JPWO2014042177A1 - Liquid feeding method, centrifugal separation method, liquid feeding device and centrifugal separator - Google Patents

Liquid feeding method, centrifugal separation method, liquid feeding device and centrifugal separator Download PDF

Info

Publication number
JPWO2014042177A1
JPWO2014042177A1 JP2014535566A JP2014535566A JPWO2014042177A1 JP WO2014042177 A1 JPWO2014042177 A1 JP WO2014042177A1 JP 2014535566 A JP2014535566 A JP 2014535566A JP 2014535566 A JP2014535566 A JP 2014535566A JP WO2014042177 A1 JPWO2014042177 A1 JP WO2014042177A1
Authority
JP
Japan
Prior art keywords
liquid
density
fine
centrifugal
liquid feeding
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
JP2014535566A
Other languages
Japanese (ja)
Other versions
JP6273510B2 (en
Inventor
芳昭 浮田
芳昭 浮田
高村 禅
禅 高村
崇之 小黒
崇之 小黒
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Yamanashi NUC
Original Assignee
University of Yamanashi NUC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Yamanashi NUC filed Critical University of Yamanashi NUC
Publication of JPWO2014042177A1 publication Critical patent/JPWO2014042177A1/en
Application granted granted Critical
Publication of JP6273510B2 publication Critical patent/JP6273510B2/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502753Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by bulk separation arrangements on lab-on-a-chip devices, e.g. for filtration or centrifugation
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06Fluid handling related problems
    • B01L2200/0636Focussing flows, e.g. to laminate flows
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2200/00Solutions for specific problems relating to chemical or physical laboratory apparatus
    • B01L2200/06Fluid handling related problems
    • B01L2200/0647Handling flowable solids, e.g. microscopic beads, cells, particles
    • B01L2200/0652Sorting or classification of particles or molecules
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0803Disc shape
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00Additional constructional details
    • B01L2300/08Geometry, shape and general structure
    • B01L2300/0861Configuration of multiple channels and/or chambers in a single devices
    • B01L2300/0864Configuration of multiple channels and/or chambers in a single devices comprising only one inlet and multiple receiving wells, e.g. for separation, splitting
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2400/00Moving or stopping fluids
    • B01L2400/04Moving fluids with specific forces or mechanical means
    • B01L2400/0403Moving fluids with specific forces or mechanical means specific forces
    • B01L2400/0409Moving fluids with specific forces or mechanical means specific forces centrifugal forces
    • B01L2400/0412Moving fluids with specific forces or mechanical means specific forces centrifugal forces using additionally coriolis forces
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B01L3/502769Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements
    • B01L3/502776Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip characterised by multiphase flow arrangements specially adapted for focusing or laminating flows

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Dispersion Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Hematology (AREA)
  • Clinical Laboratory Science (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Centrifugal Separators (AREA)
  • Physical Or Chemical Processes And Apparatus (AREA)
  • Separation Of Solids By Using Liquids Or Pneumatic Power (AREA)
  • Sampling And Sample Adjustment (AREA)
  • Automatic Analysis And Handling Materials Therefor (AREA)

Abstract

マイクロデバイス等に設けた微細流路内の層流の安定化を実現できる遠心方式による送液方法等を提供する。遠心ローターの回転方向に沿って配置した微細流路内に、密度が異なる2種類以上の液体を密度が小さい方から順に遠心ローターの半径方向外側に積層することで、微細流路内の密度勾配を階段状に変化させる。微細流路内にあえて密度勾配を形成すると共に、密度の勾配方向と遠心力の作用方向とを一致させることで、液体に作用する遠心力を利用してコリオリの力による2次流れを抑制して、層流を安定化させることができる。図3Provided is a centrifugal liquid feeding method that can stabilize a laminar flow in a microchannel provided in a microdevice or the like. By laminating two or more kinds of liquids with different densities in the fine flow path arranged along the rotation direction of the centrifugal rotor, in order from the one with the smallest density, radially outside the centrifugal rotor, the density gradient in the fine flow path Is changed stepwise. In addition to forming a density gradient in the microchannel and matching the direction of density gradient and the direction of centrifugal force, the secondary flow caused by the Coriolis force is suppressed by utilizing the centrifugal force acting on the liquid. Thus, the laminar flow can be stabilized. FIG.

Description

本発明は、微細流路内の送液方法等に関し、具体的には、マイクロデバイス等に設けた微細流路内の層流の安定化を実現できる遠心方式による送液方法等に関する。   The present invention relates to a liquid feeding method and the like in a fine channel, and specifically relates to a centrifugal liquid feeding method and the like that can realize stabilization of a laminar flow in a fine channel provided in a microdevice or the like.

近年、試料分析の各工程の手間を省き、作業時間を短縮するための技術として、マイクロ統合分析システム(Micro Total Analysis System:μTAS)あるいはラボチップ(Lab-on-a-chip)と呼ばれる数センチないし数ミリ程度の超小型の生化学分析デバイス(マイクロデバイス)が登場している。
マイクロデバイスはこれまで人手で行っていた各オペレーションやオペレーション間の試料の移動など、分析に関わる一連の工程を1つの基板上で再現するものであり、従来と比較して試料や試薬の必要量が少ない、反応時間が短い、廃棄物が少ないなどのメリットがあり、医療診断、環境や食品のオンサイト分析、医薬品や化学品の生産等、広い分野での利用が期待されている。
In recent years, a few centimeters or so-called micro total analysis system (μTAS) or lab chip (Lab-on-a-chip) has been used as a technology to save time and labor for sample analysis. An ultra-small biochemical analysis device (micro device) of several millimeters has appeared.
A microdevice reproduces a series of processes related to analysis, such as each operation that has been performed manually and sample transfer between operations, on a single substrate. There are merits such as less reaction time, shorter reaction time, and less waste, and it is expected to be used in a wide range of fields such as medical diagnosis, on-site analysis of the environment and food, and production of pharmaceuticals and chemicals.

通常、マイクロデバイスは、一つの基板上にリアクタやリザーバー等の各種容器及びこれらを繋ぐ微細流路、微細流路を通る液体の流れを制御するためのバルブ等が形成されており、液体の混合、加熱、冷却などによる反応制御や、分光学的あるいは電気的作用を応用した検出作業を可能としている。
マイクロデバイスで用いる液体としては、たとえば血液・蛋白・遺伝子などを含む溶液、微生物・動植物細胞などの固体成分を含む溶液、各種化学物質を含む環境水、土壌抽出水など、さらにはそれらの分析に使用する各種の試薬、バッファ液、洗浄水などが挙げられる。以下、本明細書中ではこれらマイクロデバイスで用いる各種液体をまとめて単に「液体」と表記する。
Usually, a microdevice is formed with various containers such as a reactor and a reservoir, a fine channel connecting them, a valve for controlling the flow of liquid through the fine channel, etc. on a single substrate. It is possible to perform reaction control by heating, cooling, etc. and detection work applying spectroscopic or electrical action.
Examples of liquids used in microdevices include solutions containing blood, proteins, genes, etc., solutions containing solid components such as microorganisms and animal and plant cells, environmental water containing various chemical substances, soil extract water, etc. Examples include various reagents used, buffer solutions, and washing water. Hereinafter, in the present specification, various liquids used in these micro devices are collectively referred to as “liquid”.

微細流路の中に複数の液体を流すことで、界面における物質・物体の移動現象を利用した新規な化学プロセスや微粒子の分離プロセスを実現できるため、微細流路内において層流を安定化させ、界面を保持する技術が求められている。
例えば特許文献1には、サンプル液導入流路とシース液導入流路との合流流路を部分的にテーパーさせることで、サンプル液層流を流路の中心に集束させて送液できるマイクロチップが開示されている。
By flowing multiple liquids through the microchannel, it is possible to realize a new chemical process and particle separation process that utilizes the phenomenon of substance / object movement at the interface, thus stabilizing the laminar flow in the microchannel. There is a need for a technique for maintaining the interface.
For example, Patent Document 1 discloses a microchip capable of concentrating a sample liquid laminar flow at the center of the flow path and feeding it by partially tapering a confluence flow path between the sample liquid introduction flow path and the sheath liquid introduction flow path. Is disclosed.

微細流路内の液体の流れを制御するには大型かつ高価な制御機やシリンジポンプを用いるのが一般的であるが、その他に、回転するディスク上に微細流路を形成し、遠心力を利用して液体を流すという簡便な液体ポンピング法も知られている。この方法ではディスクを回転させるだけで、回転中心から半径方向外向きに均一な遠心力が得られるため、複数の液体の制御を同時に実行できる送液系を簡便に実現できるという利点がある。   In general, a large and expensive controller or syringe pump is used to control the flow of liquid in the fine flow path, but in addition, a fine flow path is formed on the rotating disk to generate centrifugal force. There is also known a simple liquid pumping method in which a liquid is made to flow. This method has an advantage that a liquid feeding system capable of simultaneously controlling a plurality of liquids can be easily realized because a uniform centrifugal force is obtained radially outward from the center of rotation only by rotating the disk.

しかし、回転する微細流路内を流れる液体には遠心力と共にコリオリの力も作用する。コリオリの力は液体の移動速度とローターの回転数に比例し、微細流路の中央を流れる液体は移動速度が速く、微細流路の壁面近傍を流れる液体は移動速度が遅いため、微細流路中央の液体と壁面近傍の液体に作用するコリオリの力に差が生じてしまう。その結果、微細流路内において液体をひねるような渦挙動(2次流れ)が生じてしまい、層流の安定性が失われるという問題が知られている(特許文献2〜4、非特許文献1及び2)。   However, Coriolis force acts together with centrifugal force on the liquid flowing in the rotating fine channel. The Coriolis force is proportional to the moving speed of the liquid and the number of rotations of the rotor. The liquid flowing in the center of the fine flow path has a high moving speed, and the liquid flowing near the wall of the fine flow path has a low moving speed. There will be a difference in the Coriolis force acting on the central liquid and the liquid near the wall. As a result, vortex behavior (secondary flow) that twists the liquid in the fine flow path occurs, and the problem that the stability of the laminar flow is lost is known (Patent Documents 2 to 4, Non-Patent Documents). 1 and 2).

特開2011−179945号公報JP 2011-179945 A 特許第4836270号公報Japanese Patent No. 4836270 特許第4247390号公報Japanese Patent No. 4247390 特開2011−218348号公報JP 2011-218348 A

Jens Ducree et al., Multilamination of flows in planar networks ofrotating microchannels, Microfluidics and Nanofluidics, 2, 78-84, (2006).Jens Ducree et al., Multilamination of flows in planar networks ofrotating microchannels, Microfluidics and Nanofluidics, 2, 78-84, (2006). Jens Ducree, et al., Patterning of flow and mixing in rotatingradial microchannels, Microfluidics and Nanofluidics, 2, 97-105, (2006).Jens Ducree, et al., Patterning of flow and mixing in rotatingradial microchannels, Microfluidics and Nanofluidics, 2, 97-105, (2006).

特許文献2〜4のなかで(例えば特許文献2の[0006]参照。)、一般的な遠心分離に於いて遠心チューブ内の液相の擾乱を連続的な密度勾配が抑制する効果に関して言及されているが、上記の流れを伴う液体に対して作用するコリオリの力により生じる2次流れを抑制する手段としては必ずしも十分なものとはいえなかった。
また、特許文献2〜4に記載された発明はいずれも液相内の擾乱をある程度抑制した上で沈降速度法によって微粒子の分級(遠心分離)を行うものであり、沈降平衡法によって微粒子の分級を実現した技術は未だ存在していないのが現状である。
In Patent Documents 2 to 4 (see, for example, [0006] of Patent Document 2), mention is made of the effect of a continuous density gradient suppressing the disturbance of the liquid phase in a centrifugal tube in general centrifugation. However, it is not always sufficient as a means for suppressing the secondary flow generated by the Coriolis force acting on the liquid accompanied by the flow.
In addition, all the inventions described in Patent Documents 2 to 4 perform fine particle classification (centrifugation) by the sedimentation velocity method after suppressing disturbance in the liquid phase to some extent, and fine particle classification by the sedimentation equilibrium method. At present, there is no technology that realizes this.

本発明はこのような問題に鑑み、マイクロデバイス等に設けた微細流路内の層流の安定化を実現できる遠心方式による送液方法、遠心分離法、送液装置及び遠心分離装置を提供することを目的とする。   In view of such problems, the present invention provides a centrifugal liquid feeding method, a centrifugal separation method, a liquid feeding device, and a centrifugal separator that can realize stabilization of a laminar flow in a microchannel provided in a microdevice or the like. For the purpose.

本発明の送液方法は、遠心ローターの回転方向に沿って配置した微細流路内に、密度が異なる2種類以上の液体を密度が小さい方から順に遠心ローターの半径方向外側に積層することで、微細流路内の密度勾配を連続的ではなく階段状に変化させることを特徴とする。
また、液体に対して上記半径方向内側又は外側に向かって作用するコリオリの力による2次流れを遠心力で弱めることを特徴とする。
また、階段状に変化させた前記2種類以上の各液体の密度の勾配方向(密度小から密度大に向かう方向)と遠心力の作用方向とを一致させることで、各液体に対して作用するコリオリの力による2次流れを遠心力で弱めて層流を安定化させることを特徴とする。
また、微細流路の幅を10μm以上、1mm以下としたことを特徴とする。
また、各液体の密度が0.9g/cc〜1.2g/ccの範囲内であり、積層された2種類の液体の密度差が0.01g/cc以上であることを特徴とする。
In the liquid feeding method of the present invention, two or more kinds of liquids having different densities are stacked on the radially outer side of the centrifugal rotor in order from the smaller density in the fine flow path arranged along the rotation direction of the centrifugal rotor. The density gradient in the fine channel is changed stepwise instead of continuously.
Further, the secondary flow caused by the Coriolis force acting toward the inside or outside in the radial direction with respect to the liquid is weakened by a centrifugal force.
Moreover, it acts on each liquid by making the gradient direction (direction from low density to high density) of the density of each of the two or more types of liquids changed stepwise coincide with the direction of action of the centrifugal force. It is characterized by stabilizing the laminar flow by weakening the secondary flow caused by the Coriolis force with a centrifugal force.
Further, the width of the fine channel is set to 10 μm or more and 1 mm or less.
Further, the density of each liquid is in the range of 0.9 g / cc to 1.2 g / cc, and the difference in density between the two kinds of laminated liquid is 0.01 g / cc or more.

また、本発明の遠心分離法は、前記複数の液体のうち少なくとも一つの液体中に微粒子試料が含有されており、当該微粒子試料を密度勾配遠心分離法によって分離することを特徴とする。
また、前記微粒子が細胞、微生物、リポソームその他の生体関連微粒子、ラテックス粒子、ゲル粒子、工業用粒子その他の合成粒子のうちの少なくとも一つであることを特徴とする。
In addition, the centrifugal method of the present invention is characterized in that a fine particle sample is contained in at least one of the plurality of liquids, and the fine particle sample is separated by a density gradient centrifugal method.
The fine particles may be at least one of cells, microorganisms, liposomes and other bio-related fine particles, latex particles, gel particles, industrial particles and other synthetic particles.

本発明の送液装置は、遠心ローターの回転方向に沿って配置した微細流路を備えており、当該微細流路内に、密度が異なる2種類以上の液体を密度が小さい方から順に遠心ローターの半径方向外側に積層することで、微細流路内の密度勾配を連続的ではなく階段状に変化させることを特徴とする。
また、前記各液体に対して上記半径方向内側又は外側に向かって作用するコリオリの力による2次流れを遠心力で弱めることを特徴とする。
また、階段状に変化させた前記2種類以上の各液体の密度の勾配方向(密度小から密度大に向かう方向)と遠心力の作用方向とを一致させることで、各液体に対して作用するコリオリの力による2次流れを遠心力で弱めて層流を安定化させることを特徴とする。
また、微細流路の幅を10μm以上、1mm以下としたことを特徴とする。
また、各液体の密度が0.9g/cc〜1.2g/ccの範囲内であり、積層された2種類の液体の密度差が0.01g/cc以上であることを特徴とする。
The liquid feeding device of the present invention includes a fine flow path arranged along the rotation direction of the centrifugal rotor, and the two or more types of liquids having different densities are placed in the fine flow path in order from the smaller density. The density gradient in the fine channel is changed not in a continuous manner but in a staircase pattern by being laminated on the outside in the radial direction.
Further, the secondary flow caused by the Coriolis force acting on the liquid inward or outward in the radial direction is weakened by centrifugal force.
Moreover, it acts on each liquid by making the gradient direction (direction from low density to high density) of the density of each of the two or more types of liquids changed stepwise coincide with the direction of action of the centrifugal force. It is characterized by stabilizing the laminar flow by weakening the secondary flow caused by the Coriolis force with a centrifugal force.
Further, the width of the fine channel is set to 10 μm or more and 1 mm or less.
Further, the density of each liquid is in the range of 0.9 g / cc to 1.2 g / cc, and the difference in density between the two kinds of laminated liquid is 0.01 g / cc or more.

本発明の遠心分離装置は、上記送液装置を利用するものであり、前記複数の液体のうち少なくとも一つの液体中に微粒子試料が含有されており、当該微粒子試料を密度勾配遠心分離法によって分離することを特徴とする。
また、前記微粒子が細胞、微生物、リポソームその他の生体関連微粒子、ラテックス粒子、ゲル粒子、工業用粒子その他の合成粒子のうちの少なくとも一つであることを特徴とする。
A centrifugal separator according to the present invention uses the above-described liquid feeding device, and a fine particle sample is contained in at least one of the plurality of liquids, and the fine particle sample is separated by a density gradient centrifugal separation method. It is characterized by doing.
The fine particles may be at least one of cells, microorganisms, liposomes and other bio-related fine particles, latex particles, gel particles, industrial particles and other synthetic particles.

本願発明者らは微細流路内を流れる液体に対して具体的にどのような大きさ、方向及び位置でコリオリの力が作用しているのかを実験及びシュミレーションで解明し、得られた知見に基づいて、上記コリオリの力による2次流れを抑制し、微細流路内を流れる液体の安定化を実現できる本発明を完成させた。
本発明の送液方法によれば、密度が異なる2種類以上の液体を密度が小さい方から順に遠心ローターの半径方向外側に積層することで、微細流路内の密度勾配を階段状に変化させる。
例えば2種類の液体を用いる場合、図1(a)の流速分布に示すように、微細流路の中央P1(幅方向及び上下方向の中央)を流れる液体は移動速度が最も速く、流路の壁面P2〜P5(幅方向の壁面及び上下方向の壁面)近傍を流れる液体は移動速度が相対的に遅い。コリオリの力は液体の流速に比例するので、図1(b)に示すように、微細流路の縦断面(遠心ローターの半径方向に沿った断面)を見た場合、微細流路の中央P1から外向きに大きなコリオリの力が作用し、上下方向の壁面P4及びP5近傍には外向きに相対的に小さなコリオリの力が作用することになる。したがって、遠心ローターの半径方向内側の液体(液体1)は点P1近傍において大きなコリオリの力によって外側の液体(液体2)内に侵入することになり、液体2はコリオリの力が相対的に弱い点P4及び点P5近傍から液体1の方向に押し出される。その結果、流路断面内に液体1及び液体2の流れをひねるような渦挙動(2次流れ)が生じ、微細流路内において層流の安定性が失われてしまう。
The inventors of the present application have elucidated through experiments and simulations what size, direction and position the Coriolis force is acting on the liquid flowing in the fine channel, and obtained knowledge Based on this, the present invention has been completed which can suppress the secondary flow caused by the Coriolis force and can stabilize the liquid flowing in the fine flow path.
According to the liquid feeding method of the present invention, two or more kinds of liquids having different densities are stacked on the radially outer side of the centrifugal rotor in order from the smaller density, thereby changing the density gradient in the fine flow path in a stepped manner. .
For example, when two types of liquids are used, as shown in the flow velocity distribution of FIG. 1A, the liquid flowing through the center P1 (the center in the width direction and the vertical direction) of the fine channel has the fastest moving speed, The liquid flowing in the vicinity of the wall surfaces P2 to P5 (the wall surface in the width direction and the wall surface in the vertical direction) has a relatively low moving speed. Since the Coriolis force is proportional to the flow velocity of the liquid, as shown in FIG. 1B, when the longitudinal section of the microchannel (the section along the radial direction of the centrifugal rotor) is viewed, the center P1 of the microchannel Thus, a large Coriolis force is applied outward from the wall, and a relatively small Coriolis force is applied in the vicinity of the vertical wall surfaces P4 and P5. Therefore, the radially inner liquid (liquid 1) of the centrifugal rotor enters the outer liquid (liquid 2) by a large Coriolis force in the vicinity of the point P1, and the liquid 2 has a relatively weak Coriolis force. The liquid is pushed in the direction of the liquid 1 from the vicinity of the points P4 and P5. As a result, a vortex behavior (secondary flow) that twists the flow of the liquid 1 and the liquid 2 occurs in the cross section of the flow path, and the stability of the laminar flow is lost in the fine flow path.

そこで、本発明のように液体2の密度を液体1の密度よりも相対的に大きくして、微細流路内に階段状の密度勾配を形成することで、図1(c)に示すように、液体2に作用する遠心力を、液体1に作用する遠心力よりも相対的に大きくする。この場合、微細流路の縦断面を見ると、液体2には液体1に作用するよりも大きな遠心力が上下方向に均一に作用することになり、液体2は遠心力によって微細流路の外側の壁面W1に押し付けられることになる。
その結果、上述したような、コリオリの力に起因する液体2を点P4及び点P5近傍から液体1の側に押し出す力を、液体2に作用する遠心力が弱めることになり、上記渦挙動(2次流れ)が生じにくくなるため、層流の安定化を実現できる。
すなわち、本発明は微細流路内にあえて密度勾配を形成すると共に、密度の勾配方向(密度小から密度大に向かう方向)と遠心力の作用方向とを一致させることで、液体に作用する遠心力を利用してコリオリの力による2次流れを抑制して、層流を安定化させるものである。
なお、本明細書中において密度勾配が「階段状」に変化するとは、微細流路内の液体に関して、遠心ローターの半径方向に沿った方向への密度変化が空間的に連続していないことを指すものとする。
Therefore, as shown in FIG. 1C, the density of the liquid 2 is made relatively larger than the density of the liquid 1 as in the present invention to form a step-like density gradient in the fine channel. The centrifugal force acting on the liquid 2 is made relatively larger than the centrifugal force acting on the liquid 1. In this case, when the vertical cross section of the fine channel is viewed, a larger centrifugal force than the liquid 1 acts on the liquid 2 uniformly in the vertical direction, and the liquid 2 is outside the fine channel by the centrifugal force. Will be pressed against the wall surface W1.
As a result, the force that pushes the liquid 2 due to the Coriolis force as described above from the vicinity of the points P4 and P5 to the liquid 1 side is weakened by the centrifugal force acting on the liquid 2, and the vortex behavior ( (Secondary flow) is less likely to occur, so that the laminar flow can be stabilized.
That is, the present invention dares to form a density gradient in the fine flow path, and makes the centrifugal direction acting on the liquid by matching the direction of density gradient (direction from low density to high density) and the direction of action of centrifugal force. The secondary flow due to the Coriolis force is suppressed by using the force to stabilize the laminar flow.
In this specification, the density gradient changes in a “stepped shape” means that the density change in the direction along the radial direction of the centrifugal rotor is not spatially continuous with respect to the liquid in the fine flow path. Shall point to.

また、図2に示すように、液体に対してコリオリの力が作用する方向は、液体が流れる方向を一定にした場合、遠心ローターの回転方向によって変化する。すなわち、図2(a)に示すように液体が流れる方向と遠心ローターの回転方向を共に反時計回りにした場合、コリオリの力は遠心ローターの半径方向外側に向かって作用する。一方、図2(b)に示すように液体が流れる方向を反時計回り、遠心ローターの回転方向を時計回りにした場合、コリオリの力は遠心ローターの半径方向内側に向かって作用する。本発明ではコリオリの力が遠心ローターの半径方向内側に向かって作用する場合と外側に向かって作用する場合のいずれであっても、そのコリオリの力による2次流れを遠心力で弱めて層流を安定化させることができる。
本発明の送液方法では、微細流路に流す複数の液体の密度を同一にしていた従来法と比較して、遠心ローターの回転数や流速を速めた場合でも界面の安定性を維持できる。例えば流速であれば従来法よりも100倍程度速くできるケースがあるため、処理時間を大幅に短縮できる。
Further, as shown in FIG. 2, the direction in which the Coriolis force acts on the liquid changes depending on the rotation direction of the centrifugal rotor when the direction in which the liquid flows is constant. That is, as shown in FIG. 2A, when both the liquid flowing direction and the rotation direction of the centrifugal rotor are counterclockwise, the Coriolis force acts outward in the radial direction of the centrifugal rotor. On the other hand, as shown in FIG. 2B, when the liquid flow direction is counterclockwise and the rotation direction of the centrifugal rotor is clockwise, the Coriolis force acts inward of the centrifugal rotor in the radial direction. In the present invention, regardless of whether the Coriolis force acts inward or outward in the radial direction of the centrifugal rotor, the secondary flow caused by the Coriolis force is weakened by the centrifugal force and laminar flow is caused. Can be stabilized.
In the liquid feeding method of the present invention, the stability of the interface can be maintained even when the rotational speed and flow rate of the centrifugal rotor are increased as compared with the conventional method in which the density of a plurality of liquids flowing through the fine flow path is the same. For example, if the flow rate is about 100 times faster than the conventional method, the processing time can be greatly reduced.

本発明は、従来コリオリの力によって複数の液体が微細流路内で混ざってしまうために利用できなかった種々のプロセスに適用することができる。
例えば、細胞膜を溶かすことを目的とする液体(溶解液)の密度を細胞と細胞の核の密度の中間に調整し、これが微細流路内の外周側を流れるように細胞懸濁液と共に流すと、遠心中に細胞は溶解液に押し付けられ、界面において核が取り出される反応が進行する。細胞より取り出された核は、溶解液中に取り込まれるが、溶解されなかった細胞は溶解液中に侵入しない為に、核のみを選択して溶解液中に回収できる。
また、有機溶媒中の水溶性物質(イオン等)を水系に抽出するような場合には、これらの液体を微細流路中で接触させることで、微細流路中で物質の拡散距離が短い事を利用して高効率に有機溶媒から水溶性物質を液液抽出するなどのプロセスが実現できる。また、この逆に水系から疎水性物質を有機溶媒等に抽出する事も可能である。
The present invention can be applied to various processes that could not be used because a plurality of liquids are mixed in the fine channel by the Coriolis force.
For example, when the density of a liquid (dissolution solution) intended to dissolve the cell membrane is adjusted to the midpoint between the density of the cell and the cell nucleus, it flows along with the cell suspension so that it flows on the outer periphery of the microchannel. During the centrifugation, the cells are pressed against the lysate, and a reaction in which nuclei are removed at the interface proceeds. Nuclei removed from the cells are taken into the lysate, but cells that have not been lysed do not enter the lysate, so only the nuclei can be selected and recovered in the lysate.
In addition, when a water-soluble substance (ion or the like) in an organic solvent is extracted into an aqueous system, the diffusion distance of the substance in the fine channel is short by contacting these liquids in the fine channel. A process such as liquid-liquid extraction of a water-soluble substance from an organic solvent can be realized with high efficiency. Conversely, it is also possible to extract a hydrophobic substance from an aqueous system into an organic solvent or the like.

なお、微細流路の幅は10μm以上、1mm以下とするのが好ましい。一般的な分離処理では遠心ローターは3,000rpmで回転させることが多く、この条件において微細流路の幅を上記範囲内に設計すると、レイノルズ数はおよそ100以下となり、液体の粘性や流速等を総合的に考慮して最も分離能を高めることができる。
本発明の遠心分離法によれば、従来試験管サイズで行っていた密度勾配遠心法をマイクロデバイス化できるので分離装置を安価で構成できるという利点や、液体(試料)が少量で済むと共に試料を貯蔵するためのタンクを遠心ローター上に実装できるという利点もある。更に、連続方式にすれば遠心分離作業を自動化できるので、従来のように遠心管の中に密度勾配を形成する作業や、密度媒体界面に濃縮された分画を回収する作業等、人手による作業が最小限になり、再現性及び分離精度が向上するという利点がある。
また、本発明の遠心分離法は単分散性シリカ微粒子、単分散性ポリマー微粒子、磁気記録媒体、トナー、顔料、シリコンナノ粒子など、種々の微粒子の製造方法として利用することもできる。
The width of the fine channel is preferably 10 μm or more and 1 mm or less. In general separation processing, the centrifugal rotor is often rotated at 3,000 rpm. If the width of the fine channel is designed within the above range under these conditions, the Reynolds number will be approximately 100 or less, and the viscosity and flow rate of the liquid will be comprehensive. The highest resolution can be achieved by taking into account the above.
According to the centrifugation method of the present invention, the density gradient centrifugation method that has been conventionally performed in a test tube size can be made into a microdevice, so that the separation apparatus can be configured at low cost, and a small amount of liquid (sample) can be used and the sample can be prepared. There is also an advantage that a tank for storage can be mounted on the centrifugal rotor. Furthermore, since the centrifuge operation can be automated if the continuous method is used, manual operations such as forming a density gradient in the centrifuge tube and collecting fractions concentrated at the density medium interface as in the past. There is an advantage that reproducibility and separation accuracy are improved.
The centrifugal method of the present invention can also be used as a method for producing various fine particles such as monodisperse silica fine particles, monodisperse polymer fine particles, magnetic recording media, toners, pigments, and silicon nanoparticles.

なお、本明細書において液体中に微粒子が含まれる場合、微粒子としては例えば細胞、微生物、リポソームなどの生体関連微粒子、ラテックス粒子、ゲル粒子、工業用粒子などの合成粒子などが挙げられる。
生体関連微粒子には各種細胞を構成する染色体、リポソーム、ミトコンドリア、オルガネラ(細胞小器官)なども含まれる。細胞には動物細胞(血球系細胞など)や植物細胞が含まれる。微生物には大腸菌などの細菌類、タバコモザイクウイルスなどのウイルス類、イースト菌などの菌類などが含まれる。さらに、生体関連微粒子には、核酸、タンパク質、これらの複合体などの生体関連高分子も含まれる。
また、工業用粒子は、例えば有機もしくは無機高分子材料、金属などであってもよい。
有機高分子材料には、ポリスチレン、スチレン・ジビニルベンゼン、ポリメチルメタクリレートなどが含まれる。無機高分子材料には、ガラス、シリカ、磁性体材料などが含まれる。金属には、金コロイド、アルミなどが含まれる。これら微小粒子の形状は、一般には球形であるが、非球形であってもよく、また大きさや質量なども特に限定されない。
In the present specification, when fine particles are contained in the liquid, examples of the fine particles include bio-related fine particles such as cells, microorganisms, and liposomes, and synthetic particles such as latex particles, gel particles, and industrial particles.
Biologically related microparticles include chromosomes, liposomes, mitochondria, organelles (organelles) that constitute various cells. The cells include animal cells (such as blood cells) and plant cells. Microorganisms include bacteria such as Escherichia coli, viruses such as tobacco mosaic virus, and fungi such as yeast. Furthermore, biologically relevant microparticles include biologically relevant polymers such as nucleic acids, proteins, and complexes thereof.
The industrial particles may be, for example, an organic or inorganic polymer material, a metal, or the like.
Organic polymer materials include polystyrene, styrene / divinylbenzene, polymethyl methacrylate, and the like. Inorganic polymer materials include glass, silica, magnetic materials, and the like. Metals include gold colloid, aluminum and the like. The shape of these fine particles is generally spherical, but may be non-spherical, and the size and mass are not particularly limited.

微細流路内の液体に作用するコリオリの力と遠心力を示す概略図(a)〜(c)Schematic diagrams (a) to (c) showing Coriolis force and centrifugal force acting on the liquid in the fine channel. 液体に対してコリオリの力が作用する方向を説明するための図(a)及び(b)Figures (a) and (b) for explaining the direction in which the Coriolis force acts on the liquid 送液方法を説明するための概略図(a)〜(d)Schematics for explaining the liquid feeding method (a) to (d) 実施例1における微細流路内の状態を示す図(a)及び(b)The figure which shows the state in the microchannel in Example 1 (a) and (b) 実施例2における送液装置及び遠心分離装置の構成を示す図The figure which shows the structure of the liquid feeding apparatus in Example 2, and a centrifuge. 液体として水とPercollを用いた場合に形成されるパターンをPercoll密度及び流速を変えて比較した結果を示す図。The figure which shows the result of having compared the pattern formed when water and Percoll are used as a liquid, changing Percoll density and flow velocity. サンプル回収口L2及びR2で回収した各液体の混合状態を示す図。The figure which shows the mixed state of each liquid collect | recovered by sample collection port L2 and R2. 遠心ローターの回転数を変化させた場合の結果を示す図。The figure which shows the result at the time of changing the rotation speed of a centrifugal rotor. 血液に微粒子を懸濁したサンプルを密度勾配遠心法により分離した結果を示す図。The figure which shows the result of having isolate | separated the sample which suspended the microparticles | fine-particles in the blood by density gradient centrifugation. 流路出口を3本に分岐した構造を用いて行なった血液と樹脂微粒子の分離実験の結果を示す図。The figure which shows the result of the separation experiment of the blood and resin microparticles | fine-particles performed using the structure which branched the flow-path exit into three.

本発明の送液方法の実施の形態について説明する。
図3(a)に示すように、本実施の形態では、液体として密度が異なる2種類のパーコール10、20(第1パーコール10(密度小)及び第2パーコール20(密度大))と、微粒子試料を含有する液体として血液30を使用し、血液30中に含まれる血漿30a、有核赤血球(NRBC)30b及び赤血球30cを分離、回収するものとする。第1パーコール10の密度は血漿30aの浮遊密度より大きく、且つ有核赤血球30bの浮遊密度より小さくなるように調整されており、第2パーコール20の密度は有核赤血球30bの浮游密度より大きく、且つ赤血球30cの浮遊密度より小さくなるように調整されている。
An embodiment of the liquid feeding method of the present invention will be described.
As shown in FIG. 3A, in this embodiment, two types of Percoll 10, 20 (first Percoll 10 (low density) and second Percoll 20 (high density)) and fine particles having different densities as liquids are used. Blood 30 is used as a liquid containing a sample, and plasma 30a, nucleated red blood cells (NRBC) 30b, and red blood cells 30c contained in blood 30 are separated and collected. The density of the first percoll 10 is adjusted to be larger than the floating density of the plasma 30a and smaller than the floating density of the nucleated red blood cell 30b, and the density of the second percoll 20 is larger than the floating density of the nucleated red blood cell 30b, And it is adjusted so that it may become smaller than the floating density of the red blood cells 30c.

第1パーコール10、第2パーコール20及び血液30が入った各容器11、21、31から流路12、22、32が遠心ローターの半径方向にのびており、各流路は遠心ローターの周縁部において、回転方向に沿って配置された一本の微細流路40の上流側端部に連結されている。また、微細流路40の下流側端部は3本に枝分かれしており、各枝部分において血漿30a、有核赤血球30b及び赤血球30cが回収されることになる。
図3(b)に示すように、微細流路40内において、2種類のパーコールのうち密度が小さい第1パーコール10を遠心ローターの回転中心に近い方に配置し、密度が大きい第2パーコール20を外側に積層することで密度勾配を階段状にしている。また、血液30は第1パーコール10よりも更に回転中心に近い側に配置している。
The flow paths 12, 22, and 32 extend from the containers 11, 21, and 31 containing the first percoll 10, the second percoll 20, and the blood 30 in the radial direction of the centrifugal rotor. Are connected to the upstream end of one microchannel 40 arranged along the rotation direction. Further, the downstream end of the fine channel 40 is branched into three, and the plasma 30a, the nucleated red blood cell 30b, and the red blood cell 30c are collected at each branch portion.
As shown in FIG. 3B, in the fine channel 40, the first Percoll 10 having a lower density of the two types of Percoll is arranged closer to the rotation center of the centrifugal rotor, and the second Percoll 20 having a higher density. Are stacked on the outside to make the density gradient stepwise. Further, the blood 30 is arranged closer to the center of rotation than the first percoll 10.

遠心ロータを回転させながら微細流路40内に2種類のパーコール10、20及び血液30を流すと、遠心力によって血漿30aは微細流路40内の最も回転中心に近い側に移動し、有核赤血球30bは第1パーコール10と第2パーコール20の間に移動し、赤血球30cは第2パーコール20よりも外側に移動する。
そして、図3(c)及び(d)に示すように、第1の枝分かれ部分において血漿30aのみが回収され、第2の枝分かれ部分において有核赤血球30bのみが回収され、最後に赤血球30cが回収される。
When two types of Percoll 10, 20 and blood 30 are caused to flow in the fine flow path 40 while rotating the centrifugal rotor, the plasma 30a moves to the side closest to the center of rotation in the fine flow path 40 due to the centrifugal force, causing nucleation. The red blood cell 30b moves between the first percoll 10 and the second percoll 20, and the red blood cell 30c moves outside the second percoll 20.
As shown in FIGS. 3C and 3D, only the plasma 30a is recovered at the first branch portion, only the nucleated red blood cell 30b is recovered at the second branch portion, and finally the red blood cell 30c is recovered. Is done.

一般的に微粒子を回収する方式としては回分(バッチ)方式と連続方式がある。回分方式では少量ずつの処理しか行えず、かつ回転開始から加速、一定回転、回転減速、停止までの一回の運転ごとに諸条件が微妙に異なってしまうことから、バッチ間で回収精度にばらつきが出る等の問題がある。一方、連続方式では回分方式よりも試料処理量を増大させることができ、また回収精度を均一にすることが可能である。本発明は両方式に適用できるが、連続方式に適用することが好ましい。   Generally, there are a batch method and a continuous method for collecting fine particles. The batch method can perform only a small amount of processing, and the conditions vary slightly for each operation from the start of rotation to acceleration, constant rotation, rotation deceleration, and stop, so the collection accuracy varies from batch to batch. There is a problem such as coming out. On the other hand, in the continuous method, the sample throughput can be increased compared to the batch method, and the collection accuracy can be made uniform. The present invention can be applied to both systems, but is preferably applied to a continuous system.

なお、微細流路の材質は特に限定されるものではなく、有機材料では例えばシリコーン樹脂、アクリル樹脂、ポリスチレン、ポリオレフィン、ポリエステル、ポリカーボネート、フッ素樹脂、シリコーンゴムやフッ素ゴムなどのエラストマーなどが挙げられ、無機材料では例えばガラス、石英、アルミナ、ジルコニアなどが挙げられる。また、各容器及び流路の内壁に濡れ性等の物性を調節するための処理を施していてもよい。   The material of the fine channel is not particularly limited, and examples of the organic material include silicone resin, acrylic resin, polystyrene, polyolefin, polyester, polycarbonate, fluororesin, elastomer such as silicone rubber and fluororubber, Examples of inorganic materials include glass, quartz, alumina, zirconia, and the like. Moreover, the process for adjusting physical properties, such as wettability, may be given to each container and the inner wall of a flow path.

また、第1パーコール、第2パーコール及び血液が入った各容器から遠心ローターの半径方向にのびる各流路12、22、32に関して、その材質、長さ、形状等を調節することで、微細流路内の各液体の流速を調節することができる。例えば流路をS字状に蛇行させて、流路内を各液体が流れにくくすることで流速を抑えることができる。
また、本実施の形態では密度が異なる3種類の液体(第1パーコール、第2パーコール及び血液)を微細流路に流すものとしたが、4種類以上の液体を流すことにしてもよく、これにより多段の密度勾配を形成でき、分離能をより高精度にすることができる。
また、微細流路の一部に流れの障害となるような構造物(フィルター、支柱等)を設けることで、液体に含まれる不要物を除去したり、複数の液体を微細流路内の所定位置で撹拌するような機能を持たせてもよい。あるいは、微細流路を遠心ローターの回転方向に沿って複数本配置してもよい。
Further, by adjusting the material, length, shape, etc. of each flow path 12, 22, 32 extending in the radial direction of the centrifugal rotor from each container containing the first percoll, the second percoll, and blood, a fine flow can be obtained. The flow rate of each liquid in the channel can be adjusted. For example, the flow rate can be suppressed by making the flow path meander in an S-shape so that each liquid does not flow easily in the flow path.
In this embodiment, three types of liquids (first percoll, second percoll, and blood) having different densities are allowed to flow through the fine flow path. However, four or more types of liquids may be allowed to flow. As a result, a multi-stage density gradient can be formed, and the resolution can be made more accurate.
In addition, by providing a structure (filter, column, etc.) that obstructs the flow in a part of the fine channel, unnecessary substances contained in the liquid can be removed, or a plurality of liquids can be added to the predetermined channel in the fine channel. A function of stirring at a position may be provided. Alternatively, a plurality of fine channels may be arranged along the rotation direction of the centrifugal rotor.

溶液として、リン酸バッファー(PBS、密度1.006g/cc)とパーコールによる層流安定化効果を検証した。曲率半径55 mmのマイクロ流路をコンパクトディスク(遠心ローター)の同心円上に配置し、隣接する二つの入り口から、PBSが内周側、パーコールが外周側を流れるようにしてセットし、コンパクトディスクを回転させる事で遠心力による送液を行った。送液時の回転数は2000 rpmとし、この際のマイクロ流路内の流れはおよそ40 mm/sec(1.5ul/sec)である。パーコールの密度を1.075g/ccから徐々にPBSの密度に近づけると、密度1.02g/ccまでは2次流れの影響は表れなかった(図4(a)参照)。
また、同実験系を同じ密度の液体(着色水)に置き換えると、2次流れの影響により40%の流れが混合する事が分かっている。このため、密度差にして0.014g/cc、比率にして1.5%程度の密度差があれば層流を安定化する効果がある事が分かる。
また、上記PBS中にポリスチレンラテックス粒子(密度1.075g/cc以下)を懸濁し上記実験系においてパーコールの密度を1.075g/ccとした場合には、PBSとパーコールの合流直後においては粒子がPBSの中に分散していた状態であったの対し、合流後数cmの箇所においては粒子が遠心力により界面に収束され、パーコールとPBSの界面に維持されることが確認できた(図4(b)参照)。
As a solution, the laminar flow stabilization effect of phosphate buffer (PBS, density 1.006 g / cc) and percoll was verified. Place a micro flow path with a radius of curvature of 55 mm on the concentric circle of a compact disk (centrifugal rotor), and set the compact disk from two adjacent entrances so that PBS flows on the inner circumference side and percoll flows on the outer circumference side. The solution was fed by centrifugal force by rotating. The rotation speed at the time of liquid feeding is 2000 rpm, and the flow in the microchannel at this time is approximately 40 mm / sec (1.5 ul / sec). When the density of Percoll was gradually brought closer to that of PBS from 1.075 g / cc, the influence of the secondary flow did not appear until the density of 1.02 g / cc (see FIG. 4A).
In addition, when the same experimental system is replaced with a liquid of the same density (colored water), it is known that 40% of the flow is mixed due to the influence of the secondary flow. Therefore, it can be seen that if there is a density difference of 0.014 g / cc as a density difference and a density difference of about 1.5% as a ratio, there is an effect of stabilizing the laminar flow.
In addition, when polystyrene latex particles (density 1.075 g / cc or less) are suspended in the PBS and the density of Percoll is 1.075 g / cc in the experimental system, the particles are PBS immediately after the merge of PBS and Percoll. It was confirmed that the particles were converged at the interface by centrifugal force and maintained at the interface between Percoll and PBS at a few centimeters after the merging, as opposed to being dispersed inside (Fig. 4 (b)). )reference).

図5に示すように、サンプル注入口L1及びR1から周縁部に至る途中に流路を蛇行させることで液体の流れやすさを調節できる流路抵抗調整部を設けた送液装置を用いた。
サンプル注入口L1には青に着色したパーコール(Percoll、密度大)、サンプル注入口R1には赤に着色した水(密度小)を注入し、遠心ローターを反時計回りに回転させることで本発明の層流の安定化効果を検証した。
図6は水とPercollを同時に流し形成されるパターンをPercoll密度及び流速を変えて比較した結果である。
各写真の下にはPercollと水との密度差(パーコール密度−水密度)を示している。最も左側の写真は両者が水の場合であり、縞パターンが形成出来ていないが、Percollの密度が高くなり、密度差が大きくなるにつれて縞パターンが明瞭になっていく。なお、上段と下段では回転数を等しくして流量を異ならせている。
これらを比較すると、より高い流量の場合に縞パターンを明瞭にするには、すなわち2次流れを抑制して層流を安定化させるには2つの液体間で大きな密度差が必要である事が分かる。
As shown in FIG. 5, a liquid feeding device provided with a flow path resistance adjusting unit that can adjust the ease of liquid flow by meandering the flow path in the middle from the sample inlets L <b> 1 and R <b> 1 to the periphery.
The present invention is achieved by injecting blue-colored percoll (Percoll, high density) into the sample inlet L1 and red-colored water (low density) into the sample inlet R1, and rotating the centrifugal rotor counterclockwise. The laminar flow stabilization effect was verified.
FIG. 6 shows the result of comparing patterns formed by flowing water and Percoll at the same time while changing the Percoll density and flow rate.
Below each photograph, the density difference between Percoll and water (Percoll density-water density) is shown. The leftmost photograph shows a case where both are water, and a stripe pattern cannot be formed, but the density of Percoll increases and the stripe pattern becomes clear as the density difference increases. The upper and lower stages have the same number of rotations and different flow rates.
Comparing these, in order to clarify the fringe pattern at a higher flow rate, that is, to suppress the secondary flow and stabilize the laminar flow, it is necessary to have a large density difference between the two liquids. I understand.

図7はサンプル回収口L2及びR2で回収した各液体を分光し、混合状態を求めたものである。流路はサンプル回収口直前で2本に分岐しており、2つの液体の界面が明瞭な場合は混じりけの無い色素が回収される。
図7に示す様に、青色のPercollの密度を大きくする事でサンプルの混合が少なく、より明瞭な界面が形成されることが分かる。また、密度差が無い場合には0.1 μl/s程度に流量を抑えなければ明瞭な界面を形成する事が出来ないことが分かっているが、本発明の階段状の密度勾配を適用することで、100倍程度高い11.8μl/sの条件に於いても界面が維持されていることが分かる。
図8は、同程度の流量条件において遠心ローターの回転数を変化させた場合の結果を示している。この場合、回転数に対する顕著な依存性はなく、回転数のばらつきに対しても安定的に効果を発揮しているといえる。
FIG. 7 shows the mixed state obtained by spectroscopic analysis of the liquids collected at the sample collection ports L2 and R2. The flow path is branched into two just before the sample recovery port, and when the interface between the two liquids is clear, the unmixed dye is recovered.
As shown in FIG. 7, it can be seen that by increasing the density of the blue Percoll, the sample is less mixed and a clearer interface is formed. In addition, when there is no density difference, it is known that a clear interface cannot be formed unless the flow rate is suppressed to about 0.1 μl / s. However, by applying the stepwise density gradient of the present invention, It can be seen that the interface is maintained even under the condition of 11.8 μl / s, which is about 100 times higher.
FIG. 8 shows the results when the rotational speed of the centrifugal rotor is changed under the same flow rate condition. In this case, there is no significant dependence on the rotational speed, and it can be said that the effect is stably exhibited against variations in the rotational speed.

図9は、血液(非蛍光性)に微粒子(蛍光性)を懸濁したサンプルを密度勾配遠心法(沈降平衡法)により分離した結果である。血球細胞の密度はPercollの密度よりも大きいため、血液細胞はサンプル溶媒とPercollとの界面を通過して沈降する。一方、蛍光画像からわかる様に、微粒子の密度はPercollの密度よりも小さいため、微粒子は界面を通過する事がない。
図10は、流路出口を3本に分岐した構造を用いて血液と樹脂微粒子の分離実験を行ったものである。各グラフに示されるように、下段(図9参照)に流れるサンプルにはビーズが混入する事は無く、血球細胞のみを分離したサンプルを得る事が出来ており、沈降平衡法により粒子が分離出来ている事が分かる。
FIG. 9 shows the result of separating a sample in which fine particles (fluorescence) are suspended in blood (non-fluorescence) by density gradient centrifugation (precipitation equilibrium method). Since the density of blood cells is greater than that of Percoll, blood cells settle through the interface between the sample solvent and Percoll. On the other hand, as can be seen from the fluorescence image, the density of the fine particles is smaller than the density of Percoll, so that the fine particles do not pass through the interface.
FIG. 10 shows an experiment for separating blood and resin fine particles using a structure in which the outlet of the flow path is branched into three. As shown in each graph, the sample flowing in the lower stage (see Fig. 9) does not contain beads, and it is possible to obtain a sample in which only blood cells are separated, and particles can be separated by the sedimentation equilibrium method. I understand that

マイクロデバイス等に設けた微細流路内の層流の安定化を実現できる遠心方式による送液方法等であり、産業上の利用可能性を有する。   A liquid feeding method by a centrifugal method that can realize stabilization of a laminar flow in a fine channel provided in a microdevice or the like, and has industrial applicability.

10 第1パーコール
20 第2パーコール
30 血液
30a 血漿
30b 有核赤血球
30c 赤血球
11 容器
21 容器
31 容器
12 流路
22 流路
32 流路
40 微細流路
10 First Percoll 20 Second Percoll 30 Blood 30a Plasma 30b Nucleated Red Blood Cell 30c Red Blood Cell 11 Container 21 Container 31 Container 12 Channel 22 Channel 32 Channel 40 Channel

Claims (14)

遠心ローターの回転方向に沿って配置した微細流路内に、密度が異なる2種類以上の液体を密度が小さい方から順に遠心ローターの半径方向外側に積層することで、微細流路内の密度勾配を連続的ではなく階段状に変化させることを特徴とする送液方法。   By laminating two or more kinds of liquids with different densities in the fine flow path arranged along the rotation direction of the centrifugal rotor, in order from the one with the smallest density, radially outside the centrifugal rotor, the density gradient in the fine flow path A liquid feeding method characterized in that the step is changed stepwise instead of continuously. 液体に対して上記半径方向内側又は外側に向かって作用するコリオリの力による2次流れを遠心力で弱めることを特徴とする請求項1に記載の送液方法。   2. The liquid feeding method according to claim 1, wherein the secondary flow caused by the Coriolis force acting on the liquid inward or outward in the radial direction is weakened by centrifugal force. 階段状に変化させた前記2種類以上の各液体の密度の勾配方向(密度小から密度大に向かう方向)と遠心力の作用方向とを一致させることで、各液体に対して作用するコリオリの力による2次流れを遠心力で弱めて層流を安定化させることを特徴とする請求項1又は2に記載の送液方法。   By aligning the gradient direction of the density of each of the two or more liquids changed in a stepped manner (the direction from low density to high density) and the direction of action of the centrifugal force, Coriolis acting on each liquid The liquid feeding method according to claim 1, wherein the laminar flow is stabilized by weakening the secondary flow caused by force with a centrifugal force. 微細流路の幅を10μm以上、1mm以下としたことを特徴とする請求項1〜3のいずれか一項に記載の送液方法。   The liquid feeding method according to any one of claims 1 to 3, wherein a width of the fine channel is set to 10 µm or more and 1 mm or less. 各液体の密度が0.9g/cc〜1.2g/ccの範囲内であり、積層された2種類の液体の密度差が0.01g/cc以上であることを特徴とする請求項1〜4のいずれか一項に記載の送液方法。   The density of each liquid is in the range of 0.9 g / cc to 1.2 g / cc, and the density difference between the two kinds of laminated liquid is 0.01 g / cc or more. The liquid feeding method according to claim 1. 前記複数の液体のうち少なくとも一つの液体中に微粒子試料が含有されており、当該微粒子試料を密度勾配遠心分離法によって分離することを特徴とする請求項1〜5のいずれか一項に記載の送液方法を利用した遠心分離法。   The fine particle sample is contained in at least one liquid among the plurality of liquids, and the fine particle sample is separated by a density gradient centrifugal separation method. Centrifugation method using liquid feeding method. 前記微粒子が細胞、微生物、リポソームその他の生体関連微粒子、ラテックス粒子、ゲル粒子、工業用粒子その他の合成粒子のうちの少なくとも一つであることを特徴とする請求項6に記載の遠心分離法。   The centrifugal separation method according to claim 6, wherein the fine particles are at least one of cells, microorganisms, liposomes and other bio-related fine particles, latex particles, gel particles, industrial particles and other synthetic particles. 遠心ローターの回転方向に沿って配置した微細流路を備えており、当該微細流路内に、密度が異なる2種類以上の液体を密度が小さい方から順に遠心ローターの半径方向外側に積層することで、微細流路内の密度勾配を連続的ではなく階段状に変化させることを特徴とする送液装置。   A fine flow path arranged along the rotation direction of the centrifugal rotor is provided, and two or more types of liquids having different densities are stacked in the fine flow path in the radial direction outside the centrifugal rotor in order from the smaller density. The liquid feeding device is characterized in that the density gradient in the fine channel is changed stepwise instead of continuously. 前記各液体に対して上記半径方向内側又は外側に向かって作用するコリオリの力による2次流れを遠心力で弱めることを特徴とする請求項8に記載の送液装置。   The liquid feeding device according to claim 8, wherein a secondary flow caused by a Coriolis force acting toward the inner side or the outer side in the radial direction with respect to each liquid is weakened by a centrifugal force. 階段状に変化させた前記2種類以上の各液体の密度の勾配方向(密度小から密度大に向かう方向)と遠心力の作用方向とを一致させることで、各液体に対して作用するコリオリの力による2次流れを遠心力で弱めて層流を安定化させることを特徴とする請求項8又は9に記載の送液装置。   By aligning the gradient direction of the density of each of the two or more liquids changed in a stepped manner (the direction from low density to high density) and the direction of action of the centrifugal force, Coriolis acting on each liquid The liquid feeding device according to claim 8 or 9, wherein a secondary flow caused by force is weakened by centrifugal force to stabilize a laminar flow. 微細流路の幅を10μm以上、1mm以下としたことを特徴とする請求項8〜10のいずれか一項に記載の送液装置。   The liquid feeding device according to any one of claims 8 to 10, wherein a width of the fine channel is set to 10 µm or more and 1 mm or less. 各液体の密度が0.9g/cc〜1.2g/ccの範囲内であり、積層された2種類の液体の密度差が0.01g/cc以上であることを特徴とする請求項8〜11のいずれか一項に記載の送液装置。   The density of each liquid is in the range of 0.9 g / cc to 1.2 g / cc, and the density difference between the two kinds of laminated liquid is 0.01 g / cc or more. The liquid feeding device according to claim 1. 前記複数の液体のうち少なくとも一つの液体中に微粒子試料が含有されており、当該微粒子試料を密度勾配遠心分離法によって分離することを特徴とする請求項8〜12のいずれか一項に記載の送液装置を利用した遠心分離装置。   The fine particle sample is contained in at least one liquid among the plurality of liquids, and the fine particle sample is separated by a density gradient centrifugal separation method. Centrifugal separator using a liquid delivery device. 前記微粒子が細胞、微生物、リポソームその他の生体関連微粒子、ラテックス粒子、ゲル粒子、工業用粒子その他の合成粒子のうちの少なくとも一つであることを特徴とする請求項13に記載の遠心分離装置。



14. The centrifugal separator according to claim 13, wherein the fine particles are at least one of cells, microorganisms, liposomes and other bio-related fine particles, latex particles, gel particles, industrial particles and other synthetic particles.



JP2014535566A 2012-09-11 2013-09-11 Liquid feeding method, centrifugal separation method, liquid feeding device and centrifugal separator Active JP6273510B2 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
JP2012199476 2012-09-11
JP2012199476 2012-09-11
PCT/JP2013/074496 WO2014042177A1 (en) 2012-09-11 2013-09-11 Liquid supply method, centrifugal separation method, liquid supply device and centrifugal separation device

Publications (2)

Publication Number Publication Date
JPWO2014042177A1 true JPWO2014042177A1 (en) 2016-08-18
JP6273510B2 JP6273510B2 (en) 2018-02-07

Family

ID=50278287

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2014535566A Active JP6273510B2 (en) 2012-09-11 2013-09-11 Liquid feeding method, centrifugal separation method, liquid feeding device and centrifugal separator

Country Status (2)

Country Link
JP (1) JP6273510B2 (en)
WO (1) WO2014042177A1 (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
USD756773S1 (en) 2013-08-08 2016-05-24 Harl-Bella Holdings, Llc Lid with tear line
JP7121244B2 (en) * 2017-10-23 2022-08-18 国立大学法人山梨大学 Dispensing device, dispensing apparatus and method using the same, and inspection apparatus and method

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005224787A (en) * 2004-02-15 2005-08-25 Eiichi Tamiya Method and device for separating insoluble substance
JP2006158991A (en) * 2004-12-02 2006-06-22 Onchip Cellomics Consortium Centrifugal chip and centrifugal separation method
JP2007098223A (en) * 2005-09-30 2007-04-19 Fujifilm Corp Fluid operation method of chemical device
JP2008145420A (en) * 2006-12-11 2008-06-26 Samsung Electronics Co Ltd Component separation device and component separation method
JP2008531273A (en) * 2005-03-02 2008-08-14 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Flow switch in composite microfluidic CD (compact disc) using Coriolis force
JP2010502548A (en) * 2006-08-30 2010-01-28 ノースウェスタン ユニバーシティ A population of monodispersed single-walled carbon nanotubes and related methods for providing this population

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005224787A (en) * 2004-02-15 2005-08-25 Eiichi Tamiya Method and device for separating insoluble substance
JP2006158991A (en) * 2004-12-02 2006-06-22 Onchip Cellomics Consortium Centrifugal chip and centrifugal separation method
JP2008531273A (en) * 2005-03-02 2008-08-14 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア Flow switch in composite microfluidic CD (compact disc) using Coriolis force
JP2007098223A (en) * 2005-09-30 2007-04-19 Fujifilm Corp Fluid operation method of chemical device
JP2010502548A (en) * 2006-08-30 2010-01-28 ノースウェスタン ユニバーシティ A population of monodispersed single-walled carbon nanotubes and related methods for providing this population
JP2008145420A (en) * 2006-12-11 2008-06-26 Samsung Electronics Co Ltd Component separation device and component separation method

Also Published As

Publication number Publication date
JP6273510B2 (en) 2018-02-07
WO2014042177A1 (en) 2014-03-20

Similar Documents

Publication Publication Date Title
Sajeesh et al. Particle separation and sorting in microfluidic devices: a review
Zhou et al. Label-free microfluidic sorting of microparticles
Johnston et al. Dean flow focusing and separation of small microspheres within a narrow size range
Chung et al. Microstructure-induced helical vortices allow single-stream and long-term inertial focusing
Moloudi et al. Inertial particle focusing dynamics in a trapezoidal straight microchannel: Application to particle filtration
Park et al. Multiorifice flow fractionation: continuous size-based separation of microspheres using a series of contraction/expansion microchannels
Gossett et al. Label-free cell separation and sorting in microfluidic systems
US8563325B1 (en) Coaxial microreactor for particle synthesis
Jeon et al. Ion concentration polarization-based continuous separation device using electrical repulsion in the depletion region
Chen A triplet parallelizing spiral microfluidic chip for continuous separation of tumor cells
Kurup et al. Field-free particle focusing in microfluidic plugs
US9597692B2 (en) Micro-fluidic device for sorting particles, and methods for sorting particles
US11833508B2 (en) Multi-dimensional double spiral device and methods of use thereof
Madadelahi et al. Mathematical modeling and computational analysis of centrifugal microfluidic platforms: a review
Sollier et al. Inertial microfluidic programming of microparticle-laden flows for solution transfer around cells and particles
US20120063971A1 (en) Inertial particle focusing system
Aguirre et al. Integrated micromixer for incubation and separation of cancer cells on a centrifugal platform using inertial and dean forces
Jeon et al. Continuous particle separation using pressure-driven flow-induced miniaturizing free-flow electrophoresis (PDF-induced μ-FFE)
Alidoust et al. Emergence of microfluidic devices in sample extraction; an overview of diverse methodologies, principals, and recent advancements
Jiang et al. Centrifuge-based deterministic lateral displacement separation
Rodriguez‐Mateos et al. Inertial focusing of microparticles, bacteria, and blood in serpentine glass channels
Choi Hydrophoresis—a microfluidic principle for directed particle migration in flow
JP2006263693A (en) Mechanism and device for continuously separating particulates
Chen et al. Microfluidic chemical processing with on-chip washing by deterministic lateral displacement arrays with separator walls
JP6273510B2 (en) Liquid feeding method, centrifugal separation method, liquid feeding device and centrifugal separator

Legal Events

Date Code Title Description
A521 Request for written amendment filed

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20160831

A621 Written request for application examination

Free format text: JAPANESE INTERMEDIATE CODE: A621

Effective date: 20160909

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20170808

A521 Request for written amendment filed

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20171005

TRDD Decision of grant or rejection written
A01 Written decision to grant a patent or to grant a registration (utility model)

Free format text: JAPANESE INTERMEDIATE CODE: A01

Effective date: 20171121

A61 First payment of annual fees (during grant procedure)

Free format text: JAPANESE INTERMEDIATE CODE: A61

Effective date: 20171212

R150 Certificate of patent or registration of utility model

Ref document number: 6273510

Country of ref document: JP

Free format text: JAPANESE INTERMEDIATE CODE: R150

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250