Patent Application
20070221501
The invention relates to a method for collecting particles which are suspended in a liquid, in particular for collecting suspended biological objects, such as biological cells for example, in a fluidic microsystem, said method having the features of the preamble of claim 1. The invention also relates to a device for implementing such a method and to the uses thereof.
It is known to trap or collect particles which are suspended in a liquid in fluidic Microsystems in a dielectrophoretic field cage (see, for example, the publication “Trapping in AC octopole field cages” by T. Schnelle et al. in “Journal of Electrostatics”, vol. 50, 2000, pages 17 to 29). This technique has the disadvantage that only relatively large particles with typical dimensions >500 nm can reliably be trapped. In the case of smaller particles, such as viruses for example, the dielectrophoretic trapping forces may be too low or may be superposed by thermal distortions.
Using planar electrodes to which high-frequency AC voltages are supplied, electrohydrodynamic flows can be generated in a liquid-filled compartment by means of electroosmosis. In the publication “Optimizing Particle Collection for enhanced surface-based biosensors” (see “IEEE ENGINEERING IN MEDICINE AND BIOLOGY MAGAZINE”, November/December 2003, page 68), K. F. Hoettges et al. describe the use of circulating electrohydrodynamic flows to collect particles which are suspended in the liquid. In this method, as shown in
The technique described by K. F. Hoettges et al. has a number of disadvantages, in particular with regard to use in biology, biochemistry and medicine. The circulating eddy flow has a relatively small catchment area for the particles to be collected. Furthermore, the particles can be collected only immediately next to the electrodes. However, contact with the electrodes may be harmful for the particles, particularly if the particles comprise biological materials. Moreover, electrodes with a relatively large surface area are required in order to form suitably large collecting areas. However, undesirable heating occurs on electrodes with a large surface area. Finally, one significant disadvantage of the technique described by Hoettges et al. lies in the fact that said technique is based on electroosmosis and positive electrophoresis and therefore is restricted to low frequencies and low conductivities of the solutions used. It is therefore not possible to use this method to investigate cells in physiological solutions.
It is also known to guide viruses 1′ into the trapping area of a funnel-shaped, dielectric field cage 50′ by using electrohydrodynamic flows 30′, as shown in
Flows in fluidic Microsystems can also be induced by high electric field strengths (electric heating). However, this principle, which is used for example in traveling wave pumps in microchips (see the publication “A travelling-wave micropump for aqueous solutions: Comparison of 1 g and μg results” by T. Müller et al. in “Electrophoresis”, vol. 14, 1993, pages 764 to 772), may be disadvantageous for biological particles in particular, due to the conversion of heat.
The objective of the invention is to provide improved methods for collecting particles which are suspended in a liquid, in particular for collecting suspended biological objects, by means of which the disadvantages of the conventional methods are overcome and which in particular permit collection from a larger catchment area and without harm to the collected particles. Another objective of the invention is to provide improved devices for collecting particles which are suspended in a liquid, in particular for implementing the methods according to the invention.
This objective is achieved by methods and devices having the features of claims 1 and 25. Advantageous embodiments and applications of the invention emerge from the dependent claims.
In terms of the method, the invention is based on the general technical teaching of collecting suspended particles in at least one collecting area in a compartment with a circulating flow which runs at least partially along a longitudinal extent of at least one electrode on a lateral surface of the compartment. The collecting area is the volume into which the flow guides the particles and in which the particles may collect in particular due to a local reduction in flow. The circulating flow, which is generated according to the invention by an interaction of the liquid with high-frequency electric fields at the electrode, advantageously runs in a plane parallel to the respective lateral surface. The inventors have found that the limitation in terms of the effectiveness of collection in the conventional techniques can be overcome and the catchment area of the flow circulating at the electrode can be enlarged if the flow no longer circulates as previously about an axis parallel to the orientation of the lateral surface, but rather has a local axis of rotation perpendicular to this lateral surface. Another significant advantage of the invention consists in that even very small particles, such as viruses for example, can be effectively collected by the at least one flow.
The net liquid stream in the circulating flows is zero, since there is no source or sink in the collecting area and the liquid is incompressible. Nevertheless, a net particle stream from the outside towards the inside is observed. This can be explained by the fact that, due to negative dielectrophoresis, the particle concentration between the electrodes (liquid stream directed towards the outside) is lower than in the area surrounding the electrodes (liquid stream directed towards the inside).
If, according to one preferred embodiment of the invention, the particles are collected in the collecting area without any mechanical contact with a wall or any other part of the compartment, advantages may be obtained with regard to the manipulation of biological particles, such as biological cells for example, which in the event of mechanical contact would react with undesirable changes in state. However, if mechanical contact even is desired, then according to an alternative embodiment of the invention the particles can be arranged in the collecting area with contact with a lateral surface of the compartment. A measurement through a compartment wall can thus advantageously be simplified. Even if collection takes place with contact with the lateral surface, it is possible to prevent any contact with the electrodes and thus an undesirable electrode reaction, unlike in the case of the conventional techniques using electroosmosis. In this case, the collecting area can be formed by a part of the lateral surface in which the wall material of the compartment is exposed and no electrodes are present.
According to one particularly preferred embodiment of the invention, a number of locally circulating flows are generated at least one electrode, of which in each case at least one branch of the local circulation is directed towards the at least one collecting area. Two flows, for example, run along the electrode. This advantageously increases the effectiveness of collection.
Further increase in size of the catchment area for collection can advantageously be achieved if, according to one variant of the method according to the invention, a plurality of locally circulating flows are generated at a plurality of electrodes. This makes it possible in particular for the particles to be guided towards the at least one collecting area from a number of directions. If the flows relative to one another are designed to be symmetrical, in particular point-symmetrical, with respect to the collecting area in such a way that the latter contains a calm flow or is essentially free of flow, the situation can advantageously be achieved whereby the particles conveyed from one side to the collecting area do not leave the collecting area in another direction, e.g. at the opposite side.
Since, according to the invention, the flow is generated along the longitudinal extent of the respective electrode, the catchment area can advantageously be expanded by means of elongate or strip-shaped electrodes which preferably extend radially from the collecting area in different directions.
According to another advantageous embodiment of the invention, the particles are collected from a catchment area of the compartment which has a volume that is 102 to 109 times greater than the volume of the collecting area. This ratio indicates that the method according to the invention can be used not only to collect particles, but also to concentrate or accumulate them at a high factor. By way of example, the catchment area of a single eddy may have a volume of up to 10 μl and the collecting area may have a volume of from 1 femtoliter up to 50 picoliters, so that the invention can advantageously be implemented with fluidic microsystems.
According to one particularly preferred embodiment of the invention, high-frequency electric fields are also used to directly exert a predefined dielectrophoretic driving force on the particles. Under the effect of the high-frequency electric fields, the particles are moved towards the collecting area by means of negative dielectrophoresis. The indirect hydrodynamic force effect is advantageously further amplified as a result. It is particularly preferred if, according to the invention, high-frequency electric fields are generated which are used for electrodynamic flow generation and simultaneously for the dielectrophoretic manipulation of the particles.
The effectiveness of collection can be further increased if the high-frequency electric fields are used to generate at least one dielectrophoretic field cage with a potential minimum located in the collecting area. The dielectrophoretic trapping forces in the field cage are dependent on the particle size. Advantageously, particles which are so small that the trapping forces of the field cage would be too weak for effective collection can be bound by means of the electrohydrodynamic flows to form larger aggregates in such a way that field forces which are sufficient for reliable trapping in the field cage are achieved. According to the invention, the field cage is closed in two spatial directions (funnel-shaped field cage) or three spatial directions (field cage that is closed on all sides). The field cage can be formed with 6, 8 or more electrodes.
If, according to one advantageous variant of the invention, electrodes are arranged and supplied with high-frequency electric voltages in such a way that a plurality of field cages are formed, it is advantageously possible to further increase the size of the catchment area for particle collection according to the invention. Preferably an inner field cage and an outer field cage are provided, the potential minima of which occupy the same position in the collecting area. The field cages are arranged concentrically with respect to one another, wherein the respective outer field cage moves particles towards the inner field cage by means of negative dielectrophoresis.
It may be provided according to the invention that, in the collecting area, at least one further force acts on the particles. Additional holding and/or manipulation of the particles in the collecting area can thus advantageously be achieved. The generation of an optically active force may have advantages when combining the technique according to the invention with an optical measurement in the collecting area and for selective particle manipulation. The generation of a dielectrophoretic force may have advantages for effective cooperation with a dielectrophoretic barrier of the field cage. An additional magnetic force offers advantages when manipulating magnetic particles. Finally, the at least one further force may be a force generated by ultrasound, for example nodes of an ultrasonic field may be formed in the collecting area.
In order to exert a further force, the possibility further exists that a start object is located in the collecting area, e.g. a bead which can also be functionalized. Due to this start object, the particles are influenced not only by dielectric interactions, but rather also possibly by specific binding to the bead or hydrodynamic shielding brought about by the start object.
According to another preferred embodiment of the invention, in the collecting area, at least one measurement is carried out on the collected particles. This may bring advantages in particular when manipulating or evaluating collected biological particles. The measurement preferably comprises an electrical, electrochemical and/or optical measurement known per se for example from the field of fluidic microsystems.
According to one preferred application of the invention, the measurement is aimed at detecting a receptor/ligand binding event. For this measurement, according to the invention, the lateral surface of the compartment may be functionalized in the region of the at least one collecting area with detection spots in the form of receptor molecules (e.g. proteins, antibodies, DNA, viruses (for transfection experiments), etc.), as known per se from conventional microarrays or biochips, so that a specific receptor/ligand interaction with particles or molecules accumulated in the collecting area takes place. The interaction can then be detected in a known manner, e.g. by way of electrical, electrochemical or optical reading methods.
Advantageously, with the method according to the invention, the concentration of analyte particles or analyte molecules in the vicinity of the detection spots can be increased (increase in sensitivity) and the detection process can be accelerated compared to the purely diffusive transport of analyte particles or analyte molecules to the detection spots.
The functionalized receptor array can be applied for example to a flat electrode and, together with a second substrate containing the collecting electrodes, forms a microchamber. After accumulation of the analyte particles or analyte molecules by the method according to the invention and the binding of the same to the immobilized receptors on the array, the collecting structure can then be removed again. It can accordingly also be used multiple times.
If, according to a further modification of the invention, the particles are collected in a plurality of collecting areas in the compartment, advantages may be obtained in respect of parallel accumulation of the particles from a plurality of catchment areas in the compartment and parallel manipulation or evaluation of the collected particles.
For use in fluidic microsystems, one particular advantage of the invention is that collection can take place not just from a catchment area with a suspension liquid at rest, but even dynamically from a moving suspension liquid. The compartment may for example be passed through by a laminar flow which according to the invention is superposed with the locally circulating flow at the electrodes.
Furthermore, mutual superposition of a plurality of locally circulating flows may be provided in the compartment. A first circulating flow can guide the particles directly into a collecting area which forms part of a further circulating flow arranged downstream. This makes it possible to arrange a plurality of circulations in the manner of a cascade, in which particles are guided from an expanded catchment area into a single collecting area.
The method according to the invention is particularly suitable for collecting particles with a diameter of less than 1 μm. For biological applications, it is thus advantageously possible to collect in particular cells, viruses, bacteria, proteins, cell constituents and/or biological macromolecules, e.g. DNA.
According to further variants of the invention, it may be provided that the flows circulating locally at the electrodes are amplified by a local temperature gradient in the liquid. The temperature gradient may be formed by local heating of the liquid, which preferably takes place by exposing the liquid and/or lateral surfaces of the compartment to light and the corresponding absorption thereof and/or by means of thermoelements embedded (“buried”) in the walls. The temperature gradient may alternatively or additionally be formed by local, targeted cooling of the liquid.
The local heating of the liquid may advantageously also be used to initiate chemical reactions. The local high temperatures in the collecting area may in this case initiate e.g. heat-activated reactions, such as aggregation or precipitation.
In terms of the device, the abovementioned object of the invention is achieved by a collecting device for collecting suspended particles, which comprises, on a lateral surface of a compartment for holding a liquid, at least one electrode for generating one or more locally circulating flows in the liquid, by means of which suspended particles can be guided to at least one predetermined collecting area in the compartment, wherein the collecting device is designed to generate the at least one flow in such a way that part of the flow extends along the longitudinal extent of the electrode and the flow circulates about an axis which is oriented perpendicular to the respectively adjacent lateral surface with the electrode.
According to advantageous variants of the invention, the collecting area may be arranged at a distance from the lateral surfaces of the compartment or may be arranged in such a way that the collecting area is in contact with one of the lateral surfaces.
The electrode at which the at least one circulating flow can be generated is preferably connected to a voltage source for supplying predefined high-frequency electrical voltages. The at least one electrode which is used to generate the circulating flow is also referred to as the collecting electrode. When generating a plurality of circulating flows which are directed towards one or more collecting areas, the collecting device accordingly comprises a plurality of collecting electrodes, which form a collecting electrode array.
If, according to one preferred embodiment of the invention, the collecting device is designed to exert on the particles to be collected not just electrohydrodynamic forces but also dielectrophoretic forces, the effectiveness of collection can be improved by the additional force effect. The dielectrophoretic force effect is exerted by the interaction of the particles with high-frequency electric fields which are generated in the compartment by at least one electrode, which will be referred to hereinafter as the cage electrode. If the abovementioned field cages which are closed on one or all sides are to be generated, the compartment is equipped with a cage electrode array.
According to a particularly preferred variant of the invention, the collecting electrodes and cage electrodes are identical. The collecting electrode array and cage electrode array are formed by a common electrode arrangement. In this case, the structure of the collecting device and the activation of the electrodes is simplified.
One particular advantage of the collecting device according to the invention consists in the fact that it can be miniaturized. The compartment of the collecting device preferably forms part of a fluidic microsystem. The collecting function according to the invention can advantageously be combined with collecting, sorting, evaluation or measurement functions of the microsystem. The collecting device is arranged for example in the channel of a fluidic microsystem, which forms said compartment with the flow generator. Surprisingly, with the collecting device according to the invention, a collection of particles can also take place in the flowed-through channel.
In order to increase the collection activity, it may be provided according to one modification of the invention that a plurality of collecting areas are arranged in a row along a longitudinal direction of the channel.
Particular advantages for an expanded field of application of the collecting device are obtained when said device is equipped with a magnetic field device for exerting a magnetic holding force in said collecting area and/or with a measuring device for detecting electrical, electrochemical or optical properties of particles in the collecting area.
According to further variants of the invention, the flow generator may additionally comprise a heating device and/or a light source.
Further details and advantages of the invention will become apparent from the following description of examples of embodiments and from the appended drawings, in which:
The embodiments of the invention will be described below with reference to the application of the invention in fluidic microsystems for dielectrophoretic particle manipulation. Such fluidic microsystems, their components and their operating methods are known per se and will therefore not be described below. The invention will be discussed below by way of example with reference to a configuration in which electrodes are used both for collection and to exert a dielectrophoretic driving force, that is to say in which the collecting electrodes and cage electrodes are identical. It should be mentioned that the implementation of the invention is not restricted to this embodiment. Rather, according to the invention, collecting electrodes may be used exclusively to generate an electrohydrodynamic flow and not form part of a dielectric field cage, as illustrated for example in
Each electrode for electrohydrodynamic flow generation has the shape of a strip or band with a length (see also
Reference numeral 50 denotes a measuring device, for example a microscope with a CCD camera, by means of which for example fluorescence-marked particles in the collecting area can be optically measured and evaluated. To this end, at least one optically transparent window is provided in the lateral surface 11 of the channel (see
The cause of the electrohydrodynamic flow 30 is illustrated in
The temperature conditions of a liquid which is initially at rest in the compartment are shown in
Under the effect of the high-frequency fields in the compartment 10, dielectrophoretic forces are also exerted on the particles. The electric field conditions are accordingly illustrated in the right-hand part of
The voltage amplitude required to generate the electrohydrodynamic flow is selected as a function of the dielectric properties of the suspension liquid and the geometric properties of the electrode arrangement. It is also possible to provide for empirical selection by means of experiments. The high-frequency electric fields are preferably selected in such a way that only negative dielectrophoresis acts on the particles. The collection shown in
Under the following operating conditions, an accumulation of hepatitis-A viruses (diameter approx. 30 nm) could be achieved within 10 minutes. High-frequency AC voltages of frequency: 7.4 MHz, amplitude: 4 Vrms electrode gap: 5 μm. The initial concentration of the viruses in the compartment was approx. 109 to 1010/ml. The accumulation of fluorescence-marked hepatitis-A viruses for various observation times is shown in
Located outside the fluidic microsystem is a measuring device (not shown) by means of which the particles in the collecting areas 41, 42, 43, . . . are measured through a window 51 along a sampling line 52. For example, a fluorescence correlation measurement (FCS) takes place in order to detect receptor/ligand binding events in the collected particles.
A cascade-type combination of a plurality of circulating flows is illustrated schematically in
In the electrode arrangements shown in
According to a further modification of the invention, the collecting device may be equipped with a cooling device, e.g. a Peltier element, in order to prevent undesirable overall heating of the collecting device.
The features of the invention that are disclosed in the above description, in the drawings and in the claims may be of importance both individually and in combination with one another in order to implement the invention in its various embodiments.
| Number | Date | Country | Kind |
|---|---|---|---|
| 10 2004 023 466.3 | May 2004 | DE | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP05/04925 | 5/6/2005 | WO | 1/15/2007 |
