DE19853658A1 - Manipulation of biotic or abiotic particles suspended in fluid microsystem, useful for e.g. separation and aggregate formation of biological particles - Google Patents

Abstract

A process (I) for the manipulation of biotic or abiotic particles is new and comprises suspending the particles in a fluid microsystem which has closed ends that terminate the reference direction. A process (I) for the manipulation of biotic or abiotic particles is new and comprises suspending the particles in a fluid microsystem which has closed ends that terminate the reference direction. Particle speed relative to the microsystem, is adjusted by predetermined centrifugal and/or gravitational forces acting parallel to the reference direction. Particles are also exposed to deflection forces differing in direction from the reference direction. An Independent claim is included for the microsystem (II).

Description

The invention relates to a method for manipulating par particles in fluidic microsystems, in particular for movement of particles in microsystems along predetermined, at at least sections of straight tracks, and devices for Implementation of such a method, in particular a fluidic microsystem in which synthetic or biological Particles are manipulated in a suspension liquid, and applications of such a microsystem.

Fluidic microsystems with a structure through which fluid flows ren (e.g. channels), in which microelectrodes for influencing of particles (e.g. biological cells) in the flow Channels through high frequency fields based on negative or positive dielectrophoresis are used for example in the publication by G. Fuhr et al. in "Nature sciences "(Vol. 81, 1994, pp. 528 ff.).

Usually fluidic microsystems become liquid flows through to propel the particles. The one on both Channel longitudinal sides (top, bottom) applied microelectrodes lead to a compartmentalization of the channel using high frequency quenter electrical fields with which the suspended par article in the desired manner, e.g. B. via branches in Adjacent channels or other structural elements can be deflected can. Above all, the induction poses problems the particle at each channel end and the setting the generally low flow velocities (some µl / h), which is always serious with increasing miniaturization Bring restrictions.  

A general disadvantage of conventional fluidic microsystems is that of directed and adjustable particles movement requires a solution flow whose control problems (e.g. flow velocity).

From the publication by M. J. Madou et al. in "SPIE", volume 3259, 1998, pp. 80 ff. Is a centrifugal flow system known in which fluid flows in a microsystem not with conventional pumps and valves, but under the Effect of centrifugal forces can be set. For this be the microsystem is found in a disk-shaped carrier in the form of a CD-ROM disc. Analogous to the operation of CD Storage media, the carrier is provided with high Ge speed (in the range of 100 to 10,000 revolutions per Minute) to be rotated. The liquids in the microsystem move radially under the action of centrifugal forces outward. Simultaneously to this fluid movement certain biochemical reactions in the microsystem. It is also provided the liquid movement for particle transport, as in a conventionally pumped liquid flow use.

The centrifugal technique according to M. J. Madou et al. owns the following disadvantages. Both achieving a sufficient Fluid movement as well as a possible disability Entrainment of particles with the liquid in disc-shaped flat rotor inevitably require the high mentioned Speeds of the carrier. This results in a restriction of the conventional centrifugal flow system to certain Basic functions of conventional centrifugation or Achieve biochemical reactions. The above micro Electrode technology for the generation of high-frequency electrical Fields in the microstructures are not applicable. A wide one The disadvantage relates to the conventional Zen centrifugal technology realized particle sorting and  counts. These are only possible by using microchannels of a size that is the same size as that to be machined corresponding particle. This is a given microsy stem always limited to a certain particle size. Except that's what happens when handling biological particles (Cells, cell components) quickly to interactions between the particles and the channel wall leading to channel stop exercises.

The object of the invention is to provide an improved method for manipulating particles in fluidic microsystems specify the disadvantages of conventional microsystems be overcome and that an extended range of application owns. The object of the invention is also a verbes sertes fluidic microsystem with a directed particle Specify movement that is simplified and with high accuracy is adjustable. The object of the invention is also to apply to specify such an improved microsystem.

These tasks are performed using methods and devices 10 solved the features according to claims 1 and 10. Advantageous embodiments and application of the invention result from the dependent claims.

A first important aspect of the invention is in, deviating from the conventional centrifugal flow system coated with moving liquids to a procedure hen, in a fluidic microsystem under the we centrifugal forces only to manipulate particles are moved, essentially none Fluid flows or movements occur in the microsystem To this end, a series of measures are implemented that in particular the use of an at least one-sided ge closed fluidic microsystem, the attachment of a such microsystems on a vibratory rotor centrifuge  direction and operation of this centrifuge device with a predetermined speed at which the Parti move the microsystem in the desired way.

The method according to the invention enables centrifugation gears at low speeds. Because of the use of a Vibrating rotor system, in which a rotor as a carrier for the Microsystem from a vertical orientation (at standstill or low speeds) to a horizontal orientation (at high speeds), affect when losing weight the speed, the gravitational force, the movement particles in the microsystem. According to another Another aspect of the invention is particle movement in microsystems closed at least on one side, which is at a standstill with the mic oriented vertically systems. The particle movement takes place as Sedi mentation under the effect of gravitational force.

According to the invention, such microsystems, those with microelectrode devices for dielectrophoretic Influencing particle movement are equipped with the Combined principle of centrifugation. The suspended par particles move through the due to the centrifugal forces Microchannels or other microstructures in a microsystem, in which they (without being able to exit) under the effect of electri shear polarization forces z. B. separated into a previously fixed position brought, merged, sorted or by be avoided.

An important advantage of the invention is that Erstma lig with complex structured microsystems with dielectropho reticulate particle influence on the use of heavy controllable and fault-prone pumps or valves can be used without restricting functionality of the microsystem occurs. There are no restrictions in  Regarding the channel cross dimensions. There is a possibility speed, the microsystem simultaneously with the associated control electronics to set the tronics in rotation. Interactions of parti celn (especially biological particles) with wall areas of the microsystem can easily be avoided or else with appropriate structuring for the investigation of bin be achieved in a predetermined manner.

Details and further advantages of the invention are in the fol described with reference to the accompanying drawings ben. Show it:

Fig. 1 is a schematic perspective view of a structure of an OF INVENTION to the invention with a micro centrifuge system,

Fig. 2 is a schematic plan view of a microsystem according to the Invention, which is directed to a particle separation, and

Fig. 3 is a schematic plan view of a programmable loading microsystem according to another embodiment of the invention.

The embodiments of the invention described here relate look at the combination of a microsystem with a Microelectrode device for exercising negative or positive ver dielectrophoresis (dielectrophoretic Microsystem), with a vibratory rotor centrifuge device. Both the dielectrophoretic microsystem (apart from the duct structures can be closed at least on one side) as well as the vibratory rotor centrifuge device are each known per se, so that their technical details here is not discussed further. It is emphasized that the term the vibratory rotor centrifuge device here also in the broadest  The meaning is to be understood to mean that every centrifuge direction with at least one speed-dependent erectable Including the microsystem and the rotor associated control forms in which the microsystem and associated control integrated or on which the microsystem and the associated control are attached.

The particles manipulated according to the invention can be synthetic Particles or biological objects. The synthetic Particles are, for example, membrane-coated structures, such as Liposomes or vesicles, or so-called beads or also Macromolecules. The biological objects include, for example These are biological cells or components of these (e.g. cell organelles), bacteria or viruses. The particles can too Aggregates or agglomerations of such particles and / or Objects.

Fig. 1 is a schematic overview of an inventive device for illustrating the attachment of a dielectrophoretic system to a centrifuge device.

On a conventional or application-dependent modified rotor of a centrifuge with the axis of rotation 11, there are four recordings 12 , in each case a microsystem 15 and control electronics 13 for fitting and for the applied speeds correspondingly for controlling the microsystem with high-frequency alternating signals of different phase positions and amplitudes are used. The control electronics are connected to the microsystem 15 via cables 14 , plugs or otherwise. The control device is preferably supplied with power via an electrical connection (all-round contact) with the fixed laboratory system. The microsystem has an input depot 16 which, depending on the application, can be of different sizes and is filled with a particle or cell suspension before centrifugation. A channel structure runs from the input depot 16 , the details of which are explained below, up to collecting zones 17 a, 17 b, which form a closed end of the microsystem 15 at least during centrifugation. This means that the end of the microsystem can either be permanently closed or opened when the device comes to a standstill by means of appropriate connecting elements and connected to predetermined additional systems for sample transmission. The microsystem 15 is arranged on the receptacle 12 that when the centrifuge device is operating (rotation of the rotor about the axis of rotation 11 at the rotational frequency ω), the centrifugal forces acting on the microsystem 15 and the particles therein are in the reference direction from the input depot 18 are directed to the collecting zones 17 a, 17 b. The recordings 12 are pivotally attached to the rotor (not shown). When the centrifuge is at a standstill, the receptacle 12 is oriented essentially vertically or at a slight angle with respect to the axis of rotation. In centrifuge operation, the receptacles 12 are upright at a greater angle up to the horizontal orientation perpendicular to the axis of rotation 11 . Under the effect of gravity (when the centrifuge is at a standstill) or centrifugal forces, the particles pass through the electronically controlled microchannel system and collect in the collecting zones (e.g. at the closed end of the part of the microsystem pointing away from the rotor axis).

During this run, the particles are treated according to predetermined programs (see below). Since the particles perform various movements depending on their density and assume end positions, the advantage of centrifugal separation and movement is combined with the possibilities of programmable dielectrophoresis in the present invention. As a rule, negative dielectrophoresis is used, in exceptional cases also positive dielectrophoresis of the particles. Another advantage of the invention is the control of the particle movement via the rotational speed (ω) of the rotor 11 . Since programmable variations can also run through here, there is a second complex of definable parameters for particle manipulation.

The centrifuge device is provided with a speed control (not shown) which is set up for a reproducible and precise speed setting, particularly in low speed ranges. The speed is selected depending on the application, depending on the desired speed of the particles to be manipulated and depending on the specific centrifuge design. The particle speeds of interest for biological particles (e.g. cells) are below approx. 500 µm / s (preferably in the range of 50 to 100 µm / s) and for synthetic particles (e.g. latex beads) at higher speeds (e.g. a few mm / s). The speed of the centrifuge device is selected in accordance with the relationships between the speed and centrifugal force as a function of the size or mass density of the particles. For particle diameters in the range of 50 to 600 nm (e.g. viruses), the speeds can be in the range of 1 to 1000 rpm, for example. For particles with a diameter of approx. 5 µm speeds up to 100 rpm are preferred, although higher speeds can also be set. With particularly small particles, e.g. B. Macromolecules even higher speeds can be realized. For biological cells there is a distance of approx. 5 to 10 cm from the axis of rotation 11 speeds in the range from a few revolutions per minute to a few 100 revolutions per minute, preferably below 100 rpm. The centrifugal forces that can be achieved are in the range from pN to nN. However, the centrifuge device is also designed for higher speeds, which can be set in particular for small particles or for cleaning or rinsing purposes. These increased speeds can range up to the range of speeds of conventional laboratory centrifuges.

The speed of the centrifuge is also dependent on the dielectrophoretic forces selected on the particles act in the microsystem. The dielectrophoretic forces are as polarization forces depend on the particle type and size gig. The speed is preferably selected so that the Centrifugal forces on the particles less than or equal to dielectrophoretic forces are. If this is not known the speed can also be related to the following criterion rium can be selected. The particles have to move so slowly move through the channel structure that when entering the Microelectrode devices have enough time to dielectrophore table distraction remains. The effectiveness or ineffectiveness the dielectrophoretic deflection depending on the Speed can be optically or electrically with suitable sensors be recorded.

Fig. 2 shows schematically a microsystem for the separation of a particle mixture consisting of larger particles chen 21 (z. B. cells) and small particles 22 which are in a suspension. The centrifugal forces act in the direction of arrow 23 (reference direction). The typical dimensions of the channel structure 24 are as follows:
Width:
a few 10 µm to a few mm (typically: 200-400 µm)
Length:
a few mm to a few cm (typically: 20-50 mm)
Height:
a few µm to a few 100 µm (typically: 50 µm).

On the top 25 and bottom 26 of the channel 24 Mi microelectrodes 27 a, 27 b are arranged opposite, which generate field barriers across the channel when driven with an AC voltage (usually a frequency in the MHz range and an amplitude of a few volts) , which deflect the particles via negative (conditionally also positive) dielectrophoresis (in the case shown here the large particles).

The channel structure 24 extends from the input depot 28 to the closed channel ends 29 a, 29 b, into which the straight channel branches in a central section. A first pair of microelectrodes 27 a, 27 b is arranged directly at the channel-side end of the input depot 28 to form a field barrier, which protrudes obliquely into the channel and has the task of forcing the large particles 21 into the right part of the channel 24 in plan view . A second pair of the microelectrodes 27 a, 27 b is arranged immediately before the branching to the channel ends 29 a, 29 b and forms a field barrier that extends obliquely across the channel width into the branch leading to the channel end 29 b and is provided for this purpose, to guide the large particles 21 towards this channel end.

An inventive manipulation process that this Example is directed to a separation of the particles the following steps.

Before the centrifugation, the microsystem is filled with a suitable liquid. The microsystem is already installed in a receptacle 12 in the centrifuge (see FIG. 1). The installation can also follow after filling the microsystem. Shortly before the start of centrifugation, the electric 27 a, 27 b are driven and in the input depot 28 , for. B. added with a pipetting device, the suspension of the particles to be separated. The centrifuge device is initially still at rest, ie the microsystem is oriented vertically or slightly inclined to the vertical. The gravitational force that acts on the particles leads to a mass-dependent drop in the channel structure (sedimentation). Depending on the desired particle speed, the further movement of the particles towards the channel ends takes place exclusively under the action of the gravitational force or under the joint action of the gravitational force and the centrifugal forces. The centrifugation can thus be understood as sedimentation under the effect of an artificially increased fall acceleration. The moving particles are separated by the size of the electric field of the first pair of micro electrodes.

The illustration in FIG. 2 shows the conditions during sedimentation or centrifugation. Due to the precisely adjustable centrifugal forces via the rotation speed, the particles move into the lower part of the microsystem. According to the usual centrifugation principles, the particles with the highest density sediment first. Since the particles 21 are shifted to the right by the electrical field barrier in the channel, while the particles 22 remain unaffected by this, a separation of the two types of particles results in the channel ends 29 a, 29 b. The particles in each of the channel ends also arrange themselves according to their density, as in the usual centrifugation. The microsystem shown can be regarded as the basic form of a device according to the invention, which basic form can be enlarged, expanded or combined with other microstructures depending on the application. The advantage is that there is no solution flow and yet the particle movement is directed and adjustable. Such systems can also produce opposite movements if the particles have buoyancy.

Based on the basic form shown, a fiction appropriate microsystem can be expanded as it is  is known from the dielectrophoretic microsystems. The after the channel structure can in particular several, via Ver have branches interconnected individual channels. The Channels can be straight or curved. Curved channel shapes (e.g. arches, meanders, bends, angles, etc.) can in particular another for the investigation of bond differences of particles can be used with the channel walls.

According to a further modification, the microsystem can be rotatably attached to the receptacle 12 (see FIG. 1). During a first centrifugation process takes place in a first micro system orientation z. B. particle separation according to Fig. 2. Then, changing the orientation of the microsystem by 180 °, so that the gravitational and / or centrifugal forces opposite to the direction of the arrow 23 act. The channel ends 29 a, 29 b then take over the function of input depots, of which, in the presence of suitable channel structures (additional lateral branches), a further distribution of the separated particles into subgroups or a specific treatment (loading with substances, electroporation and the like. ) can be done. Depending on the channel structure, it is also possible to change the orientation other than the 180 ° reversal mentioned. There is also the possibility of designing the receptacle 12 in such a way that the microsystem is rotated during the centrifugation.

Another embodiment of the invention, namely a programmable loading microsystem for cells or particles, is shown in FIG. 3. Here the centrifugation channel is divided into three parts 31 a, 31 b, 31 c. Openings 32 are located in the intermediate walls, through which electrodes 33 on the top and bottom of the channel extend again. The openings are adapted to the particle size (typically 5 to 20 times larger than the diameter). At the beginning, different solutions are poured into each of the channel parts 31 a to 31 c, which serve to chemically change or load the particles. The particles are then inserted into a channel part (here, for example, 31c). By centrifugation, the particles (eg first the black, then the light ones) reach the electrodes 33 and can thus be automatically transferred through the openings 32 into the neighboring solutions via the electrical field barriers.

Here too there is a sorting in the three channel ends 31 d, 31 e, 31 f and at the same time an arrangement of the particles according to the mass differences.

Further properties of the microsystems are that they can have openings (inflows, flows, outflows) nen that can be closed, so that the particles after easily removed or inserted from centrifugation or before can be. Furthermore, all of the microelectrode elements can (Holding electrodes for particles, microfield cages etc.) installed that are used for the dielectrophoretic influence of Particles are known per se and in conventional microsy systems that work with flowing liquids become. Due to the interaction of the gravitational or This is centrifugal forces with the dielectrophoretic forces inventive method an electrically controlled or active centrifugation. In addition, combinations with the Effect of optical forces (laser tweezers), magnetic Forces (action on magnetic particles) or mechanical Forces in the form of ultrasonic forces can be provided.

Fields of application of the invention are in particular:

• - cell separation / fractionation,
• - cell sorting,
• - cell loading (molecular, nanoparticles, beads),  
• - cell discharge (molecular),
• Cell permeation (so-called electroporation),
• - cell fusion (so-called electrofusion),
• - cell pair formation, and
• - Cell aggregate formation.

The invention is not limited to specific solutions or suspensions limited fluids. It is advantageous if the Viscosity of the liquid contained in the microsystem is known is. If the viscosity is known, the speed can be turned on position of a certain particle velocity on the Based on table values or through a program algo determine rhythm. Alternatively, it is also possible to use the actual speed of the particles in the microsystem during centrifugation (e.g. with an optical Sensor) and the speed to set a specific par regulate particle speed. It can be provided that in different sections of the channel structures, e.g. B. in parallel channels that only have an opening with are interconnected, liquids with different vis costs are included. In this case, however, will be visco preferred, where it is ensured that the Diffusion of the liquids through the opening over the centri Migration period is relatively small or negligible is small.

If the mass density of the particles is smaller than the liquid speed in the microsystem, the invention can be abge accordingly converts to be implemented by particles if necessary on the side of the microsystem facing away from the axis of rotation brought and under the effect of buoyancy or under combined effect of buoyancy and centrifugal forces hike to the other end of the microsystem.  

The microsystem becomes application-dependent in terms of Channel structure and the alignment of the electrode devices customized. The channel cross dimensions are usually essential Lich larger than the diameter of the individual particles. There by clogging of the channels is advantageously ver avoided. Are only particles with particularly small dimensions manipulations (e.g. bacteria or viruses or cell or ganelles), the channel dimensions can be reduced accordingly be z. B. to amounts below 10 microns.

The invention is implemented with a microsystem that is closed at least on one side. The closed end can be a closed channel end, a closed collection zone or a closed cavity in the microsystem. At the particle manipulation according to the invention essentially takes place no liquid movement towards the closed The End. This means, especially when realizing Sam melzones or cavities at the closed end that these like the entire microsystem at the beginning of the particle manipulation the solution or suspension for the particles is filled.

If there is an aggregation when manipulating the particles or there is temporary blockage of the channel structures, so it is provided according to the invention, the speed of the centrifuge increase briefly so that the particles stick together detach and move on.

Claims (16)

1. A method for manipulating particles in a fluidic microsystem ( 15 , 24 , 31 ), in which the particles ( 21 , 22 ) are moved in a suspension liquid in a predetermined reference direction, characterized in that
the microsystem ( 15 , 24 , 31 ) is closed at least at its end lying in the reference direction ( 17 a, 17 b, 29 a, 29 b, 31 d, 31 e, 31 f), and
the particles move under the action of centrifugal and / or gravitational forces in the suspension liquid which is at rest with respect to the microsystem ( 15 , 24 , 31 ), the centrifugal and / or gravitational forces being essentially parallel to the reference direction, and the particles in the microsystem ( 15 , 24 , 31 ) are subjected to deflection forces whose direction deviates from the reference direction. 2. The method according to claim 1, wherein the microsystem ( 15 , 24 , 31 ) is attached to a Schwingrotorzentrifugeneinrichtung, the particle movement takes place when the Schwingro torzentrifugeneinrichtung as sedimentation under the effect of gravitational force and when operating the Schwingrotorzentrifu gene device under the action of centrifugal forces. 3. The method according to claim 2, wherein the deflecting forces elek trical polarization forces, optical forces, magnetic Forces or include ultrasound forces.   4. The method according to claim 3, wherein the speed of the Vibratory rotor centrifuge device is set so that the centrifugal forces on the particles smaller or equal than the deflecting forces. 5. The method according to claim 3, wherein the speed of the Vibratory rotor centrifuge device is set so that the particles move so slowly that under the effect of Deflection forces a deflection of the particles from the reference line tion takes place. 6. The method according to claim 3, wherein the speed of the Vibratory rotor centrifuge device depending on the an optical or electrical sensor particle is regulated. 7. The method according to any one of the preceding claims, in which several particle movements under the influence of centrifugal forces take place in separate centrifugation steps, whereby between the centrifugation steps an adjustment of the Microsystem to change the orientation in relation to the Centrifugal forces occur. 8. The method according to any one of the preceding claims, in which the speed of the vibratory rotor centrifuge device in dependency depending on the size or density of the particles. 9. The method according to any one of the preceding claims, in which the particles counteract under the influence of buoyancy forces set to the direction of centrifugal and / or gravitational move forces.   10. microsystem ( 15 , 24 , 31 ) with at least one channel that runs from an input depot ( 16 , 28 ) to channel ends ( 17 a, 17 b, 29 a, 29 b, 31 d, 31 e, 31 f), characterized in that the microsystem ( 15 , 24 , 31 ) is arranged for attachment to the rotor of a centrifuge in such a way that when centrifuged, the centrifugal forces acting on particles in the channel run essentially parallel to the channel orientation, and the channel ends ( 17 a , 17 b, 29 a, 29 b, 31 d, 31 e, 31 f) are closed or can be closed during centrifuge operation. 11. A microsystem according to claim 10, which is a microelectrode has device, the microelectrodes for generating Field barriers in the microsystem included. 12. The microsystem according to claim 11, wherein the microelectrodes arranged on opposite long sides of the channel and for the application of a high-frequency AC voltage are set up. 13. A microsystem according to claim 12, wherein the microelectrodes are band-shaped electrodes that are oblique to the channel alignment stretch and to create field barriers in the channel are set up. 14. Microsystem according to one of claims 10 to 13, which on Rotor of the centrifuge is pivotally attached. 15. Microsystem according to one of the preceding claims 10 to 14, in which an electronic control of the microsystem is attached to the centrifuge rotor. 16. Use of a method or a device according to any of the preceding claims for separation, fractionation tion, sorting, loading, unloading, permeation, fusion,  Pair formation and / or aggregate formation of synthetic particles and / or biological particles.