JP6843420B2 - Fine particle separation device and fine particle separation method - Google Patents
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- 239000010419 fine particle Substances 0.000 title claims description 139
- 238000000926 separation method Methods 0.000 title claims description 79
- 238000000034 method Methods 0.000 claims description 27
- 239000002861 polymer material Substances 0.000 claims description 23
- 239000000017 hydrogel Substances 0.000 claims description 12
- 229920000642 polymer Polymers 0.000 claims description 11
- -1 droplets Substances 0.000 claims description 10
- 239000007900 aqueous suspension Substances 0.000 claims description 8
- 230000008602 contraction Effects 0.000 claims description 8
- 230000008961 swelling Effects 0.000 claims description 8
- 239000002184 metal Substances 0.000 claims description 6
- 239000002923 metal particle Substances 0.000 claims description 6
- 229910052755 nonmetal Inorganic materials 0.000 claims description 6
- 239000000126 substance Substances 0.000 claims description 6
- 239000002245 particle Substances 0.000 description 46
- 210000003743 erythrocyte Anatomy 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- 239000000758 substrate Substances 0.000 description 9
- 229920000208 temperature-responsive polymer Polymers 0.000 description 9
- 239000011324 bead Substances 0.000 description 8
- QNILTEGFHQSKFF-UHFFFAOYSA-N n-propan-2-ylprop-2-enamide Chemical compound CC(C)NC(=O)C=C QNILTEGFHQSKFF-UHFFFAOYSA-N 0.000 description 8
- 239000004205 dimethyl polysiloxane Substances 0.000 description 6
- 239000011521 glass Substances 0.000 description 6
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 6
- 238000007789 sealing Methods 0.000 description 6
- 210000004027 cell Anatomy 0.000 description 5
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 4
- 238000010586 diagram Methods 0.000 description 4
- 238000006073 displacement reaction Methods 0.000 description 4
- 239000011859 microparticle Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 229920002120 photoresistant polymer Polymers 0.000 description 4
- 239000004793 Polystyrene Substances 0.000 description 3
- 239000003822 epoxy resin Substances 0.000 description 3
- 238000000206 photolithography Methods 0.000 description 3
- 229920000647 polyepoxide Polymers 0.000 description 3
- 229920002223 polystyrene Polymers 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 2
- DMLAVOWQYNRWNQ-UHFFFAOYSA-N azobenzene Chemical compound C1=CC=CC=C1N=NC1=CC=CC=C1 DMLAVOWQYNRWNQ-UHFFFAOYSA-N 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 238000003320 cell separation method Methods 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 230000005684 electric field Effects 0.000 description 2
- 229920001746 electroactive polymer Polymers 0.000 description 2
- 238000001914 filtration Methods 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 230000036571 hydration Effects 0.000 description 2
- 238000006703 hydration reaction Methods 0.000 description 2
- 125000001165 hydrophobic group Chemical group 0.000 description 2
- 239000004816 latex Substances 0.000 description 2
- 229920000126 latex Polymers 0.000 description 2
- 229910044991 metal oxide Inorganic materials 0.000 description 2
- 150000004706 metal oxides Chemical class 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 229920000136 polysorbate Polymers 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- 230000002522 swelling effect Effects 0.000 description 2
- 230000007704 transition Effects 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 1
- 210000000601 blood cell Anatomy 0.000 description 1
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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 field and the production field. 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, a centrifugal separation method, a filtration method, a fluorescence activated cell separation method (FACS), a magnetic cell separation method (MACS), and the like are used for separating such biological particles. However, the centrifugal separation method has low accuracy, and the filtration method has a problem that clogging occurs. Further, 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６）、粒子径に基づき簡便に粒子を分離できる。本手法を用いて、高分離分解能かつ高処理量の粒子分離を実現した事例（非特許文献３）も報告されているが、ＤＬＤは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（分離直径）が固定されており、汎用性の低下を招いている。
In recent years, a fine particle separation method using a microchannel device (Non-Patent Document 1) has been reported as a means for solving these problems, and the separation method is 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 hydraulic action during separation In the case of the active particle separation method, the system is complicated by using external energy, so the passive separation method is expensive. It is desired to realize separation performance. In recent years, as one of the passive separation methods, a case of fine particle separation by the deterministic lateral displacement (DLD) method has been reported (Non-Patent Documents 2 to 4). DLD is a particle separation method that utilizes the flow generated in the fluid by the columns arranged in the flow path, and takes different orbits in the DLD flow path according to the particle characteristics such as particle size, shape, and hardness (Fig. 6). , Particles can be easily separated based on the particle size. A case (Non-Patent Document 3) in which high separation resolution and high processing amount of particles are realized by using this method has also been reported, but DLD is a particle that is a boundary of change in particle orbit due to the geometric shape of the flow path. The diameter (separation diameter) is fixed, which reduces versatility.
本発明は、このような課題を解決し、刺激応答性高分子で支柱を作製し、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するものである。 The present invention solves such a problem, and by forming a strut with a stimulus-responsive polymer and changing the flow path geometric shape by stimulus control, the separation diameter is the particle diameter that is the boundary of the change of the particle orbit. It provides a fine particle separation device capable of adjusting the above and a method for separating fine particles using the same.
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。 The present invention provides the following invention in order to solve the above problems.
（１）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り、
微粒子の流入口および流出口、ならびにマイクロ流路および配列された支柱からなり；
該マイクロ流路は、該配列された支柱間の隙間で形成され；
該支柱は刺激応答性高分子材料からなり、刺激に対する収縮または膨潤による該支柱形状の変化の度合いにより、該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ことを特徴とする微粒子分離デバイス。
（２）微粒子をその大き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）微粒子をその形状に基づき分離する上記（１）に記載の微粒子分離デバイス。
（４）微粒子をその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。
（５）刺激応答性高分子材料が物理的刺激または化学的刺激に応答するハイドロゲルである上記（１）〜（４）のいずれかに記載の微粒子分離デバイス。
（６）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）から（５）のいずれかに記載の微粒子分離デバイス。
（７）流入された微粒子が水性懸濁液である上記（１）〜（６）のいずれかに記載の微粒子分離デバイス。
（８）単一の分離直径を有する支柱配列から構成される上記（１）〜（７）のいずれかに記載の微粒子分離デバイス。
（９）複数の分離直径を有する支柱配列から構成される上記（１）〜（７）のいずれかに記載の微粒子分離デバイス。
（１０）分離する微粒子を、配列された支柱間の隙間で形成されたマイクロ流路に流入させ、ついでマイクロ流路を通過させた後にマイクロ流路から流出させる微粒子の分離方法であり、
該支柱は刺激応答性高分子材料からなり、刺激に対する収縮または膨潤による該支柱形状の変化の度合いにより、該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ことを特徴とする微粒子の分離方法。
（１１）微粒子をその大きさに基づき分離する上記（１０）に記載の微粒子の分離方法。
（１２）微粒子をその形状に基づき分離する上記（１０）に記載の微粒子の分離方法。
（１３）微粒子をその硬さに基づき分離する上記（１０）に記載の微粒子の分離方法。
（１４）刺激応答性高分子材料が物理的刺激または化学的刺激に応答するハイドロゲルである上記（１０）〜（１３）のいずれかに記載の微粒子の分離方法。
（１５）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（１０）〜（１４）のいずれかに記載の微粒子の分離方法。
（１６）流入された微粒子が水性懸濁液である上記（１０）〜（１５）のいずれかに記載の微粒子の分離方法。
（１７）単一の分離直径を有する支柱配列から構成される上記（１０）〜（１６）のいずれかに記載の微粒子の分離方法。
（１８）複数の分離直径を有する支柱配列から構成される上記（１０）〜（１６）のいずれかに記載の微粒子の分離方法。
(1) A fine particle separation device for separating inflowing fine particles according to their characteristics.
Consists of inlet and outlet of fine particles, as well as microchannels and arranged struts;
The microchannels are formed in the gaps between the arranged struts;
The strut is made of a stimulus-responsive polymer material, and the trajectory of the fine particles flowing through the microchannel is controlled according to the degree of change in the shape of the strut due to contraction or swelling in response to a stimulus to separate the fine particles according to the characteristics of the fine particles. A fine particle separation device characterized by being configured in.
(2) The fine particle separation device according to (1) above, which separates fine particles based on their size.
(3) The fine particle separation device according to (1) above, which separates fine particles based on their shape.
(4) The fine particle separation device according to (1) above, which separates fine particles based on their hardness.
(5) The fine particle separation device according to any one of (1) to (4) above, wherein the stimulus-responsive polymer material is a hydrogel that responds to a physical stimulus or a chemical stimulus.
(6) The fine particle separation device according to any one of (1) to (5) above, wherein the fine particles are selected from polymer fine particles, biological fine particles, droplets, metal fine particles, and non-metal particles.
(7) The fine particle separation device according to any one of (1) to (6) above, wherein the inflowed fine particles is an aqueous suspension.
(8) The fine particle separation device according to any one of (1) to (7) above, which is composed of a strut arrangement having a single separation diameter.
(9) The fine particle separation device according to any one of (1) to (7) above, which is composed of a strut arrangement having a plurality of separation diameters.
(10) A method for separating fine particles, in which the fine particles to be separated are allowed to flow into the microchannel formed in the gap between the arranged columns, then passed through the microchannel, and then flow out from the microchannel.
The strut is made of a stimulus-responsive polymer material, and the trajectory of the fine particles flowing through the microchannel is controlled according to the degree of change in the shape of the strut due to contraction or swelling in response to a stimulus to separate the fine particles according to the characteristics of the fine particles. A method for separating fine particles, which is characterized by being composed of.
(11) The method for separating fine particles according to (10) above, which separates fine particles based on their size.
(12) The method for separating fine particles according to (10) above, which separates fine particles based on their shape.
(13) The method for separating fine particles according to (10) above, which separates fine particles based on their hardness.
(14) The method for separating fine particles according to any one of (10) to (13) above, wherein the stimulus-responsive polymer material is a hydrogel that responds to a physical stimulus or a chemical stimulus.
(15) The method for separating fine particles according to any one of (10) to (14), wherein the fine particles are selected from polymer fine particles, biological fine particles, droplets, metal fine particles and non-metal particles.
(16) The method for separating fine particles according to any one of (10) to (15) above, wherein the inflowed fine particles is an aqueous suspension.
(17) The method for separating fine particles according to any one of (10) to (16) above, which is composed of a strut arrangement having a single separation diameter.
(18) The method for separating fine particles according to any one of (10) to (16) above, which is composed of a strut arrangement having a plurality of separation diameters.
本発明によれば、刺激応答性高分子で支柱を作製し、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 strut is made of a stimulus-responsive polymer, and the separation diameter, which is the particle diameter that is the boundary of the change in the particle orbit, can be adjusted by changing the flow path geometric shape by stimulus control. A device and a method for separating fine particles using the device may 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、微粒子の流入口および流出口、ならびにマイクロ流路および配列された支柱からなり、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、支柱は刺激応答性高分子材料からなり、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 fine particle separation device of the present invention is a fine particle separation device for separating inflowing fine particles according to their characteristics. The particle separation device consists of an inlet and an outlet for the particles, as well as microchannels and arranged struts, the microchannels being formed in the gaps between the arranged struts. In the present invention, the strut is made of a stimulus-responsive polymer material, and the trajectory of the fine particles flowing through the microchannel is controlled according to the degree of change in the shape of the strut due to contraction or swelling in response to the stimulus, and the fine particles are separated according to the characteristics of the fine particles. It is composed of. 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 polymer 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, and ionic strength. For example, a temperature-responsive polymer material is a hydrogel, which is a polymer having a property of swelling when it contains a large amount of water, and has a function of causing swelling / contraction due to reversible hydration / dehydration due to temperature change. Has. Examples of the temperature-responsive polymer material include poly-N-isopropylacrylamide (PNIPAM), poly-N-vinylalkylamide, polyvinylalkyl ether, etc., but the phase transition temperature is around 30 ° C. Poly-N-isopropylacrylamide is suitable because it can be applied to the above and the shape of the strut can be finely controlled because it exhibits a gentle 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 electroactive polymer materials include polyacrylamide-2-methylpropanesulfonic acid (PAMPS) and the like.
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-metal particles such as metal oxides.
微粒子の粒径は、通常、１ｎｍ〜１０ｍｍ、好適には１０ｎｍ〜１ｍｍである。 The particle size of the fine particles is usually 1 nm to 10 mm, preferably 10 nm to 1 mm.
微粒子は水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、たとえば微粒子を界面活性剤水溶液に粒子濃度10２〜10１０/mＬ程度で懸濁した溶液とするのが好適である。 The microparticles are introduced in the form of an aqueous suspension from the inlet of the microparticle separation device. The aqueous suspension is preferably a solution in which fine particles are suspended in an aqueous surfactant solution at a particle concentration of about 10 2 to 10 10 / mL.
マイクロ流路は、配列された支柱間の隙間で形成される。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 microchannel is formed in the gap between the arranged columns. For example, a microchannel device is a polydimethylsiloxane (polydimethylsiloxane) for sealing a DLD channel formed between strut sequences made of the temperature-responsive polymer poly-N-isopropylacrylamide (PNIPAM) and the channel. It consists of a polydimethylsulfonic: PDMS) flow path. A DLD strut by PNIPAM is produced on a glass substrate by photolithography using, for example, a film mask having a strut diameter (D p ) of 20 μm, a strut gap (d) of 30 μm, and a strut arrangement inclination (θ) of 0.05 rad. Here, the inclination (θ) of the column arrangement is the inclination in the arrangement direction of the columns in the plan view. The PDMS flow path for sealing the DLD flow path transfers a pattern from a mold prepared using a negative photoresist "SU-8" based on the epoxy resin "EPON SU-8" to PDMS on a Si substrate. It is produced by. After aligning the DLD strut made on the glass substrate with the PDMS flow path, they are bonded together to form a device.
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 support column is not limited to a cylinder, and may be any columnar body. For the arrangement of the columns, the shape of the columns, the diameter of the columns, the gap between the columns (d), and the inclination (θ) of the arrangement of the columns are preferably set according to the characteristics of the particles 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 according to the size of the fine particles, the strut diameter is set from 10 nm to 10 mm, the gap between the strut is 1 nm to 10 mm, the diameter of the separated fine particles is larger, and the inclination of the strut arrangement is about 0.01 to 0.5 rad. The array of struts is configured to have a single separation diameter (ie, the particle diameter that borders the changes in the particle trajectory due to the geometry of the flow path), or to have multiple separation diameters. You may be.
本発明の微粒子の分離方法は、分離する微粒子を、配列された支柱間の隙間で形成されたマイクロ流路に流入させ、ついでマイクロ流路を通過させた後にマイクロ流路から流出させる微粒子の分離方法である。 In the method for separating fine particles of the present invention, the fine particles to be separated are allowed to flow into the microchannel formed in the gap between the arranged columns, and then the fine particles are separated after passing through the microchannel and then flowing out from the microchannel. The method.
ここで、支柱は刺激応答性高分子材料からなり、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されてなる。 Here, the strut is made of a stimulus-responsive polymer material, and the trajectory of the fine particles flowing through the microchannel is controlled according to the degree of change in the shape of the strut due to contraction or swelling in response to the stimulus so that the fine particles are separated according to the characteristics of the fine particles. It is composed of.
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 or hardness, and the fine particles will be separated based on their size, their shape, or 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。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 polymer 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, and ionic strength. For example, a temperature-responsive polymer material is a hydrogel, which is a polymer having a property of swelling when it contains a large amount of water, and has a function of causing swelling / contraction due to reversible hydration / dehydration due to temperature change. Has. Examples of the temperature-responsive polymer material include poly-N-isopropylacrylamide (PNIPAM), poly-N-vinylalkylamide, polyvinylalkyl ether, etc., but the phase transition temperature is around 30 ° C. Poly-N-isopropylacrylamide is suitable because it can be applied to the above and the shape of the strut can be finely controlled because it exhibits a gentle 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 electroactive polymer materials include polyacrylamide-2-methylpropanesulfonic acid (PAMPS) and the like.
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 erythrocytes and living cells, metal fine particles, and non-metal particles such as metal oxide.
微粒子の粒径は、通常、１ｎｍ〜１０ｍｍ、好適には１０ｎｍ〜１ｍｍである。 The particle size of the fine particles is usually 1 nm to 10 mm, preferably 10 nm to 1 mm.
微粒子は水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、たとえば微粒子を界面活性剤水溶液に粒子濃度10２〜10１０/mL程度で懸濁した溶液とするのが好適である。 The microparticles are introduced in the form of an aqueous suspension from the inlet of the microparticle separation device. The aqueous suspension is preferably a solution in which fine particles are suspended in an aqueous surfactant solution at a particle concentration of about 10 2 to 10 10 / mL.
マイクロ流路は、配列された支柱間の隙間で形成される。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。たとえば、PNIPAMによるＤＬＤ支柱は、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。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 microchannel is formed in the gap between the arranged columns. For example, a microchannel device is a polydimethylsiloxane (polydimethylsiloxane) for sealing a DLD channel formed between strut sequences made of the temperature-responsive polymer poly-N-isopropylacrylamide (PNIPAM) and the channel. It consists of a polydimethylsulfonic: PDMS) flow path. For example, a PNIPAM DLD strut is made on a glass substrate by photolithography using a film mask with strut diameter (D p ) 20 μm, strut gap (d) 30 μm, and strut array tilt (θ) 0.05 rad. .. The PDMS flow path for sealing the DLD flow path transfers a pattern from a mold prepared using a negative photoresist "SU-8" based on the epoxy resin "EPON SU-8" to PDMS on a Si substrate. It is produced by. After aligning the DLD strut made on the glass substrate with the PDMS flow path, they are bonded together to form a device.
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 support column is not limited to a cylinder, and may be any columnar body. For the arrangement of the columns, the shape of the columns, the diameter of the columns, the gap between the columns (d), and the inclination (θ) of the arrangement of the columns are preferably set according to the characteristics of the particles 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 according to the size of the fine particles, the strut diameter is set from 10 nm to 10 mm, the gap between the strut is 1 nm to 10 mm, the diameter of the separated fine particles is larger, and the inclination of the strut arrangement is about 0.01 to 0.5 rad. The array of struts may be configured to have a single separation diameter or may be configured to have multiple separation diameters.
後述するように、流入口からデバイスに導入された粒子は、ＤＬＤ流路へと流入し、ＤＬＤ流路の温度が24℃（室温）の場合は，大きい微粒子のみ支柱配列の傾きに沿う軌道(置換モード：displacement mode)を取り、小さい微粒子は流れと同一方向に進む軌道(ジグザグモード：zigzag mode)を取る。一方、ＤＬＤ流路の温度を35℃に変化させた場合は、両方の微粒子は流れと同一方向に進む軌道であるジグザグモードを取る。このようにＤＬＤ流路の温度制御を行うことで微粒子分離のオン・オフ（ON・OFF）制御が可能である。 As will be described later, the particles introduced into the device from the inflow port flow into the DLD flow path, and when the temperature of the DLD flow path is 24 ° C. (room temperature), only large fine particles follow the inclination of the column arrangement ( It takes a displacement mode), and small particles take an orbit (zigzag mode) that travels in the same direction as the flow. On the other hand, when the temperature of the DLD flow path is changed to 35 ° C., both fine particles take a zigzag mode, which is an orbit traveling in the same direction as the flow. By controlling the temperature of the DLD flow path in this way, it is possible to control the separation of fine particles on / off (ON / OFF).
支柱を形成する刺激応答性高分子材料として、光、電場もしくは磁場、またはｐＨ、溶液組成もしくはイオン強度に応答するハイドロゲルを用いる場合も、温度応答性ハイドロゲルの場合の温度と同様にして光、電場もしくは磁場、またはｐＨ、溶液組成もしくはイオン強度の制御を行うことにより微粒子分離のオン・オフ（ON・OFF）制御が可能である。 When a hydrogel that responds to light, electric or magnetic fields, or pH, solution composition, or ionic strength is used as the stimulus-responsive polymer material that forms the struts, light is applied in the same manner as in the case of the temperature-responsive hydrogel. , Electric field or magnetic field, or pH, solution composition or ionic strength can be controlled to turn on / off (ON / OFF) fine particle separation.
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, a case where separation by particle shape is performed will be described. For example, in the case of disk-shaped particles such as red blood cells (thickness 2 μm, diameter 6 μm), the apparent diameter of the particles in the DLD flow path changes depending on the flow posture of the particles, resulting in separation behavior (Displacement mode, Zigzag mode). It is known to have an impact. Therefore, we used a separation method (JP Beech et al., Lab Chip, 12, 1048-1051, 2012) in which the posture of erythrocytes (diameter direction and thickness direction of the disk) was controlled with respect to the support of DLD, and rotation of erythrocytes. 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. 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。 Further, in the present invention, a case where separation is performed based on the hardness of the particles will be described. Since shear stress acts on the particles in the DLD flow path, the degree of deformation of the particles differs according to the difference in hardness of the particles. For example, even if the particles have the same diameter in the stationary state, the particles that are more easily deformed have a smaller apparent diameter in the DLD flow path than the particles that are not deformed. That is, even if the particles have the same size, it is possible to separate the particles by utilizing the difference in the degree of deformation of the particles. For example, in the case of erythrocytes, since the hardness of normal erythrocytes and that of malaria-infected erythrocytes are different, it is expected that disease can be detected by separating erythrocytes using the hardness as an index (T. Krueger et al., Biomicrofluidics, 8, 054114, 2014). By applying the separation method of the present invention to these, separation can be performed according to the hardness of the particles.
以下、実施例により本発明をさらに詳細に説明する。
実施例１
マイクロ流路デバイスは、温度応答性高分子の一種であるポリ-N-イソプロピルアクリルアミド（poly-N-isopropylacrylamide：PNIPAM)のフォトレジストで作製したＤＬＤ支柱及び，それ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、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した。ＤＬＤ支柱を密封するための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した。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ＤＬＤマイクロ流路デバイスを形成した（図１）。図１は、温度応答性高分子を用いたＤＬＤマイクロ流路デバイスの概要図を示す。
Hereinafter, the present invention will be described in more detail with reference to Examples.
Example 1
The microchannel device is a DLD strut made of a photoresist of poly-N-isopropylacrylamide (PNIPAM), which is a kind of temperature-responsive polymer, and polydimethylsiloxane (polydimethylsiloxane) for sealing it. It consists of a polydimethylsulfonic (PDMS) flow path. The DLD strut by PNIPAM was prepared on a glass substrate by photolithography using a film mask having a strut diameter (D p ) of 20 μm, a strut gap (d) of 30 μm, and a strut arrangement inclination (θ) of 0.05 rad). The PDMS flow path for sealing the DLD strut is to transfer the pattern from a mold prepared using a negative photoresist "SU-8" based on the epoxy resin "EPON SU-8" to PDMS on a Si substrate. Made by After aligning the DLD column prepared on the glass substrate with the PDMS flow path, they were bonded together to form a DLD microchannel device (FIG. 1). FIG. 1 shows a schematic view of a DLD microchannel device using a temperature responsive polymer.
導入試料には、直径6μmと3μmのポリスチレンビーズ(Polysciences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1.0×106/mLで懸濁した溶液と、粒子を懸濁していない0.1v/v%のTween（登録商標）20水溶液(バッファー)を用意した。導入試料を入口のリザーバに滴下した後、シリンジポンプ(KD Scientific社製, KDS200)にて出口を陰圧にすることで送液した。 For the introduced sample, polystyrene beads (manufactured by Polysciences) having a diameter of 6 μm and 3 μm were suspended in a 0.1 v / v% aqueous solution of a surfactant Tween® 20 (Sigma) at a particle concentration of 1.0 × 10 6 / mL. A solution and a 0.1 v / v% Tween® 20 aqueous solution (buffer) in which the particles were not suspended were prepared. After the introduced sample was dropped into the reservoir at the inlet, the liquid was sent by making the outlet negative pressure with a syringe pump (KD Scientific, KDS200).
PNIPAMで作製したＤＬＤ支柱の温度変化(10〜50℃)に伴う直径変化を測定すると、支柱の直径が16.3μm 〜39.9μmの範囲で変化する様子が観察された(図２、３)。これに伴い，ＤＬＤの分離直径が3.4μm 〜11.2μmの範囲で可変できることが確認された(図３)。図２は、高温時（35℃）と低温時（24℃）のPNIPAMの支柱を示す図であり、図３は温度変化に伴う支柱直径と分離直径Dcの変化を示す図である。支柱の加熱と冷却にはペルチェ素子（センサーコントロールズ社製，CHP−22HS）を用いた。 When the diameter change of the DLD strut made by PNIPAM with the temperature change (10 to 50 ° C) was measured, it was observed that the diameter of the strut changed in the range of 16.3 μm to 39.9 μm (Figs. 2 and 3). Along with this, it was confirmed that the separation diameter of DLD can be changed in the range of 3.4 μm to 11.2 μm (Fig. 3). FIG. 2 is a diagram showing PNIPAM columns at high temperature (35 ° C.) and low temperature (24 ° C.), and FIG. 3 is a diagram showing changes in column diameter and separation diameter D c with temperature change. A Peltier element (CHP-22HS, manufactured by Sensor Controls) was used to heat and cool the columns.
デバイスに導入された粒子は、配列した支柱の５〜１０列目（gap number 5−10）の範囲からＤＬＤ流路へと流入した（図４：微粒子のＤＬＤ流路への流入位置を示す図）。図５は、高温時（35℃）と低温時（24℃）における６μmと３μmのポリスチレンビーズの流入位置と流出位置の関係を示す図である。
ＤＬＤ流路の温度が２４℃（室温）の場合は，直径６μmのビーズのみ支柱配列の傾きに沿う軌道(置換モード：displacement mode)を取り、直径３μmのビーズは流れと同一方向に進む軌道(ジグザグモード：zigzag mode)を取る様子が確認された(図５（a)）。一方、顕微鏡用温度管理ステージ（東海ヒット社製）を用いてＤＬＤ流路の温度を35℃に変化させた場合は、直径６μmと３μmのビーズともに流体の流れと同一方向に進む軌道であるジグザグモードを取る様子が確認された(図５（b)）。ＤＬＤ流路の温度制御を行うことでビーズ分離のオン・オフ（ON・OFF）制御が可能であることを確認した。
The particles introduced into the device flowed into the DLD flow path from the range of the 5th to 10th rows (gap number 5-10) of the arranged columns (Fig. 4: Figure showing the inflow position of the fine particles into the DLD flow path). ). FIG. 5 is a diagram showing the relationship between the inflow position and the outflow position of 6 μm and 3 μm polystyrene beads at high temperature (35 ° C.) and low temperature (24 ° C.).
When the temperature of the DLD flow path is 24 ° C. (room temperature), only beads with a diameter of 6 μm take an orbit (displacement mode) along the inclination of the strut arrangement, and beads with a diameter of 3 μm take an orbit in the same direction as the flow (replacement mode). It was confirmed that the zigzag mode) was taken (Fig. 5 (a)). On the other hand, when the temperature of the DLD flow path is changed to 35 ° C using a temperature control stage for microscopes (manufactured by Tokai Hit Co., Ltd.), beads with diameters of 6 μm and 3 μm both travel in the same direction as the fluid flow in a zigzag pattern. It was confirmed that the mode was taken (Fig. 5 (b)). It was confirmed that on / off (ON / OFF) control of bead separation is possible by controlling the temperature of the DLD flow path.
本発明によれば、分離直径を調節可能な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, it is possible to provide a fine particle separation device having an adjustable separation diameter and a fine particle separation method using the same.
Claims (18)
微粒子の流入口および流出口、ならびにマイクロ流路および配列された支柱からなり；
該マイクロ流路は、該配列された支柱間の隙間で形成され；
該支柱は刺激応答性高分子材料からなり、刺激に対する収縮または膨潤による該支柱形状の変化の度合いにより、該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ことを特徴とする微粒子分離デバイス。 It is a fine particle separation device for separating the inflowing fine particles according to their characteristics.
Consists of inlet and outlet of fine particles, as well as microchannels and arranged struts;
The microchannels are formed in the gaps between the arranged struts;
The strut is made of a stimulus-responsive polymer material, and the trajectory of the fine particles flowing through the microchannel is controlled according to the degree of change in the shape of the strut due to contraction or swelling in response to a stimulus to separate the fine particles according to the characteristics of the fine particles. A fine particle separation device characterized by being configured in.
該支柱は刺激応答性高分子材料からなり、刺激に対する収縮または膨潤による該支柱形状の変化の度合いにより、該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ことを特徴とする微粒子の分離方法。 This is a method for separating fine particles, in which fine particles to be separated are allowed to flow into a microchannel formed in a gap between arranged columns, then passed through the microchannel, and then flow out from the microchannel.
The strut is made of a stimulus-responsive polymer material, and the trajectory of the fine particles flowing through the microchannel is controlled according to the degree of change in the shape of the strut due to contraction or swelling in response to a stimulus to separate the fine particles according to the characteristics of the fine particles. A method for separating fine particles, which is characterized by being composed of.
から選ばれる請求項１０〜１４のいずれか１項に記載の微粒子の分離方法。 The method for separating fine particles according to any one of claims 10 to 14, wherein the fine particles are selected from polymer fine particles, biological fine particles, droplets, metal fine particles, and non-metal particles.
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