JP2004313028A - Bioluminescence-measuring device and kit for measuring intracellular atp - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a small simple bioluminescence-measuring device which can simultaneously and highly sensitively detect many samples. <P>SOLUTION: A reactor 1 for performing a bioluminescence reaction for measuring microorganisms and a photodetector 3 for detecting the luminescence from the reactor 1 are closely disposed to correspond to each other as a pair of 1 : 1 in the vertical direction. Furthermore, the reactor 1 and a dispenser 2 equipped with a reagent tank used for injecting reagents are arranged as a pair of 1 : 1 in the vertical direction. The collectively simultaneous injection of reagents into the reactor is performed from the upper vertical direction. The detection of the bioluminescence from the reactor 1 is carried out from the lower vertical direction. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

【０００６】
本手法は、細胞に含まれるＡＴＰ量が微生物細胞の個数に比例することを利用するもので、高感度で測定時間が短いため、微生物検査には非常に有効である。
現在、ＡＴＰ法に用いられている一般的な装置（一般名称はルミノメータ）は、光電子倍増管を用いている。本装置を用いて多数のサンプルを同時に測定する場合には、ＸＹＺ駆動式に時分割にスキャンを行なう必要がある。あるいは、ＣＣＤカメラを用いて同時計測を行なう。ＣＣＤカメラを用いる場合には、サンプルプレートとＣＣＤカメラ素子の大きさが違うため、高価なレンズを組み込んだ縮小光学系が必要である。また、測定試薬の添加は手動で行なうのが一般的である。試薬添加を自動で行なう機構も付属で装備されているが、１０μＬ以下の容量には適用できず、順次時系列に添加するようになっている。特に、手動で測定試薬を添加する場合には、反応時間の制御が難しいため、発光反応を行なう酵素反応の速度を制御し、すなわち、反応の時間を遅くして、発光強度が大きく減衰しないようにしている。通常は、発光反応に関与する酵素濃度（あるいいは活性）を低くしてある。
【非特許文献１】
Ｍｅｔｈｏｄｓ ｉｎ Ｅｎｚｙｍｏｌｏｇｙ Ｖｏｌ．３０５ Ｂｉｏｌｕｍｉｎｅｓｃｅｎｃｅ ａｎｄ Ｃｈｅｍｉｌｕｍｉｎｅｓｃｅｎｃｅ Ｐａｒｔ Ｃ ｅｄｉｔｅｄ ｂｙ Ｍｉｒｉａｍ Ｍ．Ｚｉｅｇｌｅｒ ａｎｄ Ｔｈｏｍａｓａ Ｏ．Ｂａｌｄｗｉｎ， ｐｐ．３５６−３６３
【０００７】
【発明が解決しようとする課題】
微生物検査における測定対象は、食品や飲料水等の固体・液体に存在する微生物、加工環境に存在する微生物（例えば、使用器具・容器やヒトの手指等）、空気中のちり等に存在する微生物と多様である。また、ビール・清涼飲料水・コーヒー等のように含まれる菌数の少ないものもある。すなわち、微生物検査する対象の形状や菌数に大きな違いがある。
【０００８】
例えば、食品や飲料水等の固体・液体に存在する微生物の測定は、測定対象の食品や飲料水の一部をそのまま、あるいは測定溶液に溶解して上記装置で測定できる。しかし、測定対象が使用器具・容器やヒトの手指等の場合には、滅菌した綿棒等で測定対象の表面をふき取り、ふき取った付着微生物を一定量の滅菌水あるいは生理食塩水に溶出させて上記測定装置で測定する。また、空中浮遊微生物の測定は、一定量の空気を適当なフィルターで吸引ろ過してフィルター表面に空中浮遊微生物を捕獲後、フィルター上に直接試薬を添加してＣＣＤカメラあるいは顕微鏡下で発光を測定するか、フィルターに捕獲された微生物を滅菌水あるいは生理食塩水に溶出させて測定する。その際、捕獲空中浮遊微生物が少ない場合には、寒天培地等で培養後、測定しなければならない。菌数の少ないビール・清涼飲料水・コーヒー等の場合も同様に寒天培地等で培養後、測定しなければならない。
【０００９】
上記の操作は、手動で行なわれていたため、試薬添加を含む測定の自動化が望まれていた。特に、空中浮遊微生物の測定は、操作が煩雑なため、自動化が望まれていた。しかし、装置の自動化には、複数のサンプルへの試薬添加や複数の試薬添加のためにＸＹＺ駆動式のディスペンサが必要となり、装置が大型になる問題があった。また、現状の装置では感度が低いため、菌数の少ない測定対象は培養等の操作が必要で自動化が困難であった。培養等の操作を無くすためには、装置を高感度化する必要があるが、ＣＣＤカメラや顕微鏡システムを使用するため装置が大型になる問題があった。さらに、測定場所も食品工場内、レストラン等の調理場、野外等と広範囲にわたり、大型な装置は持ち運びが不便であったり、装置設置のための新たな電源工事や信号線配線工事を必要とする問題があった。
【００１０】
本発明の目的は、多様な測定対象を広範囲な場所で、自動かつ高感度に測定できる小型で簡便な微生物測定用の生物発光測定装置及び細胞内ＡＴＰ測定キットを提供することにある。
【００１１】
【課題を解決するための手段】
上記目的を達成するために、本発明による微生物測定用発光検出装置は、培養等の操作を必要とせずに検出できる感度を有し、試薬添加を自動的に行なう必要がある。測定感度を向上させるためには、酵素濃度（あるいは酵素活性）を上げて反応速度を早くした測定試薬を用い、試薬添加後の測定時間と発光強度を正確に測定する。その際に使用する発光試薬中の酵素の発光反応の半減期が１分以内の酵素濃度で、測定試薬添加後直ちに測定を開始することが望ましい。
【００１２】
酵素濃度（あるいは酵素活性）を上げて反応速度を速くした測定試薬を用いて多数のサンプルを測定する場合には、発光反応を行なう酵素反応を正確に制御するために、順次時系列的な試薬添加及び測定法は使用できず、一括同時試薬添加を行ない、一括同時反応・検出する必要がある。一括同時反応・検出を実現する装置は、１次元的又は２次元的に配列された透明な底部を有する複数の反応セルを備える反応槽と、反応槽の上方に位置し、下面に複数の反応セルと一対一に対応付けられる複数本のキャピラリーを備える送液部と、送液部を反応槽に対して上下方向に駆動する駆動部と、反応セルと一対一に対応して反応槽の下面に近接して配列された複数の光検出素子を有する光検出部とを含み、反応槽の各反応セル内で発生する発光を光検出部の複数の光検出素子によって個別に検出する構成とするのが好ましい。
【００１３】
反応槽の複数の反応セルと反応セルから生じる生物発光等の発光を検出する検出素子を一対一で垂直方向で対応するように密着して配置する構造を採用することにより、複雑な光学系を使用せずに高集光効率で発光を検出できるため、簡便に高感度化が達成できる。また、反応セルへの試薬注入を反応槽の上部垂直方向から行い、反応セルからの生物発光等の発光検出を反応槽の下部垂直方向から行うことにより、装置の小型化及び大きな受光立体角を確保し、小型で高感度な装置を実現できる。更に、複数の反応セルと試薬注入を行う送液部を一対一で垂直方向で対応するように配置することで、複雑な駆動部を用いない簡単な装置構成で一括同時反応・検出を実現できる。その際、反応槽と検出部が密着することにより静電誘導によるノイズ（微小擬似電流）が問題となるが、反応槽と光検出部との間に光透過性の導電膜を配置することによりノイズを除去することができる。送液部は、試薬を入れる試薬容器と、試薬容器と複数本のキャピラリーとを連通する流路と、試薬容器内を加圧する加圧手段とを備え、キャピラリーを流量制御用部材とし、加圧手段によって試薬容器内を所定時間加圧する定圧加圧送液法によって複数本のキャピラリーの先端から複数の反応セルに試薬を均等に送液するのが好ましい。定圧加圧方式は、３気圧以上の高圧ボンベ、またはコンプレッサ等の圧力源からの圧縮空気（１−２気圧程度）を使用して、電磁弁等の圧力切り替え装置により数秒程度の圧力を加えることにより行なうことができる。通常の測定では、遊離ＡＴＰ消去試薬、ＡＴＰ抽出試薬、及び酵素を用いて細胞内のＡＴＰを発光反応させる発光試薬の３種類の測定試薬が必要であるため、反応セル上に配置した各々の試薬に対応した送液部が一定時間毎に反応セル上に回転移動し自動的に順次試薬添加し、発光反応させることにより自動化ができる。
【００１４】
また、内部にサンプル溶液用容器、細胞外ＡＴＰ消去試薬用容器、細胞内ＡＴＰ抽出試薬用容器、及び発光試薬用容器とを含む試薬トレイを用いて、順次下方に溶液を移動させて反応させることにより、簡単な装置構成で自動化ができる。上記試薬トレイは各容器間の仕切りで仕切られている。この仕切りは、例えば疎水性の膜等を用いてもよい。細胞外ＡＴＰ消去試薬用容器、細胞内ＡＴＰ抽出試薬用容器、発光試薬用容器には予め試薬を充填してあり、これらの試薬は上記の疎水性の膜等で仕切られている。各容器間の仕切りは、治具で簡単に破れる構造になっている。この治具は、例えばガラスや金属等を材料に含むニードル等であり、数グラムの力をかけることによって、各容器間の仕切りを破るものである。試薬トレイがこのような構造をとることにより、サンプル溶液と各種試薬を任意の時に瞬時に、また自動的に混合することが可能となる。尚、本装置で測定に使用するサンプル（例えば、食品や飲料水等の固体・液体やふき取った付着微生物）は、固体、液体、気体のいずれであっても、測定溶液に溶解した状態であれば測定対象となる。
【００１５】
空中浮遊微生物の測定は、反応セルに開閉式の蓋を設け、サンプリング時に一定時間蓋を開いて微生物サンプリングを行なう。サンプリング終了後、反応セルに試薬を添加する際も同様に蓋を開閉し行なう。複数の反応セルを有する装置の場合には、反応槽に設けられた複数の反応セルと同じ位置関係を有する複数の開口を備え反応槽の上面に配置される蓋を用意し、その蓋を、蓋の複数の開口と反応槽の複数の反応セルとが合致する位置と合致しない位置との間に駆動する駆動手段とを備えるのが好ましい。
【００１６】
光センサとしてホトダイオードを使用することにより、電池駆動可能になり、小型な装置とすることができる。さらに、無線通信としてブルーツース等を使用することにより、通信線等の配線が不要なワイヤレスで、かつ小型な微生物測定用の発光検出装置を実現できる。
【００１７】
本発明による細胞内ＡＴＰを生物発光反応で測定する生物発光測定装置は、実質的に透明な底部を有し試料が入れられる反応容器と、反応容器の上方に位置し、細胞外ＡＴＰを消去するための試薬、細胞内ＡＴＰを抽出するための試薬、発光反応のための試薬を収めた下方にキャピラリーを有する試薬容器を保持し、試薬容器を反応容器に対して上下方向に駆動する駆動部と、反応容器の下面に近接して配列された光検出素子を有する光検出部とを含み、反応容器内で生じた発光を検出することを特徴とする。
【００１８】
また、光検出器の出力をデジタル値に変換するＡ／Ｄコンバータと、Ａ／Ｄコンバータの出力したデジタル値を記憶するメモリと、無線通信部と、メモリ及び無線通信部を制御する制御部を備え、メモリの内容を無線通信装置を介して送信する機能を備えることもできる。
【００１９】
細胞を含む試料の細胞内ＡＴＰを生物発光反応で測定する本発明の生物発光測定装置は、実質的に透明な底部を有する反応容器と、反応容器の上方に位置し、試料及び試薬を保持する送液部と、反応容器の下面に近接して配列された光検出素子を有する光検出部とを有し、送液部は、サンプル溶液を保持する第１の空間と、細胞外ＡＴＰ消去試薬を保持する第２の空間と、細胞内ＡＴＰ抽出試薬を保持する第３の空間と、発光試薬を保持する第４の空間とを有する容器と、当該容器の上部に配置されたニードルとを備え、ニードルを下方に移動させることにより、第１の空間に保持された試料に第２の空間に保持された細胞外ＡＴＰ消去試薬、第３の空間に保持された細胞内ＡＴＰ抽出試薬、第４の空間に保持された発光試薬を混合し、混合液を反応容器に吐出させる。
【００２０】
本発明による細胞内ＡＴＰ測定キットは、細胞外ＡＴＰを消去させるＡＴＰ消去試薬と、細胞内ＡＴＰを抽出させるＡＴＰ抽出試薬と、ルシフェリンとルシフェラーゼを含む発光試薬とを含み、ルシフェラーゼの濃度は１ｇ／Ｌ以上２０ｇ／Ｌ以下であることを特徴とする。ルシフェラーゼの濃度を１ｇ／Ｌ以上２０ｇ／Ｌ以下である試薬を含むキットを用いることにより、半減期１分以内とし、添加後に多数検体をほぼ同時に計測することができ、測定の再現性を高めることができる。この細胞内ＡＴＰ測定キットは、ニードルによって破ることのできる仕切りによって各々区切られて一直線上に配列された第１から第４の空間を有し、第１の空間は試料を入れるための空室であり、第２の空間にＡＴＰ消去試薬を保持し、第３の空間にＡＴＰ抽出試薬を保持し、第４の空間に発光試薬を保持するのが好ましい。
【００２１】
【発明の実施の形態】
以下、図面を参照して本発明の実施の形態を説明する。
図１は、本発明による発光検出装置の一例の全体構成を示す説明図であり、図１（ａ）は測定時の断面図、図１（ｂ）は装置の扉を開いた時の断面図である。この発光検出装置は、反応槽１、反応槽１の上方に配置され、反応槽１に試薬を添加する試薬槽を具備したディスペンサ２、反応槽１の下方に密着して配置された光検出部３から構成されている。ディスペンサ２中の発光試薬の送液は、定圧加圧管４を介してディスペンサ２内を一定圧力に加圧して送液を行う定圧加圧送液法により行う。その際の加圧時間の制御は電磁弁５で行なっている。
【００２２】
操作手順を以下に示す。まず最初、密閉扉６を開き、試薬添加部７を上にあげて、反応槽１をセットする。測定を開始するとディスペンサ２を固定したＺステージ８が下方に移動し、ディスペンサ２の先端が反応槽１に浸漬し、反応槽１に一定量の試薬を添加する。反応槽１内の試料は、ディスペンサ２から添加される反応試薬との間で発光反応を起こす。反応槽１内から発生した発光は、反応槽１の下方に密着して配置された光検出部３内の光センサ９によって検出される。図１に示した装置は、複数の反応槽及びディスペンサを有することにより複数の試料を同時に測定できる構成になっている。試料は、反応槽に注入する前に、予め遊離ＡＴＰを遊離ＡＴＰ消去試薬を用いて分解する処理、微生物細胞内のＡＴＰをＡＴＰ抽出試薬を用いて抽出する処理を施してある。あるいは、装置内に遊離ＡＴＰ消去試薬の入った試薬槽及びＡＴＰ抽出試薬の入った試薬槽を装填し、それらの試薬槽の試薬もディスペンサによって反応槽に順次添加できるように構成し、前処理も装置内で行うように構成することもできる。
【００２３】
図２は、試薬槽を具備したディスペンサ１１、反応槽１２、及び光検出部１３の関係を示す詳細図である。ディスペンサ１１は、反応試薬が入った試薬槽１４と流量制御用のキャピラリー１５から構成されている。ディスペンサ１１は、Ｏリング１６を用いて固定治具１７で加圧送液管１８に固定され、取り外し可能でディスポーザブルになっている。キャピラリー１５には、内径２５μｍ×外径３６０μｍ×長さ２０ｍｍのフューズドシリカキャピラリーを使用している。反応槽１２は内径２ｍｍ、深さ３ｍｍで、使用する反応溶液の体積が約４μＬであるので、石英ガラス板１９から試料溶液２０の液面までの長さは約１．５ｍｍである。本発明で使用した光検出素子２１（浜松ホトニクス製Ｓ１１３３−０１）の受光面は２．４×２．６ｍｍである。本実施の形態の条件下では、反応溶液の底面から受光面までの距離は１．４ｍｍであり、約７％の受光効率である（この値は実験値から計算した値とほぼ同じであった）。本装置構成によると、レンズ等を使用した複雑な光学系を必要とすることなく、密着方式という簡便な方式で受光立体角を大きくし（高集光効率）、部品数低減と光軸調整不要な簡単な構造を実現できることが分かる。
【００２４】
図３は、本発明による発光検出装置の他の一例のディスペンサ３１、反応槽３２、及び光センサアレイ３３の関係を示す詳細図である。ディスペンサ３１は、厚さ１０ｍｍ×縦８０ｍｍ×横１２０ｍｍのアクリル板の厚さ方向中央に、縦方向１２本、横方向１本の直径０．８ｍｍの流路３４を加工し、アクリル板に垂直に流量制御用のキャピラリー３５を９６本垂直に差し込んで接着して作製している。流路３４は、アクリル板に、一方の端部から他方の端部に貫通する貫通穴を加工し、その後、両端部の開口をプラグで塞いで形成した。横方向１本の流路は、両端をネジ３７，３８で塞ぐ構造とし、空気抜きの機能を持たせた。キャピラリー３５は、流路３４に連通するように形成されたアクリル板底面の穴に先端を挿入し、接着剤でアクリル板に固定した。キャピラリー３５には、内径２５μｍ×外径３６０μｍ×長さ２０ｍｍのフューズドシリカキャピラリーを使用した。ディスペンサ３１は上部に試薬槽３６を備え、試薬槽３６の１ヶ所から供給する反応試薬を定圧加圧送液法によって９６本のキャピラリー３５の先端から均等に吐出することができる。
【００２５】
本発明で使用した反応槽３２は、微少量（１〜１０μＬ）用に９ｍｍピッチで８×１２列に直径２ｍｍの反応セル用穴３９を９６ヶ所に設けた黒色石英ガラス板と、鏡面に仕上げた透明な石英ガラス板４０を熱融着により張り合わせて作製したものである。石英ガラス板４０の下面にはＩＴＯ又はＳｎＯ_２からなる透明導電膜を蒸着してある。透明導電膜は接地される。
【００２６】
光センサアレイ３３は、反応槽３２を用いて９６サンプルを同時測定できるように、光検出素子（浜松ホトニクス製フォトダイオードＳ１１３３−０１）４１を９ｍｍピッチで８×１２列に配置したものである。本発明では、レンズ等を使用した複雑な光学系を使用せず、簡便な方式で集光効率を高めるために、光検出素子４１を反応槽３２に可能な限り近づけて受光角を大きくする密着型とし、部品数低減と光軸調整不要な簡単な構造で高集光効率を達成した。光センサアレイ３３は、反応槽３２の下面に密着して配置される。各光検出素子４１の出力は、電流電圧変換増幅アンプ（ＢＵＲＲ ＢＲＯＷＮ製ＯＰＡ１２９ＵＢ）及び１０ＧΩの抵抗を用いて、１×１０^１０に増幅し、２段目のオペアンプ（ＡＮＡＬＯＧ ＤＥＶＩＣＥＳ製ＯＰ０７）を用いてトータルゲイン４．５×１０^１１に増幅した。
【００２７】
反応セル用穴３９は内径２ｍｍ、深さ３ｍｍで、使用する反応溶液の体積が約４μＬであるので、石英ガラス板４０から反応溶液の液面まで長さは約１．５ｍｍである。本発明で使用した光検出素子４１（浜松ホトニクス製Ｓ１１３３−０１）の受光面は２．４×２．６ｍｍである。本実施の形態の条件下では、反応溶液の底面から受光面までの距離は１．４ｍｍであり、約７％の受光効率である（この値は実験値から計算した値とほぼ同じであった）。本装置構成によると、レンズ等を使用した複雑な光学系を必要とすることなく、密着方式という簡便な方式で受光立体角を大きくし（高集光効率）、部品数低減と光軸調整不要な簡単な構造を実現できることが分かる。
【００２８】
図３には、専用の反応槽３２を用いた装置例を示したが、反応槽として、市販されている通常のマイクロタイタープレートを用いてもよい。その場合には、クロストーク防止用の仕切りを兼ねたセルホルダを使用するとよい。
【００２９】
図４は、反応槽として市販のマイクロタイタープレートを用いた場合の反応槽の部分の分解組立図である。なお、図４には、マイクロタイタープレートの各反応セル（ウェル）に試薬を供給するディスペンサ５１も合わせて図示した。また、図５は、マイクロタイタープレート５２、セルホルダ５３、導電性光透過板５４を組み合わせた状態の断面図である。
【００３０】
クロストーク防止用の仕切りを兼ねたセルホルダ５３は、遮光性の材料からなる板状部材であり、マイクロタイタープレート５２の各反応セル５５に対応する位置に穴５６が設けられている。セルホルダ５３は、セルホルダの各穴５６に反応セル５５の下方突出部が入るようにして、マイクロタイタープレート５２に下方から押し込まれる。導電性光透過板５４は、一方の面にＩＴＯ又はＳｎＯ_２からなる透明導電膜を蒸着したガラス板であり、セルホルダ５３の下面に接触配置される。導電性光透過板５４の下には、図３に図示した光センサアレイが設置される。光センサアレイ中のそれぞれの光検出素子には、その直上に位置する反応セルから発生する発光のみが入射し、他の反応セルから発生する発光は遮光性のセルホルダ５３によって遮断されるため入射しない。
【００３１】
次に、本発明による空中浮遊微生物を測定する発光検出装置の一例について説明する。空中浮遊微生物の測定は、通常食品工場のクリーンルームで行なわれる。近年、食品工場にもクリーンルームが導入されているが、性能の維持管理を自動的に行なわなければならない。そのため、サンプリングから測定までの操作を自動で行うことが必要である。空中浮遊微生物のサンプリングは、空中浮遊微生物捕獲槽を兼ねた反応槽の蓋を一定時間を開いて、空中浮遊微生物のサンプリングを行なった後、計測を自動的に行なうようになっている。待機状態（図６ａ）では、空中浮遊微生物捕獲槽を兼ねた反応槽６１の蓋６２は閉じている。反応槽６１内には、ＡＴＰ抽出試薬６３が予め入っている。計測開始後、自動的に蓋６２が開き、空中浮遊微生物６４，６５のサンプリングを行なう（図６ｂ）。一定時間経過後、自動的にディスペンサ６６が発光試薬を反応槽６１内に添加し、発光反応が開始する。反応槽６１内の試料は、ディスペンサ６６から添加される反応試薬との間で発光反応を起こす（図６ｃ）。反応槽６１内から発生した発光は、反応槽６１の下方に密着して配置された光検出部６７内の光センサ６８によって検出される。
【００３２】
本発明による空中浮遊微生物を測定する発光検出装置の他の例について説明する。本発明では、反応槽あるいは反応槽として用いるマイクロタイタープレートの上に、各反応セルの開口部を開閉できる蓋を設ける。
【００３３】
図７は、マイクロタイタープレートの上に開閉式の蓋を設けた例を示す概略図であり、図７（ａ）は概略平面図、図７（ｂ）はその概略側断面図である。なお、セルホルダは図示を省略してある。本例の開閉式蓋は、マイクロタイタープレート７１の上部及び側部を覆う固定蓋７２と、その上を往復移動可能な移動蓋７３から構成される。固定蓋７２は、マイクロタイタープレート７１の各反応セルに対応する位置に反応試薬添加用の穴７４（８×１２＝９６個）を有する。移動蓋７３は、固定蓋７２の穴７４と同じ配置に反応セルの数だけ穴７５を有し、アクチュエータ７６によって矢印方向に往復移動可能になっている。図は、移動蓋７３の穴７５と固定蓋７２の穴７４とが一致しないように移動蓋７３が位置づけられ、各反応セル７７が密閉されている状態を示している。空中浮遊微生物のサンプリングを行なう時は、アクチュエータ７６によって移動蓋７３を、移動蓋７３の穴７５が固定蓋７２の穴７４と一致するように移動する。一定時間サンプリング後、穴７４と７５が重なってできた開口部にディスペンサのキャピラリーを挿入して試薬を注入し発光反応を行う。このように反応セルの密閉を行なうことにより、空中浮遊微生物のサンプリングを行なうことができる。
【００３４】
図８は、図３に示した装置に開閉蓋を設けた例を示す図である。図８（ａ）は待機状態を示しており、図８（ｂ）は空中浮遊微生物のサンプリング及び計測状態を示している。
【００３５】
反応槽８１は、上部に移動式の開閉式蓋８２を有する。開閉式蓋８２には、反応槽８１内の反応セル８３と同じ９ｍｍピッチで同程度の窓用穴８４（直径：約２ｍｍ）が形成されている。開閉式蓋８２はアクチュエータ８５によって図の左右方向に移動可能になっている。反応試薬注入前の状態では、図８（ａ）に示すように、ディスペンサ８６に取り付けられた流量制御用キャピラリー８７は、反応槽８１の上部に位置しており、反応セル８３の中心と開閉式蓋８２の窓用穴８４の中心はずれている。すなわち、開閉式蓋８２は閉じた状態にあり、反応槽８１内の反応セル８３は、開閉式蓋８２により密閉された状態になる。従って、待機時には反応セル８３内の溶液の蒸発は防止される。
【００３６】
空中浮遊微生物のサンプリング時には、図８（ｂ）に示すように、開閉式蓋８２がアクチュエータ８５によって約３ｍｍ横方向にずらされ、反応セル８３の中心と窓用穴８４の中心が一致することにより、反応セル８３の上部が開放される。一定時間後、ディスペンサ８６が下降し、流量制御用キャピラリー８７の先端が反応セル８３中に浸漬し、試薬槽８８から反応試薬が注入される。反応セル８３内から発生した発光は、反応槽８２の下方に密着して配置された光検出部８９によって検出される。反応終了後、ディスペンサ８６は上昇し、開閉式蓋８２が約３ｍｍ横方向にずれて密閉状態となる。
【００３７】
図９は、ＡＴＰ法で使用するルシフェラーゼ濃度（あるいは酵素活性）の発光反応速度依存性を示す図である。酵素であるルシフェラーゼの濃度を増加させると発光強度も大きくなるが、発光強度の変化も大きくなる。従来法では、手動で試薬を混合して反応を行なっていたため、発光強度の変化が少ない、低濃度の酵素濃度で測定を行っていた。すなわち、測定精度を維持するために感度を下げて測定していた。本発明では試薬添加後、直ちに測定を行なうことができ、従来より１０倍以上の高濃度の酵素を使用することが可能である。すなわち、数１０ａｍｏｌレベルの検出感度が達成できる。数１０ａｍｏｌレベルの検出感度は、従来の装置では不可能であった数１０個程度の微生物を測定可能な値である。
【００３８】
図１０に、実際に酵素濃度を変化させて測定を行なった例を示す。ルシフェラーゼ酵素濃度を従来の１０倍以上増加させても、リアルタイムで測定を行うことができ、正確に測定できる。本発明では、試薬添加を自動化し、かつ多数試料を同時計測することにより、従来では困難であった１分以内に終わる反応を正確に測定でき、高感度計測が可能である。図１０に示されるとおり、本測定ではルシフェラーゼ酵素濃度は０．５μｇ／μＬ以上、好ましくは１．０μｇ／μＬ以上とする。この酵素濃度での反応の半減期は約１分で、数１０ａｍｏｌレベルの検出感度を達成することができ、従来の方法では測定困難であった数１０個程度の微生物の測定が可能である。しかし、サンプルと試薬の混合に要する時間は３〜５秒程度であるため、あまり反応が早すぎると混合状態（例えば、攪拌の方法）の影響を受け、発光強度の再現性が悪くなる。そのため、濃度の上限は２０μｇ／μＬ程度である。このような濃度設定とすることにより発光反応を、試薬添加後直ちに測定可能とし、かつ半減期を一分以内とすることが出来る。測定の一例としてＡＴＰの検量線を図１１に示す。本測定では、測定の再現性を表す相関係数は０．９９９以上で良好であった。その際の検出限界は、８０ａｍｏｌ（Ｓ／Ｎ＝５）であった。
【００３９】
本発明で採用した空気加圧送液法を図２を参照して以下に説明する。
空気加圧送液法は定圧状態で流路系内に粘性流を発生させ、送液する方法である。そのため、使用する微小細管以外の部分の圧損が無視できるように微小細管の内径及び長さを設定すれば、加える圧力及び時間を制御することにより容易にサブμＬ以下の溶液を送液できる。本発明では、微小細管としてディスペンサ１１に取り付けた流量制御用キャピラリー１５を用いている。試薬槽を兼ねたディスペンサ１１中の反応試薬１４の送液（添加）は、定圧加圧管１８（図２参照）を介してディスペンサ１１内を一定圧力に加圧して送液を行う定圧加圧送液法により行う。その際の送液量は、次のＨａｇｅｎ−Ｐｏｉｓｅｕｉｌｌｅの式に従う。
【００４０】
【数１】

【００４１】
ここで、ΔＰ：加えた圧力、ｒ：微小細管の内径、ｔ：圧力を加えた時間、μ
：溶液の粘性、Ｌ：微小細管の長さである。
【００４２】
本発明の一例として、注入量が０．１μＬ以下の場合の実施例を以下に示す。本実施の形態では、大気圧程度（２気圧以下）の低圧力使用、加圧時間２秒以下、及び送液量０．１μＬ以下、の条件を満たすために、内径２５μｍ、長さ２０ｍｍの流量制御用キャピラリー１５を使用した。
【００４３】
本条件下での加圧圧力と加圧時間との関係を図１２に示す。圧力範囲０．５〜２．０×１０^５Ｐａで使用すれば、０．１μＬ以下の微小量の送液が定圧加圧時間２秒以下で行うことができる。注入量は１．０μＬ以上の個別再現性は、注入量を重さで計量可能であるが、注入量が０．１μＬ以下である場合には計量できない。そこで、ルシフェリン／ルシフェラーゼ系の生物発光系（信号ピーク形状を整えるためにＡＴＰ分解酵素であるアピラーゼを加えてある）を応用して同時再現性を測定した。連続測定回数は２０回である。測定は反応液に一定時間間隔（１分間隔）で試薬を注入し、発光強度の再現性を求めた。反応液の体積は４．０μＬで、試薬注入量は（ａ）０．０１μＬ、（ｂ）０．０２μＬ、（ｃ）０．０５μＬ、である。その結果を表１に示す。微少量（０．０１−０．０５μＬ）の注入量再現性は３〜５％と良好であった。
【００４４】
【表１】

【００４５】
本発明では反応槽と検出素子が密着し、高倍率のアンプを使用しているため、ディスペンサ１１の上下駆動の際に静電誘導によるノイズ（微小擬似電流）を生じる問題がある。このノイズを除去するために、反応槽１２の下部に取り付けてある石英ガラス板１９の下面にＩＴＯ又はＳｎＯ_２からなる透明導電膜をコーティングしてある。透明導電膜は接地してある。本発明で使用した光透過性導電膜をコーティングした石英ガラス板の性能は、波長４５０〜６００ｎｍにおいて透過率９０％以上、面積抵抗１０００〜１５００Ωである。
【００４６】
図１３は、光透過性導電膜をコーティングした石英ガラス板のノイズ除去効果を示す図である。通常の石英ガラス板を使用した場合には、図１３（ａ）に示すようにディスペンサ１１の上下駆動に伴い大きな擬似信号９１，９２が発生するが、光透過性導電膜をコーティングした石英ガラス板を使用した場合には、図１３（ｂ）に示すように擬似信号は全く無かった。
図１４及び１５は、本発明による発光検出装置の他の例を示す模式図である。
【００４７】
これまで説明した装置例は、予め細胞外ＡＴＰ消去等の前処理を行ない、最後に発光試薬を添加する例であった。ここに説明する装置例は、遊離ＡＴＰ消去試薬、ＡＴＰ抽出試薬、及び酵素を用いて細胞内のＡＴＰを発光反応させる発光試薬の３種類の測定試薬を個別に順次添加して反応を行なうものである。ここで説明する測定対象は、液体試料及び容器・器具等である。測定する試料が清涼飲料水やコーヒー等の液体の場合には、一部を分注して使用する。また、食品工場内の容器や器具、厨房のまな板等の場合には、通常ふき取り検査を行なう。ふき取り検査は検査個所を綿棒で用いてふき取った後、測定溶液で溶解して使用する。ふき取り用の綿棒と試薬が一体となった製品も市販させている（例えば、キッコーマン製、「ルシパック^ＴＭ」）。
【００４８】
図１４は、試薬槽として、細胞外ＡＴＰ消去試薬槽１０１、細胞内ＡＴＰ抽出試薬槽１０２、発光試薬槽１０３の３つから構成され、その下に試料槽１０４が設置してある。試料槽１０４に予め試料溶液１０５をセットし測定を開始すると、細胞外ＡＴＰ消去試薬槽１０１、細胞内ＡＴＰ抽出試薬槽１０２、発光試薬槽１０３が一定時間毎に下降し、先端のキャピラリー１０６が試料槽１０４内の試料溶液１０５に浸漬し、試薬が添加させる。発光試薬添加後、試料槽１０４内の試料溶液１０５から発生した発光は、試料槽１０４の下方に密着して配置された光検出部１０７内の光センサ１０８によって検出される。遊離ＡＴＰ消去試薬、及びＡＴＰ抽出試薬の添加のタイミングは、測定対象に大きく依存する。例えば、遊離ＡＴＰ消去試薬の反応時間は測定対象の汚れ具合に、ＡＴＰ抽出試薬との反応時間は測定対象の微生物に依存する。通常の測定では、遊離ＡＴＰ消去試薬の反応時間は５分程度、ＡＴＰ抽出試薬との反応時間は最大２０分程度である。また、発光試薬は添加・混合後直ちに測定を開始できる。すなわち、遊離ＡＴＰ消去試薬添加５分後にＡＴＰ抽出試薬を添加し、それから２０分後に発光試薬を添加し、直ちに測定を開始すれば良い。
【００４９】
また、図１５（ａ）は、サンプル溶液用容器１１１、細胞外ＡＴＰ消去試薬用容器１１２、細胞内ＡＴＰ抽出試薬用容器１１３、発光試薬用容器１１４の４つの容器、及び容器上部に配置したニードル１１５から構成される試薬トレイ１１６を示している。ニードル１１５は蓋を兼ねた治具１１７に取り付けられている。測定時には蓋を兼ねた治具１１７を試薬トレイ１１６から取り外し、サンプル溶液を試薬トレイ１１６に分注する。試薬トレイ１１６の底部を装置内の反応セル１１８に、上部をＺステージ１１９にセットする。図１５（ｂ）に示すように、試薬トレイ１１６の底部の構造はセル１１８に簡単にはめ込めるようになっている。
【００５０】
試薬溶液の移動方法としては、各試薬トレイは疎水性の膜等で各容器間を仕切られており、上部に配置したニードル１１５がＺステージ１１９により下方に移動して、各々の容器の底、すなわち上記の疎水性の膜等に穴を開けて行なう。この穴を通じて、各種試薬等は下方に向けて移動し、混合される。全ての試薬が混合した溶液は、反応槽１１８に移動して発光反応が開始する。反応槽１１８から発生した発光は、反応槽１１８の下方に密着して配置された光検出部１２０内の光センサ１４０によって検出される。図１５（ａ）（ｂ）に示す本発明例では、定圧加圧送液法を用いたディスペンサが不要で、より簡便な装置構成になる。この試薬トレイは、装置に簡単に取り付け可能で、通常の反応容器のように取り扱える。また、各種試薬が疎水性の膜等で仕切られて各容器に各々充填された状態のキットを使用しても良い。
【００５１】
以下、測定場所が狭く電源や通信線の設置が困難な場所（例えば、食品工場の小さな生産ライン、冷凍保管庫、厨房、野外保管庫、河川等）で本発明の装置を使用する方法を説明する。
【００５２】
測定場所が狭く、設置場所が限られていると大型の装置を設置することが困難である。さらに、追加の装置を設置するとなると電源工事が必要な場合も生じてくる。本装置は、内蔵の電源で動作できるように、光センサとして低消費電力のホトダイオードを使用している。さらに、低消費電力の無線装置を内蔵することにより、電源線と信号線の不要なワイヤレスで計測できる装置になっている。
【００５３】
図１６は、ワイヤレス通信機能を有する本発明の他の例を示す機能ブロック図である。本装置は、光検出装置１２１とワイヤレス制御装置１２２から構成される。光検出装置１２１は、複数の光センサ１２３を有する光検出部１２４、複数の光センサ１２３の出力を順次サンプリングするＳ／Ｈ回路１２５、光センサ１２３で検出したアナログ値をデジタル値に変換するＡ／Ｄコンバータ１２６、Ａ／Ｄコンバータ１２６で変換された値を記憶するメモリ１２７、あるいは表示する表示部１２８、試薬の添加を自動的に行なうディスペンサ制御部１２９、ワイヤレス制御装置１２２との通信を行なう無線装置１３０、電源１３１、及びＣＰＵ１３２から構成されている。ＣＰＵ１３２は、とワイヤレス制御装置１２２からの命令を受け取り、自動的に計測を行なう。計測終了後、計測データは表示部に表示し、無線装置１３０を介してワイヤレス制御装置１２２に転送する。尚、ワイヤレス制御装置１２２に対して複数の光検出装置を制御することも可能である。
【００５４】
【発明の効果】
本発明によると、小型で簡便な微生物測定用の生物発光測定装置及び細胞内ＡＴＰ測定キットが得られる。
【図面の簡単な説明】
【図１（ａ）】本発明による発光検出装置の一例の全体構成を示す測定時の断面図。
【図１（ｂ）】装置の扉を開いた時の断面図。
【図２】試薬槽を具備したディスペンサ、反応槽、及び光検出部の関係を示す詳細図。
【図３】ディスペンサ、反応槽、及び光センサアレイの関係を示す詳細図。
【図４】反応槽として市販のマイクロタイタープレートを用いた場合の反応槽の部分の分解組立図。
【図５】マイクロタイタープレート、セルホルダ、導電性光透過板を組み合わせた状態の断面図。
【図６】空中浮遊微生物を測定する発光検出装置の概略図であり、（ａ）は待機状態を表す図、（ｂ）はサンプリング時の図、（ｃ）は測定時の図。
【図７】マイクロタイタープレートの上に開閉式の蓋を設けた例を示す概略図であり、（ａ）は概略平面図、（ｂ）はその概略側断面図。
【図８】図３に示した光検出装置に開閉蓋を設けた例を示す概略図であり、（ａ）は待機状態を表す図、（ｂ）は空中浮遊微生物のサンプリング及び計測状態を表す図。
【図９】ＡＴＰ法で使用するルシフェラーゼ濃度（あるいは酵素活性）の発光反応速度依存性を示す図。
【図１０】ルシフェラーゼ酵素濃度と発光強度時間変化の測定値を示す図。
【図１１】本装置を用いて測定したＡＴＰの検量線を示す図。
【図１２】本装置の一例の使用条件下の加圧圧力と加圧時間との関係を示す図。
【図１３】光透過性導電膜をコーティングした石英ガラス板のノイズ除去効果を示す図。
【図１４】本発明による３つの試薬を用いた発光検出装置の他の例を示す模式図。
【図１５（ａ）】本発明によるニードルを用いた一体型試薬トレイの例を示す模式図。
【図１５（ｂ）】本発明による一体型試薬トレイをセットした発光検出装置の他の例を示す模式図。
【図１６】ワイヤレス通信機能を有する本発明の他の例を示す機能ブロック図。
【符号の説明】
１，１２，３２，６１，８１…反応槽、２，１１，３１，５１，６６，８６…ディスペンサ、３，１３，６７，８９，１０７，１１８，１２４…光検出部、４…定圧加圧管、５…電磁弁、６…密閉扉、７…試薬添加部、８，１１９…Ｚステージ、９，６８，１０８，１２３…光センサ、１４，３６，８８…試薬槽、１５，３５，８７，１０６…キャピラリー、１６…Ｏリング、１７…固定治具、１８…加圧送液管、１９…石英ガラス板、２０，１０５…試料溶液、２１，４１…光検出素子、３３…光センサアレイ、３４…流路、３７，３８…ネジ、３９…反応セル用穴、４０…石英ガラス板、５２，７１…マイクロタイタープレート、５３…セルホルダ、５４…導電性光透過板、５５，８３…反応セル、５６…穴、６２…蓋、６３…試薬、６４，６５…空中浮遊微生物、７２…固定蓋、７３…移動蓋、７４…固定蓋の穴、７５…移動蓋の穴、７６，８５…アクチュエータ、８２…開閉式蓋、８４…窓用穴、９１，９２…擬似信号、１０１…細胞外ＡＴＰ消去試薬槽、１０２…細胞内ＡＴＰ抽出試薬槽、１０３…発光試薬槽、１０４…試料槽、１１１…サンプル溶液用容器、１１２…細胞外ＡＴＰ消去試薬用容器、１１３…細胞内ＡＴＰ抽出試薬用容器、１１４…発光試薬用容器、１１５…ニードル、１１６…試薬トレイ、１１７…治具、１２１…光検出装置、１２２…ワイヤレス制御装置、１２５…Ｓ／Ｈ回路、１２６…Ａ／Ｄコンバータ、１２７…メモリ、１２８…表示部、１２９…ディスペンサ制御部、１３０…無線装置、１３１…電源、１３２…ＣＰＵ[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a bioluminescence measuring device and an intracellular ATP measurement kit for measuring ATP in microbial cells using a bioluminescence method.
[0002]
[Prior art]
Measurement of E. coli, yeast, lactic acid bacteria and other viable cell counts is very important in the fields of food hygiene, clinical examination, environment and the like. In particular, in the food industry, it is important to conduct microbial contamination tests on pathogenic Escherichia coli O-157, Salmonella, Staphylococcus aureus, and the like, which cause food poisoning.
[0003]
Conventional methods for counting the number of cells of microorganisms include a colony counting method in a growth medium, a cell counter method using a microscope, and a turbidity measuring method. The method for measuring the number of colonies in a medium can measure viable cells with high sensitivity, and can also identify microorganisms by using a selective medium. However, cell culture takes more than one day. Further, a cell counter using a microscope requires colony culture, and the operation is complicated, and automation is difficult. The turbidity measurement method has a problem in that the sensitivity is low and live cells and dead cells cannot be distinguished. In recent years, the ATP method has been attracting attention as a method for overcoming these problems and measuring microorganism cells with high sensitivity and convenience. The ATP method is a method for measuring ATP (adenosine 5'-triphosphate), which is always present in living cells, by a bioluminescence method using luciferase.
[0004]
In the measurement procedure of the ATP method, first, free ATP is decomposed from a sample using a free ATP eliminating reagent. Next, after extracting ATP in the microbial cells using the ATP extraction reagent, the number of the microbial cells is counted by causing the intracellular ATP to emit light using the luminescent reagent. As the free ATP elimination reagent, decomposing enzymes such as apyrase, alkaline phosphatase, adenosine triphosphatase, hexokinase, and adenosine deaminase can be used alone or in combination. As the ATP extraction reagent, a surfactant, methanol, ethanol and the like are generally used. As a luminescent reagent, a reagent containing luciferase, which is a source of firefly emission, is generally used. These reagents are commercially available as an ATP scavenger and a reagent kit for ATP measurement. For example, Kikkoman ATP scavenger “Lucifer ATP scavenger” ^TM ", Lucifer HS Plus which is an ATP extraction reagent and a luminescent reagent ^TM And so on. The bioluminescence reaction at that time occurs as described below in the presence of luciferase and magnesium ions.
[0005]
Embedded image

[0006]
This technique utilizes the fact that the amount of ATP contained in cells is proportional to the number of microbial cells, and is very effective for microbial testing because of its high sensitivity and short measurement time.
At present, a general device (common name is a luminometer) used for the ATP method uses a photomultiplier tube. When a large number of samples are measured at the same time using this apparatus, it is necessary to perform time-division scanning in an XYZ drive system. Alternatively, simultaneous measurement is performed using a CCD camera. When a CCD camera is used, a reduction optical system incorporating an expensive lens is required because the sample plate and the CCD camera element are different in size. In addition, it is general that the measurement reagent is added manually. A mechanism for automatically adding reagent is also provided as an accessory, but cannot be applied to a volume of 10 μL or less, and is added sequentially in a time series. In particular, when the measurement reagent is manually added, it is difficult to control the reaction time.Therefore, the speed of the enzyme reaction for performing the luminescence reaction is controlled, that is, the reaction time is reduced so that the luminescence intensity does not greatly decrease. I have to. Usually, the concentration (or activity) of the enzyme involved in the luminescence reaction is reduced.
[Non-patent document 1]
Methods in Enzymology Vol. 305 Bioluminescence and Chemiluminescence Part Cedited by Mirai M.M. Ziegler and Thomasa O.M. Baldwin, pp. 356-363
[0007]
[Problems to be solved by the invention]
The microorganisms to be measured in the microbial test include microorganisms present in solids and liquids such as food and drinking water, microorganisms present in the processing environment (for example, tools and containers used, human fingers, etc.), and microorganisms present in dust in the air. And diverse. In addition, there is also a beer, soft drink, coffee, or the like, which has a small number of bacteria contained. That is, there is a great difference in the shape and the number of bacteria to be tested for microorganisms.
[0008]
For example, the measurement of microorganisms present in solids and liquids such as foods and drinking water can be measured by the above-described apparatus by using a part of the food or drinking water to be measured as it is or by dissolving it in a measurement solution. However, when the measurement target is an instrument / container used or a human finger, the surface of the measurement target is wiped off with a sterilized cotton swab or the like, and the wiped adhered microorganisms are eluted into a certain amount of sterilized water or physiological saline to perform the above. Measure with a measuring device. In the measurement of airborne microorganisms, a certain amount of air is suction-filtered with an appropriate filter to capture airborne microorganisms on the filter surface, and then a reagent is directly added to the filter and luminescence is measured using a CCD camera or microscope. Alternatively, the microorganisms captured by the filter are eluted in sterile water or physiological saline for measurement. At that time, when the amount of suspended airborne microorganisms is small, the measurement must be performed after culturing on an agar medium or the like. Similarly, in the case of beer, soft drink, coffee, etc. having a low number of bacteria, the measurement must be performed after culturing on an agar medium or the like.
[0009]
Since the above operation was performed manually, automation of measurement including addition of a reagent was desired. In particular, automation of measurement of airborne microorganisms has been desired because the operation is complicated. However, automation of the apparatus requires an XYZ-driven dispenser for adding reagents to a plurality of samples or for adding a plurality of reagents, and has a problem that the apparatus becomes large. In addition, since the current apparatus has low sensitivity, it is difficult to automate a measurement target having a small number of bacteria, such as culture. In order to eliminate operations such as culture, it is necessary to increase the sensitivity of the apparatus, but there has been a problem that the apparatus becomes large because a CCD camera and a microscope system are used. In addition, the measurement location is wide, such as in a food factory, a kitchen such as a restaurant, and the outdoors. Large equipment is inconvenient to carry, and requires new power supply work and signal wire wiring work for equipment installation. There was a problem.
[0010]
An object of the present invention is to provide a small and simple bioluminescence measuring apparatus for measuring microorganisms and an intracellular ATP measuring kit which can automatically and highly sensitively measure various measuring objects in a wide range of places.
[0011]
[Means for Solving the Problems]
In order to achieve the above object, the luminescence detection device for measuring microorganisms according to the present invention has a sensitivity that can be detected without requiring an operation such as culturing, and it is necessary to automatically add a reagent. In order to improve the measurement sensitivity, a measurement reagent whose reaction rate has been increased by increasing the enzyme concentration (or enzyme activity) is used, and the measurement time and luminescence intensity after the addition of the reagent are accurately measured. It is desirable to start the measurement immediately after the addition of the measurement reagent at an enzyme concentration at which the half life of the luminescence reaction of the enzyme in the luminescence reagent used at that time is within 1 minute.
[0012]
When measuring a large number of samples using a measurement reagent whose reaction rate has been increased by increasing the enzyme concentration (or enzyme activity), in order to accurately control the enzyme reaction that performs the luminescence reaction, the reagents must be sequentially time-series. Since addition and measurement methods cannot be used, it is necessary to perform simultaneous simultaneous reagent addition and simultaneous simultaneous reaction and detection. The device for realizing simultaneous simultaneous reaction and detection includes a reaction tank having a plurality of reaction cells having transparent bottoms arranged one-dimensionally or two-dimensionally, and a plurality of reaction cells located above the reaction tank and provided on a lower surface. A liquid sending unit including a plurality of capillaries associated with the cells one-to-one, a driving unit that drives the liquid sending unit in the vertical direction with respect to the reaction tank, and a lower surface of the reaction tank in one-to-one correspondence with the reaction cells And a photodetector having a plurality of photodetectors arranged in close proximity to each other, wherein the light emission generated in each reaction cell of the reaction tank is individually detected by the plurality of photodetectors of the photodetector. Is preferred.
[0013]
By adopting a structure in which a plurality of reaction cells in a reaction tank and detection elements for detecting light emission such as bioluminescence generated from the reaction cells are closely arranged in a one-to-one correspondence with each other in a vertical direction, a complicated optical system can be realized. Since light emission can be detected with high light collection efficiency without using it, high sensitivity can be easily achieved. In addition, by injecting reagents into the reaction cell from the upper vertical direction of the reaction tank and detecting luminescence such as bioluminescence from the reaction cell from the lower vertical direction of the reaction tank, it is possible to reduce the size of the apparatus and increase the solid angle of light reception. As a result, a small and highly sensitive device can be realized. Furthermore, by simultaneously arranging a plurality of reaction cells and a liquid sending section for injecting reagents in a one-to-one correspondence in the vertical direction, simultaneous simultaneous reaction and detection can be realized with a simple apparatus configuration without using a complicated driving section. . At that time, noise (small pseudo current) due to electrostatic induction due to close contact between the reaction tank and the detection unit causes a problem. However, by disposing a light-transmitting conductive film between the reaction tank and the light detection unit. Noise can be removed. The liquid sending unit includes a reagent container for storing the reagent, a flow path communicating the reagent container with the plurality of capillaries, and a pressurizing unit that pressurizes the inside of the reagent container. It is preferable that the reagent is uniformly sent from the tips of the plurality of capillaries to the plurality of reaction cells by a constant pressure pressurized liquid sending method in which the inside of the reagent container is pressurized for a predetermined time by the means. The constant pressure pressurization method uses a high-pressure cylinder of 3 atm or more, or compressed air (about 1-2 atm) from a pressure source such as a compressor, and applies a pressure of about several seconds using a pressure switching device such as an electromagnetic valve. Can be performed. In normal measurement, three kinds of measurement reagents are required: a free ATP elimination reagent, an ATP extraction reagent, and a luminescence reagent for causing luminescence reaction of ATP in cells using an enzyme. Can be automated by rotating the liquid sending unit corresponding to the above every predetermined time and automatically adding reagents and causing luminescence reaction.
[0014]
Further, using a reagent tray including a sample solution container, an extracellular ATP elimination reagent container, an intracellular ATP extraction reagent container, and a luminescent reagent container therein, the solution is sequentially moved downward to cause a reaction. Thereby, automation can be performed with a simple device configuration. The reagent tray is partitioned by partitions between the containers. This partition may use, for example, a hydrophobic film or the like. The extracellular ATP elimination reagent container, the intracellular ATP extraction reagent container, and the luminescent reagent container are pre-filled with reagents, and these reagents are separated by the above-mentioned hydrophobic film or the like. The partition between each container has a structure which can be easily broken by a jig. This jig is, for example, a needle or the like containing glass, metal, or the like as a material, and breaks a partition between containers by applying a force of several grams. With the reagent tray having such a structure, the sample solution and various reagents can be mixed instantaneously and automatically at any time. The sample used for measurement with this apparatus (for example, solid or liquid such as food or drinking water, or adhered microorganisms wiped off), whether solid, liquid, or gas, is dissolved in the measurement solution. If it is measured.
[0015]
For the measurement of airborne microorganisms, an openable / closable lid is provided in the reaction cell, and the microorganism is sampled by opening the lid for a certain time during sampling. After the sampling is completed, the lid is similarly opened and closed when adding the reagent to the reaction cell. In the case of an apparatus having a plurality of reaction cells, a lid is provided on the upper surface of the reaction tank with a plurality of openings having the same positional relationship as the plurality of reaction cells provided in the reaction tank, and the lid is provided. It is preferable to provide a driving unit for driving between a position where the plurality of openings of the lid and a plurality of reaction cells of the reaction tank match and a position where the plurality of reaction cells do not match.
[0016]
By using a photodiode as an optical sensor, a battery can be driven and a small device can be obtained. Further, by using Bluetooth or the like as the wireless communication, a wireless and compact light-emitting detection device for measuring microorganisms that does not require wiring such as a communication line can be realized.
[0017]
A bioluminescence measuring apparatus for measuring intracellular ATP by a bioluminescence reaction according to the present invention includes a reaction vessel having a substantially transparent bottom and into which a sample is placed, and a reaction vessel located above the reaction vessel to eliminate extracellular ATP. And a drive unit for holding a reagent container having a capillary below the reagent container, a reagent for extracting intracellular ATP, and a reagent for a luminescence reaction, and driving the reagent container up and down with respect to the reaction container. And a light detecting section having a light detecting element arranged close to the lower surface of the reaction container, and detects light emission generated in the reaction container.
[0018]
An A / D converter for converting the output of the photodetector into a digital value; a memory for storing the digital value output from the A / D converter; a wireless communication unit; and a control unit for controlling the memory and the wireless communication unit. And a function of transmitting the contents of the memory via the wireless communication device.
[0019]
The bioluminescence measuring device of the present invention for measuring intracellular ATP of a sample containing cells by a bioluminescent reaction is provided with a reaction container having a substantially transparent bottom and a sample container and a reagent which are located above the reaction container. A liquid sending section, and a light detecting section having a light detecting element arranged in proximity to the lower surface of the reaction vessel, wherein the liquid sending section has a first space for holding a sample solution, and an extracellular ATP elimination reagent. And a container having a third space for holding an intracellular ATP extraction reagent, a fourth space for holding a luminescent reagent, and a needle disposed above the container. By moving the needle downward, the extracellular ATP elimination reagent held in the second space, the intracellular ATP extraction reagent held in the third space, and the fourth sample are held in the sample held in the first space. Mix the luminescent reagent held in the space The ejected into the reaction vessel.
[0020]
The kit for measuring intracellular ATP according to the present invention comprises an ATP elimination reagent for erasing extracellular ATP, an ATP extraction reagent for extracting intracellular ATP, and a luminescent reagent containing luciferin and luciferase, and the concentration of luciferase is 1 g / L. It is characterized by being at least 20 g / L or less. By using a kit containing a reagent having a luciferase concentration of 1 g / L or more and 20 g / L or less, a half-life can be reduced to 1 minute or less, and a large number of samples can be measured almost simultaneously after addition, thereby improving the reproducibility of measurement. Can be. This intracellular ATP measurement kit has first to fourth spaces which are separated from each other by a partition which can be broken by a needle and arranged in a straight line, and the first space is an empty room for receiving a sample. Yes, it is preferable to hold the ATP elimination reagent in the second space, hold the ATP extraction reagent in the third space, and hold the luminescent reagent in the fourth space.
[0021]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, embodiments of the present invention will be described with reference to the drawings.
FIG. 1 is an explanatory view showing the overall configuration of an example of a light emission detection device according to the present invention. FIG. 1 (a) is a cross-sectional view at the time of measurement, and FIG. 1 (b) is a cross-sectional view when a door of the device is opened. It is. The luminescence detection device includes a reaction tank 1, a dispenser 2 disposed above the reaction tank 1 and having a reagent tank for adding a reagent to the reaction tank 1, and a light detection unit disposed closely below the reaction tank 1. 3 is comprised. The luminous reagent in the dispenser 2 is sent by a constant-pressure pressurized liquid sending method in which the inside of the dispenser 2 is pressurized to a constant pressure via a constant-pressure pressurizing pipe 4 to send the solution. The control of the pressurizing time at that time is performed by the solenoid valve 5.
[0022]
The operation procedure is shown below. First, the sealing door 6 is opened, the reagent adding section 7 is raised, and the reaction tank 1 is set. When the measurement is started, the Z stage 8 on which the dispenser 2 is fixed moves downward, the tip of the dispenser 2 is immersed in the reaction tank 1, and a certain amount of reagent is added to the reaction tank 1. The sample in the reaction tank 1 causes a luminescence reaction with the reaction reagent added from the dispenser 2. Light emitted from the inside of the reaction tank 1 is detected by an optical sensor 9 in a light detection unit 3 arranged closely below the reaction tank 1. The apparatus shown in FIG. 1 has a configuration in which a plurality of samples can be measured simultaneously by having a plurality of reaction vessels and dispensers. Prior to injecting the sample into the reaction tank, a treatment for decomposing free ATP using a free ATP elimination reagent and a treatment for extracting ATP in microbial cells using an ATP extraction reagent have been performed in advance. Alternatively, a reagent tank containing a free ATP-eliminating reagent and a reagent tank containing an ATP extraction reagent are loaded into the apparatus, and the reagents in those reagent tanks can be sequentially added to the reaction tank by a dispenser. It can be configured to be performed in the device.
[0023]
FIG. 2 is a detailed view showing the relationship between the dispenser 11 having the reagent tank, the reaction tank 12, and the light detection unit 13. The dispenser 11 includes a reagent tank 14 containing a reaction reagent and a capillary 15 for controlling a flow rate. The dispenser 11 is fixed to a pressurized liquid sending pipe 18 by a fixing jig 17 using an O-ring 16 and is detachable and disposable. As the capillary 15, a fused silica capillary having an inner diameter of 25 μm, an outer diameter of 360 μm, and a length of 20 mm is used. Since the reaction tank 12 has an inner diameter of 2 mm and a depth of 3 mm, and the volume of the reaction solution used is about 4 μL, the length from the quartz glass plate 19 to the liquid surface of the sample solution 20 is about 1.5 mm. The light receiving surface of the light detecting element 21 (S1133--01 manufactured by Hamamatsu Photonics) used in the present invention is 2.4 × 2.6 mm. Under the conditions of the present embodiment, the distance from the bottom surface of the reaction solution to the light receiving surface is 1.4 mm, and the light receiving efficiency is about 7% (this value was almost the same as the value calculated from the experimental value. ). According to this device configuration, the solid-state light receiving angle is increased by a simple method called the contact method (high light collection efficiency) without the need for a complicated optical system using lenses, etc., and the number of parts and the optical axis adjustment are not required. It can be seen that a simple structure can be realized.
[0024]
FIG. 3 is a detailed view showing a relationship between a dispenser 31, a reaction tank 32, and an optical sensor array 33 as another example of the light emission detecting device according to the present invention. The dispenser 31 processes 12 vertical channels and 1 horizontal channel of a 0.8 mm diameter channel 34 at the center in the thickness direction of an acrylic plate having a thickness of 10 mm × 80 mm × 120 mm, and perpendicularly to the acrylic plate. It is manufactured by vertically inserting and bonding 96 capillaries 35 for flow rate control. The flow path 34 was formed by processing a through hole penetrating from one end to the other end of the acrylic plate, and then closing the openings at both ends with plugs. One passage in the lateral direction has a structure in which both ends are closed with screws 37 and 38, and has a function of bleeding air. The end of the capillary 35 was inserted into a hole on the bottom surface of the acrylic plate formed so as to communicate with the flow path 34, and was fixed to the acrylic plate with an adhesive. As the capillary 35, a fused silica capillary having an inner diameter of 25 μm, an outer diameter of 360 μm, and a length of 20 mm was used. The dispenser 31 is provided with a reagent tank 36 on the upper part, and can uniformly discharge the reaction reagent supplied from one place of the reagent tank 36 from the tips of the 96 capillaries 35 by the constant pressure and pressure liquid sending method.
[0025]
The reaction tank 32 used in the present invention is a black quartz glass plate provided with 96 holes of reaction cell holes 39 having a diameter of 2 mm in 8 × 12 rows at a pitch of 9 mm for a minute amount (1 to 10 μL), and a mirror-finished surface. A transparent quartz glass plate 40 is bonded by heat fusion. On the lower surface of the quartz glass plate 40, ITO or SnO ₂ A transparent conductive film made of The transparent conductive film is grounded.
[0026]
The optical sensor array 33 has photodetectors (photodiodes S1133-1-01 manufactured by Hamamatsu Photonics) 41 arranged in 8 × 12 rows at a pitch of 9 mm so that 96 samples can be simultaneously measured using the reaction tank 32. In the present invention, in order to increase the light-collecting efficiency in a simple manner without using a complicated optical system using a lens or the like, in order to increase the light receiving angle by bringing the light detecting element 41 as close to the reaction tank 32 as possible. High light collection efficiency is achieved with a simple structure that requires no parts and does not require optical axis adjustment. The optical sensor array 33 is disposed in close contact with the lower surface of the reaction tank 32. The output of each photodetector 41 is 1 × 10 using a current-voltage conversion amplifier (OPA129UB manufactured by BURR BROWN) and a resistor of 10 GΩ. ¹⁰ And a total gain of 4.5 × 10 using a second-stage operational amplifier (OP07 manufactured by ANALOG DEVICES). ¹¹ Amplified.
[0027]
Since the reaction cell hole 39 has an inner diameter of 2 mm and a depth of 3 mm, and the volume of the reaction solution used is about 4 μL, the length from the quartz glass plate 40 to the liquid surface of the reaction solution is about 1.5 mm. The light receiving surface of the light detecting element 41 (S1133-1 manufactured by Hamamatsu Photonics) used in the present invention is 2.4 × 2.6 mm. Under the conditions of the present embodiment, the distance from the bottom surface of the reaction solution to the light receiving surface is 1.4 mm, and the light receiving efficiency is about 7% (this value was almost the same as the value calculated from the experimental value. ). According to this device configuration, the solid-state light receiving angle is increased by a simple method called the contact method (high light collection efficiency) without the need for a complicated optical system using lenses, etc., and the number of parts and the optical axis adjustment are not required. It can be seen that a simple structure can be realized.
[0028]
FIG. 3 shows an example of the apparatus using the dedicated reaction tank 32, but a commercially available ordinary microtiter plate may be used as the reaction tank. In that case, it is preferable to use a cell holder that also serves as a partition for preventing crosstalk.
[0029]
FIG. 4 is an exploded view of the reaction tank when a commercially available microtiter plate is used as the reaction tank. FIG. 4 also shows a dispenser 51 that supplies a reagent to each reaction cell (well) of the microtiter plate. FIG. 5 is a cross-sectional view of a state where the microtiter plate 52, the cell holder 53, and the conductive light transmitting plate 54 are combined.
[0030]
The cell holder 53 also serving as a partition for preventing crosstalk is a plate-shaped member made of a light-shielding material, and has a hole 56 at a position corresponding to each reaction cell 55 of the microtiter plate 52. The cell holder 53 is pushed into the microtiter plate 52 from below so that the lower projecting portion of the reaction cell 55 enters each hole 56 of the cell holder. The conductive light transmitting plate 54 has ITO or SnO on one surface. ₂ A glass plate on which a transparent conductive film made of is deposited, and is disposed in contact with the lower surface of the cell holder 53. The optical sensor array shown in FIG. 3 is provided below the conductive light transmitting plate 54. Only light emitted from the reaction cell located immediately above the light detection element in the light sensor array is incident, and light emitted from the other reaction cells is not incident because it is blocked by the light-shielding cell holder 53. .
[0031]
Next, an example of a luminescence detection device for measuring airborne microorganisms according to the present invention will be described. Measurement of airborne microorganisms is usually performed in a clean room of a food factory. In recent years, clean rooms have been introduced in food factories, but the performance must be maintained and managed automatically. Therefore, it is necessary to automatically perform operations from sampling to measurement. Sampling of airborne microorganisms is performed by automatically opening a lid of a reaction tank also serving as an airborne microorganism capture tank for a certain period of time, sampling airborne microorganisms, and then automatically performing measurement. In the standby state (FIG. 6a), the lid 62 of the reaction tank 61 also serving as the tank for capturing suspended microorganisms in the air is closed. An ATP extraction reagent 63 is previously contained in the reaction tank 61. After the start of the measurement, the lid 62 is automatically opened, and the microorganisms 64 and 65 suspended in the air are sampled (FIG. 6B). After a certain period of time, the dispenser 66 automatically adds the luminescent reagent into the reaction tank 61, and the luminescent reaction starts. The sample in the reaction tank 61 causes a luminescence reaction with the reaction reagent added from the dispenser 66 (FIG. 6c). Light emitted from the inside of the reaction tank 61 is detected by an optical sensor 68 in a light detection section 67 which is disposed closely below the reaction tank 61.
[0032]
Another example of the luminescence detecting device for measuring airborne microorganisms according to the present invention will be described. In the present invention, a lid capable of opening and closing the opening of each reaction cell is provided on a reaction tank or a microtiter plate used as a reaction tank.
[0033]
FIG. 7 is a schematic view showing an example in which an openable / closable lid is provided on a microtiter plate. FIG. 7 (a) is a schematic plan view, and FIG. 7 (b) is a schematic side sectional view thereof. The cell holder is not shown. The openable lid according to the present embodiment includes a fixed lid 72 that covers the upper and side portions of the microtiter plate 71, and a movable lid 73 that can reciprocate on the fixed lid 72. The fixed lid 72 has a hole 74 (8 × 12 = 96) for adding a reaction reagent at a position corresponding to each reaction cell of the microtiter plate 71. The movable lid 73 has the same number of holes 75 as the number of reaction cells in the same arrangement as the holes 74 of the fixed lid 72, and can be reciprocated in the direction of the arrow by an actuator 76. The figure shows a state in which the movable lid 73 is positioned so that the hole 75 of the movable lid 73 does not coincide with the hole 74 of the fixed lid 72, and each reaction cell 77 is sealed. When sampling airborne microorganisms, the movable lid 73 is moved by the actuator 76 such that the hole 75 of the movable lid 73 matches the hole 74 of the fixed lid 72. After sampling for a certain period of time, the capillary of the dispenser is inserted into the opening formed by overlapping the holes 74 and 75, and a reagent is injected to perform a luminescence reaction. By sealing the reaction cell in this manner, sampling of microorganisms suspended in the air can be performed.
[0034]
FIG. 8 is a diagram showing an example in which an opening / closing lid is provided in the device shown in FIG. FIG. 8A shows a standby state, and FIG. 8B shows a sampling and measurement state of airborne microorganisms.
[0035]
The reaction tank 81 has a movable opening / closing lid 82 at the upper part. The opening / closing lid 82 is formed with a window hole 84 (diameter: about 2 mm) having the same 9 mm pitch and the same size as the reaction cell 83 in the reaction tank 81. The opening / closing lid 82 can be moved in the left-right direction in FIG. In the state before the injection of the reaction reagent, as shown in FIG. 8A, the flow control capillary 87 attached to the dispenser 86 is located at the upper part of the reaction tank 81, and is opened and closed with the center of the reaction cell 83. The center of the window hole 84 of the lid 82 is off. That is, the openable lid 82 is in a closed state, and the reaction cell 83 in the reaction tank 81 is closed by the openable lid 82. Therefore, evaporation of the solution in the reaction cell 83 during standby is prevented.
[0036]
At the time of sampling of airborne microorganisms, as shown in FIG. 8 (b), the openable lid 82 is shifted laterally by about 3 mm by the actuator 85, and the center of the reaction cell 83 and the center of the window hole 84 match. The upper part of the reaction cell 83 is opened. After a certain time, the dispenser 86 descends, the tip of the flow control capillary 87 is immersed in the reaction cell 83, and the reaction reagent is injected from the reagent tank 88. Light emitted from the inside of the reaction cell 83 is detected by a light detection unit 89 disposed closely below the reaction tank 82. After completion of the reaction, the dispenser 86 is raised, and the openable lid 82 is shifted about 3 mm in the lateral direction to be in a sealed state.
[0037]
FIG. 9 is a diagram showing the luminescence reaction rate dependence of the luciferase concentration (or enzyme activity) used in the ATP method. Increasing the concentration of the enzyme luciferase increases the luminescence intensity, but also increases the change in luminescence intensity. In the conventional method, the reaction was carried out by manually mixing the reagents, so that the measurement was performed at a low enzyme concentration with little change in luminescence intensity. That is, in order to maintain the measurement accuracy, the measurement was performed with the sensitivity lowered. In the present invention, the measurement can be performed immediately after the addition of the reagent, and it is possible to use a 10 times or more higher concentration of enzyme than in the past. That is, a detection sensitivity of several tens amol level can be achieved. The detection sensitivity at the level of several tens amol is a value that can measure about several tens of microorganisms, which was impossible with a conventional apparatus.
[0038]
FIG. 10 shows an example in which the measurement was performed while actually changing the enzyme concentration. Even if the luciferase enzyme concentration is increased by 10 times or more, measurement can be performed in real time and accurate measurement can be performed. According to the present invention, by automatically adding reagents and simultaneously measuring a large number of samples, a reaction that takes less than one minute, which has been difficult in the past, can be accurately measured, and high-sensitivity measurement is possible. As shown in FIG. 10, in this measurement, the luciferase enzyme concentration is 0.5 μg / μL or more, preferably 1.0 μg / μL or more. The half-life of the reaction at this enzyme concentration is about 1 minute, a detection sensitivity of several tens amol level can be achieved, and it is possible to measure about several tens of microorganisms which were difficult to measure by the conventional method. However, since the time required for mixing the sample and the reagent is about 3 to 5 seconds, if the reaction is too fast, the reproducibility of the luminescence intensity deteriorates due to the influence of the mixing state (eg, a stirring method). Therefore, the upper limit of the concentration is about 20 μg / μL. By setting such a concentration, the luminescence reaction can be measured immediately after the addition of the reagent, and the half-life can be reduced to within one minute. FIG. 11 shows a calibration curve of ATP as an example of the measurement. In this measurement, the correlation coefficient indicating the reproducibility of the measurement was 0.999 or more, which was good. The detection limit at that time was 80 amol (S / N = 5).
[0039]
The air pressurized liquid feeding method employed in the present invention will be described below with reference to FIG.
The air pressurized liquid sending method is a method of sending a liquid by generating a viscous flow in a flow path system under a constant pressure state. Therefore, if the inner diameter and the length of the microcapillary tube are set such that the pressure loss of the portion other than the microcapillary tube to be used can be neglected, the sub-μL or less solution can be easily sent by controlling the applied pressure and time. In the present invention, a capillary 15 for controlling the flow rate attached to the dispenser 11 is used as a microcapillary. Dispensing (addition) of the reaction reagent 14 in the dispenser 11 also serving as a reagent tank is performed by constant-pressure pressurized liquid supply in which the inside of the dispenser 11 is pressurized to a constant pressure via a constant-pressure pressurizing pipe 18 (see FIG. 2) to perform liquid supply. Perform by the method. The amount of liquid sent at that time follows the Hagen-Poiseuille equation below.
[0040]
(Equation 1)

[0041]
Here, ΔP: applied pressure, r: inner diameter of the microcapillary, t: time during which pressure was applied, μ
: Viscosity of the solution, L: length of the microcapillary.
[0042]
As an example of the present invention, an example in which the injection amount is 0.1 μL or less will be described below. In the present embodiment, in order to satisfy the conditions of using a low pressure of about atmospheric pressure (2 atm or less), pressurizing time of 2 sec or less, and liquid sending amount of 0.1 μL or less, a flow rate of 25 μm in inner diameter and 20 mm in length is used. A control capillary 15 was used.
[0043]
FIG. 12 shows the relationship between the pressurizing pressure and the pressurizing time under this condition. Pressure range 0.5 to 2.0 × 10 ⁵ When used at Pa, a minute amount of liquid supply of 0.1 μL or less can be performed in a constant-pressure pressurization time of 2 seconds or less. The individual reproducibility of an injection volume of 1.0 μL or more can be measured by weight, but cannot be measured if the injection volume is 0.1 μL or less. Therefore, simultaneous reproducibility was measured using a luciferin / luciferase-based bioluminescence system (apyrase, which is an ATPase, was added to adjust the signal peak shape). The number of continuous measurements is 20 times. In the measurement, a reagent was injected into the reaction solution at regular time intervals (1 minute intervals), and reproducibility of luminescence intensity was determined. The volume of the reaction solution was 4.0 μL, and the reagent injection amounts were (a) 0.01 μL, (b) 0.02 μL, and (c) 0.05 μL. Table 1 shows the results. The injection volume reproducibility of a very small amount (0.01-0.05 μL) was as good as 3-5%.
[0044]
[Table 1]

[0045]
In the present invention, since the reaction tank and the detection element are in close contact with each other and a high-magnification amplifier is used, there is a problem that noise (small pseudo current) due to electrostatic induction occurs when the dispenser 11 is driven up and down. In order to eliminate this noise, ITO or SnO was placed on the lower surface of the quartz glass plate 19 attached to the lower part of the reaction tank 12. ₂ Is coated with a transparent conductive film. The transparent conductive film is grounded. The performance of the quartz glass plate coated with the light-transmitting conductive film used in the present invention is 90% or more in transmittance at a wavelength of 450 to 600 nm and the sheet resistance is 1000 to 1500 Ω.
[0046]
FIG. 13 is a diagram showing a noise removing effect of a quartz glass plate coated with a light transmitting conductive film. When a normal quartz glass plate is used, large pseudo signals 91 and 92 are generated as the dispenser 11 is driven up and down as shown in FIG. 13A, but the quartz glass plate coated with a light-transmitting conductive film is used. When no was used, there was no pseudo signal as shown in FIG.
14 and 15 are schematic views showing another example of the light emission detecting device according to the present invention.
[0047]
The apparatus described so far is an example in which pretreatment such as elimination of extracellular ATP is performed in advance, and finally a luminescent reagent is added. The apparatus described here performs a reaction by sequentially adding three types of measurement reagents, a free ATP elimination reagent, an ATP extraction reagent, and a luminescence reagent that causes luminescence of ATP in cells using an enzyme. is there. The measurement target described here is a liquid sample, a container, an instrument, and the like. When the sample to be measured is a liquid such as soft drink or coffee, a part of the sample is used. In the case of containers and utensils in food factories, chopping boards in kitchens, etc., a wiping test is usually performed. For the wiping inspection, use a cotton swab to wipe off the inspection area and then dissolve it in the measurement solution before use. Products that integrate a swab and a reagent for wiping are also on the market (for example, Kikkoman's "LuciPack" ^TM )).
[0048]
FIG. 14 is composed of three reagent tanks: an extracellular ATP elimination reagent tank 101, an intracellular ATP extraction reagent tank 102, and a luminescent reagent tank 103, and a sample tank 104 is provided thereunder. When a sample solution 105 is set in the sample tank 104 in advance and measurement is started, the extracellular ATP erasing reagent tank 101, the intracellular ATP extraction reagent tank 102, and the luminescent reagent tank 103 are lowered at regular intervals, and the capillary 106 at the tip is moved to the sample. It is immersed in the sample solution 105 in the tank 104, and the reagent is added. After the addition of the luminescent reagent, the luminescence generated from the sample solution 105 in the sample tank 104 is detected by an optical sensor 108 in a light detection unit 107 arranged in close contact with the lower part of the sample tank 104. The timing of adding the free ATP elimination reagent and the ATP extraction reagent largely depends on the measurement target. For example, the reaction time of the free ATP eliminating reagent depends on the degree of contamination of the measurement target, and the reaction time with the ATP extraction reagent depends on the microorganism to be measured. In a usual measurement, the reaction time of the free ATP elimination reagent is about 5 minutes, and the reaction time with the ATP extraction reagent is about 20 minutes at the maximum. The measurement can be started immediately after the addition and mixing of the luminescent reagent. That is, the ATP extraction reagent is added 5 minutes after the addition of the free ATP elimination reagent, and the luminescent reagent is added 20 minutes after the addition, and the measurement may be started immediately.
[0049]
FIG. 15A shows four containers, a sample solution container 111, an extracellular ATP elimination reagent container 112, an intracellular ATP extraction reagent container 113, and a luminescent reagent container 114, and a needle disposed at the top of the container. Shown is a reagent tray 116 composed of 115. The needle 115 is attached to a jig 117 also serving as a lid. At the time of measurement, the jig 117 also serving as a lid is removed from the reagent tray 116, and the sample solution is dispensed to the reagent tray 116. The bottom of the reagent tray 116 is set on the reaction cell 118 in the apparatus, and the top is set on the Z stage 119. As shown in FIG. 15B, the structure of the bottom of the reagent tray 116 can be easily fitted into the cell 118.
[0050]
As for the method of moving the reagent solution, each reagent tray is partitioned between the containers by a hydrophobic film or the like, and the needle 115 disposed at the top is moved downward by the Z stage 119, so that the bottom of each container, That is, a hole is formed in the above hydrophobic film or the like. Through these holes, various reagents and the like move downward and are mixed. The solution in which all the reagents are mixed moves to the reaction tank 118 and the luminescence reaction starts. The light emitted from the reaction tank 118 is detected by an optical sensor 140 in a light detection unit 120 disposed closely below the reaction tank 118. In the example of the present invention shown in FIGS. 15A and 15B, a dispenser using a constant-pressure and pressure-feeding method is not required, and a simpler device configuration is obtained. This reagent tray can be easily attached to the apparatus and can be handled like a normal reaction vessel. Alternatively, a kit in which various reagents are partitioned by a hydrophobic membrane or the like and filled in each container may be used.
[0051]
Hereinafter, a method of using the device of the present invention in a place where a measurement place is small and it is difficult to install a power supply and a communication line (for example, a small production line of a food factory, a freezer storage, a kitchen, an outdoor storage, a river, etc.) I do.
[0052]
If the measurement place is small and the installation place is limited, it is difficult to install a large device. Furthermore, when an additional device is installed, power supply work may be required. This device uses a low power consumption photodiode as an optical sensor so that it can operate with a built-in power supply. Further, by incorporating a low-power-consumption wireless device, the device can wirelessly measure without a power line and a signal line.
[0053]
FIG. 16 is a functional block diagram showing another example of the present invention having a wireless communication function. This device includes a light detection device 121 and a wireless control device 122. The photodetector 121 includes a photodetector 124 having a plurality of photosensors 123, an S / H circuit 125 for sequentially sampling the outputs of the plurality of photosensors 123, and an A for converting an analog value detected by the photosensor 123 into a digital value. A / D converter 126, memory 127 for storing the value converted by A / D converter 126, or display unit 128 for display, dispenser control unit 129 for automatically adding a reagent, and communication with wireless control device 122 It comprises a wireless device 130, a power supply 131, and a CPU 132. The CPU 132 receives the command from the wireless control device 122 and automatically performs measurement. After the measurement, the measurement data is displayed on the display unit and transferred to the wireless control device 122 via the wireless device 130. Note that it is also possible to control a plurality of light detection devices with respect to the wireless control device 122.
[0054]
【The invention's effect】
ADVANTAGE OF THE INVENTION According to this invention, the bioluminescence measuring apparatus and microcellular ATP measurement kit for small and easy microbe measurement can be obtained.
[Brief description of the drawings]
FIG. 1A is a cross-sectional view at the time of measurement showing the entire configuration of an example of a light emission detection device according to the present invention.
FIG. 1B is a cross-sectional view when the door of the apparatus is opened.
FIG. 2 is a detailed view showing the relationship between a dispenser having a reagent tank, a reaction tank, and a light detection unit.
FIG. 3 is a detailed view showing a relationship between a dispenser, a reaction tank, and an optical sensor array.
FIG. 4 is an exploded view of the reaction tank when a commercially available microtiter plate is used as the reaction tank.
FIG. 5 is a cross-sectional view showing a state where a microtiter plate, a cell holder, and a conductive light transmitting plate are combined.
FIGS. 6A and 6B are schematic diagrams of a luminescence detecting device for measuring airborne microorganisms, wherein FIG. 6A is a diagram showing a standby state, FIG. 6B is a diagram at the time of sampling, and FIG. 6C is a diagram at the time of measurement.
FIGS. 7A and 7B are schematic views showing an example in which an openable / closable lid is provided on a microtiter plate. FIG. 7A is a schematic plan view, and FIG.
8A and 8B are schematic diagrams illustrating an example in which an opening / closing lid is provided on the photodetector illustrated in FIG. 3, wherein FIG. 8A illustrates a standby state, and FIG. 8B illustrates a sampling and measurement state of airborne microorganisms. FIG.
FIG. 9 is a graph showing the luminescence reaction rate dependence of luciferase concentration (or enzyme activity) used in the ATP method.
FIG. 10 is a graph showing measured values of luciferase enzyme concentration and luminescence intensity change over time.
FIG. 11 is a diagram showing a calibration curve of ATP measured using the present apparatus.
FIG. 12 is a diagram showing a relationship between a pressurizing pressure and a pressurizing time under use conditions of an example of the present apparatus.
FIG. 13 is a view showing a noise removing effect of a quartz glass plate coated with a light transmitting conductive film.
FIG. 14 is a schematic diagram showing another example of a luminescence detection device using three reagents according to the present invention.
FIG. 15A is a schematic view showing an example of an integrated reagent tray using a needle according to the present invention.
FIG. 15 (b) is a schematic view showing another example of the luminescence detecting device in which the integrated reagent tray according to the present invention is set.
FIG. 16 is a functional block diagram showing another example of the present invention having a wireless communication function.
[Explanation of symbols]
1, 12, 32, 61, 81 ... reaction tank, 2, 11, 31, 51, 66, 86 ... dispenser, 3, 13, 67, 89, 107, 118, 124 ... light detection unit, 4 ... constant pressure pressurizing tube 5, a solenoid valve, 6 a closed door, 7 a reagent adding section, 8, 119 a Z stage, 9, 68, 108, 123 ... an optical sensor, 14, 36, 88 ... a reagent tank, 15, 35, 87, 106: Capillary, 16: O-ring, 17: Fixing jig, 18: Pressurized liquid sending tube, 19: Quartz glass plate, 20, 105: Sample solution, 21, 41: Photodetector, 33: Optical sensor array, 34 ... flow paths, 37, 38 ... screws, 39 ... reaction cell holes, 40 ... quartz glass plates, 52, 71 ... microtiter plates, 53 ... cell holders, 54 ... conductive light transmitting plates, 55, 83 ... reaction cells, 56 ... hole, 62 ... lid, 63 ... reagent, 6 72, fixed lid, 73, movable lid, 74, fixed lid hole, 75, movable lid hole, 76, 85, actuator, 82, openable lid, 84, window hole, 91 , 92: pseudo signal, 101: extracellular ATP elimination reagent tank, 102: intracellular ATP extraction reagent tank, 103: luminescence reagent tank, 104: sample tank, 111: sample solution container, 112: extracellular ATP elimination reagent Container, 113: Container for intracellular ATP extraction reagent, 114: Container for luminescent reagent, 115: Needle, 116: Reagent tray, 117: Jig, 121: Photodetector, 122: Wireless controller, 125: S / H Circuit, 126 A / D converter, 127 memory, 128 display unit, 129 dispenser control unit, 130 wireless device, 131 power supply, 132 CPU

Claims (5)

In a bioluminescence measurement device for measuring intracellular ATP by a bioluminescence reaction,
A reaction vessel having a substantially transparent bottom and containing a sample;
A reagent container having a capillary located below the reaction container and containing a reagent for erasing extracellular ATP, a reagent for extracting intracellular ATP, and a reagent for luminescence reaction, A driving unit for driving the reagent container in the vertical direction with respect to the reaction container,
A light detection unit having a light detection element arranged close to the lower surface of the reaction vessel,
A bioluminescence measuring device for detecting luminescence generated in the reaction container. The bioluminescence measurement device according to claim 1, wherein an A / D converter that converts an output of the photodetector into a digital value, a memory that stores the digital value output from the A / D converter, and a wireless communication unit; A bioluminescence measurement device, comprising: a control unit that controls the memory and the wireless communication unit; and a function of transmitting the content of the memory via the wireless communication device. In a bioluminescence measuring device for measuring intracellular ATP of a sample containing cells by a bioluminescence reaction,
A reaction vessel having a substantially transparent bottom;
A liquid sending unit that is located above the reaction container and holds a sample and a reagent,
Having a light detection unit having a light detection element arranged close to the lower surface of the reaction vessel,
The liquid sending section holds a first space for holding a sample solution, a second space for holding an extracellular ATP elimination reagent, a third space for holding an intracellular ATP extraction reagent, and a luminescent reagent. A container having a fourth space, and a needle disposed at an upper portion of the container, and moving the needle downward to allow the sample held in the first space to move into the second space. Mixing the retained extracellular ATP elimination reagent, the intracellular ATP extraction reagent retained in the third space, and the luminescent reagent retained in the fourth space, and discharging the mixture to the reaction vessel. Characteristic bioluminescence measuring device. An ATP eliminating reagent for eliminating extracellular ATP;
An ATP extraction reagent for extracting intracellular ATP,
Luciferin and a luminescent reagent containing luciferase,
A kit for measuring intracellular ATP, wherein the concentration of the luciferase is 1 g / L or more and 20 g / L or less. 5. The kit for measuring intracellular ATP according to claim 4, comprising first to fourth spaces which are separated from each other by a partition which can be broken by a needle and are arranged in a straight line, and wherein the first space contains a sample. And the second space holds the ATP elimination reagent, the third space holds the ATP extraction reagent, and the fourth space holds the luminescent reagent. Intracellular ATP measurement kit.

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