JP2018089557A - Fine particle separation device and separation method of fine particle - Google Patents
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Abstract
Description
本発明は、微粒子分離デバイスおよび微粒子の分離方法に関する。 The present invention relates to a fine particle separation device and a fine particle separation method.
微粒子分離は、医療・生化学分野、生産分野などで幅広く必要とされている。例えば、生物学研究や診断医療、再生医療では、ある細胞集団から特定の細胞（癌細胞、血球、生細胞など）のみを分離、回収することが求められる。現在、こうした生体粒子の分離には遠心分離法、濾過法、蛍光活性化細胞分離法(ＦＡＣＳ)、磁気細胞分離法(ＭＡＣＳ)などが用いられている。しかしながら、遠心分離法は精度が低く、濾過法では目詰りが生じる難点がある。また、ＦＡＣＳ、ＭＡＣＳでは蛍光や磁気ビーズによる標識が必要であり、複雑な前処理が求められ、装置も大型かつ高価である。 Fine particle separation is widely required in the medical / biochemical and production fields. For example, in biological research, diagnostic medicine, and regenerative medicine, it is required to separate and collect only specific cells (cancer cells, blood cells, living cells, etc.) from a certain cell population. At present, centrifugal separation, filtration, fluorescence activated cell separation (FACS), magnetic cell separation (MACS), and the like are used for separation of such biological particles. However, the centrifugal separation method has low accuracy, and the filtration method has a drawback that clogging occurs. FACS and MACS require labeling with fluorescence or magnetic beads, require complicated pretreatment, and the apparatus is large and expensive.
これらの課題の解決手段の一つとして近年、マイクロ流路デバイスによる微粒子分離手法（非特許文献１）が報告されており、分離手法は以下の２種類に大別される。
(1)能動的粒子分離手法：分離の際に、電場・音場・磁場などの外部エネルギーを必要とする手法
(2)受動的粒子分離手法：分離の際に、水力学的作用のみを用いる手法
能動的粒子分離手法の場合、外部エネルギーを用いることでシステムが複雑化するため、受動的分離手法にて高い分離性能を実現することが望まれる。近年，受動的分離手法の一つとして、deterministic lateral displacement (ＤＬＤ)法による微粒子分離事例が報告されている（非特許文献２〜４）。ＤＬＤは流路に配列する支柱によって流体に生じる流れを利用した粒子分離法であり、ＤＬＤ流路内において粒子の大きさ、形状、硬さなどの粒子特性に従って異なる軌道を取るため（図９）、こうした微粒子の特性に基づき簡便に粒子を分離できる。本手法を用いて、10 nmの高分離分解能や，10 mL/min高処理量の粒子分離を実現した事例（非特許文献３）も報告されている。
In recent years, a microparticle separation method using a micro-channel device (Non-Patent Document 1) has been reported as one of means for solving these problems, and the separation methods are roughly classified into the following two types.
(1) Active particle separation method: A method that requires external energy such as electric field, sound field, and magnetic field for separation.
(2) Passive particle separation method: A method that uses only hydrodynamic action during separation In the case of the active particle separation method, the system becomes complicated by using external energy. It is desirable to achieve separation performance. In recent years, fine particle separation cases by deterministic lateral displacement (DLD) method have been reported as one of passive separation methods (Non-Patent Documents 2 to 4). DLD is a particle separation method that uses the flow generated in the fluid by the struts arranged in the flow path, and takes different trajectories in the DLD flow path according to the particle characteristics such as particle size, shape, hardness, etc. (Fig. 9) Based on the characteristics of such fine particles, the particles can be easily separated. An example (Non-patent Document 3) has been reported in which high separation resolution of 10 nm and particle separation at a high throughput of 10 mL / min are realized using this method.
一方、ＤＬＤによる微粒子分離には、微粒子懸濁溶液をＤＬＤ流路に導入する際、特定の位置から微粒子を流入させるために，微粒子が懸濁されていない溶液（バッファ）を用い，両脇から微粒子懸濁溶液を挟み込むシースフロー型の流路構造（図１０）、あるいはバッファの流れと流路壁面の間に微粒子懸濁溶液を挟み込む流路構造（非特許文献４）が必要とされてきた。従来、こうした流路構造がない場合、微粒子がＤＬＤ流路全域に幅広く分散してしまうので（図１１）、ＤＬＤによる微粒子分離はできなかった。しかしながら、こうした流路構造を用いる場合、バッファによる分析試料の希釈、送液口の増加に伴う操作性の低下、デバイス構造の複雑化等の課題がある。 On the other hand, in the fine particle separation by DLD, when introducing the fine particle suspension into the DLD flow path, a solution (buffer) in which the fine particles are not suspended is used in order to allow the fine particles to flow from a specific position. There has been a need for a sheath flow type flow path structure (FIG. 10) for sandwiching the fine particle suspension solution, or a flow path structure (Non-patent Document 4) for sandwiching the fine particle suspension solution between the flow of the buffer and the wall surface of the flow path. . Conventionally, in the absence of such a flow channel structure, the fine particles are widely dispersed throughout the DLD flow channel (FIG. 11), so that the fine particles cannot be separated by DLD. However, when such a flow channel structure is used, there are problems such as dilution of the analysis sample with a buffer, deterioration in operability associated with an increase in the number of liquid feeding ports, and complication of the device structure.
本発明は、このような課題を解決し、バッファを用いた微粒子懸濁溶液の挟み込みのための流路構造を用いない、微粒子懸濁試料流入用の送液流路のみを導入路として有する微粒子分離デバイスおよびそれを用いる微粒子の分離方法を提供するものである。 The present invention solves such a problem, and does not use a flow channel structure for sandwiching a fine particle suspension using a buffer, and has a fine particle having only a liquid supply channel for inflow of a fine particle suspension sample as an introduction channel. A separation device and a method for separating fine particles using the separation device are provided.
本発明は上記の問題を解決するために、以下の発明を提供するものである。
（１）微粒子の分散された液体を流入させて，該微粒子をその特性にしたがって分離するための微粒子分離デバイスであり、
微粒子の流入口および流出口、微粒子フォーカス用マイクロ流路ならびに微粒子分離用マイクロ流路からなり；
該微粒子分離用マイクロ流路は、配列された支柱間の隙間で形成され、該微粒子分離用マイクロ流路を流れる微粒子の軌道を制御して微粒子の特性にしたがって微粒子を分離するように構成されてなり；ならびに
該微粒子フォーカス用マイクロ流路は，該微粒子分離用マイクロ流路の前段に導入路として設けられ、
流入口から導入された液体中の微粒子を，慣性力により，単一流路中の流れに沿った単一または複数本の線状に局所的に配列させるように構成されてなり、
該微粒子分離用マイクロ流路の単一または複数の特定部位に微粒子が集中して配列された状態で流入するように構成されてなる；ことを特徴とする微粒子分離デバイス。
The present invention provides the following inventions in order to solve the above problems.
(1) A fine particle separation device for flowing a liquid in which fine particles are dispersed and separating the fine particles according to their characteristics,
Consisting of a fine particle inlet and outlet, a fine particle focusing microchannel and a microparticle separation microchannel;
The microparticle separation microchannel is formed by a gap between the arranged support columns, and is configured to control the microparticle trajectory flowing through the microparticle separation microchannel to separate the microparticles according to the characteristics of the microparticles. And the fine particle focusing micro-channel is provided as an introduction path in front of the micro-particle separation micro-channel,
The fine particles in the liquid introduced from the inlet are configured to be locally arranged in a single or multiple lines along the flow in the single flow path by inertial force.
A particulate separation device, characterized in that the particulate separation device is configured to flow in a state in which the particulates are concentrated and arranged at one or a plurality of specific parts of the particulate separation microchannel.
（２）微粒子をその大きさ、形状または硬さに基づき分離する上記（１）に記載の微粒子分離デバイス。
（３）微粒子フォーカス用マイクロ流路において、流れに沿った単一本、２本または３本の線状に微粒子が慣性力によりフォーカスされる上記（１）または（２）に記載の微粒子分離デバイス。
（４）微粒子が、ポリマー微粒子、生物系微粒子、液滴、金属微粒子、および非金属粒子から選ばれる上記（１）〜（３）のいずれかに記載の微粒子分離デバイス。
(2) The fine particle separation device according to (1), wherein fine particles are separated based on the size, shape, or hardness.
(3) The fine particle separation device according to (1) or (2), wherein the fine particles are focused in a single, two, or three linear form along the flow by an inertial force in the fine particle focusing microchannel. .
(4) The fine particle separation device according to any one of (1) to (3), wherein the fine particles are selected from polymer fine particles, biological fine particles, liquid droplets, metal fine particles, and non-metallic particles.
（５）微粒子の分散された液体が水性懸濁液である上記（１）〜（４）のいずれかに記載の微粒子分離デバイス。
（６）該微粒子分離用マイクロ流路が、配列された、単一の分離直径を有する支柱間の隙間で形成される上記（１）〜（５）のいずれかに記載の微粒子分離デバイス。
（７）該微粒子分離用マイクロ流路が、配列された、複数の分離直径を有する支柱間の隙間で形成される上記（１）〜（５）のいずれかに記載の微粒子分離デバイス。
(5) The fine particle separation device according to any one of (1) to (4), wherein the liquid in which the fine particles are dispersed is an aqueous suspension.
(6) The microparticle separation device according to any one of (1) to (5), wherein the microchannel for microparticle separation is formed by gaps between columns having a single separation diameter.
(7) The microparticle separation device according to any one of (1) to (5), wherein the microchannel for microparticle separation is formed by gaps between columns having a plurality of separation diameters arranged.
（８）微粒子の分散された液体を微粒子フォーカス用マイクロ流路に導入し、導入された液体中の微粒子を，慣性力により，単一流路中の流れに沿った単一または複数本の線状に局所的に配列させ；
配列された支柱間の隙間で形成された微粒子分離用マイクロ流路の単一または複数の特定部位に微粒子が集中して配列された状態で、微粒子の分散された液体を流入させ；
該微粒子分離用マイクロ流路を流れる微粒子の軌道を制御して微粒子の特性にしたがって微粒子を分離し、該微粒子分離用マイクロ流路から流出させることを特徴とする微粒子の分離方法。
(8) A liquid in which fine particles are dispersed is introduced into a micro flow channel for fine particle focusing, and the fine particles in the introduced liquid are made into single or plural linear shapes along the flow in the single flow channel by inertial force. To arrange locally;
Flowing the liquid in which the fine particles are dispersed in a state where the fine particles are concentrated and arranged in one or a plurality of specific parts of the micro flow path for separating fine particles formed by the gaps between the arranged support columns;
A method for separating fine particles, comprising controlling the trajectory of fine particles flowing through the fine particle separation microchannel to separate the fine particles according to the characteristics of the fine particles and letting the fine particles flow out from the fine particle separation microchannel.
（９）微粒子をその大きさ、形状または硬さに基づき分離する上記（８）に記載の微粒子の分離方法。
（１０）微粒子フォーカス用マイクロ流路において、流れに沿った単一本、２本または３本の線状に微粒子が慣性力によりフォーカスされる上記（８）または（９）に記載の微粒子の分離方法。
（１１）微粒子が、ポリマー微粒子、生物系微粒子、液滴、金属微粒子、および非金属粒子から選ばれる上記（８）〜（１０）のいずれかに記載の微粒子の分離方法。
(9) The method for separating fine particles according to (8), wherein the fine particles are separated based on the size, shape, or hardness thereof.
(10) Separation of microparticles according to (8) or (9) above, in which microparticles are focused in a single, two, or three linear form along the flow by inertia force Method.
(11) The method for separating fine particles according to any one of (8) to (10), wherein the fine particles are selected from polymer fine particles, biological fine particles, droplets, metal fine particles, and non-metallic particles.
（１２）微粒子の分散された液体が水性懸濁液である上記（８）〜（１１）のいずれかに記載の微粒子の分離方法。
（１３）該微粒子分離用マイクロ流路が、配列された、単一の分離直径を有する支柱間の隙間で形成される上記（８）〜（１２）のいずれかに記載の微粒子の分離方法。
（１４）該微粒子分離用マイクロ流路が、配列された、複数の分離直径を有する支柱間の隙間で形成される上記（８）〜（１２）のいずれかに記載の微粒子の分離方法。
(12) The method for separating fine particles according to any one of (8) to (11), wherein the liquid in which the fine particles are dispersed is an aqueous suspension.
(13) The method for separating fine particles according to any one of (8) to (12), wherein the microchannel for separating fine particles is formed by gaps between columns having a single separation diameter.
(14) The method for separating fine particles according to any one of (8) to (12), wherein the microchannel for separating fine particles is formed by gaps between columns having a plurality of separation diameters arranged.
本発明によれば、バッファを用いた微粒子懸濁溶液の挟み込みのための流路構造を用いない、微粒子懸濁試料流入用の送液流路のみを導入路として有する微粒子分離デバイスおよびそれを用いる微粒子の分離方法を提供するものである。さらに、刺激応答性高分子で支柱を作製する場合、刺激制御によって流路幾何形状を変化させることにより、粒子軌道の変化の境界となる粒子直径である分離直径を調節し得る微粒子分離デバイスおよびそれを用いる微粒子の分離方法を提供し得る。 According to the present invention, a particle separation device having only a liquid flow channel for inflow of a fine particle suspension sample as an introduction channel without using a flow channel structure for sandwiching a fine particle suspension using a buffer, and the same are used. A method for separating fine particles is provided. Furthermore, when producing a strut with a stimulus-responsive polymer, a fine particle separation device capable of adjusting a separation diameter, which is a particle diameter that becomes a boundary of a change in particle trajectory, by changing a flow path geometry by stimulus control, and the same Can be provided.
本発明の微粒子分離デバイスは、流入された微粒子をその特性にしたがって分離するための微粒子分離デバイスである。微粒子分離デバイスは、微粒子懸濁液の流入口および流出口、微粒子フォーカス用マイクロ流路ならびに微粒子分離用マイクロ流路からなり、微粒子分離用マイクロ流路は、配列された支柱間の隙間で形成される。本発明においては、微粒子分離用マイクロ流路を流れる微粒子の軌道を制御して微粒子の特性にしたがって微粒子を分離するように構成されてなる。 The fine particle separation device of the present invention is a fine particle separation device for separating inflow fine particles according to their characteristics. The particle separation device is composed of an inlet and an outlet of a particle suspension, a particle focusing microchannel, and a particle separation microchannel, and the particle separation microchannel is formed by gaps between arranged columns. The In the present invention, the orbit of the fine particles flowing through the fine particle separation microchannel is controlled to separate the fine particles according to the characteristics of the fine particles.
本発明の微粒子分離デバイスにおいては、微粒子フォーカス用マイクロ流路が、微粒子分離用マイクロ流路の前段に導入路として設けられ、流入口から導入された液体中の微粒子を，慣性力により，単一流路中の流れに沿った単一または複数本（たとえば、２本または３本）の線状に局所的に配列させるように構成されてなり、微粒子分離用マイクロ流路の単一または複数の特定部位に微粒子が集中して配列された状態で流入するように構成されてなる。 In the fine particle separation device of the present invention, the fine particle focusing micro-channel is provided as an introduction path in front of the micro-particle separation micro-channel, and the fine particles in the liquid introduced from the inflow port are made to flow in a single flow by inertia force. Single or plural identification of microchannel for fine particle separation, which is configured to be locally arranged in single or plural (for example, two or three) lines along the flow in the channel It is configured to flow in a state in which fine particles are concentrated and arranged at the site.
支柱は、好適には、従来のＤＬＤにおいて一般的に用いられている材料、たとえばポリジメチルシロキサン（PDMS）等のポリマー樹脂，またはSi，ガラス等から構成され得る。さらに、刺激応答性高分子材料が用いられ得、刺激に対する収縮または膨潤による支柱形状の変化の度合いにより、マイクロ流路を流れる微粒子の軌道を制御して微粒子の特性により微粒子を分離するように構成されてなる。微粒子の特性としては、好適には大きさ，形状，または硬さが挙げられる。 The struts are preferably made of a material commonly used in conventional DLD, for example, a polymer resin such as polydimethylsiloxane (PDMS), Si, glass, or the like. Furthermore, a stimuli-responsive polymer material can be used, and the trajectory of the microparticles flowing through the microchannel is controlled according to the degree of change in the columnar shape due to contraction or swelling with respect to the stimulus, and the microparticles are separated according to the characteristics of the microparticles. Being done. The characteristics of the fine particles preferably include size, shape, or hardness.
支柱を形成する刺激応答性高分子材料は、温度、光、電場もしくは磁場等の物理的刺激、またはｐＨ、溶液組成、イオン強度等の化学的刺激に応答するハイドロゲルである。たとえば、温度応答性高分子材料は、ハイドロゲルであり、水分を大量に含むことにより膨潤する性質を有するポリマーであり、温度変化による可逆的な水和・脱水和に伴う膨潤・収縮を生じる機能を有する。温度応答性高分子材料としては、ポリ-N-イソプロピルアクリルアミド（PNIPAM)、ポリ−N-ビニルアルキルアミド、ポリビニルアルキルエーテル、等が挙げられるが、相転移温度が30℃付近であり生物系微粒子への適用が可能である点や，緩やかな温度応答性を示すため支柱形状を細く制御可能である点からポリ-N-イソプロピルアクリルアミドが好適である。光応答性高分子材料としては、アゾベンゼン含有架橋構造を有するポリアクリルアミドハイドロゲル、等が挙げられる。ｐＨ応答性高分子材料としては、側鎖に嵩高い疎水性基を有するカルボキシ基含有ポリマー、等が挙げられる。電場応答性高分子材料としては、ポリアクリルアミド−２−メチルプロパンスルホン酸（ＰＡＭＰＳ）、等が挙げられる。 The stimulus-responsive polymeric material that forms the struts is a hydrogel that responds to physical stimuli such as temperature, light, electric or magnetic fields, or chemical stimuli such as pH, solution composition, ionic strength. For example, a temperature-responsive polymer material is a hydrogel, a polymer that swells when it contains a large amount of water, and has a function of causing swelling and shrinkage due to reversible hydration and dehydration due to temperature changes. Have Examples of the temperature-responsive polymer material include poly-N-isopropylacrylamide (PNIPAM), poly-N-vinylalkylamide, and polyvinylalkylether. Poly-N-isopropylacrylamide is preferred because it can be applied and the column shape can be finely controlled to exhibit a moderate temperature response. Examples of the photoresponsive polymer material include polyacrylamide hydrogel having an azobenzene-containing crosslinked structure. Examples of the pH-responsive polymer material include a carboxy group-containing polymer having a bulky hydrophobic group in the side chain. Examples of the electric field responsive polymer material include polyacrylamide-2-methylpropanesulfonic acid (PAMPS).
分離される微粒子は、ラテックス等のポリマー微粒子、赤血球、生細胞等の生物系微粒子、液滴、金属微粒子、および金属酸化物等の非金属粒子、から選ばれる。 The fine particles to be separated are selected from polymer fine particles such as latex, biological fine particles such as erythrocytes and living cells, droplets, metal fine particles, and non-metallic particles such as metal oxides.
微粒子の粒径は、通常、１ｎｍ〜１ｍｍ、好適には１０ｎｍ〜１００μｍである。 The particle diameter of the fine particles is usually 1 nm to 1 mm, preferably 10 nm to 100 μm.
微粒子は例えば水性または油性懸濁液の形態で微粒子分離デバイスの流入口から導入される。水性懸濁液は、たとえば界面活性剤を添加した水溶液に微粒子を粒子濃度10２〜10１０/mＬ程度で懸濁した溶液とするのが好適である。水性懸濁液は、水を主成分とし、水と相溶性のあるアルコール類等の有機溶媒を含んでいてもよい。 The microparticles are introduced from the inlet of the microparticle separation device, for example in the form of an aqueous or oily suspension. The aqueous suspension is preferably, for example, a solution in which fine particles are suspended in an aqueous solution to which a surfactant is added at a particle concentration of about 10 2 to 10 10 / mL. The aqueous suspension may contain water and an organic solvent such as alcohol that is compatible with water.
本発明の微粒子分離デバイスは、流入口から導入された微粒子の配列を慣性力により整えるための微粒子フォーカス用マイクロ流路を微粒子分離用マイクロ流路の前段に設け、微粒子分離用マイクロ流路の特定位置に微粒子を集中させて導入するように構成されてなる。 The particulate separation device of the present invention is provided with a particulate focusing micro-channel for adjusting the arrangement of the particulates introduced from the inlet by an inertial force in front of the particulate separation micro-channel so that the particulate separation micro-channel is specified. It is configured to concentrate and introduce the fine particles at the position.
この微粒子フォーカス用マイクロ流路の形状は、
断面形状：長方形，正方形，半円形，三角形；
流路断面の縦横比（アスペクト比，AR)：断面形状が横長長方形の場合、0 < AR < 1 ；断面形状が縦長長方形の場合、1 < AR；断面形状が，正方形の場合、AR ＝１
流路形状：直線または蛇行流路；
流路長さ：1 μm〜1 m、好適には１ｍｍ〜１０ｃｍ；
流路幅：50nm 〜10 mm；
流路高さ：50nm 〜10 mm程度
から選ばれる。
（慣性力による粒子のフォーカシングに関する参考文献としては、H. Amini et al.,
Lab Chip, 2014,2739-2761およびJ. Kim et al., Lab Chip, 2016,16,992-1001が挙げら
れる。）
The shape of the micro-channel for fine particle focus is
Cross-sectional shape: rectangle, square, semi-circle, triangle;
Aspect ratio of channel cross section (aspect ratio, AR): 0 <AR <1 when the cross-sectional shape is a horizontally long rectangle; 1 <AR when the cross-sectional shape is a vertically long rectangle; AR = 1 when the cross-sectional shape is square
Channel shape: straight or serpentine channel;
Channel length: 1 μm to 1 m, preferably 1 mm to 10 cm;
Channel width: 50nm to 10mm;
Channel height: selected from about 50 nm to 10 mm.
(For references on particle focusing by inertial forces, see H. Amini et al.,
Lab Chip, 2014, 2739-2761 and J. Kim et al., Lab Chip, 2016, 16, 992-1001. )
本発明の慣性力による微粒子フォーカス用マイクロ流路の断面形状，微粒子のフォーカス位置，および観察方向の関係を図1に示す。図１において、 (a) 横長長方形 0 < AR < 1； (b) 縦長長方形1 < AR,； (c) 正方形1 = AR,；(d) 三角形,；(e) 半円、 (上段) 流路断面上の微粒子フォーカス位置、 (下段)マイクロ流路を流路断面上の上辺と流れに垂直な方向から観察（上面視）した場合の粒子フォーカス位置を示す。 FIG. 1 shows the relationship between the cross-sectional shape of the micro-channel for fine particle focusing by the inertia force of the present invention, the focus position of the fine particles, and the observation direction. In Fig. 1, (a) Horizontal rectangle 0 <AR <1; (b) Vertical rectangle 1 <AR ,; (c) Square 1 = AR ,; (d) Triangle, (e) Semicircle, (Upper) flow Particulate focus position on the channel cross section, (bottom) Particle focus position when the microchannel is observed from the direction perpendicular to the upper side and flow on the channel cross section (top view).
微粒子フォーカス用マイクロ流路の断面形状が横長長方形の場合、粒子は流路断面上の長辺に沿って流路中央２箇所にフォーカスされる(図1 a)。なお，図に示すように，上面視した場合には，流路中央に単一の線状に粒子がフォーカスされた状態で観察される（図1 a）。この場合，微粒子分離部であるDLD流路には，粒子は単一の線状にフォーカスされた状態で導入された後，DLD流路内において分離される(図2)。図２は、微粒子フォーカス用マイクロ流路の断面形状が横長長方形(0 < AR <1)または半円断面形状の場合におけるDLD流路での微粒子分離の概念図を示し、(a)分離直径より大きい粒子が出口1に移動し回収され，分離直径より小さい粒子は出口2から回収される 、(b) 分離直径より大きい粒子が出口3に移動し回収され，分離直径より小さい粒子は出口2から回収される。この場合の長方形断面のアスペクト比(AR)は0 < AR < 1，好適には，0.3 < AR < 0.7である。アスペクト比ARは図１においてb/aで示される。 When the cross-sectional shape of the micro-channel for fine particle focusing is a horizontally long rectangle, the particles are focused at two locations in the center of the channel along the long side on the channel cross-section (FIG. 1a). As shown in the figure, when viewed from the top, the particles are observed in a state of being focused in a single line at the center of the flow path (FIG. 1a). In this case, the particles are introduced into the DLD channel, which is the fine particle separation unit, in a focused state in a single line, and then separated in the DLD channel (Fig. 2). Figure 2 shows a conceptual diagram of fine particle separation in the DLD flow path when the cross-sectional shape of the fine particle focusing micro flow path is a horizontally long rectangle (0 <AR <1) or a semicircular cross-sectional shape. Large particles move to outlet 1 and are collected, particles smaller than the separation diameter are collected from outlet 2. (b) Particles larger than the separation diameter move to outlet 3 and are collected, and particles smaller than the separation diameter are collected from outlet 2. Collected. In this case, the aspect ratio (AR) of the rectangular cross section is 0 <AR <1, preferably 0.3 <AR <0.7. The aspect ratio AR is indicated by b / a in FIG.
一方，微粒子フォーカス用マイクロ流路の断面形状が縦長長方形の場合，粒子は流路断面上の長辺に沿って2箇所にフォーカスされる．なお，流路を上面視した場合には左右の流路壁面付近に1箇所ずつ計2箇所に粒子がフォーカスされた状態で観察される（図1b）．この場合，微粒子分離用マイクロ流路であるDLD流路には，粒子は2箇所にフォーカスされた状態で導入された後，DLD流路の幾何形状を調節することによりDLD流路内で分離される(図3)。図３は、微粒子フォーカス用マイクロ流路の断面形状が縦長長方形(1 < AR)の場合におけるDLD流路での微粒子分離の概念図を示す。分離直径より大きい粒子は出口2に移動し，分離直径より小さい粒子は出口1, 3から回収される。この場合の長方形断面のアスペクト比(AR)は1 < AR，好適には，1.4 < AR < 3.3である。 On the other hand, when the cross-sectional shape of the micro-channel for fine particle focusing is a vertically long rectangle, the particles are focused at two locations along the long side of the cross-section of the channel. When the channel is viewed from the top, particles are observed in a focused state at two locations, one near each channel wall (Fig. 1b). In this case, the particles are introduced into the DLD channel, which is a microchannel for fine particle separation, after being introduced in a focused state at two locations, and then separated in the DLD channel by adjusting the geometry of the DLD channel. (Fig. 3). FIG. 3 is a conceptual diagram of fine particle separation in the DLD flow path when the cross-sectional shape of the fine particle focus micro flow path is a vertically long rectangle (1 <AR). Particles larger than the separation diameter move to outlet 2, and particles smaller than the separation diameter are collected from outlets 1 and 3. In this case, the aspect ratio (AR) of the rectangular cross section is 1 <AR, preferably 1.4 <AR <3.3.
また，微粒子フォーカス用マイクロ流路の断面形状が正方形の場合，粒子は流路断面上の各辺に沿って4箇所にフォーカスされる(図1 c)．なお，流路を上面視した場合には左右の流路壁面付近に1箇所ずつ，流路中央に1箇所の計3箇所に粒子がフォーカスされた状態で観察される(図1c)。この場合，粒子分離部であるDLD流路には，粒子は3箇所にフォーカスされた状態で導入された後，DLD流路内において分離される(図4)。図４は、微粒子フォーカス用マイクロ流路の断面形状が正方形 (AR = 1)または三角形の場合におけるDLD流路での微粒子分離の概念図である。(a, b) 分離直径より大きい粒子は出口2，4に移動し回収され，分離直径より小さい粒子は出口1, 3, 5から回収される。この場合の断面のアスペクト比(AR)はAR = 1である。 In addition, when the cross-sectional shape of the micro-channel for fine particle focusing is a square, the particles are focused at four locations along each side on the cross-section of the channel (Fig. 1c). When the channel is viewed from above, the particles are observed in a focused state at three locations, one near the left and right channel walls and one at the center of the channel (Fig. 1c). In this case, particles are introduced into the DLD channel, which is the particle separation unit, in a focused state at three locations, and then separated in the DLD channel (Fig. 4). FIG. 4 is a conceptual diagram of fine particle separation in the DLD flow channel when the cross-sectional shape of the micro flow channel for fine particle focus is square (AR = 1) or a triangle. (a, b) Particles larger than the separation diameter move to outlets 2 and 4 and are collected, and particles smaller than the separation diameter are collected from outlets 1, 3, and 5. In this case, the cross-sectional aspect ratio (AR) is AR = 1.
また，微粒子フォーカス用マイクロ流路の断面形状が上述の形状以外の場合であっても，粒子は流路断面上の特定の位置にフォーカスされ得る。例えば，図１（ｄ）に示すように断面が三角形である場合，断面上では３箇所に粒子をフォーカスさせることができ，図中の観察方向からは３本の線状に粒子がフォーカスされた状態が観察される。また，図１（e）に示すように断面が半円状である場合，断面上では２箇所に粒子をフォーカスさせることができ，図中の観察方向からは１本の線状に粒子がフォーカスされた状態が観察される。 Even when the cross-sectional shape of the micro-channel for fine particle focusing is other than the above-mentioned shape, the particles can be focused on a specific position on the cross-section of the channel. For example, as shown in FIG. 1 (d), when the cross section is triangular, the particles can be focused at three locations on the cross section, and the particles are focused in three lines from the observation direction in the figure. The condition is observed. In addition, when the cross section is semicircular as shown in FIG. 1 (e), the particles can be focused at two locations on the cross section, and the particles are focused on one line from the observation direction in the figure. The observed state is observed.
また，微粒子フォーカス用マイクロ流路の断面形状が同じであってもその向きを変えることで，上面視した場合のフォーカスの本数は変わり得る。例えば、図１（ｃ）の正方形断面の流路を４５度回転させる，あるいは図１（ｄ）の三角形断面の流路を３０度回転させる，あるいは図１（e）に示した半円断面の流路を９０度回転させることで，上面視した場合のフォーカス位置を２本にすることができる。 Further, even if the cross-sectional shape of the micro-channel for fine particle focus is the same, the number of the focus when viewed from above can be changed by changing the direction. For example, the square cross section channel of FIG. 1 (c) is rotated 45 degrees, the triangular section channel of FIG. 1 (d) is rotated 30 degrees, or the semicircular cross section shown in FIG. By rotating the flow path by 90 degrees, the focus position when viewed from above can be made two.
微粒子フォーカス用マイクロ流路の流れは、レイノルズ数Reが0.01〜2400あるいは層流状態が好適である。 The flow of the micro-channel for fine particle focusing is preferably a Reynolds number Re of 0.01 to 2400 or a laminar flow state.
微粒子が慣性力による微粒子フォーカス用マイクロ流路から支柱配列構造を有する微粒子分離用マイクロ流路に導入される際の収束幅は、微粒子分離用マイクロ流路に配列された支柱間の隙間(d)と同等の収束幅であるのが好適である。たとえば、マイクロ流路デバイスは、温度応答性高分子であるポリ-N-イソプロピルアクリルアミド（PNIPAM)で作製した支柱配列間で形成されるＤＬＤ流路およびその流路を密封するためのポリジメチルシロキサン (polydimethylsiloxane:PDMS)流路から構成される。PNIPAMによるＤＬＤ支柱は、たとえば支柱直径(Dp) 20μm，支柱間隙間(d) 30μm、支柱配列の傾き(θ) 0.05 radのフィルムマスクを用いて、フォトリソグラフィによりガラス基板上に作製される。ここで、支柱配列の傾き(θ)は、平面図における支柱の配列方向の傾きである。ＤＬＤ流路を密封するためのPDMS流路は、Ｓｉ基板にエポキシ樹脂「EPON SU-8」をベースにしたネガ型フォトレジスト「SU-8」を用いて作製した鋳型からPDMSにパターンを転写することにより作製される。ガラス基板上に作製したDLD支柱とPDMS流路の位置合わせを行った後、貼り合わせてデバイスを形成する。 The convergence width when the fine particles are introduced from the micro-channel for focusing particles by the inertial force into the micro-channel for separating particles having a column arrangement structure is the gap between the columns arranged in the micro-channel for separating particles (d) It is preferable that the convergence width is the same as that. For example, a microchannel device includes a DLD channel formed between strut arrays made of poly-N-isopropylacrylamide (PNIPAM), which is a temperature-responsive polymer, and polydimethylsiloxane for sealing the channel ( polydimethylsiloxane (PDMS) flow path. The DLD support by PNIPAM is manufactured on a glass substrate by photolithography using, for example, a film mask having a support diameter (D p ) of 20 μm, a space between supports (d) of 30 μm, and a support array inclination (θ) of 0.05 rad. Here, the inclination (θ) of the support arrangement is an inclination in the arrangement direction of the support in the plan view. The PDMS channel for sealing the DLD channel transfers the pattern from the template made using the negative photoresist “SU-8” based on the epoxy resin “EPON SU-8” to the PDMS. It is produced by this. After aligning the DLD support fabricated on the glass substrate and the PDMS channel, they are bonded together to form the device.
本発明の１つの実施態様において、支柱としてPDMSを用いた流路の場合，エポキシ樹脂「EPON SU-8」やSiで流路の鋳型を作製した後，PDMSに流路形状パターンを転写し、Siを用いた流路の場合，Siをエッチングして用いるのが好適である。 In one embodiment of the present invention, in the case of a flow path using PDMS as a support column, a flow path shape pattern is transferred to PDMS after a flow path mold is made of epoxy resin “EPON SU-8” or Si, In the case of a channel using Si, it is preferable to use Si after etching.
支柱の形状は、円柱に限るものではなく、いかなる柱状体であってもよい。支柱の配列は、分離する微粒子の大きさ、形状，硬さ等の粒子の特性に応じて、支柱形状，支柱直径、支柱間隙間(d)、支柱配列の傾き(θ)を好適に設定することによりなされ得る。 The shape of the column is not limited to a cylinder, and may be any columnar body. For the arrangement of the columns, the column shape, the column diameter, the gap between columns (d), and the inclination (θ) of the columns are suitably set according to the particle characteristics such as the size, shape, and hardness of the fine particles to be separated. Can be done by
たとえば、微粒子の大きさによる分離の場合、支柱直径１０ｎｍ〜１０ｍｍ、支柱間隙間１ｎｍ〜１０ｍｍでかつ分離する微粒子直径より大、支柱配列の傾き０．０１〜０．５rad.程度から設定される。支柱の配列は、単一の分離直径（すなわち流路の幾何形状によって粒子軌道の変化の境界となる粒子直径）を有するように構成されていても、または複数の分離直径を有するように構成されていてもよい。 For example, in the case of separation based on the size of the fine particles, the column diameter is set to 10 nm to 10 mm, the gap between the columns is 1 nm to 10 mm, larger than the fine particle diameter to be separated, and the inclination of the column arrangement is about 0.01 to 0.5 rad. The array of struts may be configured to have a single separation diameter (ie, the particle diameter that is the boundary of particle trajectory changes due to flow path geometry) or to have multiple separation diameters. It may be.
本発明の微粒子の分離方法においては、微粒子の分散された液体を微粒子フォーカス用マイクロ流路に導入し、導入された液体中の微粒子を、慣性力により，単一流路中の流れに沿った単一または複数本の線状に局所的に配列させ；配列された支柱間の隙間で形成された微粒子分離用マイクロ流路の単一または複数の特定部位に微粒子が集中して配列された状態で、微粒子の分散された液体を流入させ；微粒子分離用マイクロ流路を流れる微粒子の軌道を制御して微粒子の特性にしたがって微粒子を分離し、該微粒子分離用マイクロ流路から流出させる。 In the fine particle separation method of the present invention, a liquid in which fine particles are dispersed is introduced into a fine particle focusing micro-channel, and the microparticles in the introduced liquid are simply moved along a flow in a single channel by inertial force. One or a plurality of lines are locally arranged; in a state where particles are concentrated and arranged at one or a plurality of specific parts of a microchannel for particle separation formed by gaps between arranged columns. The liquid in which the fine particles are dispersed is introduced; the orbits of the fine particles flowing through the fine particle separation microchannel are controlled to separate the fine particles according to the characteristics of the fine particles, and the fine particles are discharged from the fine particle separation microchannel.
ここで、支柱は、好適には、従来のＤＬＤにおいて一般的に用いられている材料、たとえばポリジメチルシロキサン（PDMS）等のポリマー樹脂，またはSi，ガラス等から構成され得る。さらに、刺激応答性高分子材料を用いて、刺激に対する収縮または膨潤による支柱形状の変化の度合いにより、マイクロ流路を流れる微粒子の軌道を制御して微粒子の特性にしたがって微粒子を分離するように構成してもよい。 Here, the support column can be preferably made of a material generally used in conventional DLD, for example, a polymer resin such as polydimethylsiloxane (PDMS), Si, glass, or the like. Furthermore, using a stimuli-responsive polymer material, the microparticle trajectory is controlled according to the characteristics of the microparticles by controlling the trajectory of the microparticles flowing through the microchannel according to the degree of change in the columnar shape due to contraction or swelling with respect to the stimulus. May be.
微粒子の特性としては、その大きさ、形状または硬さが挙げられ、微粒子は、その大きさに基づき、その形状に基づき、またはその硬さに基づき分離されることになる。 The characteristics of the fine particles include their size, shape, and hardness, and the fine particles are separated based on their size, based on their shape, or based on their hardness.
支柱を形成する刺激応答性高分子材料は、温度、光、電場もしくは磁場等の物理的刺激、またはｐＨ、溶液組成、イオン強度等の化学的刺激に応答するハイドロゲルである。たとえば、温度応答性高分子材料は、ハイドロゲルであり、水分を大量に含むことにより膨潤する性質を有するポリマーであり、温度変化による可逆的な水和・脱水和に伴う膨潤・収縮を生じる機能を有する。 The stimulus-responsive polymeric material that forms the struts is a hydrogel that responds to physical stimuli such as temperature, light, electric or magnetic fields, or chemical stimuli such as pH, solution composition, ionic strength. For example, a temperature-responsive polymer material is a hydrogel, a polymer that swells when it contains a large amount of water, and has a function of causing swelling and shrinkage due to reversible hydration and dehydration due to temperature changes. Have
分離される微粒子は、ラテックス等のポリマー微粒子、赤血球、生細胞等の生物系微粒子、金属微粒子、および金属酸化物等の非金属粒子、から選ばれる。 The fine particles to be separated are selected from polymer fine particles such as latex, biological fine particles such as red blood cells and living cells, metal fine particles, and non-metallic particles such as metal oxides.
微粒子の粒径は、通常、１ｎｍ〜１ｍｍ、好適には１０ｎｍ〜１００μｍである。 The particle diameter of the fine particles is usually 1 nm to 1 mm, preferably 10 nm to 100 μm.
微粒子は例えば水性または油性懸濁液の形態で微粒子分離デバイスの流入口から導入される。水性懸濁液は、たとえば界面活性剤を添加した水溶液に微粒子を粒子濃度10２〜10１０/mＬ程度で懸濁した溶液とするのが好適である。水性懸濁液は、水を主成分とし、水と相溶性のあるアルコール類等の有機溶媒を含んでいてもよい。 The microparticles are introduced from the inlet of the microparticle separation device, for example in the form of an aqueous or oily suspension. The aqueous suspension is preferably, for example, a solution in which fine particles are suspended in an aqueous solution to which a surfactant is added at a particle concentration of about 10 2 to 10 10 / mL. The aqueous suspension may contain water and an organic solvent such as alcohol that is compatible with water.
本発明の微粒子分離方法においては、流入口から導入された微粒子の配列を慣性力により整えるための微粒子フォーカス用マイクロ流路を微粒子分離用マイクロ流路の前段に設け、微粒子分離用マイクロ流路の特定位置に微粒子を集中させるように構成されてなる。 In the fine particle separation method of the present invention, a fine particle focusing microchannel for adjusting the arrangement of the fine particles introduced from the inlet by inertia force is provided in the front stage of the fine particle separation microchannel. It is configured to concentrate fine particles at a specific position.
この微粒子フォーカス用マイクロ流路の形状は、
断面形状：長方形，正方形，半円形，三角形；
流路断面の縦横比（アスペクト比，AR)：断面形状が横長長方形の場合、0 < AR < 1 ；断面形状が縦長長方形の場合、1 < AR；断面形状が，正方形の場合、AR ＝１
流路形状：直線または蛇行流路；
流路長さ：1 μm〜1 m、好適には１ｍｍ〜１０ｃｍ；
流路幅：50nm 〜10 mm；
流路高さ：50nm 〜10 mm程度
から選ばれる。
The shape of the micro-channel for fine particle focus is
Cross-sectional shape: rectangle, square, semi-circle, triangle;
Aspect ratio of channel cross section (aspect ratio, AR): 0 <AR <1 when the cross-sectional shape is a horizontally long rectangle; 1 <AR when the cross-sectional shape is a vertically long rectangle; AR = 1 when the cross-sectional shape is square
Channel shape: straight or serpentine channel;
Channel length: 1 μm to 1 m, preferably 1 mm to 10 cm;
Channel width: 50nm to 10mm;
Channel height: selected from about 50 nm to 10 mm.
本発明の慣性力による微粒子フォーカス用マイクロ流路の断面形状，微粒子のフォーカス位置，および観察方向の関係を図1に示す。図１において、 (a) 横長長方形 0 < AR < 1； (b) 縦長長方形1 < AR,； (c) 正方形1 = AR,；(d) 三角形,；(e) 半円、 (上段) 流路断面上の粒子フォーカス位置、 (下段)マイクロ流路を流路断面の上辺と流れに垂直な方向から観察(上面視)した場合の粒子フォーカス位置を示す。 FIG. 1 shows the relationship between the cross-sectional shape of the micro-channel for fine particle focusing by the inertia force of the present invention, the focus position of the fine particles, and the observation direction. In Fig. 1, (a) Horizontal rectangle 0 <AR <1; (b) Vertical rectangle 1 <AR ,; (c) Square 1 = AR ,; (d) Triangle, (e) Semicircle, (Upper) flow The particle focus position on the cross section of the channel, (bottom) shows the particle focus position when the microchannel is observed from the direction perpendicular to the upper side and the flow of the channel cross section (top view).
マイクロ流路の断面形状が横長長方形の場合、粒子は流路断面上の長辺に沿って流路中央２箇所にフォーカスされる(図1 a)。なお，図に示すように，上面視した場合には，流路中央に単一の線状に粒子がフォーカスされた状態で観察される（図1 a）。この場合，微粒子分離部であるDLD流路には，粒子は単一の線状にフォーカスされた状態で導入された後，DLD流路内において分離される(図2)。図２は、微粒子フォーカス用マイクロ流路の断面形状が横長長方形(0 < AR <1)または半円断面形状の場合におけるDLD流路での微粒子分離の概念図を示し、(a)分離直径より大きい粒子が出口1に移動し回収され，分離直径より小さい粒子は出口2から回収される 、(b) 分離直径より大きい粒子が出口3に移動し回収され，分離直径より小さい粒子は出口2から回収される。この場合の長方形断面のアスペクト比(AR)は0 < AR < 1，好適には，0.3 < AR < 0.7である。アスペクト比ARは図１においてb/aで示される。 When the cross-sectional shape of the microchannel is a horizontally long rectangle, the particles are focused at two locations in the center of the channel along the long side of the channel cross-section (FIG. 1a). As shown in the figure, when viewed from the top, the particles are observed in a state of being focused in a single line at the center of the flow path (FIG. 1a). In this case, the particles are introduced into the DLD channel, which is the fine particle separation unit, in a focused state in a single line, and then separated in the DLD channel (Fig. 2). Figure 2 shows a conceptual diagram of fine particle separation in the DLD flow path when the cross-sectional shape of the fine particle focusing micro flow path is a horizontally long rectangle (0 <AR <1) or a semicircular cross-sectional shape. Large particles move to outlet 1 and are collected, particles smaller than the separation diameter are collected from outlet 2. (b) Particles larger than the separation diameter move to outlet 3 and are collected, and particles smaller than the separation diameter are collected from outlet 2. Collected. In this case, the aspect ratio (AR) of the rectangular cross section is 0 <AR <1, preferably 0.3 <AR <0.7. The aspect ratio AR is indicated by b / a in FIG.
一方，微粒子フォーカス用マイクロ流路の断面形状が縦長長方形の場合，粒子は流路断面上の長辺に沿って2箇所にフォーカスされる．なお，流路を上面視した場合には左右の流路壁面付近に1箇所ずつ計2箇所に粒子がフォーカスされた状態で観察される（図1b）．この場合，微粒子分離用マイクロ流路であるDLD流路には，粒子は2箇所にフォーカスされた状態で導入された後，DLD流路の幾何形状を調節することによりDLD流路内で分離される(図3)。図３は、微粒子フォーカス用マイクロ流路の断面形状が縦長長方形(1 < AR)の場合におけるDLD流路での微粒子分離の概念図を示す。分離直径より大きい粒子は出口2に移動し，分離直径より小さい粒子は出口1, 3から回収される。この場合の長方形断面のアスペクト比(AR)は1 < AR，好適には，1.4 < AR < 3.3である。 On the other hand, when the cross-sectional shape of the micro-channel for fine particle focusing is a vertically long rectangle, the particles are focused at two locations along the long side of the cross-section of the channel. When the channel is viewed from the top, particles are observed in a focused state at two locations, one near each channel wall (Fig. 1b). In this case, the particles are introduced into the DLD channel, which is a microchannel for fine particle separation, after being introduced in a focused state at two locations, and then separated in the DLD channel by adjusting the geometry of the DLD channel. (Fig. 3). FIG. 3 is a conceptual diagram of fine particle separation in the DLD flow path when the cross-sectional shape of the fine particle focus micro flow path is a vertically long rectangle (1 <AR). Particles larger than the separation diameter move to outlet 2, and particles smaller than the separation diameter are collected from outlets 1 and 3. In this case, the aspect ratio (AR) of the rectangular cross section is 1 <AR, preferably 1.4 <AR <3.3.
また，微粒子フォーカス用マイクロ流路の断面形状が正方形の場合，粒子は流路断面上の各辺に沿って4箇所にフォーカスされる(図1 c)．なお，流路を上面視した場合には左右の流路壁面付近に1箇所ずつ，流路中央に1箇所の計3箇所に粒子がフォーカスされた状態で観察される(図1c)。この場合，粒子分離部であるDLD流路には，粒子は3箇所にフォーカスされた状態で導入された後，DLD流路内において分離される(図4)。図４は、微粒子フォーカス用マイクロ流路の断面形状が正方形 (AR = 1)または三角形の場合におけるDLD流路での微粒子分離の概念図である。(a, b) 分離直径より大きい粒子は出口2，4に移動し回収され，分離直径より小さい粒子は出口1, 3, 5から回収される。この場合の断面のアスペクト比(AR)はAR = 1である。 In addition, when the cross-sectional shape of the micro-channel for fine particle focusing is a square, the particles are focused at four locations along each side on the cross-section of the channel (Fig. 1c). When the channel is viewed from above, the particles are observed in a focused state at three locations, one near the left and right channel walls and one at the center of the channel (Fig. 1c). In this case, particles are introduced into the DLD channel, which is the particle separation unit, in a focused state at three locations, and then separated in the DLD channel (Fig. 4). FIG. 4 is a conceptual diagram of fine particle separation in the DLD flow channel when the cross-sectional shape of the micro flow channel for fine particle focus is square (AR = 1) or a triangle. (a, b) Particles larger than the separation diameter move to outlets 2 and 4 and are collected, and particles smaller than the separation diameter are collected from outlets 1, 3, and 5. In this case, the cross-sectional aspect ratio (AR) is AR = 1.
また，微粒子フォーカス用マイクロ流路の断面形状が上述の形状以外の場合であっても，粒子は流路断面上の特定の位置にフォーカスされ得る。例えば，図１（ｄ）に示すように断面が三角形である場合，断面上では３箇所に粒子をフォーカスさせることができ，図中の観察方向からは３本の線状に粒子がフォーカスされた状態が観察される。また，図１（e）に示すように断面が半円状である場合，断面上では２箇所に粒子をフォーカスさせることができ，図中の観察方向からは１本の線状に粒子がフォーカスされた状態が観察される。 Even when the cross-sectional shape of the micro-channel for fine particle focusing is other than the above-mentioned shape, the particles can be focused on a specific position on the cross-section of the channel. For example, as shown in FIG. 1 (d), when the cross section is triangular, the particles can be focused at three locations on the cross section, and the particles are focused in three lines from the observation direction in the figure. The condition is observed. In addition, when the cross section is semicircular as shown in FIG. 1 (e), the particles can be focused at two locations on the cross section, and the particles are focused on one line from the observation direction in the figure. The observed state is observed.
また，微粒子フォーカス用マイクロ流路の断面形状が同じであってもその向きを変えることで，上面視した場合のフォーカスの本数は変わり得る。例えば、図１（ｃ）の正方形断面の流路を４５度回転させる，あるいは図１（ｄ）の三角形断面の流路を３０度回転させる，あるいは図１（e）に示した半円断面の流路を９０度回転させることで，上面視した場合のフォーカス位置を２本にすることができる。 Further, even if the cross-sectional shape of the micro-channel for fine particle focus is the same, the number of the focus when viewed from above can be changed by changing the direction. For example, the square cross section channel of FIG. 1 (c) is rotated 45 degrees, the triangular section channel of FIG. 1 (d) is rotated 30 degrees, or the semicircular cross section shown in FIG. By rotating the flow path by 90 degrees, the focus position when viewed from above can be made two.
微粒子フォーカス用マイクロ流路の流れは、レイノルズ数Reが0.01〜2400あるいは層流状態が好適である。 The flow of the micro-channel for fine particle focusing is preferably a Reynolds number Re of 0.01 to 2400 or a laminar flow state.
微粒子が慣性力による微粒子フォーカス用マイクロ流路から支柱配列構造を有する微粒子分離用マイクロ流路に導入される際の収束幅は、微粒子分離用マイクロ流路に配列された支柱間の隙間(d)と同等の収束幅であるのが好適である。たとえば、マイクロ流路デバイスは、温度応答性高分子であるポリ-N-イソプロピルアクリルアミド（PNIPAM)で作製した支柱配列間で形成されるＤＬＤ流路およびその流路を密封するためのポリジメチルシロキサン (polydimethylsiloxane:PDMS)流路から構成される。PNIPAMによるＤＬＤ支柱は、たとえば支柱直径(Dp) 20μm，支柱間隙間(d) 30μm、支柱配列の傾き(θ) 0.05 radのフィルムマスクを用いて、フォトリソグラフィによりガラス基板上に作製される。ここで、支柱配列の傾き(θ)は、平面図における支柱の配列方向の傾きである。ＤＬＤ流路を密封するためのPDMS流路は、Ｓｉ基板にエポキシ樹脂「EPON SU-8」をベースにしたネガ型フォトレジスト「SU-8」を用いて作製した鋳型からPDMSにパターンを転写することにより作製される。ガラス基板上に作製したDLD支柱とPDMS流路の位置合わせを行った後、貼り合わせてデバイスを形成する。 The convergence width when the fine particles are introduced from the micro-channel for focusing particles by the inertial force into the micro-channel for separating particles having a column arrangement structure is the gap between the columns arranged in the micro-channel for separating particles (d) It is preferable that the convergence width is the same as that. For example, a microchannel device includes a DLD channel formed between strut arrays made of poly-N-isopropylacrylamide (PNIPAM), which is a temperature-responsive polymer, and polydimethylsiloxane for sealing the channel ( polydimethylsiloxane (PDMS) flow path. The DLD support by PNIPAM is manufactured on a glass substrate by photolithography using, for example, a film mask having a support diameter (D p ) of 20 μm, a space between supports (d) of 30 μm, and a support array inclination (θ) of 0.05 rad. Here, the inclination (θ) of the support arrangement is an inclination in the arrangement direction of the support in the plan view. The PDMS channel for sealing the DLD channel transfers the pattern from the template made using the negative photoresist “SU-8” based on the epoxy resin “EPON SU-8” to the PDMS. It is produced by this. After aligning the DLD support fabricated on the glass substrate and the PDMS channel, they are bonded together to form the device.
本発明の１つの実施態様において、支柱としてPDMSを用いた流路の場合，エポキシ樹脂「EPON SU-8」やSiで流路の鋳型を作製した後，PDMSに流路形状パターンを転写し、Siを用いた流路の場合，Siをエッチングして用いるのが好適である。 In one embodiment of the present invention, in the case of a flow path using PDMS as a support column, a flow path shape pattern is transferred to PDMS after a flow path mold is made of epoxy resin “EPON SU-8” or Si, In the case of a channel using Si, it is preferable to use Si after etching.
支柱の形状は、円柱に限るものではなく、いかなる柱状体であってもよい。支柱の配列は、分離する微粒子の大きさ、形状，硬さ等の粒子の特性に応じて、支柱形状，支柱直径、支柱間隙間(d)、支柱配列の傾き(θ)を好適に設定することによりなされ得る。 The shape of the column is not limited to a cylinder, and may be any columnar body. For the arrangement of the columns, the column shape, the column diameter, the gap between columns (d), and the inclination (θ) of the columns are suitably set according to the particle characteristics such as the size, shape, and hardness of the fine particles to be separated. Can be done by
たとえば、微粒子の大きさによる分離の場合、支柱直径１０ｎｍ〜１０ｍｍ、支柱間隙間１ｎｍ〜１０ｍｍでかつ分離する微粒子直径より大、支柱配列の傾き０．０１〜０．５rad.程度から設定される。支柱の配列は、単一の分離直径（すなわち流路の幾何形状によって粒子軌道の変化の境界となる粒子直径）を有するように構成されていても、または複数の分離直径を有するように構成されていてもよい。 For example, in the case of separation based on the size of the fine particles, the column diameter is set to 10 nm to 10 mm, the gap between the columns is 1 nm to 10 mm, larger than the fine particle diameter to be separated, and the inclination of the column arrangement is about 0.01 to 0.5 rad. The array of struts may be configured to have a single separation diameter (ie, the particle diameter that is the boundary of particle trajectory changes due to flow path geometry) or to have multiple separation diameters. It may be.
本発明の微粒子の分離方法においては、微粒子の分散された液体を微粒子フォーカス用マイクロ流路に導入し、導入された液体中の微粒子を、慣性力により，単一流路中の流れに沿った単一または複数本の線状に局所的に配列させ；配列された支柱間の隙間で形成された微粒子分離用マイクロ流路の単一または複数の特定部位に微粒子が集中して配列された状態で、微粒子の分散された液体を流入させ；微粒子分離用マイクロ流路を流れる微粒子の軌道を制御して微粒子の特性にしたがって微粒子を分離し、該微粒子分離用マイクロ流路から流出させる。 In the fine particle separation method of the present invention, a liquid in which fine particles are dispersed is introduced into a fine particle focusing micro-channel, and the microparticles in the introduced liquid are simply moved along a flow in a single channel by inertial force. One or a plurality of lines are locally arranged; in a state where particles are concentrated and arranged at one or a plurality of specific parts of a microchannel for particle separation formed by gaps between arranged columns. The liquid in which the fine particles are dispersed is introduced; the orbits of the fine particles flowing through the fine particle separation microchannel are controlled to separate the fine particles according to the characteristics of the fine particles, and the fine particles are discharged from the fine particle separation microchannel.
ここで、支柱は、好適には、従来のＤＬＤにおいて一般的に用いられている材料、たとえばポリジメチルシロキサン（PDMS）等のポリマー樹脂，またはSi，ガラス等から構成され得る。さらに、刺激応答性高分子材料を用いて、刺激に対する収縮または膨潤による支柱形状の変化の度合いにより、マイクロ流路を流れる微粒子の軌道を制御して微粒子の特性にしたがって微粒子を分離するように構成してもよい。 Here, the support column can be preferably made of a material generally used in conventional DLD, for example, a polymer resin such as polydimethylsiloxane (PDMS), Si, glass, or the like. Furthermore, using a stimuli-responsive polymer material, the microparticle trajectory is controlled according to the characteristics of the microparticles by controlling the trajectory of the microparticles flowing through the microchannel according to the degree of change in the columnar shape due to contraction or swelling with respect to the stimulus. May be.
微粒子の特性としては、その大きさ、形状または硬さが挙げられ、微粒子は、その大きさに基づき、その形状に基づき、またはその硬さに基づき分離されることになる。 The characteristics of the fine particles include their size, shape, and hardness, and the fine particles are separated based on their size, based on their shape, or based on their hardness.
支柱を形成する刺激応答性高分子材料は、温度、光、電場もしくは磁場等の物理的刺激、またはｐＨ、溶液組成、イオン強度等の化学的刺激に応答するハイドロゲルである。たとえば、温度応答性高分子材料は、ハイドロゲルであり、水分を大量に含むことにより膨潤する性質を有するポリマーであり、温度変化による可逆的な水和・脱水和に伴う膨潤・収縮を生じる機能を有する。 The stimulus-responsive polymeric material that forms the struts is a hydrogel that responds to physical stimuli such as temperature, light, electric or magnetic fields, or chemical stimuli such as pH, solution composition, ionic strength. For example, a temperature-responsive polymer material is a hydrogel, a polymer that swells when it contains a large amount of water, and has a function of causing swelling and shrinkage due to reversible hydration and dehydration due to temperature changes. Have
分離される微粒子は、ラテックス等のポリマー微粒子、赤血球、生細胞等の生物系微粒子、金属微粒子、および金属酸化物等の非金属粒子、から選ばれる。 The fine particles to be separated are selected from polymer fine particles such as latex, biological fine particles such as red blood cells and living cells, metal fine particles, and non-metallic particles such as metal oxides.
微粒子の粒径は、通常、１ｎｍ〜１ｍｍ、好適には１０ｎｍ〜１００μｍである。 The particle diameter of the fine particles is usually 1 nm to 1 mm, preferably 10 nm to 100 μm.
微粒子は例えば水性または油性懸濁液の形態で微粒子分離デバイスの流入口から導入される。水性懸濁液は、たとえば界面活性剤を添加した水溶液に微粒子を粒子濃度10２〜10１０/mＬ程度で懸濁した溶液とするのが好適である。水性懸濁液は、水を主成分とし、水と相溶性のあるアルコール類等の有機溶媒を含んでいてもよい。 The microparticles are introduced from the inlet of the microparticle separation device, for example in the form of an aqueous or oily suspension. The aqueous suspension is preferably, for example, a solution in which fine particles are suspended in an aqueous solution to which a surfactant is added at a particle concentration of about 10 2 to 10 10 / mL. The aqueous suspension may contain water and an organic solvent such as alcohol that is compatible with water.
つぎに、本発明において、粒子形状による分離を行う場合について説明する。たとえば、赤血球（厚み２μm、 直径６μm）などの円盤形状の粒子の場合、粒子の流れる姿勢によって、ＤＬＤ流路内での粒子の見掛けの直径が変化し、分離挙動（Displacement mode, Zigzag mode）に影響を与えることが知られている。そのため，ＤＬＤの支柱に対して赤血球の姿勢（円盤の直径方向，厚み方向）を制御した分離手法（J. P. Beech et al., Lab Chip, 12, 1048-1051, 2012）や赤血球の回転を用いた分離手法（K. K. Zeming et al., Nat. Commun., 4, 1625, 2013）がこれまでに報告されているので、本発明の分離方法をこれらに適用することにより粒子形状による分離を行うことができる。 Next, in the present invention, the case of performing separation based on particle shape will be described. For example, in the case of disc-shaped particles such as red blood cells (thickness 2μm, diameter 6μm), the apparent diameter of the particles in the DLD channel changes depending on the flow direction of the particles, resulting in separation behavior (Displacement mode, Zigzag mode). It is known to have an impact. Therefore, a separation method (JP Beech et al., Lab Chip, 12, 1048-1051, 2012) in which the posture of the red blood cells (diameter direction and thickness direction of the disc) was controlled with respect to the DLD column and rotation of the red blood cells was used. Since separation methods (KK Zeming et al., Nat. Commun., 4, 1625, 2013) have been reported so far, it is possible to perform separation by particle shape by applying the separation method of the present invention to these methods. it can.
さらに、本発明において、粒子の硬さによる分離を行う場合について説明する。ＤＬＤ流路内では，粒子にせん断応力が働くため、粒子の硬さの違いに従って、粒子が変形する度合いが異なる。たとえば、静止状態では同じ直径の粒子の場合であっても，より変形しやすい粒子は，変形しない粒子と比較して，ＤＬＤ流路内での見かけの直径が小さくなる。すなわち，サイズが同じ粒子であっても，粒子の変形度合いの違いを利用した粒子分離が可能になる。たとえば，赤血球の場合において、通常の赤血球とマラリアに感染した赤血球とでは硬さが異なるため、硬さを指標とした赤血球の分離によって病気の検出などが期待される（T. Krueger et al., Biomicrofluidics, 8, 054114, 2014）。本発明の分離方法をこれらに適用することにより粒子の硬さによる分離を行うことができる。 Furthermore, in the present invention, a case where separation is performed based on particle hardness will be described. Since shear stress acts on the particles in the DLD flow path, the degree of deformation of the particles varies according to the difference in particle hardness. For example, even in the case of particles having the same diameter in a stationary state, particles that are more likely to be deformed have a smaller apparent diameter in the DLD channel than particles that are not deformed. That is, even if the particles have the same size, particle separation utilizing the difference in the degree of deformation of the particles becomes possible. For example, in the case of red blood cells, normal red blood cells and red blood cells infected with malaria have different hardness, so it is expected to detect diseases by separating red blood cells using hardness as an index (T. Krueger et al., Biomicrofluidics, 8, 054114, 2014). By applying the separation method of the present invention to these, separation based on particle hardness can be performed.
実施例１
図５は、本発明の微粒子分離デバイスの１実施態様を示す概要図である。慣性力による微粒子フォーカス用マイクロ流路(導入路)とdeterministic lateral displacement (ＤＬＤ)支柱配列を有する微粒子分離用マイクロ流路から構成される。導入路である微粒子フォーカス用マイクロ流路は、矩形断面（アスペクト比0.5）を有する直線流路（幅50μｍ、高さ25μｍ、長さ2.5cm）であり、下流のＤＬＤ流路（微粒子分離用マイクロ流路）のパラメータは、流路長23mm,流路幅3mm,支柱直径(Ｄｐ) 30μｍ、支柱間距離(d) 20μｍ、支柱配列の傾き0.05radとした。デバイスはＳｉ基板にエポキシ樹脂「EPON SU-8」をベースにしたネガ型フォトレジスト「SU-8」を用いて作製した鋳型からポリジメチルシロキサン （PDMS)にパターンを転写することにより作製した。作製したPDMS流路とガラス基板を酸素プラズマ処理後に接着することにより形成した。
Example 1
FIG. 5 is a schematic view showing one embodiment of the particulate separation device of the present invention. It is composed of a micro-channel for fine particle focusing (introduction channel) by inertial force and a micro-channel for particle separation having a deterministic lateral displacement (DLD) column arrangement. The micro-channel for fine particle focusing, which is an introduction channel, is a straight channel (width 50 μm, height 25 μm, length 2.5 cm) having a rectangular cross section (aspect ratio 0.5), and a downstream DLD channel (micro particle separation micro-channel). The parameters of the flow path were as follows: flow path length 23 mm, flow path width 3 mm, strut diameter (D p ) 30 μm, strut distance (d) 20 μm, strut array inclination 0.05 rad. The device was fabricated by transferring a pattern from a template made using a negative photoresist "SU-8" based on the epoxy resin "EPON SU-8" to a polydimethylsiloxane (PDMS) on a Si substrate. The produced PDMS channel and glass substrate were formed by bonding after oxygen plasma treatment.
導入試料には、直径13μmと7μmのポリスチレンビーズ(Thermo Fisher社製，Bangs Laboratories社製)を0.1v/v%の界面活性剤Tween（登録商標）20(Sigma)水溶液に粒子濃度1.0×10５/mLで懸濁した溶液を用意した。粒子懸濁溶液は、シリンジポンプ(KD Scientific社製, KDS200)にて送液した。 As an introduction sample, polystyrene beads having diameters of 13 μm and 7 μm (manufactured by Thermo Fisher, Bangs Laboratories) were added to an aqueous solution of a surfactant Tween (registered trademark) 20 (Sigma) with a concentration of 1.0 × 10 5 in 0.1 v / v%. A solution suspended at / mL was prepared. The particle suspension was fed with a syringe pump (KD Scientific, KDS200).
粒子懸濁溶液を流量（Ｑ）１mL/hでデバイスに導入した際の慣性力による微粒子のフォーカシングの様子を図６に示す。導入された微粒子は、微粒子フォーカス用マイクロ流路上流部では、ビーズが流路全体に分散した状態で流れる様子が確認された（図６ａ）。微粒子フォーカス用流路中流部では直径13μmのビーズが流路中央へと移動し（図６ｂ）、微粒子フォーカス用流路下流部では直径13μmと7μmのビーズがいずれも流路中央へと移動した（図６ｃ）様子が確認された。これは、微粒子が流路中央へと移動するために必要な流路長さは粒子直径の３乗に反比例するためである。一方、低流量（Ｑ＝0.5mL/h、0.１mL/h）に設定した際には、微粒子が流路中央へと十分には移動せず、Ｑ＝１mL/hで直径13μmと7μmのビーズが十分に流路中央に移動することが確認された（図７ａ〜ｃ；図７は、異なる流量条件下における微粒子フォーカス用マイクロ流路での微粒子フォーカシングの様子を示す図である。）。これは、粒子が流路中央へと移動するために必要な流路長さは流速に反比例するためと考えられる。 FIG. 6 shows the state of fine particle focusing by inertia force when the particle suspension is introduced into the device at a flow rate (Q) of 1 mL / h. It was confirmed that the introduced fine particles flowed in a state where the beads are dispersed in the whole flow channel in the upstream portion of the fine particle focusing micro flow channel (FIG. 6a). In the middle part of the fine particle focusing channel, beads having a diameter of 13 μm moved to the center of the channel (FIG. 6B), and in the downstream part of the fine particle focusing channel, beads having diameters of 13 μm and 7 μm moved to the center of the channel ( FIG. 6 c) was confirmed. This is because the channel length necessary for the fine particles to move to the center of the channel is inversely proportional to the cube of the particle diameter. On the other hand, when the flow rate is set low (Q = 0.5 mL / h, 0.1 mL / h), the fine particles do not move sufficiently to the center of the flow path, and the diameter is 13 μm and 7 μm at Q = 1 mL / h. It was confirmed that the beads sufficiently moved to the center of the flow channel (FIGS. 7a to 7c; FIG. 7 is a diagram showing a state of fine particle focusing in the micro flow channel for fine particle focusing under different flow rate conditions). This is presumably because the channel length necessary for the particles to move to the center of the channel is inversely proportional to the flow velocity.
流量を１mL/hに設定した際の、微粒子分離用マイクロ流路（ＤＬＤ流路）での微粒子分離の様子を図８に示す。ＤＬＤ流路の特定位置にフォーカスされた状態で微粒子が導入された後（図８ａ）、直径13μmのビーズはＤＬＤの支柱配列の傾きに沿って進む置換モード（displacement mode)の軌道を取り、直径7μmのビーズは流れと同一方向に進むジグザグモード（zigzag mode）の軌道を取る様子が確認された（図８ｂ）。ＤＬＤ流路下流部では直径13μmと7μmのビーズが分離された後、出口１と出口２からそれぞれ回収される様子が確認された（図８ｃ）。すなわち、シースフロー型のような，バッファにより微粒子懸濁溶液を挟み込むマイクロ流路構造を用いることなく、微粒子分離デバイスにより微粒子分離を実現した。 FIG. 8 shows the state of particle separation in the microparticle separation microchannel (DLD channel) when the flow rate is set to 1 mL / h. After the microparticles are introduced in a focused state in the DLD channel (FIG. 8a), the 13 μm diameter beads take a displacement mode trajectory that travels along the inclination of the DLD strut array, It was confirmed that the 7 μm beads took a zigzag mode orbit in the same direction as the flow (FIG. 8 b). It was confirmed that beads having diameters of 13 μm and 7 μm were separated from the downstream portion of the DLD channel and then recovered from the outlet 1 and the outlet 2 respectively (FIG. 8c). That is, fine particle separation was realized by a fine particle separation device without using a micro flow channel structure in which a fine particle suspension solution was sandwiched between buffers, such as a sheath flow type.
本発明によれば、バッファを用いた微粒子懸濁溶液の挟み込みのための流路構造を用いない、微粒子懸濁試料流入用の送液流路のみを導入路として有する微粒子分離デバイスおよびそれを用いる微粒子の分離方法を提供し得る。 According to the present invention, a particle separation device having only a liquid flow channel for inflow of a fine particle suspension sample as an introduction channel without using a flow channel structure for sandwiching a fine particle suspension using a buffer, and the same are used. A method for separating particulates may be provided.
Claims (14)
微粒子の流入口および流出口、微粒子フォーカス用マイクロ流路ならびに微粒子分離用マイクロ流路からなり；
該微粒子分離用マイクロ流路は、配列された支柱間の隙間で形成され、該微粒子分離用マイクロ流路を流れる微粒子の軌道を制御して微粒子の特性にしたがって微粒子を分離するように構成されてなり；ならびに
該微粒子フォーカス用マイクロ流路は，該微粒子分離用マイクロ流路の前段に導入路として設けられ、
流入口から導入された液体中の微粒子を，慣性力により，単一流路中の流れに沿った単一または複数本の線状に局所的に配列させるように構成されてなり、
該微粒子分離用マイクロ流路の単一または複数の特定部位に微粒子が集中して配列された状態で流入するように構成されてなる；ことを特徴とする微粒子分離デバイス。 A fine particle separation device for flowing a liquid in which fine particles are dispersed and separating the fine particles according to their characteristics;
Consisting of a fine particle inlet and outlet, a fine particle focusing microchannel and a microparticle separation microchannel;
The microparticle separation microchannel is formed by a gap between the arranged support columns, and is configured to control the microparticle trajectory flowing through the microparticle separation microchannel to separate the microparticles according to the characteristics of the microparticles. And the fine particle focusing micro-channel is provided as an introduction path in front of the micro-particle separation micro-channel,
The fine particles in the liquid introduced from the inlet are configured to be locally arranged in a single or multiple lines along the flow in the single flow path by inertial force.
A particulate separation device, characterized in that the particulate separation device is configured to flow in a state in which the particulates are concentrated and arranged at one or a plurality of specific parts of the particulate separation microchannel.
配列された支柱間の隙間で形成された微粒子分離用マイクロ流路の単一または複数の特定部位に微粒子が集中して配列された状態で、微粒子の分散された液体を流入させ；
該微粒子分離用マイクロ流路を流れる微粒子の軌道を制御して微粒子の特性にしたがって微粒子を分離し、該微粒子分離用マイクロ流路から流出させることを特徴とする微粒子の分離方法。 The liquid in which the fine particles are dispersed is introduced into the micro flow channel for fine particle focusing, and the fine particles in the introduced liquid are locally localized in a single or multiple lines along the flow in the single flow channel by inertial force. To arrange;
Flowing the liquid in which the fine particles are dispersed in a state where the fine particles are concentrated and arranged in one or a plurality of specific parts of the micro flow path for separating fine particles formed by the gaps between the arranged support columns;
A method for separating fine particles, comprising controlling the trajectory of fine particles flowing through the fine particle separation microchannel to separate the fine particles according to the characteristics of the fine particles and letting the fine particles flow out from the fine particle separation microchannel.
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