JP2006158991A - Centrifugal chip and centrifugal separation method - Google Patents

Centrifugal chip and centrifugal separation method Download PDF

Info

Publication number
JP2006158991A
JP2006158991A JP2004349609A JP2004349609A JP2006158991A JP 2006158991 A JP2006158991 A JP 2006158991A JP 2004349609 A JP2004349609 A JP 2004349609A JP 2004349609 A JP2004349609 A JP 2004349609A JP 2006158991 A JP2006158991 A JP 2006158991A
Authority
JP
Japan
Prior art keywords
separation chamber
specific gravity
solution
reservoir
centrifuge
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
JP2004349609A
Other languages
Japanese (ja)
Other versions
JP4485926B2 (en
Inventor
Kazunobu Okano
和宣 岡野
Kenji Yasuda
賢二 安田
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
On-chip Cellomics Consortium
Original Assignee
On-chip Cellomics Consortium
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by On-chip Cellomics Consortium filed Critical On-chip Cellomics Consortium
Priority to JP2004349609A priority Critical patent/JP4485926B2/en
Priority to EP07013611A priority patent/EP1901067A3/en
Priority to EP05016714A priority patent/EP1626278A3/en
Priority to US11/195,662 priority patent/US7569354B2/en
Publication of JP2006158991A publication Critical patent/JP2006158991A/en
Priority to US12/143,181 priority patent/US20090042739A1/en
Priority to US12/143,156 priority patent/US20090042200A1/en
Priority to US12/471,853 priority patent/US20100021933A1/en
Priority to US12/472,037 priority patent/US20090325215A1/en
Priority to US12/472,010 priority patent/US20100016569A1/en
Priority to US12/471,993 priority patent/US20100018862A1/en
Priority to US12/471,947 priority patent/US20100016568A1/en
Application granted granted Critical
Publication of JP4485926B2 publication Critical patent/JP4485926B2/en
Priority to US13/755,079 priority patent/US20130252848A1/en
Priority to US14/640,471 priority patent/US20150231635A1/en
Expired - Fee Related legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Landscapes

  • Centrifugal Separators (AREA)

Abstract

<P>PROBLEM TO BE SOLVED: To provide a centrifugal chip and a centrifugal separation method by which a cell or granulated powder is separated from a trace quantity of a sample liquid by centrifugal separation. <P>SOLUTION: The chip for centrifugal separation is attached to a rotary plate rotating around a rotary shaft. The chip for centrifugal separation is formed from flow passages for supplying a plurality of solutions each having different specific gravity onto a substrate, a separation chamber functioning as a separation area with the flow passages collected and a plurality of flow passages branched from the separation chamber. A reservoir is arranged at each of inlets and outlets of the respective flow passages and the plurality of the solutions each having different specific gravity are supplied to the flow passages. The distance from the rotary shaft to the respective reservoirs of the inlet side connected to the flow passages is made equal and the distance from the rotary shaft to a liquid level of the respective reservoirs of the outlet side connected to the plurality of the flow passages branched from the separation chamber is made equal. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

本発明は遠心を用いて細胞などを分離する技術に関する。   The present invention relates to a technique for separating cells and the like using centrifugation.

細胞などの分離を行うには比重の異なる溶液を重層して遠心することで、細胞をその比重で分離することが生化学分野では一般的に行われている。   In order to separate cells and the like, it is a common practice in the biochemical field to separate cells by their specific gravity by layering and centrifuging solutions having different specific gravity.

他方、微細加工技術を用いることで従来の生化学レベル・細胞レベルにおける分析・反応を小型集積・高速化する様々なマイクロ流体チップが開発されている。これらのチップの特徴の一つは、マイクロメートルの微小な流路に流体を流すことができ、そのために層流の形成が容易になることである。従って２つの層流は壁がなくても境界を維持して互いに混ざり合うことは無い。   On the other hand, various microfluidic chips have been developed that use microfabrication technology to miniaturize and accelerate conventional analysis and reaction at the biochemical level and cell level. One of the features of these chips is that a fluid can flow through a minute flow path of micrometer, which facilitates the formation of a laminar flow. Therefore, even if there are no walls, the two laminar flows maintain their boundaries and do not mix with each other.

一般に遠心を用いる分離では、チューブ内にパーコールなどの比重の異なる溶液を積層して用いる関係上、あまり小さな体積の分離には適していない。一般にはｍＬオーダーのチューブを用いる。しかしながら、近年の生化学の発展は、分析機器の高感度化が進み、また、検体を同時に検査するマルチプルアッセイとハイスループットスクリーニングが主流となり、このため、試料用液の微量化が進んでいる。最も微量化が遅れているのが遠心を用いる分離分野である。   In general, separation using centrifugation is not suitable for separation of a very small volume because a solution having different specific gravity such as Percoll is laminated in a tube. Generally, mL order tubes are used. However, the recent development of biochemistry has led to higher sensitivity of analytical instruments, and multiple assays and high-throughput screening that simultaneously test specimens have become mainstream. For this reason, the amount of sample liquid has been reduced. The separation is most delayed in the separation field using centrifugation.

本発明では、極微量の試料用液からの遠心分離による細胞や顆粒の分離を可能にする遠心チップと遠心分離方法を提供する。   The present invention provides a centrifuge chip and a centrifuge method that enable separation of cells and granules by centrifugation from a very small amount of sample liquid.

回転軸を中心に回転する回転板に遠心分離用チップを取り付ける。遠心分離用チップは、基板上に比重の異なる複数の溶液を供給する流路と、これらの流路が集合して分離領域として機能するひとつの分離チャンバーと、前記分離チャンバーから分岐する複数の流路から形成されるものとする。すべての流路の入口と出口にリザーバを配し、前記流路に比重の異なる複数の溶液を供給するとともに、流路に接合するすべての入口のリザーバの回転軸からの距離が等しくかつ、及び前記分離チャンバーから分岐する複数の流路に接合する出口側のリザーバの液面がそれぞれ回転軸からの距離を等しくする。   A centrifuge chip is attached to a rotating plate that rotates around a rotating shaft. The centrifuge chip has a flow path for supplying a plurality of solutions having different specific gravities on a substrate, a single separation chamber in which these flow paths gather to function as a separation region, and a plurality of flow branches from the separation chamber. It shall be formed from the road. Reservoirs are arranged at the inlets and outlets of all channels, a plurality of solutions having different specific gravities are supplied to the channels, and the distances from the rotation axes of the reservoirs of all the inlets joined to the channels are equal, and The liquid levels of the outlet-side reservoirs joined to the plurality of flow paths branched from the separation chamber have the same distance from the rotation axis.

（実施例１）
図１は本発明に係わる遠心分離装置の概要を示す構成図である。１は回転板であり、表面に本発明に係わる遠心チップを実装するスペース２が形成されている。スペース２は遠心チップが容易に着脱可能な構成とされる。回転板１はモータ３により、所定の速度で水平に回転させられる。４は光源であり、回転板１に実装された遠心チップの分離部を照射する。５はレンズであり、遠心チップの分離部を透過した光を集光する。レンズ５で集光された光はミラー６で反射されて、高速カメラ７で撮像される。８はパソコンであり、高速カメラ７撮像された遠心チップの分離部を分析し、モータ３に対する速度信号を計算して、モータ３の速度を制御する。 Example 1
FIG. 1 is a configuration diagram showing an outline of a centrifugal separator according to the present invention. Reference numeral 1 denotes a rotating plate, on which a space 2 for mounting a centrifugal tip according to the present invention is formed. The space 2 is configured such that the centrifugal tip can be easily attached and detached. The rotating plate 1 is rotated horizontally by a motor 3 at a predetermined speed. Reference numeral 4 denotes a light source that irradiates the separation portion of the centrifugal chip mounted on the rotating plate 1. Reference numeral 5 denotes a lens that collects the light transmitted through the separation portion of the centrifugal tip. The light collected by the lens 5 is reflected by the mirror 6 and picked up by the high-speed camera 7. 8 is a personal computer, which analyzes the separation part of the centrifuge chip imaged by the high-speed camera 7, calculates a speed signal for the motor 3, and controls the speed of the motor 3.

実施例１によれば、遠心中にカメラ７で試料の分離状態を観察しながら分離ができる。分離の度合いをパソコン８が備えるモニター（図示しない）を見ながら、あるいは、パソコン８の備えるプログラムでモータ３の回転速度を制御し、最適な分離を行うことができる。観測の一例を示すと以下のようである。例えば、モータ３を１８００ｒｐｍで回転させ、光源４、レンズ５、カメラ７よりなる光学系で、スペース２に実装された遠心チップの分離部の画像観察を行う。ここで、遠心チップが光源を横切るときの１秒あたりの数が高速カメラ１３の画像取り込みレートの倍数になるように回転数を制御する。これにより、回転している遠心チップの画像を静止状態のように撮影することができる。たとえば３０フレーム／秒のカメラを用いて、撮像すると、３０×Ｎ／秒（Ｎ：整数および整数の分数）の回転数で遠心を行えばよい。よって、上記１８００ｒｐｍで遠心を行えば、ちょうど静止したような画像が得られる。複数のチップを同時に撮像する場合は、取り込んだ画像を遠心チップごとの画像にパソコン８上で切り分けることで、各チップの画像を得ることができる。   According to Example 1, separation can be performed while observing the separation state of the sample with the camera 7 during centrifugation. Optimum separation can be performed by observing the degree of separation on a monitor (not shown) provided in the personal computer 8 or by controlling the rotational speed of the motor 3 with a program provided in the personal computer 8. An example of observation is as follows. For example, the motor 3 is rotated at 1800 rpm, and the image observation of the separation part of the centrifuge chip mounted in the space 2 is performed by an optical system including the light source 4, the lens 5, and the camera 7. Here, the number of rotations is controlled so that the number per second when the centrifuge chip crosses the light source is a multiple of the image capturing rate of the high-speed camera 13. Thereby, the image of the rotating centrifuge tip can be taken like a stationary state. For example, when imaging is performed using a camera of 30 frames / second, centrifugation may be performed at a rotation speed of 30 × N / second (N: an integer and an integer fraction). Therefore, if the centrifugation is performed at 1800 rpm, an image that seems to be still can be obtained. When imaging a plurality of chips simultaneously, an image of each chip can be obtained by dividing the captured image into images for each centrifugal chip on the personal computer 8.

（実施例２）
図２は、実施例１の遠心分離装置に適用するのに好適な遠心チップ１００の構成を模式的に示す平面図である。図３は、図２に示した遠心チップ１００のリザーバ部分に着目して断面にして示した構成を模式的に示す斜視図である。 (Example 2)
FIG. 2 is a plan view schematically showing a configuration of a centrifuge chip 100 suitable for application to the centrifuge of the first embodiment. FIG. 3 is a perspective view schematically showing a configuration shown in a cross section focusing on the reservoir portion of the centrifugal tip 100 shown in FIG.

１１、１２、１３は複数の独立した比重の異なる溶液を供給する流路であり、一端が、それぞれ、リザーバ２１−２３に接続され、他端が分離チャンバー１７に接続される。分離チャンバー１７の他端には、排出用流路１４，１５，１６が接続され、排出用流路１４，１５，１６の他端には、それぞれ、リザーバ２４−２６が接続される。各流路に連通したリザーバ２１−２６のうち、リザーバ２３には試料を含む最も低比重の溶液、２２、２１はその順に比重の高い溶液が入っている。この状態でモータ３を回転させ、遠心をかけると、分離チャンバー１７には、遠心の加速度のかかる方から順に３１，３２，３３のように、比重の高い順の溶液の層ができる。試料の成分の内、最も比重の高い溶液層３１の比重より高い比重の成分は溶液層３１の中に入り、その次に比重が低いが、最も低い比重より比重の高い成分は溶液層３２の中に入り、最も低い比重より比重の低い成分は溶液層３３の中に入る。それぞれの成分は、対応する層に対応する排出用流路１４，１５，１６を介して、リザーバ２４−２６に回収される。ここで、３０は、遠心初期の遠心の加速度の方向に見た液面高さの差である。この液面高さの差により、各溶液は層流状態で矢印の方向３５に流れ、分離チャンバー１７で層流の中に分離された成分が排出用流路１４，１５，１６に流出してくる。ここで重要なのは、最も比重の低い溶液でリザーバ２１−２３の液面高さおよび２４−２６の液面高さが揃うようにすることで各流路内の液の流れの乱れを防ぐことである。   Reference numerals 11, 12, and 13 are flow paths that supply a plurality of independent solutions having different specific gravities. One end of each is connected to the reservoir 21-23 and the other end is connected to the separation chamber 17. The other ends of the separation chamber 17 are connected to the discharge channels 14, 15, and 16, and the other ends of the discharge channels 14, 15, and 16 are connected to the reservoirs 24 to 26, respectively. Of the reservoirs 21-26 communicating with each flow path, the reservoir 23 contains a solution having the lowest specific gravity including the sample, and 22 and 21 contain solutions having a higher specific gravity in that order. When the motor 3 is rotated in this state and centrifuged, the solution chambers in order of decreasing specific gravity are formed in the separation chamber 17 in the order of 31, 32, 33 from the direction where the centrifugal acceleration is applied. Among the components of the sample, components having a specific gravity higher than the specific gravity of the solution layer 31 having the highest specific gravity enter the solution layer 31, and then the specific gravity is low, but components having a specific gravity higher than the lowest specific gravity are those of the solution layer 32. A component having a specific gravity lower than the lowest specific gravity enters the solution layer 33. Each component is recovered in the reservoirs 24-26 via the discharge channels 14, 15, 16 corresponding to the corresponding layers. Here, 30 is the difference in liquid level seen in the direction of the acceleration of the centrifuge at the initial stage of the centrifuge. Due to the difference in liquid level, each solution flows in the direction indicated by the arrow 35 in a laminar flow state, and the components separated in the laminar flow in the separation chamber 17 flow out into the discharge channels 14, 15, 16. come. What is important here is that the liquid level of the reservoir 21-23 and the liquid level of 24-26 are aligned with the solution having the lowest specific gravity, thereby preventing disturbance of the liquid flow in each channel. is there.

この遠心チップ１００の構成は以下のようである。流路１１−１６および分離チャンバー１７は、例えば、ＰＤＭＳの基板の片面に鋳型で形成され、リザーバ２１−２６は、当該基板の他面にガラス加工により形成されて貼り付けられる。前記流路１１−１６とリザーバ２１−２６とは、基板を貫通する穴により連通するものとされる。チップの外寸はほぼ３０×３０ｍｍとする。図では扇形をしているが、回転板１のスペース２に装填できる形であれば良くチップの外形の制約は無い。まず、基板に流路１１−１６および分離チャンバー１７を形成するための鋳型について説明する。   The configuration of the centrifugal chip 100 is as follows. For example, the flow path 11-16 and the separation chamber 17 are formed of a mold on one surface of a PDMS substrate, and the reservoirs 21-26 are formed and attached to the other surface of the substrate by glass processing. The flow path 11-16 and the reservoir 21-26 communicate with each other through a hole penetrating the substrate. The outer dimension of the chip is approximately 30 × 30 mm. Although it is fan-shaped in the figure, there is no restriction on the outer shape of the chip as long as it can be loaded into the space 2 of the rotating plate 1. First, a mold for forming the flow path 11-16 and the separation chamber 17 on the substrate will be described.

鋳型は量産のためのものである。洗浄したガラス基板あるいはシリコン基板を酸素プラズマで５分間アッシングし、表面に付着している有機物を除去する。光感光性レジストであるＳＵ８−２５をスピンコートする。スピンコートは５００ｒｐｍで１０秒間、続いて２０００ｒｐｍ３０秒間で良好な結果を得ている。ＳＵ８−２５をスピンコートでガラス基板上に一様に展開したものを、７５℃で１分間続いて１００℃５分間ホットプレート上でプレベークして、結果として、２５μｍの厚さのＳＵ８−２５層を形成する。図２に示す流路１１−１６と分離チャンバー１７に対応する形を透かしにしたクロムマスクを用いて１５秒間紫外線露光する。７５℃で３分間、続いて１００℃で５分間ホットプレートでベークする。ＳＵ−８デベロッパーを用い、所定のマニュアルに従い現像する。イソプロパノールで未重合部分を除去し、１６０℃で３０分間ベークして鋳型を得る。結果として、ガラス基板あるいはシリコン基板上に、高さが２５μｍの流路１１−１６と分離チャンバー１７の突起の形ができる。ここで、流路１１−１６の幅は５０μｍとし、分離チャンバー１７の幅（入出流路の取り付け位置間）は４ｍｍ、分離チャンバー１７の遠心方向の幅は１５０μｍ（＝５０μｍ×３）とする。   The mold is for mass production. The cleaned glass substrate or silicon substrate is ashed with oxygen plasma for 5 minutes to remove organic substances adhering to the surface. A photo resist, SU8-25, is spin-coated. The spin coat has good results at 500 rpm for 10 seconds, followed by 2000 rpm for 30 seconds. A SU8-25 uniformly spread on a glass substrate by spin coating is pre-baked on a hot plate at 75 ° C. for 1 minute and then at 100 ° C. for 5 minutes, resulting in a 25 μm thick SU8-25 layer. Form. UV exposure is performed for 15 seconds using a chrome mask with a watermark corresponding to the flow path 11-16 and the separation chamber 17 shown in FIG. Bake on hot plate at 75 ° C. for 3 minutes followed by 100 ° C. for 5 minutes. Using a SU-8 developer, develop according to a predetermined manual. Unpolymerized portions are removed with isopropanol, and baked at 160 ° C. for 30 minutes to obtain a mold. As a result, the projections of the flow path 11-16 and the separation chamber 17 having a height of 25 μm are formed on the glass substrate or the silicon substrate. Here, the width of the flow path 11-16 is set to 50 μm, the width of the separation chamber 17 (between the positions where the input and output flow paths are attached) is 4 mm, and the width of the separation chamber 17 in the centrifugal direction is 150 μm (= 50 μm × 3).

次に、この鋳型を用いた遠心チップの作成法を述べる。ガラス基板あるいはシリコン基板上に突起の高さが２５μｍの流路１１−１６と分離チャンバー１７の形を持つ鋳型の周辺を、１．５ｍｍの高さの壁で囲う。この壁の内側のサイズは、チップの外寸の３０×３０ｍｍとする。この壁の内側に、マニュアルどおりに調製したＰＤＭＳモノマー混合液を脱気し、７５℃３０分間空気恒温槽で加熱重合させ、ＰＤＭＳを重合させる。この際、ＰＤＭＳモノマー混合液層の厚さが均一になるように、壁の上面にシリコンウエハーを載せてはさみつけるのが良い。なお、壁は、単にＰＤＭＳモノマー混合液を保持するためだけであるから、ガラス板でも、シリコン板でも良い。重合したＰＤＭＳから上記壁、シリコンウエハーおよび鋳型を剥がすと、ＰＤＭＳの一面に流路１１−１６と分離チャンバー１７の部分が凹んだ基板４１が得られる。図３では、流路１１−１３が基板４１の一面に形成されている状態を示す。   Next, a method for producing a centrifugal tip using this template will be described. The periphery of the mold having the shape of the flow path 11-16 having a protrusion height of 25 μm and the separation chamber 17 on the glass substrate or silicon substrate is surrounded by a wall having a height of 1.5 mm. The inner size of the wall is 30 × 30 mm which is the outer dimension of the chip. Inside this wall, the PDMS monomer mixed solution prepared according to the manual is degassed and heated and polymerized in an air thermostat at 75 ° C. for 30 minutes to polymerize PDMS. At this time, a silicon wafer is preferably placed on the upper surface of the wall and sandwiched so that the thickness of the PDMS monomer mixture layer is uniform. In addition, since the wall is only for holding the PDMS monomer mixture, it may be a glass plate or a silicon plate. When the wall, silicon wafer, and mold are peeled from the polymerized PDMS, a substrate 41 having a channel 11-16 and a separation chamber 17 portion recessed on one surface of the PDMS is obtained. FIG. 3 shows a state where the flow path 11-13 is formed on one surface of the substrate 41.

次に、基板４１の流路とリザーバとの接続位置にポンチで２ｍｍφの貫通孔４３、４４、４５を開ける。次いで、基板４１（ＰＤＭＳ）の流路１１−１６と分離チャンバー１７が形成された面に、別途用意した３０ｍｍ角の１ｍｍ厚のガラス板４２を酸素プラズマで１０秒間アッシングし両者を貼り付ける。これで基板４１の一面の流路１１−１６と分離チャンバー１７対応の凹部がガラス板４２で塞がれて流路１１−１６と分離チャンバー１７が完成する。   Next, through holes 43, 44 and 45 of 2 mmφ are opened with punches at the connection positions between the flow path of the substrate 41 and the reservoir. Next, a separately prepared 30 mm square 1 mm thick glass plate 42 is ashed with oxygen plasma for 10 seconds on the surface of the substrate 41 (PDMS) where the flow path 11-16 and the separation chamber 17 are formed, and both are attached. As a result, the channel 11-16 on one surface of the substrate 41 and the recess corresponding to the separation chamber 17 are closed by the glass plate 42, and the channel 11-16 and the separation chamber 17 are completed.

次に、基板４１の流路と分離チャンバーの形成された面と反対の面に、ガラス板を張り合わせて形成したリザーバ２１−２３を貼り付ける。リザーバとＰＤＭＳは共有結合で貼り付けることができる。この際、基板４１に形成した貫通孔４３，４４，４５で、それぞれの流路１１−１３とリザーバ２１−２３とが連通するようになされる。図３では、流路１１−１３が基板４１の貫通孔４３，４４，４５を介して、基板４１の他面に貼り付けられたリザーバ２１−２３と連通している状態を示す。図３は、基板４１の両面が使用されていることが分かりやすいように、一部についての断面で示したので、リザーバ２４−２６と流路１４−１６の関係が表れていないが、図３と同様であることは容易に分かる。また、分離チャンバー１７が、流路１１−１３と同様に、基板４１の片面に形成されていることも容易に分かる。なお、リザーバ２１−２３はガラス板で形成したから、図３では、これらは、厚みを持っている形で表示されるべきであるが、煩雑になるので、輪郭の線の表示にとどめた。さらに、ここでは、リザーバ２１−２３はガラス板を張り合わせて形成するものとしたが、所定の厚さのガラス板にリザーバ２１−２３をモールドした形で形成して、この板を貼り付けるものとしても良い。   Next, a reservoir 21-23 formed by bonding a glass plate to the surface opposite to the surface where the flow path of the substrate 41 and the separation chamber are formed is attached. The reservoir and PDMS can be covalently attached. At this time, the flow paths 11-13 and the reservoirs 21-23 are communicated with each other through the through holes 43, 44, 45 formed in the substrate 41. FIG. 3 shows a state in which the flow path 11-13 communicates with the reservoir 21-23 attached to the other surface of the substrate 41 through the through holes 43, 44 and 45 of the substrate 41. 3 shows a part of the cross section so that it is easy to understand that both surfaces of the substrate 41 are used, the relationship between the reservoir 24-26 and the flow path 14-16 does not appear. It is easy to see that Further, it can be easily seen that the separation chamber 17 is formed on one side of the substrate 41 in the same manner as the flow path 11-13. Since the reservoirs 21 to 23 are formed of a glass plate, in FIG. 3, these should be displayed in a form having a thickness. However, since they are complicated, only the outline lines are displayed. Further, here, the reservoir 21-23 is formed by bonding glass plates, but the reservoir 21-23 is formed by molding a glass plate of a predetermined thickness, and this plate is attached. Also good.

リザーバ２１−２３について見ると、リザーバ２１−２３の回転中心側の上面には、溶液注入用の穴４６，４７，４８が設けられるとともに、リザーバ２１−２３は基本的には分離壁５１，５２で独立したものとされるが、この分離壁５１，５２は、溶液注入用の穴４６，４７，４８の近辺で上部が切り欠かれている。   Looking at the reservoir 21-23, holes 46, 47, and 48 for injecting the solution are provided on the upper surface of the reservoir 21-23 on the rotation center side, and the reservoir 21-23 basically includes the separation walls 51 and 52. However, the separation walls 51 and 52 are notched at the upper part in the vicinity of the solution injection holes 46, 47 and 48.

図３を参照しながら、図２に示したような状態になるように、遠心チップ１００のリザーバ２１−２３に溶液を入れる方法について説明する。まず、溶液注入用の穴４８から、最も比重の低い溶液をリザーバ２３に注入する。このとき、リザーバ２１−２３の分離壁５１，５２の切り欠きから、他のリザーバ２２、２１にも流入するように多量の溶液をリザーバ２３に注入する。リザーバ２１−２３が全て最も比重の低い溶液で満たされた状態で遠心をかけると流路１１−１６の全て、分離チャンバー１７に最も比重の低い溶液が行き渡る。出口側のリザーバ２４−２６の液面が共通になったところで遠心を停止する。この状態で、リザーバ２２，２１に溶液注入用の穴４７，４６から、それぞれ、より比重の高い溶液をそれぞれリザーバの容積と同程度の量だけ注入する。その結果、リザーバ２２，２１では、最も比重の低い溶液が溢れ出して、より比重の高い溶液に置換される。さらに、リザーバ２３には、分離対象を含む試料溶液を注入する。この段階が、図２に示す状態である。この状態で遠心チップ１００を回転板１のスペース２に実装してモータ３により遠心をかけると、図２に示すように、比重に応じた溶液層ができ、試料溶液中の成分が、その比重に応じて溶液層に分離される。   With reference to FIG. 3, a method for putting the solution into the reservoirs 21 to 23 of the centrifuge chip 100 so as to be in the state shown in FIG. First, the solution having the lowest specific gravity is injected into the reservoir 23 from the solution injection hole 48. At this time, a large amount of solution is injected into the reservoir 23 so as to flow into the other reservoirs 22 and 21 from the cutouts of the separation walls 51 and 52 of the reservoir 21-23. When centrifugation is performed in a state where all of the reservoirs 21 to 23 are filled with the solution having the lowest specific gravity, the solution having the lowest specific gravity is distributed to all of the flow paths 11 to 16 and the separation chamber 17. Centrifugation is stopped when the liquid level of the reservoirs 24-26 on the outlet side becomes common. In this state, the solutions having higher specific gravity are respectively injected into the reservoirs 22 and 21 through the solution injection holes 47 and 46 by the same amount as the volume of the reservoirs. As a result, in the reservoirs 22 and 21, the solution having the lowest specific gravity overflows and is replaced with a solution having a higher specific gravity. Further, the sample solution containing the separation target is injected into the reservoir 23. This stage is the state shown in FIG. When the centrifuge chip 100 is mounted in the space 2 of the rotating plate 1 and centrifuged by the motor 3 in this state, a solution layer corresponding to the specific gravity is formed as shown in FIG. According to the solution layer.

（実施例３）
図４は、実施例３の遠心チップ１００の構成を模式的に示す平面図である。図２に示した実施例２の遠心チップ１００と対比して明らかなように、実施例３の遠心チップ１００は試料側リザーバ６１，６２と回収側リザーバ６３，６４が回転中心１０からの距離が異なる。このため、回転させた場合のＧはリザーバ６１，６２のほうがリザーバ６３，６４よりも大きくかかる。このため液は矢印６９の方向に流れる。それぞれのリザーバからの流路６５−６８は分離チャンバー７０に連結される。この例では、溶液は２種類とした例であり、遠心中に溶液は試料側リザーバ６１，６２から回収側リザーバ６３，６４に液落差に対応した遠心力差により流れる。このときも入口側および出口側における高比重溶液面と試料側の低比重溶液面の遠心中心からの距離が等しいことが重要となる。そのため、実施例２と同様、低比重溶液が高比重溶液を覆う形でリザーバ６３，６４に入れられるのが良い。同様に回収部のリザーバ６３，６４の液面の高さも揃える必要がある。これらに差があると、分離チャンバー７０での２液層が形成されない。 (Example 3)
FIG. 4 is a plan view schematically showing the configuration of the centrifugal tip 100 of the third embodiment. As apparent from the comparison with the centrifugal tip 100 of the second embodiment shown in FIG. 2, the centrifugal tip 100 of the third embodiment has a distance between the sample side reservoirs 61 and 62 and the recovery side reservoirs 63 and 64 from the rotation center 10. Different. For this reason, the G when rotated is larger in the reservoirs 61 and 62 than in the reservoirs 63 and 64. For this reason, the liquid flows in the direction of arrow 69. The flow paths 65-68 from the respective reservoirs are connected to the separation chamber 70. In this example, two types of solutions are used. During centrifugation, the solution flows from the sample side reservoirs 61 and 62 to the recovery side reservoirs 63 and 64 due to a centrifugal force difference corresponding to the liquid drop. Also at this time, it is important that the distance from the centrifugal center of the high specific gravity solution surface on the inlet side and the outlet side and the low specific gravity solution surface on the sample side are equal. Therefore, as in the second embodiment, the low specific gravity solution may be placed in the reservoirs 63 and 64 so as to cover the high specific gravity solution. Similarly, it is necessary to make the liquid level of the reservoirs 63 and 64 of the recovery unit uniform. If there is a difference between them, the two-liquid layer in the separation chamber 70 is not formed.

実施例３でも、遠心チップ１００のディメンジョンは、実施例２と同等である。図５は実施例３の分離チャンバー７０を取り出して模式的に示す斜視図である。遠心によりＧのかかる方向に１００μｍで厚みは２５μｍである。分離部の両端には厚みは２５μｍで幅が５０μｍの流路６５，６６および６７，６８に連結されている。すなわち、図３に示す基板４１の片面に形成される流路１１−１３および、図示しなかったが、分離チャンバー１７と同じである。
（分離部の動作説明）
図６は低比重溶液流路５５と高比重溶液流路５６との２流路が結合した分離チャンバー７０における被分離物質の移動の様子を模式的に示す図である。ここでは、実施例３の遠心チップでヒト赤血球とヒトリンパ球の分離を試みた例について説明する。 Also in Example 3, the dimensions of the centrifugal tip 100 are the same as in Example 2. FIG. 5 is a perspective view schematically showing the separation chamber 70 of Example 3 taken out. The thickness is 100 μm and the thickness is 25 μm in the direction of G by centrifugation. Both ends of the separation part are connected to flow paths 65, 66 and 67, 68 having a thickness of 25 μm and a width of 50 μm. That is, the flow path 11-13 formed on one side of the substrate 41 shown in FIG.
(Explanation of separation unit operation)
FIG. 6 is a diagram schematically showing the state of movement of the substance to be separated in the separation chamber 70 in which the two channels of the low specific gravity solution channel 55 and the high specific gravity solution channel 56 are combined. Here, an example in which separation of human erythrocytes and human lymphocytes using the centrifuge chip of Example 3 is described.

まず、チップのリザーバ６１，６２に２−メタクリロキシエチルホスホリルコリンあるいはＢＡＳを含むＰＢＳ（ｐＨ７．４）を入れ、流路６５−６８および分離チャンバー７０全体に上記溶液を満たし、３０分間放置し、流路表面を２−メタクリロキシエチルホスホリルコリンあるいはＢＳＡでコーティングする。この操作は細胞の非特異吸着を防止する上で重要な操作である。次に、ＰＢＳで洗浄し、流路６５−６８および分離チャンバー７０内の過剰なＢＳＡなどを除去する。次に、試料側（低比重溶液）のリザーバ６２と回収側（高比重溶液）のリザーバ６１にＰＢＳを満たす。このとき、２本の流路に一定の圧がかかる（２本の流路に同じ液量の液が流れる）様に、上述したリザーバ間の分離壁の切り欠きの上まで液を入れる。次に、回収側（高比重溶液）のリザーバ６１に比重を１．０７７に調製した溶液を添加し、１８００ｒｐｍで回転、遠心をかけ、予め流路中に低比重溶液と高比重溶液を満たしておく。操作はすべて室温で行う。その後、試料側（低比重溶液）に試料混合液を添加し、１８００ｒｐｍで回転、遠心をかける。このとき、図１で説明した光学系で分離チャンバー７０の画像観察を行うと、図６に示すように、分離チャンバー７０においては、低比重溶液と高比重溶液は２層の層流となり、大きな黒丸で示す赤血球は高比重溶液に移動し、小さい白丸で示すリンパ球は低比重溶液に残ることが観察できる。   First, PBS (pH 7.4) containing 2-methacryloxyethyl phosphorylcholine or BAS is placed in the chip reservoirs 61 and 62, the flow path 65-68 and the entire separation chamber 70 are filled with the above solution, and left for 30 minutes. The road surface is coated with 2-methacryloxyethyl phosphorylcholine or BSA. This operation is an important operation for preventing nonspecific adsorption of cells. Next, it wash | cleans with PBS and the excess BSA etc. in the flow path 65-68 and the separation chamber 70 are removed. Next, the sample side (low specific gravity solution) reservoir 62 and the collection side (high specific gravity solution) reservoir 61 are filled with PBS. At this time, the liquid is poured onto the above-described notch of the separation wall between the reservoirs so that a constant pressure is applied to the two flow paths (the same amount of liquid flows in the two flow paths). Next, a solution prepared with a specific gravity of 1.077 is added to the reservoir 61 on the collection side (high specific gravity solution), rotated at 1800 rpm, centrifuged, and previously filled the low specific gravity solution and high specific gravity solution into the flow path. deep. All operations are performed at room temperature. Thereafter, the sample mixed solution is added to the sample side (low specific gravity solution), and rotated and centrifuged at 1800 rpm. At this time, when the image of the separation chamber 70 is observed with the optical system described with reference to FIG. 1, in the separation chamber 70, the low specific gravity solution and the high specific gravity solution become two-layer laminar flows as shown in FIG. It can be observed that red blood cells indicated by black circles migrate to a high density solution, and lymphocytes indicated by small white circles remain in the low density solution.

図７は低比重溶液流路１３と中比重溶液流路１２と高比重溶液流路１１の３流路が結合した分離チャンバー１７における被分離物質の移動の様子を模式的に示す図である。ここでは、実施例１の遠心チップで血清の分離を試みた例について説明する。   FIG. 7 is a diagram schematically showing the movement of the substance to be separated in the separation chamber 17 in which the three specific gravity solution flow paths 13, the medium specific gravity solution flow path 12, and the high specific gravity solution flow path 11 are combined. Here, an example in which serum separation is attempted with the centrifugal tip of Example 1 will be described.

まず、図６を参照して説明したのと同様にして、試料側（低比重溶液）流路１３と２つの回収側（中比重溶液および高比重溶液）流路１２，１１および分離チャンバー１７を洗浄する。ここでは、低比重溶液の比重は、ほぼ、１に、中比重溶液の比重は１．０７７に、高比重溶液の比重は１．１１３に調整して用いる。洗浄後、試料側（低比重溶液）のリザーバ２３と回収側（中、高比重溶液）のリザーバ２２，２１に低比重溶液を満たす。このとき、３本の流路に一定の圧がかかる（３本の流路に同じ液量の液が流れる）様に、上述したリザーバ間の分離壁５１の切り欠きの上まで液を入れる。次に、回収側（中、高比重溶液）のリザーバ２２，２１に比重を１．０７７および１．１１３に調製した溶液を、それぞれ、添加し、１８００ｒｐｍで回転、遠心をかけ、予め流路中に低比重溶液、中、高比重溶液を満たしておく。操作はすべて室温で行う。その後、試料側（低比重溶液）リザーバ２３に試料（血清）混合液を添加し、１８００ｒｐｍで回転、遠心をかける。このとき、図１で説明した光学系で分離チャンバー７０の画像観察を行うと、図７に示すように、分離チャンバー１７においては、低比重溶液、中高比重溶液および高比重溶液が３層の層流となり、大きな黒丸で示す赤血球は高比重溶液に移動し、星印で示す多核球は中高比重溶液に移動し、小さい白丸で示すヒト単核球は低比重溶液に残ることが観察できる。   First, in the same manner as described with reference to FIG. 6, the sample side (low specific gravity solution) flow path 13, the two collection side (medium specific gravity solution and high specific gravity solution) flow paths 12 and 11, and the separation chamber 17 are provided. Wash. Here, the specific gravity of the low specific gravity solution is adjusted to approximately 1, the specific gravity of the medium specific gravity solution is adjusted to 1.077, and the specific gravity of the high specific gravity solution is adjusted to 1.113. After washing, the low specific gravity solution is filled in the reservoir 23 on the sample side (low specific gravity solution) and the reservoirs 22 and 21 on the collection side (medium, high specific gravity solution). At this time, the liquid is poured onto the above-described notch of the separation wall 51 between the reservoirs so that a constant pressure is applied to the three flow paths (the same amount of liquid flows in the three flow paths). Next, the solutions prepared with specific gravity of 1.077 and 1.113 are added to the reservoirs 22 and 21 on the collection side (medium, high specific gravity solution), respectively, and rotated and centrifuged at 1800 rpm, respectively. In addition, a low specific gravity solution, a medium specific gravity solution and a high specific gravity solution are filled. All operations are performed at room temperature. Thereafter, the sample (serum) mixed solution is added to the sample side (low specific gravity solution) reservoir 23, and rotated and centrifuged at 1800 rpm. At this time, when the image of the separation chamber 70 is observed with the optical system described with reference to FIG. 1, as shown in FIG. 7, the separation chamber 17 has three layers of a low specific gravity solution, a medium high specific gravity solution, and a high specific gravity solution. It can be observed that red blood cells indicated by large black circles move to a high density solution, polynuclear cells indicated by asterisks move to a medium / high density solution, and human mononuclear cells indicated by small white circles remain in the low density solution.

ここで、図１を参照して説明したように、遠心チップ１００の回転速度、高速カメラ１０１の画像取り込みレートを適当に調整すれば、これらの分離状態を静止画像の状態で撮影することができる。   Here, as described with reference to FIG. 1, if the rotational speed of the centrifugal tip 100 and the image capture rate of the high-speed camera 101 are appropriately adjusted, these separated states can be photographed as still images. .

本発明に係わる遠心分離装置の概要を示す構成図である。It is a block diagram which shows the outline | summary of the centrifuge concerning this invention. 実施例１の遠心分離装置に適用するのに好適な遠心チップ１００の構成を模式的に示す平面図である。It is a top view which shows typically the structure of the centrifuge chip 100 suitable for applying to the centrifuge of Example 1. FIG. 図２に示した遠心チップ１００のリザーバ部分に着目して断面にして示した構成を模式的に示す斜視図である。本発明に係る第２の製造工程を示す図である。FIG. 3 is a perspective view schematically showing a configuration shown in a cross-section focusing on a reservoir portion of the centrifugal tip 100 shown in FIG. 2. It is a figure which shows the 2nd manufacturing process which concerns on this invention. 実施例３の遠心チップ１００の構成を模式的に示す平面図である。6 is a plan view schematically showing the configuration of a centrifugal tip 100 of Example 3. FIG. 実施例３の分離チャンバー７０を取り出して模式的に示す斜視図である。It is the perspective view which takes out the separation chamber 70 of Example 3, and shows typically. 低比重溶液流路５５と高比重溶液流路５６との２流路が結合した分離チャンバー７０における被分離物質の移動の様子を模式的に示す図である。It is a figure which shows typically the mode of the movement of the to-be-separated substance in the separation chamber 70 which 2 flow paths, the low specific gravity solution flow path 55 and the high specific gravity solution flow path 56 couple | bonded. 低比重溶液流路１３と中比重溶液流路１２と高比重溶液流路１１の３流路が結合した分離チャンバー１７における被分離物質の移動の様子を模式的に示す図である。FIG. 3 is a diagram schematically showing a state of movement of a substance to be separated in a separation chamber 17 in which three flow paths of a low specific gravity solution flow path 13, a medium specific gravity solution flow path 12 and a high specific gravity solution flow path 11 are combined.

符号の説明Explanation of symbols

１…回転板、２…スペース、３…モータ、４…光源、５…レンズ、６…ミラー、７…高速カメラ、８…パソコン、１０…回転中心、１１，１２，１３，１４，１５，１６，６５，６６，６７，６８，７１，７２，７３，７４，７５，７６…流路、１７，７０…分離チャンバー、２１，２２，２３，２４，２５，２６，６１，６２．６３，６４…リザーバ、４１…基板、４２…ガラス板、４３，４４，４５…貫通孔、４６，４７，４８…溶液注入用の穴、５１，５２…分離壁、１００…遠心チップ。
DESCRIPTION OF SYMBOLS 1 ... Rotary plate, 2 ... Space, 3 ... Motor, 4 ... Light source, 5 ... Lens, 6 ... Mirror, 7 ... High-speed camera, 8 ... Personal computer, 10 ... Center of rotation, 11, 12, 13, 14, 15, 16 , 65, 66, 67, 68, 71, 72, 73, 74, 75, 76 ... flow path, 17, 70 ... separation chamber, 21, 22, 23, 24, 25, 26, 61, 62.63, 64 DESCRIPTION OF SYMBOLS ... Reservoir, 41 ... Substrate, 42 ... Glass plate, 43, 44, 45 ... Through hole, 46, 47, 48 ... Hole for solution injection, 51, 52 ... Separation wall, 100 ... Centrifugal tip.

Claims (7)

回転板を回転させるモータと、
前記モータに回転させられる軸を中心に回転する回転板と、
前記回転板の面に取り付けられる遠心分離用チップと、
を備える遠心分離装置であって、
前記遠心分離用チップは比重の異なる複数の溶液を供給される流路と、
前記流路が集合するひとつの分離チャンバーと、
前記分離チャンバーから分岐する複数の流路と、
を備えるとともに、
前記分離チャンバーに溶液を供給する流路と、前記分離チャンバーから分岐する複数の流路の端部にはリザーバを備え、かつ、前記分離チャンバーに溶液を供給する流路に連なるリザーバに所定の比重の溶液を保持するとともに、リザーバの一つに被分離試料を入れる、
ことを特徴とする遠心分離装置。 A motor for rotating the rotating plate;
A rotating plate that rotates about an axis that is rotated by the motor;
A centrifuge chip attached to the surface of the rotating plate;
A centrifuge comprising:
The centrifuge chip is supplied with a plurality of solutions having different specific gravity;
One separation chamber in which the flow paths gather;
A plurality of flow paths branched from the separation chamber;
With
A flow path for supplying the solution to the separation chamber and a plurality of flow paths branched from the separation chamber are provided with a reservoir, and the reservoir connected to the flow path for supplying the solution to the separation chamber has a predetermined specific gravity. And hold the sample to be separated in one of the reservoirs.
A centrifugal separator characterized by that. 前記比重の異なる複数の溶液を供給される流路につながる前記リザーバの位置が回転軸からの距離が等しく、かつ、前記前記分離チャンバーから分岐する複数の流路につながる前記リザーバの位置が回転軸からの距離が等しい構造であることを特徴とする請求項1記載の遠心分離装置。 The position of the reservoir connected to the flow path to which the plurality of solutions having different specific gravities are supplied has the same distance from the rotation axis, and the position of the reservoir connected to the plurality of flow paths branched from the separation chamber is the rotation axis. 2. The centrifugal separator according to claim 1, wherein the distance from the centrifugal separator is equal. 回転板を回転させるモータと、
前記モータに回転させられる軸を中心に回転する回転板と、
前記回転板の面に取り付けられる遠心分離用チップと、
を備える遠心分離装置による分離方法であって、
前記遠心分離用チップは比重の異なる複数の溶液を供給される流路と、
前記流路が集合するひとつの分離チャンバーと、
前記分離チャンバーから分岐する複数の流路と、
を備えるとともに、
前記分離チャンバーに溶液を供給する流路と、前記分離チャンバーから分岐する複数の流路の端部にはリザーバを備え、かつ、前記分離チャンバーに溶液を供給する流路に連なるリザーバに所定の比重の溶液を保持させるとともに、リザーバの一つに被分離試料を入れ、前記リザーバから分離チャンバーに遠心により搬送される溶液が、前記比重に対応した層を形成して、前記被分離試料を比重に対応して分離する、
ことを特徴とする遠心分離法。 A motor for rotating the rotating plate;
A rotating plate that rotates about an axis that is rotated by the motor;
A centrifuge chip attached to the surface of the rotating plate;
A separation method using a centrifuge comprising:
The centrifuge chip is supplied with a plurality of solutions having different specific gravity;
One separation chamber in which the flow paths gather;
A plurality of flow paths branched from the separation chamber;
With
A flow path for supplying the solution to the separation chamber and a plurality of flow paths branched from the separation chamber are provided with a reservoir, and the reservoir connected to the flow path for supplying the solution to the separation chamber has a predetermined specific gravity. The sample to be separated is placed in one of the reservoirs, and the solution transported by centrifugation from the reservoir to the separation chamber forms a layer corresponding to the specific gravity so that the sample to be separated has a specific gravity. Correspondingly separate,
Centrifugation method characterized by that. 回転板を回転させるモータと、
前記モータに回転させられる軸を中心に回転する回転板と、
前記回転板の面に取り付けられる遠心分離用チップと、
を備える遠心分離装置に適用できる遠心分離用チップであって、
前記遠心分離用チップは比重の異なる複数の溶液を供給される流路と、
前記流路が集合するひとつの分離チャンバーと、
前記分離チャンバーから分岐する複数の流路と、
を備えるとともに、
前記分離チャンバーに溶液を供給する流路と、前記分離チャンバーから分岐する複数の流路の端部にはリザーバを備える、
ことを特徴とする遠心分離用チップ。 A motor for rotating the rotating plate;
A rotating plate that rotates about an axis that is rotated by the motor;
A centrifuge chip attached to the surface of the rotating plate;
A centrifuge chip applicable to a centrifuge comprising:
The centrifuge chip is supplied with a plurality of solutions having different specific gravity;
One separation chamber in which the flow paths gather;
A plurality of flow paths branched from the separation chamber;
With
A flow path for supplying a solution to the separation chamber, and a reservoir at an end of a plurality of flow paths branched from the separation chamber,
A centrifuge chip characterized by that. 前記分離チャンバーに溶液を供給する流路につながる前記リザーバの位置が回転軸からの距離が等しく、かつ、前記分離チャンバーから分岐する複数の流路の端部につながるリザーバの位置が回転軸からの距離が等しい構造であることを特徴とする遠心分離用チップ。 The position of the reservoir connected to the flow path for supplying the solution to the separation chamber is the same distance from the rotation axis, and the position of the reservoir connected to the ends of the plurality of flow paths branched from the separation chamber is different from the rotation axis. A centrifuge chip characterized by having a structure having an equal distance. 前記遠心分離用チップの流路と前記分離チャンバーが形成される基板の面と反対の面に前記リザーバが形成され、前記リザーバの一端と前記流路の一端とが基板を貫通する孔により連通されている請求項３記載の遠心分離用チップ。   The reservoir is formed on the surface of the centrifuge chip opposite to the surface of the substrate on which the separation chamber is formed, and one end of the reservoir and the one end of the channel are communicated with each other through a hole penetrating the substrate. The centrifuge chip according to claim 3. 前記遠心分離用チップの比重の異なる複数の溶液を供給される流路の一端に連通されているリザーバは、前記連通している孔の反対位置の一部で、複数のリザーバを分離している分離壁が切り欠かれている請求項３記載の遠心分離用チップ。
The reservoir connected to one end of the flow path to which a plurality of solutions having different specific gravity of the centrifuge chip are supplied separates the plurality of reservoirs at a part of the position opposite to the communicating hole. The centrifuge chip according to claim 3, wherein the separation wall is cut out.
JP2004349609A 2004-08-03 2004-12-02 Centrifugal tip and centrifugation Expired - Fee Related JP4485926B2 (en)

Priority Applications (13)

Application Number Priority Date Filing Date Title
JP2004349609A JP4485926B2 (en) 2004-12-02 2004-12-02 Centrifugal tip and centrifugation
EP07013611A EP1901067A3 (en) 2004-08-03 2005-08-01 Cellomics system
EP05016714A EP1626278A3 (en) 2004-08-03 2005-08-01 Cellomics system
US11/195,662 US7569354B2 (en) 2004-08-03 2005-08-03 Cellomics system
US12/143,181 US20090042739A1 (en) 2004-08-03 2008-06-20 Cellomics system
US12/143,156 US20090042200A1 (en) 2004-08-03 2008-06-20 Cellomics system
US12/471,853 US20100021933A1 (en) 2004-08-03 2009-05-26 Cellomics systems
US12/472,037 US20090325215A1 (en) 2004-08-03 2009-05-26 Cellomics system
US12/472,010 US20100016569A1 (en) 2004-08-03 2009-05-26 Cellomics system
US12/471,993 US20100018862A1 (en) 2004-08-03 2009-05-26 Cellomics system
US12/471,947 US20100016568A1 (en) 2004-08-03 2009-05-26 Cellomics system
US13/755,079 US20130252848A1 (en) 2004-08-03 2013-01-31 Cellomics system
US14/640,471 US20150231635A1 (en) 2004-08-03 2015-03-06 Cellomics system

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
JP2004349609A JP4485926B2 (en) 2004-12-02 2004-12-02 Centrifugal tip and centrifugation

Publications (2)

Publication Number Publication Date
JP2006158991A true JP2006158991A (en) 2006-06-22
JP4485926B2 JP4485926B2 (en) 2010-06-23

Family

ID=36661581

Family Applications (1)

Application Number Title Priority Date Filing Date
JP2004349609A Expired - Fee Related JP4485926B2 (en) 2004-08-03 2004-12-02 Centrifugal tip and centrifugation

Country Status (1)

Country Link
JP (1) JP4485926B2 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007017342A (en) * 2005-07-08 2007-01-25 Rohm Co Ltd Chip having metering part
JP2007315832A (en) * 2006-05-24 2007-12-06 Hitachi High-Technologies Corp Biochemical analyzer and examination cartridge used therefor
JP2009162643A (en) * 2008-01-08 2009-07-23 Sony Corp Sending with flowing method and analysis method of fine particle, and substrate for analyzing fine particle
JP2012076017A (en) * 2010-10-01 2012-04-19 Chiba Univ Microchannel system for elutriator and particle separation method
WO2014042177A1 (en) * 2012-09-11 2014-03-20 国立大学法人北陸先端科学技術大学院大学 Liquid supply method, centrifugal separation method, liquid supply device and centrifugal separation device
JP2016057303A (en) * 2014-09-09 2016-04-21 鷲津　正夫 Device for separating and detecting sample using continuous density gradient
WO2023018395A1 (en) * 2021-08-09 2023-02-16 İzmi̇r Yüksek Teknoloji̇ Ensti̇tüsü Centrifugal microfluid chip integrated with vacuum to separate microparticles and cells depending on densities

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2007017342A (en) * 2005-07-08 2007-01-25 Rohm Co Ltd Chip having metering part
JP4546889B2 (en) * 2005-07-08 2010-09-22 ローム株式会社 Chip with weighing unit
JP2007315832A (en) * 2006-05-24 2007-12-06 Hitachi High-Technologies Corp Biochemical analyzer and examination cartridge used therefor
JP4597091B2 (en) * 2006-05-24 2010-12-15 株式会社日立ハイテクノロジーズ Biochemical analyzer and inspection cartridge used therefor
JP2009162643A (en) * 2008-01-08 2009-07-23 Sony Corp Sending with flowing method and analysis method of fine particle, and substrate for analyzing fine particle
JP2012076017A (en) * 2010-10-01 2012-04-19 Chiba Univ Microchannel system for elutriator and particle separation method
WO2014042177A1 (en) * 2012-09-11 2014-03-20 国立大学法人北陸先端科学技術大学院大学 Liquid supply method, centrifugal separation method, liquid supply device and centrifugal separation device
JPWO2014042177A1 (en) * 2012-09-11 2016-08-18 国立大学法人山梨大学 Liquid feeding method, centrifugal separation method, liquid feeding device and centrifugal separator
JP2016057303A (en) * 2014-09-09 2016-04-21 鷲津　正夫 Device for separating and detecting sample using continuous density gradient
WO2023018395A1 (en) * 2021-08-09 2023-02-16 İzmi̇r Yüksek Teknoloji̇ Ensti̇tüsü Centrifugal microfluid chip integrated with vacuum to separate microparticles and cells depending on densities

Also Published As

Publication number Publication date
JP4485926B2 (en) 2010-06-23

Similar Documents

Publication Publication Date Title
US7743928B2 (en) Integrated apparatus and methods for treating liquids
JP3749991B2 (en) Micro liquid weighing structure and microchip having the structure
KR101257975B1 (en) Methods and systems for delivery of fluidic samples to sensor arrays
TWI409459B (en) Microchannelarray, method of manufacturing the same and blood test method using the same
US9470609B2 (en) Preparation of thin layers of a fluid containing cells for analysis
US20160051986A1 (en) Microfluidic device and method for operating thereof
WO2009121037A2 (en) Three-dimensional microfluidic devices
JP2007526108A (en) Fluidic MEMS device
JP4485926B2 (en) Centrifugal tip and centrifugation
JP7163558B2 (en) Separation Apparatus and Separation Method, Separation Device, Inspection Apparatus and Inspection Method
US20080190503A1 (en) Flow Switching on a Multi-Structured Microfluidic Cd (Compact Disc) Using Coriolis Force
JP2005114438A (en) Method of using chip, and inspection chip
Kuo et al. Microfluidic blood-plasma separation chip using channel size filtration effect
CN108080043A (en) The multichannel micro-fluidic chip device and preparation method of negative pressure of vacuum sample introduction and application
KR101199505B1 (en) Fabrication Method of Micro-fluidic Chip Using Ultrasonic Bonding
US20040228774A1 (en) Microreactor, its production method, and sample screening device
D'Silva High-Throughput Microfluidic Capture of Rare Cells from Large Volumes of Blood
KR20100048507A (en) Plasma separation device using microfluidic channel and plasma separation method using microfluidic channel
CN112113906A (en) Sample detection device and manufacturing method thereof
JP4255891B2 (en) Protein crystallization method
CN213181226U (en) Sample detection device
JP2009174891A (en) Microchip
KR101528154B1 (en) Apparatus for Measuring Cholesterol and Method thereof
JP2004301733A (en) Analyzer and analyzing method
Brenner Polymer fabrication and microfluidic unit operations for medical diagnostics on a rotating disk

Legal Events

Date Code Title Description
RD04 Notification of resignation of power of attorney

Free format text: JAPANESE INTERMEDIATE CODE: A7424

Effective date: 20070125

RD04 Notification of resignation of power of attorney

Free format text: JAPANESE INTERMEDIATE CODE: A7424

Effective date: 20070125

RD03 Notification of appointment of power of attorney

Free format text: JAPANESE INTERMEDIATE CODE: A7423

Effective date: 20070319

A621 Written request for application examination

Free format text: JAPANESE INTERMEDIATE CODE: A621

Effective date: 20070528

A711 Notification of change in applicant

Free format text: JAPANESE INTERMEDIATE CODE: A711

Effective date: 20070703

A711 Notification of change in applicant

Free format text: JAPANESE INTERMEDIATE CODE: A711

Effective date: 20080213

A977 Report on retrieval

Free format text: JAPANESE INTERMEDIATE CODE: A971007

Effective date: 20090415

A131 Notification of reasons for refusal

Free format text: JAPANESE INTERMEDIATE CODE: A131

Effective date: 20090428

A521 Written amendment

Free format text: JAPANESE INTERMEDIATE CODE: A523

Effective date: 20090624

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

Free format text: JAPANESE INTERMEDIATE CODE: A01

Effective date: 20100302

A01 Written decision to grant a patent or to grant a registration (utility model)

Free format text: JAPANESE INTERMEDIATE CODE: A01

A61 First payment of annual fees (during grant procedure)

Free format text: JAPANESE INTERMEDIATE CODE: A61

Effective date: 20100325

R150 Certificate of patent or registration of utility model

Free format text: JAPANESE INTERMEDIATE CODE: R150

FPAY Renewal fee payment (event date is renewal date of database)

Free format text: PAYMENT UNTIL: 20130402

Year of fee payment: 3

FPAY Renewal fee payment (event date is renewal date of database)

Free format text: PAYMENT UNTIL: 20140402

Year of fee payment: 4

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

R250 Receipt of annual fees

Free format text: JAPANESE INTERMEDIATE CODE: R250

LAPS Cancellation because of no payment of annual fees