JPH0687953B2 - METHOD FOR SEPARATING PARTICLE AND SEPARATOR - Google Patents
METHOD FOR SEPARATING PARTICLE AND SEPARATORInfo
- Publication number
- JPH0687953B2 JPH0687953B2 JP1191911A JP19191189A JPH0687953B2 JP H0687953 B2 JPH0687953 B2 JP H0687953B2 JP 1191911 A JP1191911 A JP 1191911A JP 19191189 A JP19191189 A JP 19191189A JP H0687953 B2 JPH0687953 B2 JP H0687953B2
- Authority
- JP
- Japan
- Prior art keywords
- fine particles
- laser light
- separation
- medium
- cell
- 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.)
- Expired - Lifetime
Links
- 238000000034 method Methods 0.000 title claims description 15
- 239000002245 particle Substances 0.000 title description 17
- 239000010419 fine particle Substances 0.000 claims description 79
- 238000011084 recovery Methods 0.000 claims description 31
- 230000003287 optical effect Effects 0.000 claims description 20
- 230000005684 electric field Effects 0.000 claims description 17
- 238000005192 partition Methods 0.000 claims description 16
- 239000011810 insulating material Substances 0.000 claims description 4
- 238000000926 separation method Methods 0.000 description 47
- 210000004027 cell Anatomy 0.000 description 29
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 17
- 241000894006 Bacteria Species 0.000 description 6
- XKRFYHLGVUSROY-UHFFFAOYSA-N Argon Chemical compound [Ar] XKRFYHLGVUSROY-UHFFFAOYSA-N 0.000 description 4
- 238000001962 electrophoresis Methods 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 239000011521 glass Substances 0.000 description 3
- 230000005484 gravity Effects 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 229910052782 aluminium Inorganic materials 0.000 description 2
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 description 2
- 229910052786 argon Inorganic materials 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 210000000601 blood cell Anatomy 0.000 description 2
- 239000003990 capacitor Substances 0.000 description 2
- 239000000919 ceramic Substances 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000000684 flow cytometry Methods 0.000 description 2
- 239000004816 latex Substances 0.000 description 2
- 229920000126 latex Polymers 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 239000004033 plastic Substances 0.000 description 2
- 239000004743 Polypropylene Substances 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 238000005868 electrolysis reaction Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000010030 laminating Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000002923 metal particle Substances 0.000 description 1
- 230000005486 microgravity Effects 0.000 description 1
- -1 polypropylene Polymers 0.000 description 1
- 229920001155 polypropylene Polymers 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
Description
【発明の詳細な説明】 〔産業上の利用分野〕 この発明は、細胞、細菌、血球、タンパク粒子、ポリマ
ーラテックス、セラミック微粒子などの微粒子を、その
電荷、誘電率、体積、屈折率、吸収率などの物理量に応
じて分離することのできる微粒子の分離方法およびこの
分離方法を実施するための分離装置に関する。DETAILED DESCRIPTION OF THE INVENTION [Industrial field of application] The present invention relates to fine particles such as cells, bacteria, blood cells, protein particles, polymer latex, and ceramic fine particles, whose electric charge, dielectric constant, volume, refractive index and absorptivity are used. The present invention relates to a method for separating fine particles that can be separated according to a physical quantity such as, and a separation apparatus for carrying out this separation method.
従来、このような微粒子の分離方法の１つに、電気泳動
法がある。この方法は荷電微粒子の媒質中での電気的移
動度の差を利用して荷電微粒子の分離を行うもので、タ
ンパク質やアミノ酸の分離などに用いられている。Conventionally, one of the methods for separating such fine particles is an electrophoresis method. This method separates charged fine particles by utilizing the difference in electric mobility of charged fine particles in a medium, and is used for separating proteins and amino acids.
また、特に細胞などを分離、分別する方法として、フロ
ーサイトメトリーが知られている。このものは細胞など
を懸濁させた媒質を極細の細流とし、この細流をオリフ
ィスから液滴として滴下する際に、液滴に電界を作用さ
せて液滴の物理量に応じて滴下位置を変化させ、これに
より細胞等を分離するものである。Flow cytometry is known as a method for separating and sorting cells and the like. This is a medium in which cells and the like are suspended into an extremely fine stream, and when this stream is dropped as a droplet from an orifice, an electric field is applied to the droplet to change the dropping position according to the physical quantity of the droplet. , Thereby separating cells and the like.
しかしながら、電気泳動法によるものでは、分離精度が
低く、また分離に長時間を要する不都合がある。また、
フローサイトメトリーでは、液滴化が必須であり、応用
範囲、適用範囲が限られる問題がある。However, the electrophoresis method has a disadvantage that the separation accuracy is low and the separation requires a long time. Also,
In flow cytometry, droplet formation is indispensable, and there is a problem that the application range and application range are limited.
よって、この発明では分離精度が高くかつ分離時間が短
時間であり、適用範囲が広範囲にわたる微粒子の分離方
法およびその装置を提供することを課題する。Therefore, it is an object of the present invention to provide a method and apparatus for separating fine particles, which have a high separation accuracy, a short separation time, and have a wide range of application.
この発明では、微粒子を分散した媒質にレーザ光を入射
するとともに該媒質にレーザ光入射方向と同一方向成分
を有する電界をレーザ光軸上で形成することにより、上
記課題を解決するようにした。In the present invention, the above problem is solved by making laser light incident on a medium in which fine particles are dispersed and forming an electric field having the same direction component as the laser light incident direction on the medium on the laser optical axis.
微粒子を分散させた媒質中にレーザ光を入射すると、レ
ーザ光の光軸上に微粒子が閉じ込められるとともにレー
ザ光の入射方向に微粒子が光圧力を受けて移動する。こ
の光圧力はレーザ光の強度、微粒子の光吸収率、粒子径
等によって左右される。この際、同時にレーザ光の光軸
上に電界を印加することによって、微粒子には同時に電
気力が作用し、その電気力の方向にも微粒子が移動す
る。この電気力は微粒子の電荷量、双極モーメント等に
左右される。したがって、レーザ光の強度、電界の強
度、およびその向きを変化させることにより、微粒子の
有する物理量に応じた移動量を伴って微粒子をレーザ光
軸上に移動させることができ、微粒子の分離を行うこと
ができる。When laser light is incident on a medium in which fine particles are dispersed, the fine particles are confined on the optical axis of the laser light, and the fine particles move in the incident direction of the laser light under light pressure. This light pressure depends on the intensity of laser light, the light absorption rate of fine particles, the particle diameter, and the like. At this time, by simultaneously applying an electric field on the optical axis of the laser light, an electric force acts on the fine particles at the same time, and the fine particles also move in the direction of the electric force. This electric force depends on the charge amount of the fine particles, the dipole moment, and the like. Therefore, by changing the intensity of the laser beam, the intensity of the electric field, and the direction thereof, the fine particles can be moved along the laser optical axis with a movement amount according to the physical quantity of the fine particles, and the fine particles are separated. be able to.
以下、この発明を詳細に説明する。The present invention will be described in detail below.
第１図は、この発明の分離装置の一例を模式的に示すも
のである。第１図中符号１はセルである。このセ１はガ
ラス、プラスチックなどから作られており、その内部に
はセル１内の空間を二分するように仕切部材２が設けら
れている。この仕切部材２はプラスチックなどの絶縁材
料からなる板状のもので、その中央部にはピンホール状
の円形の小孔（空隙）３が穿けられている。この小孔３
の径は10〜200μｍ程度である。また、セル１の相対向
する内面にはそれぞれアルミニウムなどの金属膜からな
る１対の電極4,4が設けられている。これらの電極4,4の
ほぼ中央部には分離用レーザ光Ａを通すための窓5,5が
形成されており、これら窓5,5と仕切部材２の小孔３と
が一直線上に正確に並ぶようにその位置が定められてい
る。１対の電極4,4間の間隔は約2mm程度となっている。FIG. 1 schematically shows an example of the separation device of the present invention. Reference numeral 1 in FIG. 1 is a cell. The cell 1 is made of glass, plastic, or the like, and a partition member 2 is provided inside the cell 1 so as to divide the space in the cell 1 into two. The partition member 2 is a plate-shaped member made of an insulating material such as plastic, and has a pinhole-shaped circular small hole (gap) 3 in the center thereof. This small hole 3
Has a diameter of about 10 to 200 μm. Further, a pair of electrodes 4 and 4 made of a metal film such as aluminum are provided on the inner surfaces of the cell 1 which face each other. Windows 5, 5 for passing the separation laser beam A are formed in substantially the center of these electrodes 4, 4, and these windows 5, 5 and the small hole 3 of the partition member 2 are aligned in a straight line. The position is set so as to line up with. The distance between the pair of electrodes 4, 4 is about 2 mm.
また、セル１の外部とは、アルゴンレーザ、YAGレーザ
などの分離用レーザ光源６が設けられており、この分離
用レーザ光源６からの分離用レーザ光Ａがセル１の壁を
通過し、窓５、小孔３および窓５を通るようになってい
る。さらに、１対の電極4,4は図示しない直流電源に接
続されている。Further, a separation laser light source 6 such as an argon laser or a YAG laser is provided outside the cell 1, and the separation laser light A from the separation laser light source 6 passes through the wall of the cell 1 and a window. 5, through the small hole 3 and the window 5. Further, the pair of electrodes 4, 4 is connected to a DC power source (not shown).
そして、セル１内の仕切部材２で二分された空間のう
ち、分離用レーザ光源６側の領域は供給エリヤ７とさ
れ、これの反対側は回収エリヤ８とされている。In the space divided into two by the partition member 2 in the cell 1, a region on the side of the separation laser light source 6 is a supply area 7, and the opposite side is a recovery area 8.
次に、この装置によって微粒子を分離する方法を説明す
る。Next, a method of separating fine particles by this device will be described.
まず、セル１の供給エリヤ７に、微粒子を分散した媒質
を供給する。ここでの媒質としては、水、水溶液、有機
溶剤、有機溶剤溶液などが用いられる。また、回収エリ
ヤ８には微粒子を含まない媒質を供給しておく。First, a medium in which fine particles are dispersed is supplied to the supply area 7 of the cell 1. As the medium here, water, an aqueous solution, an organic solvent, an organic solvent solution, or the like is used. Further, a medium containing no fine particles is supplied to the recovery area 8.
次に、１対の電極4,4間に直流電圧を印加し、電極4,4間
の電界を形成する。この時、微粒子の電荷が負の場合は
供給エリヤ７側の電極を正極に、回収エリヤ８側の電極
を負極となるように電源に接続する。微粒子の電荷が正
の場合には、これと反対の極性となることは言うまでも
ない。Next, a DC voltage is applied between the pair of electrodes 4 and 4 to form an electric field between the electrodes 4 and 4. At this time, if the charge of the particles is negative, the electrode on the supply area 7 side is connected to the positive electrode and the electrode on the recovery area 8 side is connected to the power source so as to be the negative electrode. It goes without saying that when the charge of the fine particles is positive, the polarity is opposite to this.
この極性の電界の形成により、供給エリヤ７に存在する
微粒子は供給エリヤ７側の電極４の方向に引かれ、仕切
部材２の小孔３を通って回収エリヤ８に拡散することが
防止される。電極4,4間の印加電圧は数Ｖ程度でよい。
この電圧印加によって、仕切部材２が絶縁材料からなる
ので電流は小孔３に集中した状態となって高電界が低印
加電圧で得られる。Due to the formation of the electric field of this polarity, fine particles existing in the supply area 7 are attracted toward the electrode 4 on the supply area 7 side, and are prevented from diffusing into the recovery area 8 through the small holes 3 of the partition member 2. . The applied voltage between the electrodes 4 and 4 may be about several volts.
By applying this voltage, since the partition member 2 is made of an insulating material, the current is concentrated in the small holes 3 and a high electric field can be obtained with a low applied voltage.
この電圧印加と同時に分離用レーザ光源６からの分離用
レーザ光Ａをセル１の一方の窓５から入射し、供給エリ
ヤ７、小孔３、回収エリヤ８を通り、他方の窓５に導
く。分離用レーザ光Ａの強度は上述のように微粒子の分
離度合と関係するので、特に限定されないが、微粒子が
細胞などの生物である場合は、そのダメージを避けるた
め100mW以下が好ましいが無機粒子や高分子粒子では1W
程度までの出力を適用することもできる。Simultaneously with the application of this voltage, the separation laser light A from the separation laser light source 6 enters through one window 5 of the cell 1, passes through the supply area 7, the small hole 3, and the recovery area 8 and is guided to the other window 5. Since the intensity of the separation laser beam A is related to the degree of separation of the fine particles as described above, it is not particularly limited, but when the fine particles are organisms such as cells, 100 mW or less is preferable in order to avoid damage, 1W for polymer particles
Outputs up to a degree can also be applied.
この電圧印加と分離用レーザ光Ａの入射とによって、供
給エリヤ７中に分散している微粒子は分離用レーザ光Ａ
の光軸方向の閉じ込め力を受けてレーザ光Ａの光軸上に
移動し、かつレーザ光Ａの光圧力を受けて同光軸上をレ
ーザ光Ａの入射方向側から出射方向側に向けて移動しよ
うとする。By applying this voltage and entering the separation laser light A, the fine particles dispersed in the supply area 7 are separated by the separation laser light A.
Is moved on the optical axis of the laser light A by receiving the confining force in the optical axis direction of the laser light, and the optical pressure of the laser light A is received on the optical axis from the incident direction side of the laser light A toward the emitting direction side. Try to move.
これと同時に微粒子には直流電界による電気泳動力が作
用する。この電気泳動力の向きは、微粒子の電荷の正負
および電極4,4の極性によって定まり、供給エリヤ７側
に向く力が作用する場合と、回収エリヤ８側に向く力が
作用する場合とがあり、微粒子はしたがって電気泳動力
を受けて供給エリヤ７側あるいは回収エリヤ８側に向け
て移動しようとする。At the same time, an electrophoretic force due to a DC electric field acts on the particles. The direction of this electrophoretic force is determined by the positive / negative of the charge of the particles and the polarities of the electrodes 4, 4, and there are cases where a force that faces the supply area 7 side acts and where a force that faces the recovery area 8 side acts. Therefore, the particles tend to move toward the supply area 7 side or the recovery area 8 side by receiving the electrophoretic force.
この状態で、分離用レーザ光Ａの強度と電極4,4の極性
ならびに印加電圧をそれぞれ制御し、微粒子に作用する
光圧力と電気泳動力およびその向きを変化させることに
より、レーザ光Ａ光軸上での微粒子の移動を制御させる
ことができ、微粒子を供給エリヤ７から回収エリヤ８に
移送することもできれば、小孔３上に停止させておくこ
ともでき、また供給エリヤ７内にそのままとどめておく
こともできる。微粒子に作用する光圧力が電気泳動力よ
りも大きい場合、電気泳動法の向きが供給エリヤ７側の
向きであればそれらの力の差に応じた移動速度で微粒子
が回収エリヤ７の方へ移動し、また電気泳動力の向きが
回収エリヤ８側の向きであればそれらの力の和に応じた
移動速度で微粒子が回収エリヤ７の方へ移動する。ま
た、微粒子に作用する光圧力が電気泳動法よりも小さい
場合、電気泳動力の向きが供給エリヤ７側の向きであれ
ば、それらの力の差に応じた移動速度で微粒子が供給エ
リヤ７側に移動し、電気泳動力の向きが回収エリヤ８側
の向きであれば、それらの力の和に応じた移動速度で回
収エリヤ８側に移動する。In this state, the intensity of the separation laser beam A, the polarities of the electrodes 4, 4 and the applied voltage are respectively controlled to change the optical pressure and the electrophoretic force acting on the particles and the direction thereof, whereby the optical axis of the laser beam A is changed. It is possible to control the movement of the fine particles above, and to transfer the fine particles from the supply area 7 to the recovery area 8 or to stop them on the small holes 3 and to keep them in the supply area 7 as they are. You can also keep it. When the light pressure acting on the fine particles is larger than the electrophoretic force, and if the direction of the electrophoresis method is the supply area 7 side, the fine particles move toward the recovery area 7 at a moving speed according to the difference between these forces. If the direction of the electrophoretic force is toward the collecting area 8, the fine particles move toward the collecting area 7 at a moving speed corresponding to the sum of these forces. When the light pressure acting on the fine particles is smaller than that in the electrophoresis method, and the direction of the electrophoretic force is the direction of the supply area 7, the fine particles are supplied to the supply area 7 side at a moving speed corresponding to the difference between the forces. If the direction of the electrophoretic force is toward the recovery area 8 side, the electrophoretic force moves to the recovery area 8 side at a moving speed corresponding to the sum of these forces.
したがって、異種の微粒子が複雑種分散している媒質を
セル１内の供給エリヤ７に供給すれば、ある特定の微粒
子のみをそれが有する物理量に対応した移動量で回収エ
リヤ８側に移送することができ、微粒子の分離が行え
る。また、１種の微粒子のみを分散した媒質を供給すれ
ば、微粒子を１個ずつ回収エリヤ８側に移送し、分散す
ることもできる。Therefore, if a medium in which different kinds of fine particles are dispersed in a complicated manner is supplied to the supply area 7 in the cell 1, only certain specific fine particles are transferred to the recovery area 8 side with a movement amount corresponding to the physical quantity of the specific fine particles. It is possible to separate fine particles. Further, if a medium in which only one kind of fine particles is dispersed is supplied, the fine particles can be transferred one by one to the recovery area 8 side and dispersed.
また、１対の電極4,4間に交流を印加しても同様に微粒
子の分離が可能である。微粒子に交流電界が印加される
と、微粒子に誘電泳動力が働く。この誘電泳動力は、微
粒子に生じた電気双極モーメントとその体積と電界の強
さによって定まり、その力の向きは微粒子の誘電率が媒
質の誘電率より大きい場合は強電界方向が向かい、微粒
子の誘電率が媒質のそれより小さい場合は弱電界方向に
向かう。Further, fine particles can be similarly separated by applying an alternating current between the pair of electrodes 4, 4. When an AC electric field is applied to the particles, the dielectrophoretic force acts on the particles. This dielectrophoretic force is determined by the electric dipole moment generated in the particles, its volume and the strength of the electric field. The direction of the force is the direction of the strong electric field when the dielectric constant of the particles is greater than the dielectric constant of the medium, If the permittivity is smaller than that of the medium, the direction is toward the weak electric field.
したがって、直流印加の場合の電気泳動力に代えて、交
流印加の場合には誘電泳動力を用いることにより、同様
の取り扱いを行うことができる。Therefore, the same handling can be performed by using the dielectrophoretic force in the case of applying the AC instead of the electrophoretic force in the case of applying the DC.
また、電極4,4間に交流重畳電圧を印加してもよい。こ
の場合は、微粒子に直流分による電気泳動力と交流分に
よる誘電泳動力とが同時に働くことになる。Alternatively, an AC superimposed voltage may be applied between the electrodes 4, 4. In this case, the electrophoretic force due to the direct current component and the dielectrophoretic force due to the alternating current component act on the fine particles at the same time.
印加電圧として、交流電圧あるいは交直重畳電圧を用い
る場合には、セル１中の媒質の電気分解が生じない点で
好ましい。It is preferable to use an AC voltage or an AC / DC superimposed voltage as the applied voltage because electrolysis of the medium in the cell 1 does not occur.
なお、微粒子の分離用レーザ光Ａの光軸上での移動に際
しては、当該微粒子と媒質との間の粘性に基づく粘性力
が移動方向の反対方向に働き、この粘性力も電気泳動力
および光圧力と同様に微粒子の移動を決める要素となり
うるが、実際の操作では、この粘性力を加味して分離用
レーザ光Ａの光強度、印加電圧の向きおよび強さを設定
すればよく、特に微粒子の分離には問題となることはな
い。When the fine particles are moved on the optical axis of the separation laser beam A, a viscous force based on the viscosity between the fine particles and the medium acts in a direction opposite to the moving direction, and the viscous force also causes the electrophoretic force and the optical pressure. Although it can be a factor that determines the movement of the fine particles, in the actual operation, the light intensity of the separation laser beam A, the direction and strength of the applied voltage may be set in consideration of this viscous force, and particularly Separation is not a problem.
第２図に示した装置は、上述のようにして分離された微
粒子を分取する際に用いられるものの一例である。The apparatus shown in FIG. 2 is an example of an apparatus used when separating the fine particles separated as described above.
この装置は、セル１の回収エリヤ８に２つの分取口9,9
を形成し、この分取口9,9に向けて分取用レーザ光源10,
10からの分取用レーザ光B,Bを回収エリヤ８の分離用レ
ーザ光Ａとそれぞれ交差して入射するように構成されて
いる。This device consists of two collection ports 9 and 9 in the recovery area 8 of the cell 1.
Laser light source 10 for preparative separation toward this preparatory opening 9, 9.
The separation laser beams B and B from 10 are configured to enter the separation laser beam A of the recovery area 8 while intersecting with each other.
この装置では、上述のように分離用レーザ光Ａの強度お
よび印加電圧ならびにその極性を調節した微粒子をレー
ザ光Ａの光軸上を回収エリヤ８側に移動させる。この微
粒子の移動中において、微粒子が発する散乱光や蛍光を
観察、計測して微粒子を固定、識別し、その微粒子が分
取したいものであれば、分取用レーザ光源10からの分取
用レーザ光Ｂの強度を分離用レーザ光Ａのそれよりも大
きくする。これにより、分離用レーザ光Ａに乗ってその
光軸上を移動してきた微粒子は分離用レーザ光Ａと分取
用レーザ光Ｂとが交差する交差点で光強度の強い分別用
レーザ光Ｂに乗り移り、回収エリヤ８から分取口９を経
て分取されることになる。この際、図示したように２つ
の分取用レーザ光B,Bを用いれば２種の微粒子を分取す
ることができ、当然２つ以上の分取用レーザ光Ｂ…を用
いれば２種以上の微粒子を分取できる。また、交差点を
１点とし、交差角度を変えて２以上の分取用レーザ光Ｂ
…を照射するようにしてもよい。In this apparatus, the fine particles whose intensity and applied voltage of the separation laser light A and their polarities are adjusted as described above are moved to the recovery area 8 side on the optical axis of the laser light A. During the movement of the fine particles, the scattered light or fluorescence emitted by the fine particles is observed and measured to fix and identify the fine particles, and if the fine particles are desired to be separated, the preparative laser from the preparative laser light source 10 is used. The intensity of the light B is made higher than that of the separating laser light A. As a result, the fine particles that have moved along the optical axis of the separation laser light A are transferred to the separation laser light B having a high light intensity at the intersection where the separation laser light A and the separation laser light B intersect. , The collection area 8 is collected through the collection port 9. At this time, as shown in the figure, two sorts of laser light B, B can be used to sort out two kinds of fine particles, and naturally two or more sorts of laser light B ... Fine particles can be collected. In addition, the number of preparative laser beams B is 2 or more by changing the crossing angle with one crossing point.
You may make it irradiate.
また、第１図および第２図に示した装置において、供給
エリヤ７の一端部に微粒子を分散した媒質を連続的に供
給し、供給エリヤ７の他端部から媒質のみを連続的に排
出し、連続的に微粒子の分離を行うことも可能である。
さらに、仕切部材２の小孔３に代えて、開き幅の狭いス
リットを用いてもよい。In the apparatus shown in FIGS. 1 and 2, a medium in which fine particles are dispersed is continuously supplied to one end of the supply area 7, and only the medium is continuously discharged from the other end of the supply area 7. It is also possible to continuously separate fine particles.
Further, instead of the small hole 3 of the partition member 2, a slit having a narrow opening width may be used.
第３図は、この発明の分離装置の他を示すもので、第１
図に示した基本型を多段に重ねたものである。FIG. 3 shows another separation device of the present invention.
The basic model shown in the figure is stacked in multiple stages.
この装置は、セル１内に４つの仕切部材４…が設けら
れ、これによって回収エリヤが第１の回収エリヤから第
４の回収エリヤ8a,8b,8c,8dに細分されている。また、
５つの電極4,4…が供給エリヤ７および各回収エリヤ8a,
8b,…のそれぞれにおいて分離用レーザ光Ａの光軸をは
さんで対峙した状態で設けられており、これらの電極4,
4…間には、図示のように抵抗R1,R2,R3,R4およびコンデ
ンサC1,C2,C3,C4を並列に接続して１つの電源11に接続
されている。In this device, four partition members 4 ... Are provided in the cell 1, whereby the recovery area is subdivided from the first recovery area into the fourth recovery areas 8a, 8b, 8c, 8d. Also,
The five electrodes 4, 4 ... Are provided by the supply area 7 and the recovery areas 8a,
8b, ... Are provided so as to face each other with the optical axis of the separation laser beam A interposed therebetween, and these electrodes 4,
Between the four terminals, resistors R 1 , R 2 , R 3 , R 4 and capacitors C 1 , C 2 , C 3 , C 4 are connected in parallel and connected to one power source 11, as shown in the figure. .
また、各回収エリヤ8a,8b,…には、それぞれ分取口9,9
…が設けられ、これに対応して分取用レーザ光源10,10
…からの分取用レーザ光B,B…が各回収エリヤ8a,8b,…
に入射するように構成されている。さらに、この例の装
置では各仕切部材2,2…間の間隙を数十μｍ程度と狭く
することもできる。Further, the collecting ports 8a, 8b, ...
... is provided and corresponding to this, the preparative laser light sources 10, 10
Preparative laser beams B, B from… are collected area 8a, 8b,…
Is configured to be incident on. Further, in the device of this example, the gap between the partition members 2, 2, ... Can be narrowed to about several tens of μm.
この装置では、電源11に直流電源を用いれば、各抵抗
R1,R2,…の抵抗値に応じた直流電圧がそれぞれ各電極4,
4…間に印加され、交流電源を用いれば各コンデンサC1,
C2,…の容量値に応じた交流電圧がそれぞれ印加され、
それぞれの抵抗値あるいは容量値を調節することによっ
て各電極4,4…間への印加電圧を調節することができ
る。勿論、電極4,4……間毎に別々に電源を接続しても
よい。In this device, if a DC power source is used as the power source 11,
The DC voltage corresponding to the resistance value of R 1 , R 2 , ...
Applied between 4 and each capacitor C 1 ,
AC voltage is applied according to the capacitance of C 2 ,.
By adjusting the resistance value or the capacitance value of each, the applied voltage between the electrodes 4, 4 ... Can be adjusted. Of course, a power source may be separately connected for each of the electrodes 4, 4.
この装置によれば、第１の回収エリヤ8aが次段の供給エ
リヤ７として機能し、第２の回収エリヤ8bがさらに次の
段の供給エリヤ７として働き、微粒子の分離が多段に行
われることになる。この際、後段に行くにしたがって微
粒子の移動できる条件を厳しく設定すれば、各段毎に微
粒子を分離することができ、先の第２図に示したものと
同様にこれらの微粒子を各段毎に分取することができ
る。According to this device, the first recovery area 8a functions as the supply area 7 of the next stage, and the second recovery area 8b functions as the supply area 7 of the next stage, so that fine particles are separated in multiple stages. become. At this time, if the conditions under which the fine particles can be moved are set severely in the subsequent stages, the fine particles can be separated in each stage, and these fine particles can be separated in each stage as in the case shown in FIG. Can be sorted into
このような微粒子の分離方法によれば、分離用レーザ光
Ａの光軸上での電界強度を正確に求めることができるの
で、高精度の分離が可能である。また、微粒しが光軸上
に閉じ込められて移動するので、媒質の対流や流動等に
よって微粒子の移動が乱されることがほとんどなく、外
部からの攪乱に対する許容度が大きいものとなる。さら
に、分離用レーザ光Ａの光圧力と電気力とによって微粒
子の移動が決められので、微粒子分離のための条件の選
択範囲が広くなり、多種の微粒子を分離でき、適用範囲
が広範囲となる。According to such a method of separating fine particles, the electric field strength of the separation laser beam A on the optical axis can be accurately obtained, and therefore highly accurate separation is possible. Further, since the fine particles move while being confined on the optical axis, the movement of the fine particles is hardly disturbed by convection or flow of the medium, and the tolerance to external disturbance is large. Furthermore, since the movement of the fine particles is determined by the light pressure and the electric force of the separation laser beam A, the selection range of the conditions for separating the fine particles is widened, various kinds of fine particles can be separated, and the applicable range is wide.
また、この発明の方法あるいは装置によって分離された
微粒子はセル１内の回収エリヤ８においてその分析を行
うこともできる。例えば、細菌、細胞などの場合にはこ
れらを予め蛍光色素で特異的に染色しておき、分離され
た細菌、細胞などからの蛍光を計測したり、あるいは吸
光度を測定したりして分析を行うことができる。また、
電気抵抗の変化からも微粒子の分析が行える。Further, the fine particles separated by the method or apparatus of the present invention can be analyzed in the recovery area 8 in the cell 1. For example, in the case of bacteria, cells, etc., these are specifically stained beforehand with a fluorescent dye, and the fluorescence from the separated bacteria, cells, etc. is measured, or the absorbance is measured and analyzed. be able to. Also,
Fine particles can also be analyzed from changes in electrical resistance.
このように、この発明は、タンパク、細胞、細菌、血
球、ウィルスなどの分離はもとより、ポリマーラテック
スなどの高分子微粒子やセラミック微粒子、金属微粒
子、ガラス微粒子などの分離、分級などにも適用でき
る。As described above, the present invention can be applied not only to separation of proteins, cells, bacteria, blood cells, viruses and the like, but also to separation and classification of polymer particles such as polymer latex, ceramic particles, metal particles and glass particles.
さらに、この発明の装置を無重力下あるいは微小重力下
（例えば、スペースシャトル，スペースラボなど）に設
置し、重力の作用しない場で分離操作を行うこともで
き、この場合、重力が無視できない比較的重量の大きな
微粒子の分離に有利となる。Further, the device of the present invention can be installed under zero gravity or under microgravity (for example, space shuttle, space lab, etc.) and separation operation can be performed in a place where gravity does not act. In this case, gravity cannot be ignored. This is advantageous for separating heavy particles.
以下、実施例を示してこの発明を具体的に説明する。Hereinafter, the present invention will be specifically described with reference to examples.
（実施例） 第１図に示した装置を作成した。セル１は厚さ1mmのガ
ラス板を貼り合わせて作り、電極4,4にはアルミニウム
膜を使用し、電極4,4間の距離を2mmとした。仕切部材２
には、厚さ100μｍのポリプロピレンからなるシートを
用い、開き幅が150μｍのスリットを形成した。分離用
レーザ光源６には出力可変型のアルゴンレーザを使っ
た。(Example) The apparatus shown in FIG. 1 was prepared. The cell 1 was made by laminating glass plates with a thickness of 1 mm, an aluminum film was used for the electrodes 4 and 4, and the distance between the electrodes 4 and 4 was 2 mm. Partition member 2
As the sheet, a sheet made of polypropylene having a thickness of 100 μm was used, and a slit having an opening width of 150 μm was formed. A variable output type argon laser was used as the separation laser light source 6.
微粒子として、径５〜８μｍのイースト菌を用い、これ
を純水に分散したものを分離用試料とした。Yeast bacteria having a diameter of 5 to 8 μm were used as the fine particles, and those dispersed in pure water were used as a separation sample.
供給エリヤ７側の電極４を陽極に、回収エリヤ８側の電
極５を陰極になるように直流電源に接続し、直流2Vを印
加した状態で、分離用試料をセル１の供給エリヤ７にマ
イクロピペットで注入し、純水を回収エリヤ８に供給し
た。ついで、イースト菌の挙動を観察するために、出力
10mW未満の弱いレーザ光を入射し、この時のセル１内の
状況をセル１の上方から顕微鏡を介してビデオカメラで
撮影し、観察した。The electrode 4 on the supply area 7 side is connected to the anode and the electrode 5 on the recovery area 8 side is connected to the DC power source so that the electrode 5 becomes the cathode. It was injected with a pipette and pure water was supplied to the recovery area 8. Then, output to observe the behavior of yeast.
A weak laser beam of less than 10 mW was incident, and the condition inside cell 1 at this time was photographed with a video camera from above cell 1 through a microscope and observed.
この映像を第４図に模式的に示す。図中矢印はレーザ光
の入射位置を示し、符号12はスリット、２は仕切部材、
７は供給エリヤ、８は回収エリヤを示す。図中供給エリ
ヤ７において一例にならんでいる多数の点状物Ｍは、イ
ースト菌にレーザ光が照射されて散乱した散乱光を示
し、イースト菌の存在を示す。したがって、実物のイー
スト菌よりも大きく映っている。第４図に示した状態で
はレーザ光が弱いので、イースト菌は電気泳動力で供給
エリヤ７にのみ存在し、回収エリヤ８に拡散してゆかな
いことがわかる。This image is schematically shown in FIG. In the figure, the arrow indicates the incident position of the laser beam, reference numeral 12 is a slit, 2 is a partition member,
Reference numeral 7 indicates a supply area, and 8 indicates a recovery area. A large number of spots M, which follow the example in the supply area 7 in the figure, represent scattered light scattered by irradiation of the yeast with laser light, indicating the presence of yeast. Therefore, it looks larger than the real yeast. Since the laser light is weak in the state shown in FIG. 4, it is understood that the yeast exist only in the supply area 7 due to the electrophoretic force and do not diffuse to the recovery area 8.
次に、印加電圧を1Vに降下するとともにレーザ光源の出
力を80mWに上昇し、分離用レーザＡとした時のイースト
菌の移動の状況を、レーザ光源の出力を上昇した時を起
点として４秒おきに撮影したものを第５図ないし第８図
に示した。第５図は時間０秒のものを、第６図は時間４
秒のものを、第７図は時間８秒のものを、第８図は時間
12秒のものをそれぞれ示す。Next, the applied voltage was lowered to 1V, the output of the laser light source was increased to 80 mW, and the movement status of the yeast bacteria when the separation laser A was used was taken as a starting point when the output of the laser light source was raised, and every 4 seconds. The photographs taken in Fig. 5 are shown in Figs. Figure 5 shows time 0 seconds, Figure 6 shows time 4 seconds.
Seconds, Figure 7 is time 8 seconds, Figure 8 is time
12 seconds are shown respectively.
時間０秒（第５図）では、イースト菌はまだ供給エリヤ
７に存在し、そのうちのいくつかがスリット12に近づい
ている。At time 0 seconds (Fig. 5), yeast are still present in the feeding area 7, some of which are approaching the slit 12.
時間４秒（第６図）では、イースト菌のうちの先頭のも
のがスリット12の間に入り、他のイースト菌もこれにつ
づいている。時間８秒（第７図）では先頭のイースト菌
がスリット12を通り抜け、時間12秒（第８図）には完全
に供給エリヤ８に移動している。そして、このイースト
菌の移動は分離用レーザ光Ａの光軸上で行われているこ
とがわかる。At a time of 4 seconds (Fig. 6), the first yeast among the yeasts entered between the slits 12, and the other yeasts also followed it. At the time of 8 seconds (Fig. 7), the leading yeast fungus passed through the slit 12 and completely moved to the supply area 8 at the time of 12 seconds (Fig. 8). Then, it is understood that the movement of this yeast is carried out on the optical axis of the separation laser beam A.
イースト菌に働くスリット12付近での電気泳動力は、移
動速度から求めると1.5×10-12Nであり、その向きは供
給エリヤ７側であった。また、分離用レーザ光Ａ（出力
80mW）の光圧力は7.5×10-12Nであった。The electrophoretic force in the vicinity of the slit 12 acting on the yeast was 1.5 × 10 −12 N when calculated from the moving speed, and the direction was the supply area 7 side. Also, the separation laser light A (output
The light pressure of 80 mW) was 7.5 × 10 -12 N.
この実験より、イースト菌の分離を数十秒の短時間で行
えることが確認された。さらに、イースト菌を１個ずつ
回収エリヤ８に取り出すことが可能であることも確認さ
れた。From this experiment, it was confirmed that yeast can be separated in a short time of several tens of seconds. Furthermore, it was also confirmed that it is possible to take out the yeasts one by one to the recovery area 8.
以上説明したように、この発明の微粒子の分離方法は微
粒子を分散した媒質にレーザ光を入射するとともに該媒
質にレーザ光入射方向と同一方向成分を有する電界をレ
ーザ光軸上に形成するものであり、またその分離装置は
セルと、このセル内の空間を、微粒子を分散した媒質を
収容する供給エリヤと分離された微粒子を回収する回収
エリヤとに分けるとともに、レーザ光を通過させる空隙
が形成された絶縁材料からなる仕切部材と、この仕切部
材の空隙に電気力線を通過させるように電界を印加する
電極を有するものであるので、微粒子を高精度で、かつ
短時間で分離することができ、しかも媒質の対流などの
外乱を受けることなく分離が可能である。また、微粒子
の種類として多岐にわたるものが適用可能であり、応用
範囲が極めて広いものとなるなどの効果を有する。As described above, the method of separating fine particles of the present invention is one in which laser light is made incident on a medium in which fine particles are dispersed and an electric field having the same direction component as the laser light incident direction is formed on the medium on the laser optical axis. In addition, the separation device divides the cell and the space inside the cell into a supply area that contains a medium in which fine particles are dispersed and a recovery area that collects the separated fine particles, and forms a gap through which laser light passes. Since it has a partition member made of an insulating material and an electrode for applying an electric field so that electric lines of force pass through the space of the partition member, it is possible to separate fine particles with high accuracy in a short time. In addition, the separation is possible without being affected by disturbance such as convection of the medium. In addition, various types of fine particles can be applied, which has an effect that the range of application is extremely wide.
第１図ないし第３図はいずれもこの発明の分離装置の例
を示す概略構成図、 第４図ないし第８図は実施例において観察されたイース
ト菌の移動の状況を模式的に示す説明図である。 １……セル、２……仕切部材、３……小孔、４……電
極、Ａ……分離用レーザ光、７……供給エリヤ、８……
回収エリヤ。1 to 3 are all schematic diagrams showing examples of the separation apparatus of the present invention, and FIGS. 4 to 8 are explanatory diagrams schematically showing the movement of yeast observed in the examples. is there. 1 ... Cell, 2 ... Partition member, 3 ... Small hole, 4 ... Electrode, A ... Separation laser light, 7 ... Supply area, 8 ...
Recovery area.
Claims (2)
るとともに該媒質にレーザ光入射方向と同一方向成分を
有する電界をレーザ光軸上で形成することを特徴とする
微粒子の分離方法。1. A method of separating fine particles, characterized in that laser light is made incident on a medium in which fine particles are dispersed and an electric field having a component in the same direction as the laser light incident direction is formed on the medium on the laser optical axis.
供給エリヤと分離された微粒子を回収する回収エリヤと
に分けるとともに、レーザ光を通過させる空隙が形成さ
れた絶縁材料からなる仕切部材と、 この仕切部材の空隙に電気力線を通過させるように電界
を印加する電極を有することを特徴とする微粒子の分離
装置。2. A cell and a space in the cell are divided into a supply area for containing a medium in which fine particles are dispersed and a recovery area for collecting the separated fine particles, and a gap for passing a laser beam is formed. A device for separating fine particles, comprising: a partition member made of an insulating material; and an electrode for applying an electric field so that lines of electric force pass through the voids of the partition member.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1191911A JPH0687953B2 (en) | 1989-07-25 | 1989-07-25 | METHOD FOR SEPARATING PARTICLE AND SEPARATOR |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP1191911A JPH0687953B2 (en) | 1989-07-25 | 1989-07-25 | METHOD FOR SEPARATING PARTICLE AND SEPARATOR |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH0356842A JPH0356842A (en) | 1991-03-12 |
| JPH0687953B2 true JPH0687953B2 (en) | 1994-11-09 |
Family
ID=16282499
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP1191911A Expired - Lifetime JPH0687953B2 (en) | 1989-07-25 | 1989-07-25 | METHOD FOR SEPARATING PARTICLE AND SEPARATOR |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0687953B2 (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4816906B2 (en) * | 2005-12-27 | 2011-11-16 | 独立行政法人産業技術総合研究所 | Particulate collection device |
| US8274654B2 (en) * | 2007-06-13 | 2012-09-25 | Shimadzu Corporation | Apparatus for measuring nanoparticles |
| WO2017069260A1 (en) * | 2015-10-23 | 2017-04-27 | 株式会社カワノラボ | Particle analysis device |
-
1989
- 1989-07-25 JP JP1191911A patent/JPH0687953B2/en not_active Expired - Lifetime
Also Published As
| Publication number | Publication date |
|---|---|
| JPH0356842A (en) | 1991-03-12 |
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