JP2007502979A - マイクロ流体装置における混合 - Google Patents
マイクロ流体装置における混合 Download PDFInfo
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- JP2007502979A JP2007502979A JP2006523888A JP2006523888A JP2007502979A JP 2007502979 A JP2007502979 A JP 2007502979A JP 2006523888 A JP2006523888 A JP 2006523888A JP 2006523888 A JP2006523888 A JP 2006523888A JP 2007502979 A JP2007502979 A JP 2007502979A
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Abstract
Description
本発明は、一般にマイクロ流体装置に関し、特に、血液、尿などのような生物学的試料の分析に使用される装置に関する。このようなマイクロ流体装置は、少量の液体試料を試薬と接触させて、分析対象物の有無の定性的又は定量的計測を提供する。通常、計量された試料が、試薬又は試薬との接触に備えて試料を準備するために使用されるコンディショニング剤を含有する1個以上のチャンバを通過する。試料の量は普通、10μL未満であり、チャンバは同程度のサイズである。チャンバは毛管通路によって相互接続され、これらの毛管通路中を試料が毛管力又は加えられる力、たとえば遠心力によって移動する。
液体は、マイクロ流体装置中、少なくとも二つの液体を第一のチャンバに小出しして液体どうしを合わせる方法によって混合される。好ましい実施態様では、液体は、液体を含有する溜めから第一のチャンバに小出しされる。第二工程で、合わせた液体を第一のチャンバから少なくとも一つの毛管通路を介して第二のチャンバに放出して液体どうしを混合する。一部の実施態様では、二つ以上の平行な毛管通路が使用される。もう一つの実施態様では、第二のチャンバは、少なくとも一つの毛管通路を介して少なくとも第三の混合チャンバと液的に連絡している。
マイクロチャネル中の流れ
本発明を使用するマイクロ流体装置は通常、約1〜2000μm、好ましくは約200〜1000μmの範囲の断面寸法を有する流路を使用する。流路が、ほぼ長方形である断面を有する場合、寸法とは、長方形の対角線をいうことができる。小さめの流路が分析される試料中の成分を効果的にろ別することができるため、このような流路の最小寸法は、多くの実際の用途で、約5μmであると考えられる。問題なければ、より小さな寸法を使用してもよい。好ましい範囲の流路は、毛管力だけで液体試料を移動させることを可能にする。また、試料流体に対して疎水性になるように処理しておいた毛管壁によって、又は流路寸法の顕著な変化によって移動を止めることも可能である。流れに対する抵抗には、たとえばポンピング、真空、電気浸透、加熱、吸収材、追加的な毛管力又は遠心力によって圧力差を加えることによって打ち勝つことができる。その結果、マイクロ流体装置中で実施される分析のための要求に応じて、液体を計量し、装置の一つの領域から別の領域に移動させることができる。
本発明の分析装置は「チップ」と呼ぶこともできる。チップは一般に小さく平坦であり、典型的には約1〜2インチ四方(25〜50mm四方)であるか、半径が約40〜80mmのディスクである。試料の量は小さくなる。たとえば、試験ごとに約0.1〜10μLしか含まないが、試料の総量は10〜200μLの範囲であることができる。試料流体及び試薬を保持するチャンバは、試料を容易に見て、試料の反応から生じる変化を適当な器具によって計測することができるよう、比較的幅広く浅くなる。相互接続する毛管通路は通常、1〜2000μm、好ましくは200〜500μmの範囲の断面寸法を有する。形状は、通路を形成するために使用される方法によって決まるが、長方形の断面を有する通路が好ましい。通路の深さは、試料が粒子を含有する多くの実際の用途では、少なくとも5μmであるが、試料の性質が許すならば、より小さくてもよい。
正確な分析結果を得なければならないならば、試料と多量の液体試薬又はコンディショニング剤との混合が重要である。本明細書で記載するチャンバと毛管との組み合わせで徹底的な混合が起こることは証明されているが、混合が起こる過程は十分には解明されていない。従来技術の多くは、ラミナー流が効率的な混合を阻止すると仮定し、したがって、平行に流れる薄い液層を形成して拡散混合を促進することを重視している。本発明者らは、彼らの方法で、混合に有利な局在性効果が起こるが、それを測ることは困難であると考えている。液体は毛管を通過するが、それはラミナー流としてであり、したがって、ほとんど混合は起こらないと予想するであろう。しかし、液体が、比較的大きなチャンバを接続する毛管に入り、そこを出るとき、液体が加速又は減速し、別個の縁の周囲を流れるとき何らかの局在性の渦流又は乱流が発生するということは考えられる。したがって、流れは呼称的にはラミナー性であるかもしれないが、毛管及びチャンバの壁と液体との交点で生じる効果が液体の混合に貢献するのかもしれない。さらには、遠心(又は他の)力の適用によって液体にエネルギーを加えて、液体を付勢して毛管ストッパに打ち勝たせる。液体は、その初期位置から毛管を通過して大きなチャンバに入るとき、加速し、減速する。液体が毛管から出るとき小滴がしばしば形成することが観測されている。異なる液体を合わせる小滴の形成が液体の混合を誘発するのかもしれない。個々の小滴を合わせることが、成層と類似した過程でさらなる混合を提供すると推測される。すなわち、二つの不相溶性の液体が、それらを連続的に分割し、成層することによって合わされるならば、最終的に、層は、区別不可能であるほど薄くなる。したがって、何千もの小滴が合わされるならば、二つの液体の分離ははっきりせず、液体は実質的に完全に混合する。また、液体の細分化が進み、分子が移動しなければならない距離が減るにつれ、分子拡散によってある程度の混合が起こると推測される。混合が起こったのち混合の程度を決定することができるが、必要なマイクロ流体機構の設計は、混合される液体の相対量及びそれらの物性に依存して異なり、実験的確認を要するかもしれない。
本発明のマイクロ流体装置には数多くの用途がある。分析は、血液、尿、水、唾液、髄液、腸液、食品及び血漿をはじめとする多くの生物学的流体の試料に対して実施することができる。血液及び尿が特に対象である。試験する流体の試料を試料溜めに入れたのち、一つ以上の計量毛管又は溜めの中で分析量になるまで計量する。計量した試料を、タンパク質、細胞、小さな有機分子又は金属をはじめとする分析対象物に関して試験する。そのようなタンパク質の例は、アルブミン、HbAlc、プロテアーゼ、プロテアーゼ抑制因子、CRP、エステラーゼ及びBNPを含む。分析することができる細胞としては、大腸菌、シュードモナス、白血球、赤血球、ピロリ菌、A群連鎖球菌、クラミジア及び単核症がある。検出することができる金属としては、鉄、マンガン、ナトリウム、カリウム、リチウム、カルシウム及びマグネシウムがある。
図1に示す全体構造を有する、チャンバ18及び22を接続する五つの平行な毛管20等を含むマイクロ流体装置を製造した。
一つの毛管通路だけで第一のチャンバ18と第二のチャンバ22とを接続するという点で実施例1の装置とは異なるもう一つのマイクロ流体装置を製造した。また、第二のチャンバ22には、遠心力が加えられる方向に下がる5連続の段を設けた。第一のチャンバ18は、直径5.5mm、深さ1.5mmであり、容量が約36μLであった。第二のチャンバ22は、幅5mm、長さ7mm、平均深さ約1.2mmであり、容量が約46μLであった。一つの毛管(長さ3mm、深さ200μm、幅500μm)が段付き斜面の上に出ていた。混合チャンバと、希釈チャンバに供給する二つの毛管とは、実施例1と同じ寸法であった。
この実施例では、図3に示すタイプのマイクロ流体チップでHbAlcの試験を実施した。内部構造の界面エネルギーは、表面に対して25°の水の接触角を提供するように調節し、ポリプロピレンフィルムのふた(Excel 2930)で覆った。血液試料を試料導入口10から導入すると、そこから毛管作用によってプレチャンバ12まで進み、さらに計量毛管14に達した。補助的な計量溜め16があってもよいが、試料サイズがさらなる容積を要する場合だけ設けられる。毛管14及び溜め16中の試料の量は0.3μLであった。変性剤/酸化液(9.62μL)(Sigma哺乳動物細胞溶解/抽出剤)を溜め18に収容した。それを第一のチャンバ20(18.84μL)に空け、同時に、距離29mmのところで1200rpmで回転させることによる遠心力の適用によって毛管ストッパ(図示せず)に打ち勝つことにより、計量溜め16及び対応する計量毛管14を空にした。第一のチャンバ20が血液試料及び変性剤/酸化剤のための空間を提供した。合わせた液を、30×30μmの断面寸法を有する長さ2000μmの毛管通路3本のセットに通して移した。第二のチャンバ30が液体を受け、混合した。力を解除すると、流体がチャンバ30の上から出て、毛管通路23を通ってチャンバ24に入った。43mmの距離のところで2500rpmで回転させることにより、過剰な液体を廃棄留め31に移した。
図3のチップを2種類の溶液で試験した。第一の試験では、pH4のリン酸緩衝剤を試料導入口10に導入し、そこから試料毛管14及び溜め16に移した。次いで、この溶液を、第一の混合チャンバ20の中で、チャンバ18中のpH10のリン酸緩衝剤と合わせた。染料計測により、pH7のチャンバ30中で二つの緩衝溶液の混合が実質的に完了したことがわかった。緩衝剤よりも粘稠な液体である血液が試料である場合、チャンバ18からの溶解緩衝剤(チオシアン酸リチウム)との混合は、図3の第一のチャンバ20及び第二のチャンバ30の両方の使用を要した。
図1の全体構造及び設計要素を有するマイクロ流体装置における血液と緩衝剤水溶液との混合をシミュレーションするため、25%のポリエチレングリコール(PEG20,000mw)を0.5N NaOH溶液に加えた。粘度はほぼヒト血液の粘度であった。PEG/NaOH溶液10μLを溜め10に加え、pH4の緩衝剤(50mmリン酸)100μLを溜め14に加えた。フェノールレッドpH指示薬を使用して、二つの溶液をチャンバ18及び22の中で合わせたときの混合の進行を示した。高めの遠心力を加えた場合、目視では、液体は別個の液体として見えるようであったが、遠心力を減らした場合、完全な混合が起こっていることがわかった。
混合した血液と緩衝剤との希釈試料を遠心分離し、混合した血液と緩衝剤をBayer潜血試薬パッドで試験することにより、緩衝剤で血液を溶解させる効率を試験した。溶解が不完全であるならば、遠心分離器中に赤い血液のペレットが形成するか、潜血パッド中に濃緑色のスポットが現れる。比較のため、血液50μL及び希釈又は非希釈溶解緩衝剤(チオシアン酸リチウム)500μLの溶液を3分間インキュベートしたのち、1300rpmで10分間、遠心分離器中で回転させた。次に、溶液をリン酸緩衝食塩溶液(pH7.0)中で100倍希釈し、再び1300rpmで10分間遠心分離した。マイクロ流体装置中で溶解緩衝剤への血液の混合を実施し、その後、混合物を混合チャンバから取り出し、リン酸緩衝剤で100倍及び10,000倍に希釈し、遠心分離及び潜血試薬法によって試験した。血液がマイクロ流体装置中で実質的に完全に溶解していることがわかった。
Claims (39)
- マイクロ流体装置中で液体を混合する方法であって、
(a)少なくとも第一の液体及び第二の液体を第一のチャンバに小出しして、合わせた液体を形成することと、
(b)(a)の前記合わせた液体を、前記第一のチャンバから、前記第一のチャンバと液的に連絡した少なくとも一つの毛管通路を介して第二のチャンバに放出して、前記合わせた液体の混合を完成することと
を含む方法。 - (a)の前記合わせた液体を二つ以上の毛管通路に通して前記第二のチャンバに放出する、請求項1記載の液体混合方法。
- (a)の前記合わせた液体を少なくとも二つの毛管通路に通して前記第二のチャンバに放出する、請求項2記載の液体混合方法。
- 前記第二のチャンバが少なくとも一つの毛管通路を介して少なくとも第三のチャンバと液的に連絡している、請求項1記載の液体混合方法。
- (a)の前記合わせた液体を小滴の形態で前記第二のチャンバに放出する、請求項1記載の方法。
- 前記第一のチャンバが、(a)の合わせた液体の量の少なくとも約2倍の容積を有する、請求項1記載の方法。
- 前記第二のチャンバが、(a)の合わせた液体の量の少なくとも約2倍の容積を有する、請求項1記載の方法。
- 前記第一のチャンバが、(a)の合わせた量を保持するために必要な深さの少なくとも約2倍の深さを有する、請求項6記載の方法。
- 前記第二のチャンバが、(a)の合わせた量を保持するために必要な深さの少なくとも約2倍の深さを有する、請求項7記載の方法。
- 第一のチャンバ中の液位の上に少なくとも100μmの空間がある、請求項1記載の方法。
- 第二のチャンバ中の液位の上に少なくとも100μmの空間がある、請求項1記載の方法。
- 前記少なくとも一つの毛管通路が1〜2000μmの断面寸法を有する、請求項1記載の方法。
- 前記少なくとも一つの毛管通路が200〜1000μmの断面寸法を有する、請求項12記載の方法。
- 前記少なくとも一つの毛管通路が0.5〜100mmの長さを有する、請求項1記載の方法。
- 前記少なくとも一つの毛管通路が1〜50mmの長さを有する、請求項14記載の方法。
- 三つ以上の毛管通路が前記第一及び第二のチャンバの間で液的に連絡している、請求項1記載の方法。
- 前記第一及び第二のチャンバの少なくとも一方が、前記合わせた液体の混合を支援するための段又は斜面を含む、請求項1記載の方法。
- (a)の前記合わせた液体の前記少なくとも一つの毛管通路中の速度が少なくとも1mm/secである、請求項1記載の方法。
- 前記第一及び第二の液体を溜めから毛管通路に通して前記第一のチャンバに小出しする、請求項1記載の方法。
- 合わせた液体を完全に混合したのち、さらなる処理のために下流のチャンバに移す、請求項1記載の方法。
- (a)少なくとも第一の液体及び第二の液体を受け、合わせるための第一のチャンバと、
(b)前記少なくとも第一及び第二の液体の完全な混合のための、少なくとも一つの毛管通路を介して前記第一のチャンバと液的に連絡している第二のチャンバと
を含むマイクロ流体装置。 - 前記第一及び第二のチャンバが二つ以上の毛管通路を介して液的に連絡している、請求項21記載のマイクロ流体装置。
- 前記第一及び第二のチャンバが少なくとも二つの毛管通路を介して液的に連絡している、請求項22記載のマイクロ流体装置。
- 前記第二のチャンバが少なくとも一つの毛管通路を介して少なくとも第三のチャンバと液的に連絡している、請求項21記載のマイクロ流体装置。
- 前記第一のチャンバが、前記第一及び第二の容器の合わせた容積の少なくとも約2倍の容積を有する、請求項21記載のマイクロ流体装置。
- 前記第二のチャンバが、前記第一及び第二の容器の合わせた容積の少なくとも約2倍の容積を有する、請求項21記載のマイクロ流体装置。
- 前記第一のチャンバが、前記第一及び第二の容器の合わせた容積を保持するために必要な深さの少なくとも約2倍の深さを有する、請求項25記載のマイクロ流体装置。
- 前記第二のチャンバが、前記第一及び第二の容器の合わせた容積を保持するために必要な深さの少なくとも約2倍の深さを有する、請求項26記載のマイクロ流体装置。
- 第一のチャンバ中の液位の上に少なくとも100μmの空間がある、請求項21記載のマイクロ流体装置。
- 第二のチャンバ中の液位の上に少なくとも100μmの空間がある、請求項21記載のマイクロ流体装置。
- 前記少なくとも一つの毛管通路が1〜2000μmの断面寸法を有する、請求項21記載のマイクロ流体装置。
- 前記少なくとも一つの毛管通路が200〜1000μmの断面寸法を有する、請求項31記載のマイクロ流体装置。
- 前記少なくとも一つの毛管通路が0.5〜100mmの長さを有する、請求項21記載のマイクロ流体装置。
- 前記少なくとも一つの毛管通路が1〜50mmの長さを有する、請求項33記載のマイクロ流体装置。
- 三つ以上の毛管通路が前記第一及び第二のチャンバの間で液的に連絡している、請求項21記載のマイクロ流体装置。
- 前記少なくとも一つの通路が、合わせた液体の少なくとも1mm/secの速度を提供するサイズである、請求項21記載のマイクロ流体装置。
- 前記第一及び第二のチャンバの少なくとも一方が、前記第一及び第二の液体の混合又は取り出しを支援するための段又は斜面を含む、請求項21記載のマイクロ流体装置。
- 前記第一のチャンバが、毛管通路を介して、前記少なくとも第一及び第二の液体を収容する溜めと液的に連絡している、請求項21記載のマイクロ流体装置。
- 前記第二のチャンバが、混合が完了する前の前記液体の早期移動を防ぐための手段を含む、請求項21記載のマイクロ流体装置。
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JP2006523888A Active JP4707035B2 (ja) | 2003-08-19 | 2004-08-05 | マイクロ流体装置における混合 |
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US20050041525A1 (en) | 2005-02-24 |
CA2533107A1 (en) | 2005-03-03 |
JP4707035B2 (ja) | 2011-06-22 |
PL1658130T3 (pl) | 2008-10-31 |
CA2533107C (en) | 2012-04-17 |
ATE392947T1 (de) | 2008-05-15 |
ES2305856T3 (es) | 2008-11-01 |
EP1658130A1 (en) | 2006-05-24 |
US7347617B2 (en) | 2008-03-25 |
DE602004013339D1 (de) | 2008-06-05 |
TW200516253A (en) | 2005-05-16 |
PT1658130E (pt) | 2008-07-16 |
DK1658130T3 (da) | 2008-09-01 |
EP1658130B1 (en) | 2008-04-23 |
WO2005018787A1 (en) | 2005-03-03 |
DE602004013339T2 (de) | 2009-07-02 |
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