms by introducing fluid and stirring fluid force. Since the microbial concentration necessary for performing the antigen-antibody reaction is obtained by the dielectrophoretic force without culturing as in the prior art, it can be detected in a short time. Since the number of microorganisms is detected as an electrical impedance signal, quantitative measurement is possible.
[0046]
The shape of the electrodes 2a and 2b is such that an unequal electric field is generated to perform dielectrophoresis, and a large number of microbial chains called pearl chains can be bridged between the electrodes 2a and 2b, for example, castle wall type electrodes, comb teeth A shape such as a mold electrode is preferred. The castle wall type electrode is formed by disposing electrodes 2a and 2b each having a plurality of rectangular electrode pieces protruding at intervals of one pitch (for example, 50 μm to 100 μm) on both sides, with a pitch of 5 μm to 10 μm. The electric field concentrates between the edges of the rectangular electrode pieces 2a and 2b. In addition, the comb-shaped electrode has a pair of electrodes in which teeth (for example, 30 μm to 100 μm width) formed like a comb are nested and inserted into the groove, and is opposed by a narrow gap (for example, 5 μm to 10 μm width). It is an electrode, and the electric field is an uneven electric field formed between edges in the thickness direction, and a large number of pearl chains are formed. It is desirable to select the most appropriate value for the gap and pitch depending on the size of the microorganism.
[0047]
The electrodes 2a and 2b are preferably formed as thin film electrodes such as chromium and platinum, formed on a glass substrate or plastic substrate by sputtering, vapor deposition, plating or the like, and etched by photolithography or the like. A thin film having a thickness of about 50 nm to 200 nm is desirable for forming a large number of pearl chains. The material of the electrodes 2a and 2b is not limited to chromium or platinum, but may be any metal that does not cause electrolysis when an AC voltage is applied and has a low ionization tendency.
[0048]
  As shown in FIG. 1, the measurement chamber 1 can be configured to cover the substrate of the electrode unit 2 in addition to housing the electrode unit 2 as a separate body. For example, if a spacer is arranged around the electrodes 2a and 2b and a glass plate, a plastic plate or the like is placed on the spacer and adhered, the minute measurement chamber 1 can be made compact. Effective when sample solution is small, instead of stirringIn a closed flowFlowTheGuide the microorganisms near the electrodes 2a and 2b to make the microorganism concentration uniformbe able to.
[0049]
Next, 3 is a power supply unit for dielectrophoresis that applies an alternating voltage to generate dielectrophoresis between the electrodes 2a and 2b, 4 is a measurement unit that can measure the impedance between the electrodes 2a and 2b, and 5 is a micro An arithmetic control unit configured by a processor and the like, functions by loading a control program and various data into the work area, controls at least the dielectrophoresis power supply unit 3 and the measurement unit 4, and includes a clock means. It is. The power source unit 3 for dielectrophoresis is controlled by the arithmetic control unit 5 with respect to the voltage and frequency of the alternating voltage applied between the electrodes 2a and 2b. In the first embodiment, a function generator that can change the frequency between 1 kHz and 10 MHz and can apply a peak-to-peak voltage (hereinafter referred to as pp) of 1 Vpp to 20 Vpp is employed. In this specification, the AC voltage means a voltage such as a sine wave, a triangular wave, a square wave, etc. that changes the flow direction at a substantially constant cycle, and the average value of the currents on both the positive and negative sides is equal. is there. The frequency varies depending on the type of microorganism and the conductivity of the suspension, and the voltage is selected so that electrolysis does not occur between the electrodes 2a and 2b and the dielectrophoretic force increases.
[0050]
The measurement unit 4 is provided with a resistance for current detection of about 500Ω, and is inserted in series in the voltage application circuit shown in FIG. 1, and the current flowing through this is measured to determine the conductance (resistance) between the electrodes 2a and 2b. The reciprocal of the component is calculated. That is, the arithmetic control unit 5 controls the frequency and voltage of the dielectrophoresis power supply unit 3 at a timing of 10 sec, for example, which is measured by the clock means 6, and applies an AC voltage having an optimal frequency between the electrodes 2a and 2b. At the same time, the current flowing through the voltage application circuit is measured by the measurement unit 4 at this time, the resistance component due to the concentration of microorganisms in the sample solution is measured, and the conductance is calculated by taking the reciprocal. . The clock means 6 performs not only the sampling timing but also time management for scheduling for automatic operation of the microorganism measuring apparatus in the first embodiment.
[0051]
Now, 7 is a memory unit storing a program to be loaded into the arithmetic control unit 5 and various data, 7a is provided in the memory unit 7, and measurement control data for collecting and concentrating a specific microorganism, It is the measurement table which stored the calibration curve data etc. for converting the number of microorganisms from the output (henceforth, DEPIM output) of the measurement part 4 which shows conductance. For the control data for measurement, the suspension conductivity of the sample solution in which the microorganism is suspended and the optimum frequency at which a positive dielectrophoretic force can be applied to the microorganism are converted into data for each microorganism. Similarly, as shown in (Table 1), the measurement table 7a includes the type of antibody that causes an antigen-antibody reaction for each microorganism, and the voltage V applied for collection._C1, Voltage V applied when separating using fluid resistance_{C 2}, Set time t for aggregation by antigen-antibody reaction_c1Cleaning time t for cleaning_c2Is stored.
[0052]
[Table 1]

Subsequently, in FIG. 1, 8 is a data input unit for inputting a microorganism name, an antibody type, and the like, 9 is a display for data input, and the calculation control unit 5 calculates based on the calibration curve data. It is a display unit such as an LCD for displaying the number of microorganisms. Reference numeral 10 denotes a sample solution tank that stores a sample solution containing microorganisms, and 11 denotes a supply pipe that connects the sample solution tank 10 and the measurement chamber 1 and guides the sample solution in the sample solution tank 10 to the measurement chamber 1. Reference numeral 12 denotes a discharge pipe for discharging the measured sample solution from the measurement chamber 1, 13 denotes a supply valve such as an electromagnetic valve provided in the supply pipe 11, and 14 denotes a discharge valve provided in the discharge pipe 12.
[0053]
Reference numeral 15 denotes a stirrer such as a stirrer, which stirs the sample liquid before the arithmetic control unit 5 performs dielectrophoresis based on the time measured by the clock means 6. As a result, when the sample solution is introduced from the supply tube 11 and subjected to dielectrophoresis, the concentration of microorganisms can be made uniform and many microorganisms can be led to the vicinity of the electrodes 2a and 2b. Furthermore, if necessary, after the antigen-antibody reaction is completed, the stirrer 15 causes a flow to separate other microorganisms while leaving the specific microorganisms aggregated by the reaction. It has the effect of washing away unwanted microorganisms.
[0054]
Further, in the case of a minute measurement chamber 1 in which a spacer is arranged around the electrodes 2a and 2b and a glass plate or the like is placed on and adhered to the measurement chamber 1, the measurement chamber 1 is discharged instead of the stirring device 15. It is appropriate to connect the tube 12 to the sample liquid layer 10 and flow as a closed circuit to guide the microorganisms to the vicinity of the electrodes 2a and 2b and to use in combination with a method for equalizing the microorganism concentration.
[0055]
Reference numeral 16 is a cleaning liquid tank, 17 is a cleaning pipe, and 18 is a cleaning valve. This specific microorganism is agglutinated by the antigen-antibody reaction, and a flow is generated by the stirring device 15 to cause the unreacted microorganism to flow to the fluid force (the fluid force applied against the dielectrophoretic force described later, based on the viscous force. This is separated with a force based on the fluid resistance and washed away. If the sample liquid tank 10 and the cleaning liquid tank 16 cannot secure a head (water head) or are difficult to adjust, it is also preferable to provide a pump. In the time management of the clock means 6, when the predetermined time tc2 elapses, the arithmetic control unit 5 opens the cleaning valve 18, supplies the cleaning liquid to the measurement chamber 1, and cleans the space between the electrodes 2a and 2b. As described above, a cleaning solution is used to separate and wash off unreacted microorganisms. However, in the process of separating unreacted microorganisms, the stirring action of the stirring device 15 is used in combination instead of separation with the cleaning solution. In some cases, it is possible to separate by the stirring action of the stirring device 15 alone.
[0056]
Reference numeral 19 denotes an antibody solution tank containing an antibody solution containing an antibody that specifically reacts with a specific microorganism, 20 denotes an antibody introduction tube for introducing the antibody solution into the measurement chamber 1, and 21 denotes an antibody supply valve. The antibody solution tank 19, the antibody introduction tube 20, and the antibody supply valve 21 constitute the antibody supply unit of the first embodiment. In the first embodiment, a sample solution containing a plurality of microorganisms is concentrated between electrodes 2a and 2b as a pretreatment before an antigen-antibody reaction by dielectrophoresis, and the microorganisms concentrated in this pretreatment are only specific microorganisms. An antibody solution that reacts specifically is introduced. The specific microorganisms are aggregated by the antigen-antibody reaction, the microorganisms that have not been aggregated by the stirring device 15 or the cleaning liquid are separated by fluid force, and finally washed with the cleaning liquid, and then the number of specific microorganisms is counted between the electrodes 2a and 2b. All of these processes are performed on the surface of the electrode part 2.
[0057]
Furthermore, the microorganism measuring apparatus according to the first embodiment includes a measurement self-checking mechanism so that it can correctly measure without much knowledge. In other words, if the microorganism measuring apparatus is not properly prepared, it will be useless even if measurement is performed. Therefore, this microorganism measuring apparatus performs a self-check immediately after the start of measurement. Microorganisms are not collected in the state where measurement has just started. When the DEPIM signal is detected by measuring the impedance between the electrodes 2a and 2b in this state, the initial value depends only on the conductivity and dielectric constant of the suspension. If the calculation control unit 5 detects the DEPIM signal immediately after the start of measurement and determines that the initial value indicated by the suspension is significantly different from the normal value range, the electrode unit 2 is damaged and short-circuited. The alarm is sounded or a warning is displayed on the display unit 9 to prompt the user to check or replace the electrode or the device. The normal value of the initial value indicated by the suspension may be determined for each suspension in the measurement table 7a or may be determined using stored calibration curve data. By performing such a self-check, the number of microorganisms can be easily and reliably measured without being an expert.
[0058]
Subsequently, on the basis of FIGS. 2 (a), (b), (c), FIG. 3, and FIG. 4, a procedure for selectively measuring only specific microorganisms by the microorganism count measuring apparatus and the microorganism count measuring method of the first embodiment. Will be described. In FIGS. 2A, 2B, and 2C, for simplicity, it is assumed that only two types of microorganisms A and B are suspended, and the microorganism concentrations are approximately equal. As shown in FIG. 2A, pre-collection by dielectrophoresis is performed in step 1. Voltage V between electrodes 2a and 2b installed in the sample solution_c1Is applied to collect all microorganisms A and B by the dielectrophoretic force. When the DEPIM signal shows a saturation tendency, the pre-collection is terminated. When there is no significant difference in the sizes and dielectric properties of these microorganisms A and B, as is understood from (Equation 1), approximately the same dielectrophoretic force acts on both, and both are collected by the electrodes. At this time, when impedance measurement by the DEPIM method is performed, collection of both microorganisms A and B brings about the same impedance change, and in such a case, the two cannot be distinguished.
[0059]
In step 2, only a specific microorganism A is selected from a plurality of types of microorganisms A and B by antigen-antibody reaction. That is, an antibody solution containing an antibody that specifically reacts with the microorganism A is added in a state where the pre-collection of the microorganisms A and B is completed. At this time, the microorganisms A are densely brought into contact with each other by dielectrophoresis, and when an antibody is allowed to act on the microorganisms in such a state, the antibodies are cross-linked and only the microorganism A forms an aggregate, so-called aggregation. A reaction takes place. The clock means 6 has a predetermined time t_c1It is determined that the agglutination reaction has been completed.
[0060]
Subsequently, in step 3, the time t_c1When the time elapses, the arithmetic control unit 5 sets the voltage V to a level at which no more microorganisms are collected by the dielectrophoretic force._c2To lower. However, since a sufficiently high electric field is maintained between the electrodes 2a and 2b, the already collected microorganisms are held as they are without diffusing. Then, the microorganisms are selected by fluid force. As is clear from (Equation 1), the dielectrophoretic force F_DEPIs basically proportional to the square of the electric field (voltage V) and the cube of the size (radius a) of the microorganism (Equation 4).
[0061]
[Expression 4]

However, A is a proportionality constant. When performing the dielectrophoresis, if the sample liquid in the measurement chamber 1 is stirred to generate a flow, fluid resistance based on viscous force is generated in the microorganisms in the sample liquid. Since the flow rate generated by the flow of stirring and the washing liquid is low, when the relative speed between the microorganism and the sample liquid is constant from the relationship with the Reynolds number, the normal viscous force F_VIs proportional to the size of the microorganism (radius a), and (Equation 5) holds.
[0062]
[Equation 5]

However, B is a proportionality constant.
From (Equation 4) and (Equation 5), the viscous force F_VDielectrophoretic force F against_DEPThe relative dielectrophoretic force F *_DEPF *_DEPIs represented by (Equation 6).
[0063]
[Formula 6]

The relationship of (Equation 6) is illustrated in FIG. When the microorganism causes an agglutination reaction rather than adding the antibody solution, the apparent radius increases and the square of the voltage V²The rate of increase relative to increases. That is, the relative dielectrophoretic force F * acting on the microorganisms before aggregation._DEPChanges along the straight line OA in FIG. 3, but changes along the straight line OB after aggregation.
[0064]
When there is a flow on the surface of the electrodes 2a and 2b, the viscous force F_VRelative dielectrophoretic force F * in order to overcome the above and collect and hold microorganisms on the electrode with dielectrophoretic force_DEPMust be larger than a certain size. As a guide, when the dielectrophoretic force is greater than the viscous force, that is, the relative dielectrophoretic force F *_DEP> 1 can be considered as the condition. This relative dielectrophoretic force F *_DEPIs represented by a straight line CD in the figure, and in the region above the straight line (hatched part in the figure), the dielectrophoretic force is relatively influenced by the flow F._VIs better than
[0065]
In FIG. 3, the voltage is V_C1Relative dielectrophoretic force F * when set to_DEPAgglomerated microorganisms (radius a₁), Non-aggregated microorganisms (radius a₂) Is above the straight line CD, it can overcome the viscous force and collect microorganisms. On the other hand, the voltage is V_{C 2}Relative dielectrophoretic force F * when set to_DEPIs a point E for the agglomerated microorganism and is above the straight line CD, so that the microorganism can be collected. However, for a non-aggregated microorganism, the point F is below the straight line CD and the viscous force F_VBecause of the predominance, microorganisms cannot be collected by dielectrophoretic force.
[0066]
Therefore, the pre-collection in step 1 is the voltage V_C1Then, both microorganisms A and B are collected by dielectrophoresis. However, after proceeding to step 2 and performing an agglutination reaction, the voltage is set to V in step 3._{C 2}When the sample solution is made to flow down to the flow rate, the microorganism A that has formed aggregates remains collected by the electrodes 2a and 2b, but the microorganism B that has not aggregated has a viscous force F._VDetaches from the electrodes 2a and 2b and is washed away. That is, only the microorganism A that is the detection target finally remains in the state of being collected by the electrode. If the impedance is measured in this state, only the microorganism A can be selectively detected by the DEPIM method.
[0067]
FIG. 4 shows the measurement results when Escherichia coli is selectively measured from a sample solution obtained by mixing equal amounts of Escherichia coli and Pseudomonas aeruginosa according to Steps 1 to 3 described above. In FIG. 4, the measurement was performed under the conditions of a suspension conductivity of 0.1 mS / m and a dielectrophoresis frequency of 1 MHz. E. coli and Pseudomonas aeruginosa are similar in size and dielectric properties, and the conventional DEPIM method cannot selectively detect only one of them from the sample solution.
[0068]
In the measurement of FIG. 4, pre-collection by dielectrophoresis is performed at step 1 at a voltage of 10V. The transient increase in conductance observed at this time is the result of the collection of both E. coli and Pseudomonas aeruginosa in the sample solution between the electrode gaps by the dielectrophoretic force as shown in FIG. 2 (a). Next, in Step 2, 10 ml of E. coli antibody was added to the sample solution, and an E. coli aggregate was formed between the electrodes 2a and 2b as shown in FIG. 2 (b). At this time, the conductance remains constant. After completion of the agglutination reaction, the voltage is reduced to 1 V in step 3, and the sample solution is stirred and the washing solution is introduced as shown in FIG. As a result, the conductance rapidly decreases by about 50%. The conductance (residual conductance) observed after step 3 corresponds to the concentration of E. coli that aggregates and is retained between the electrodes 2a and 2b, and the E. coli concentration in the sample solution was quantified by this residual conductance. It will be.
[0069]
Although the above description is directed to a sample solution in which two types of microorganisms are mixed, even if there are three or more types, only specific microorganisms can be selectively detected by the same method.
(Embodiment 2)
Hereinafter, a microorganism detection apparatus and a microorganism detection method according to Embodiment 2 of the present invention will be described. FIG. 5 is a configuration diagram of the microorganism measuring device according to the second embodiment of the present invention, FIG. 6A is an electrode chip diagram before the measurement of the microorganism measuring device according to the second embodiment of the present invention, and FIG. FIG. 6 (c) is an agglomeration process diagram for the electrode part of the microorganism measuring apparatus according to the second embodiment of the present invention to selectively measure a specific microorganism, and FIG. 6 (c) is an electrode part of the microorganism measuring apparatus according to the second embodiment of the present invention. 7 is a cleaning process diagram for selectively measuring a specific microorganism, and FIG. 7 is a measurement result diagram of the specific microorganism of the microbiological analyzer in the second embodiment of the present invention. The members having the same reference numerals as those of the microorganism measuring apparatus of the first embodiment also indicate the same members in the microorganism measuring apparatus of the second embodiment, and the detailed description will be given to the microorganism measuring apparatus of the first embodiment. Description is omitted.
[0070]
  In FIG. 5, 1 is a measurement chamber for storing a sample solution in which a plurality of microorganisms are mixed, 2 ′ is a microbe measurement electrode chip for measuring the number of microbes, and 2a and 2b are provided on the microbe measurement electrode chip 2 ′. A pair of electrodes. In FIGS. 6A, 6B, and 6C, reference numeral 2c denotes an antibody solid-phased between the electrodes 2a and 2b on the microbe measurement electrode chip 2 '.In this specification,Adsorbing an antibody on the surface of a glass substrate and utilizing it for antigen-antibody reaction is called antibody solid-phase. In the second embodiment, a sample solution containing a plurality of microorganisms is concentrated between electrodes 2a and 2b as a pretreatment for an antigen-antibody reaction by dielectrophoresis, and the microorganisms concentrated in this pretreatment The immobilized antibody 2c that reacts specifically is allowed to act. By the antigen-antibody reaction, the specific microorganism is immobilized on the microbe measurement electrode chip 2 'via the antibody 2c. As will be described later, the sample solution is flowed by the agitator 15 to separate unreacted microorganisms, and a washing solution is introduced to wash away unreacted microorganisms. Thereafter, the number of specific microorganisms is counted between the electrodes 2a and 2b. All of these treatments are performed on the surface of the microbe measurement electrode tip 2 '.
[0071]
There are various solid-phase methods such as a physical method and a chemical method. In the second embodiment, the antibody 2c is obtained by cleaning the electrode substrate of the microbe measurement electrode chip 2 'with acetone and then dialyzing it. The antibody solution is dropped onto the electrode substrate, kept at 4 ° C. as it is, and allowed to stand for 15 to 18 hours to be solid-phased. The microbe measurement electrode chip 2 ′ can be a disposable electrode that is replaced for each measurement.
[0072]
3 is a power supply unit for dielectrophoresis, 4 is a measurement unit capable of measuring the impedance between the electrodes 2a and 2b, 5 is an arithmetic control unit, and 6 is a clock means. Reference numeral 7 denotes a memory unit, and 7a denotes a measurement table that is provided in the memory unit 7 and stores measurement control data for concentrating specific microorganisms, calibration curve data for converting the number of microorganisms, and the like. is there. The control data for measurement of the second embodiment is as shown in (Table 2). The measurement table 7a includes the type of antibody that causes an antigen-antibody reaction for each microorganism, and the voltage V applied for collection._C1, Voltage V to be applied when separating by fluid force_{C 2}, Set time t for aggregation by antigen-antibody reaction_clCleaning time t for cleaning_c2Is at least stored.
[0073]
[Table 2]

8 is a data input unit for inputting the name of microorganism and suspension conductivity, 9 is a display unit, 10 is a sample solution tank, and 11 is a supply pipe for guiding the sample solution to the measurement chamber 1. Reference numeral 12 denotes a discharge pipe for discharging the measured sample solution from the measurement chamber 1, 13 denotes a supply valve such as an electromagnetic valve provided in the supply pipe 11, and 14 denotes a discharge valve provided in the discharge pipe 12. Reference numeral 15 denotes a stirring device. Reference numeral 16 is a cleaning liquid tank, 17 is a cleaning pipe, and 18 is a cleaning valve.
[0074]
Subsequently, a procedure for selectively measuring only a specific microorganism by the microorganism count measuring apparatus and the microorganism count measuring method of the second embodiment will be described with reference to FIGS. 6 (a), (b), (c), and FIG. . In FIGS. 6A, 6B, and 6C, for simplicity, it is assumed that only two types of microorganisms A and B are suspended, and the microorganism concentrations are approximately equal. As shown in FIG. 6A, in step 1, the antibody 2c is immobilized on the microbe measurement electrode chip 2 '. The microbe measurement electrode chip 2 ′ used in the DEPIM method is a pattern formed by photolithography after forming a metal thin film such as chromium on the surface of the glass substrate, and the glass substrate is exposed between the electrodes 2a and 2b. Yes, the antibody 2c is immobilized on this gap portion.
[0075]
Next, in step 2, pre-collection of microorganisms by dielectrophoresis and fixation of specific microorganisms to the electrode chip 2 'for microorganism measurement by the immobilized antibody 2c are performed. That is, the voltage V is applied to the electrode installed in the sample solution._c1First, all microorganisms A and B are collected by the dielectrophoretic force. If the DEPIM signal shows a saturation tendency, the pre-collection is completed. When there is no significant difference in the sizes and dielectric properties of the microorganisms A and B, almost equal dielectrophoretic force acts on both as expected from (Equation 1), and both are collected by the electrodes 2a and 2b. At this time, when impedance measurement is performed by the DEPIM method, collection of both microorganisms A and B brings about the same impedance change, and the two cannot be distinguished. Part of the dielectrophoretic microorganisms A and B is held in contact with the glass substrate surface between the electrodes 2a and 2b. In Step 1, since the antibody of the microorganism A is immobilized on the glass substrate, the microorganism A that has contacted the glass substrate surface binds to the antibody and is consequently fixed to the glass surface.
[0076]
Subsequently, in step 3, the cleaning liquid is sorted by fluid resistance (viscosity). Time t set in advance by the clock means 6_c1Is passed, it is determined that the antigen-antibody reaction has been completed, and the voltage V_c2To reduce the dielectrophoretic force, and the sample solution is flowed by stirring or introducing a cleaning solution, and fluid force (viscous force) is applied to the microorganism. If the viscous force is an appropriate value, the microorganism A fixed on the glass surface by the antibody 2c solid-phased in Step 2 is held between the electrodes 2a and 2b even when the voltage is lowered. On the other hand, the microorganisms B that are not immobilized are separated from the electrodes 2a and 2b by the viscous force of the flowing liquid and are finally washed away with the cleaning liquid. That is, finally, only the microorganism A to be detected remains collected in the electrode. If the impedance is measured in this state, only the microorganism A is selectively detected by the microorganism measuring apparatus of the second embodiment.
[0077]
FIG. 7 shows the measurement results when Escherichia coli was selectively measured from a sample solution in which equal amounts of Escherichia coli and Pseudomonas aeruginosa were mixed according to steps 1 to 3 described above. In FIG. 7, the measurement was performed under the conditions of a suspension conductivity of 0.1 mS / m and a dielectrophoresis frequency of 1 MHz. E. coli and Pseudomonas aeruginosa are similar in size and dielectric properties, and the conventional DEPIM method cannot selectively detect only one of them from the mixed suspension.
[0078]
First, in Step 1, an E. coli antibody is immobilized on the surface of the glass substrate of the electrode by physical adsorption. In step 2, pre-collection by dielectrophoresis is performed at a voltage of 10V. The transient increase in conductance observed at this time is a result of both E. coli and Pseudomonas aeruginosa in the suspension being collected between the electrode gaps by the dielectrophoretic force. In the DEPIM method, the bacterial concentration can be quantitatively estimated from the initial increase rate of the conductance, but in the mixed suspension, a signal corresponding to the mixed concentration of both bacteria is obtained. In addition, a part of the E. coli collected at this time binds to the antibody 2c immobilized on the surface of the glass substrate.
[0079]
After completion of the pre-collection, the voltage is reduced to 1V in step 3, and as shown in FIG. 6 (c), the flow is caused by the stirring and the introduction of the washing liquid, and the green that is not bound to the antibody 2c solidified by the viscous force When only Pseudomonas aeruginosa was detached from the electrode, the conductance rapidly decreased by about 50%. The conductance (residual conductance) observed after step 3 corresponds to the concentration of E. coli held between the electrodes 2a and 2b by the immobilized antibody 2c, and this residual conductance causes the mixed suspension to Can be quantified.
[0080]
Although the above description is directed to sample liquids of two types of microorganisms, only specific microorganisms can be selectively detected by the same method even in the case of three or more types.
[0081]
【The invention's effect】
  According to a first aspect of the present invention, there is provided a microorganism measuring apparatus comprising a plurality of types of microorganisms.By the first dielectrophoresisConcentrate, supply antibody solution from antibody supply unit, and react specifically with specific microorganisms,thisBy adding a second dielectrophoresis and flow action toAggregates and separates other microorganisms, and calculates and outputs the number of microorganisms from the change in conductance between the electrodes,There is no need to spend a long time for concentration, and this concentrated microorganism is supplied with an antibody solution that reacts specifically with a specific microorganism.By adding the second dielectrophoresis and flow actionIt is possible to select only a specific microorganism to be measured from a plurality of types of microorganisms, and to measure the number of microorganisms from a change in conductance by impedance measurement of the specific microorganism after selection, Among them, a specific microorganism can be quantified with high sensitivity and quickness, without using a specialist.
[0082]
In the microorganism measuring apparatus according to claim 2 of the present invention, since the antibody solution supply unit includes the antibody solution tank, the antibody solution supply pipe, and the antibody solution valve, a plurality of types concentrated by operating the antibody solution valve. The antibody solution can be automatically supplied to the microorganisms.
[0083]
In the microorganism measuring apparatus according to claim 3 of the present invention, a stirring device is provided in the measurement chamber, and after the arithmetic and control unit performs dielectrophoresis, stirring of the sample liquid for separating microorganisms other than the specific microorganism. Therefore, the microorganism concentration can be made uniform and repaired in the vicinity of the electrode. Furthermore, after the antigen-antibody reaction, microorganisms other than specific microorganisms that have specifically reacted with the antibody can be separated by a flow by stirring.
[0084]
According to a fourth aspect of the present invention, on the basis of the time measured by the clock means, the calculation control unit opens the cleaning valve, supplies the cleaning liquid to the measurement chamber, discharges the sample liquid, and then cleans the chamber. Therefore, microorganisms other than specific microorganisms that have specifically reacted with the antibody can be separated by the fluid force of the washing liquid, and the separated unreacted microorganisms can be washed away to enable impedance measurement.
[0085]
  The microorganism measuring apparatus according to claim 5 of the present invention is an electrode chip for measuring microorganisms.In addition,An antibody that specifically reacts with a specific microorganism is immobilized on a pair of electrode substrates, an AC voltage is applied to concentrate multiple types of microorganisms, and the immobilized antibody and the specific microorganism are specifically identified. Reaction and separation, and calculate and output a specific number of microorganisms from the change in conductance between the electrodes.A plurality of types of microorganisms contained in the sample solutionSince concentration is performed by dielectrophoresis, it is not necessary to spend a long time for concentration. An antibody that specifically reacts with a specific microorganism is immobilized on a solid phase, and only the specific microorganism is immobilized on a microbe measurement electrode chip.By adding a second dielectrophoresis and flow actionSince other microorganisms are separated, only specific microorganisms to be measured can be selected from multiple types of microorganisms, and the number of microorganisms is measured from changes in conductance by impedance measurement of specific microorganisms after selection. Therefore, specific microorganisms in the mixed suspension can be quantified with high sensitivity and speed, without using an expert.
[0086]
  The microorganism measuring apparatus according to claim 6 of the present invention isWith certain microorganismsApplying an alternating voltage to other microorganisms to cause the dielectrophoretic force to act on each microorganism, the dielectrophoretic forceAnd fluid force due to fluid actionSeparation using the difference ofDoFrom the difference in the size of the specific microorganism that reacted and the size of other microorganisms, the dielectrophoretic forceAnd fluid forceIs converted to the difference ofDoTherefore, separation can be performed easily and quickly.
[0087]
The electrode chip for measuring a microorganism according to claim 7 of the present invention has an antibody that specifically reacts with a specific microorganism because an antibody that specifically reacts with the specific microorganism is immobilized on a substrate between the electrodes. Can be fixed on a microbe measurement electrode chip and other microorganisms can be washed, and it is possible to select only a specific microorganism to be measured from multiple types of microorganisms at low cost. it can.
[0088]
  The method for measuring microorganisms according to claim 8 of the present invention comprises a plurality of types of microorganisms.First dielectrophoresisThe antibody solution is supplied to the concentrated microorganism, and the antibody solution reacts specifically with the specific microorganism.Furthermore, by adding a second dielectrophoresis and flow action to this microorganismIsolate other unreacted microorganisms, then measure the impedance between the electrodes and calculate the number of microorganisms of only specific microorganisms, then apply an AC voltage between the electrodes.A plurality of types of microorganisms contained in the sample solutionSince it is concentrated by dielectrophoresis, it is not necessary to spend a long time for concentration, and an antibody solution that specifically reacts with a specific microorganism is supplied to this concentrated microorganism.Add the second dielectrophoresis and flow actionSince only specific microorganisms are aggregated and other microorganisms are separated, only a specific microorganism to be measured can be selected from a plurality of types of microorganisms, and by impedance measurement of the selected specific microorganism, Since the number of microorganisms is measured from the change in conductance, specific microorganisms in the mixed suspension can be quantified with high sensitivity and speed, without using a specialist.
[0089]
  According to a ninth aspect of the present invention, there is provided a microorganism measuring method comprising:FirstAntibody concentrated by electrophoresis and immobilized between electrodesByReacts specifically with specific microorganismsThis is added to the second dielectrophoresis and flow action, and the second dielectrophoretic force and fluid forceAfter separation, the impedance between the electrodes is measured to calculate the number of microorganisms of only specific microorganisms.A plurality of types of microorganisms contained in the sample solutionBecause it is concentrated by dielectrophoresis, there is no need to spend a long time for concentration. An antibody that specifically reacts with a specific microorganism is immobilized on a solid phase, and only a specific microorganism is immobilized on a microbe measurement electrode chip.By adding a second dielectrophoresis and flow actionSince other microorganisms are separated, only specific microorganisms to be measured can be selected from multiple types of microorganisms, and the number of microorganisms is measured from changes in conductance by impedance measurement of specific microorganisms after selection. Therefore, specific microorganisms in the mixed suspension can be quantified with high sensitivity and speed, without using an expert.
[Brief description of the drawings]
FIG. 1 is a configuration diagram of a microorganism measuring apparatus according to Embodiment 1 of the present invention.
FIG. 2 (a) Concentration process diagram for the electrode part of the microorganism measuring apparatus according to the first embodiment of the present invention to selectively measure a specific microorganism.
(B) Process of antigen-antibody reaction for the electrode part of the microorganism measuring apparatus in Embodiment 1 of the present invention to selectively measure a specific microorganism
(C) is a cleaning process diagram for the electrode unit of the microorganism measuring apparatus according to Embodiment 1 of the present invention to selectively measure a specific microorganism.
FIG. 3 is a microbe selectable region diagram of the microbiological determination device according to the first embodiment of the present invention.
FIG. 4 is a measurement result diagram of specific microorganisms in the microbiological measuring device according to the first embodiment of the present invention.
FIG. 5 is a configuration diagram of a microorganism measuring apparatus according to Embodiment 2 of the present invention.
FIG. 6 (a) Electrode chip diagram when the microorganism measuring apparatus in the second embodiment of the present invention is not yet processed.
(B) Aggregation process diagram for the electrode unit of the microorganism measuring apparatus according to the second embodiment of the present invention to selectively measure a specific microorganism.
(C) Cleaning process diagram for selectively measuring a specific microorganism by the electrode unit of the microorganism measuring apparatus according to the second embodiment of the present invention.
FIG. 7 is a measurement result diagram of specific microorganisms in the microbiological measuring device according to the second embodiment of the present invention.
FIG. 8 is a frequency characteristic diagram of Re [K] using cytoplasmic conductivity as a parameter.
[Explanation of symbols]
1 Measurement chamber
2 electrodes
2 'Electrode chip for microorganism measurement
2a, 2b electrode
2c antibody
3 Power supply for dielectrophoresis
4 Measurement section
5 Operation control unit
6 Clock means
7 Memory part
7a Measurement table
8 Data input part
9 Display
10 Sample solution tank
11 Supply pipe
12 Discharge pipe
13 Supply valve
14 Discharge valve
15 Stirrer
16 Cleaning liquid tank
17 Washing tube
18 Cleaning valve
19 Antibody tank
20 Antibody introduction tube
21 Antibody supply valve

Claims (9)

A measurement chamber capable of containing a sample solution;
It is immersed in the sample solution in the measurement chamber, generates an unequal electric field, concentrates a plurality of types of microorganisms contained in the sample solution by a first dielectrophoresis , and reacts the concentrated microorganisms with antibodies. A pair of electrodes capable of measuring the impedance at this time by separating the other microorganisms leaving only the specific microorganisms that have reacted specifically with the antibody by applying a second dielectrophoresis and fluidity action When,
A dielectrophoresis power supply for applying an alternating voltage between the electrodes;
A measurement unit capable of measuring impedance between the electrodes;
A calculation control unit for controlling the power supply unit for dielectrophoresis and the measurement unit;
A memory unit storing a measurement table storing measurement control data and calibration curve data for each microorganism to be measured;
An antibody solution supply unit for supplying an antibody solution for antigen-antibody reaction to the microorganisms concentrated between the electrodes to the measurement chamber;
The arithmetic control unit applies a first AC voltage by the dielectrophoresis power supply unit to concentrate a plurality of types of microorganisms, supplies an antibody solution from the antibody supply unit, and specifically reacts with the specific microorganisms. A second alternating current that causes a dielectrophoretic force greater than the fluid force associated with the flow action to act on the specific microorganism and a dielectrophoretic force less than the fluid force associated with the flow action acting on the other microorganism. A voltage is applied, the specific microorganism is aggregated and the other microorganisms are separated, and the specific number of microorganisms is calculated and output from a change in conductance between the electrodes measured by the measurement unit. Microorganism measuring device.   The antibody solution supply unit includes an antibody solution tank containing the antibody solution, an antibody solution supply pipe for introducing the antibody solution into the measurement chamber, and an antibody solution valve provided in the antibody supply tube. 2. The microorganism measuring apparatus according to claim 1, wherein the antibody solution is supplied to the measurement chamber after the microorganisms are concentrated.   The microorganism measuring apparatus according to claim 1 or 2, wherein a stirring device is provided in the measurement chamber to stir the sample solution.   The measurement chamber is provided with a cleaning liquid cleaning pipe, and the cleaning pipe is provided with a cleaning valve. Based on the time measured by the clock controller, the arithmetic control unit opens the cleaning valve to supply the cleaning liquid to the measurement chamber. The microorganism measuring apparatus according to any one of claims 1 to 3, wherein the microorganism is supplied and separated to wash away specific microorganisms by fluid force. A measurement chamber capable of containing a sample solution;
It is immersed in the sample solution in the measurement chamber, generates an unequal electric field, concentrates a plurality of types of microorganisms contained in the sample solution by a first dielectrophoresis , and reacts the concentrated microorganisms with antibodies. A pair of electrodes capable of measuring the impedance at this time by separating the other microorganisms leaving only the specific microorganisms that have reacted specifically with the antibody by applying a second dielectrophoresis and fluidity action A microbe measurement electrode chip comprising:
A dielectrophoresis power supply for applying an alternating voltage between the electrodes;
A measurement unit capable of measuring impedance between the electrodes;
A calculation control unit for controlling the power supply unit for dielectrophoresis and the measurement unit;
A memory unit storing a measurement table storing measurement control data and calibration curve data for each microorganism to be measured;
In the microbe measurement electrode chip, an antibody that specifically reacts with a specific microbe is immobilized on a substrate of the pair of electrodes,
The arithmetic control unit, said dielectric by electrophoresis power supply unit by applying an AC voltage to concentrate the plurality of types of microorganisms, the immobilized antibody and the specific microorganism and specifically reacted with the second A microorganism measuring apparatus which is separated by a dielectrophoretic force and a fluid force accompanying the flow action, calculates and outputs the specific number of microorganisms from a change in conductance between the electrodes measured by the measuring unit. When separating other microorganisms while leaving only specific microorganisms that have reacted specifically with the antibody, an alternating voltage is applied to each of the specific microorganisms and the other microorganisms so that a dielectrophoretic force acts on each of the microorganisms. microorganism measuring apparatus according to claim 5, wherein the separating by utilizing the difference in fluid force caused by the flow action and force. A microbe measurement electrode chip used in the microbe measurement apparatus according to claim 6,
An antibody that specifically reacts with a specific microorganism is immobilized on a substrate between the electrodes,
Microorganisms other than the specific microorganism that reacted are separated by a fluid force greater than the dielectrophoretic force,
An electrode chip for measuring microorganisms, which calculates and outputs the specific number of microorganisms from a change in conductance between the electrodes. Applying an alternating voltage between a pair of electrodes to concentrate a plurality of types of microorganisms by first dielectrophoresis , supplying an antibody solution to the concentrated microorganisms, causing the antibody solution to specifically react with a specific microorganism , Further, the second dielectrophoresis and flow action are applied to the microorganism, and the specific microorganism is acted on by the dielectrophoretic force larger than the fluid force, and the other microorganism is acted on by the dielectrophoretic force smaller than the fluid force. other microbial unreacted were separated, microbial measuring method characterized by subsequently calculating the number of microorganisms impedance to only specific microorganism measurement between the electrodes with flocculating microorganisms. By applying an AC voltage between a pair of electrodes was concentrated a plurality of types of microorganisms in the first dielectrophoresis, the immobilized antibody is specifically reactive with a particular microorganism between the electrodes, further to the microorganism The second dielectrophoresis and fluid action are applied, and the other dielectrophoresis force and fluid force separate only the specific microorganisms that have reacted specifically with the antibody, and then separate the other microorganisms. A method for measuring microorganisms, comprising measuring and calculating the number of microorganisms of only specific microorganisms.

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