Patent Grant
8388823
This invention pertains to methods and apparatuses to separate particles with different physical properties, and/or to measure the quantity or size of particles. The invention mainly finds application in the analysis and separation of biological samples.
The patent application PCT/WO 00/69565 to G. Medoro describes an apparatus and method for manipulating and locating particles using closed cages of dielectrophoretic potential. Besides this, PCT/WO 00/69565 teaches methods of separation. A first method is based on the differential action, on populations of different cells, of dielectrophoresis (DEP) negative (NDEP) and positive (PDEP), and does not permit discriminating particles if they are both subject to NDEP or PDEP. Other methods of separation instead require using information from sensors integrated in the device, and cannot be implemented when there are no such sensors.
Many other methods of separation are known that nevertheless require using flows of liquid, which is an undesirable characteristic in many applications, since it requires generating well controlled flows. A review of these methods, above all as concerns the use of dielectrophoresis is given in Electrophoresis 2002, 23, 2569-2582 M P. Hughes, and in Electrophoresis 2002 23, 1973-1983 P. R. C. Gascoyne, J Vykoukal, and in the related bibliographical references.
In particular, the patent U.S. Pat. No. 5,888,370 to Becker et al. teaches among other things to use dielectrophoresis to determine the height of particles in a speed-profile of liquid flow, to which the separation of the particles follows.
Other methods of separation based on dielectrophoresis do not require using flows of liquid, but they utilize so-called Travelling waves (TWD). These methods are based on the application to an array of electrodes of sinusoidal voltages at suitably phase-shifted radio frequencies (e.g. 0, 90, 180, 270 degrees). The result is an electric field that travels in space at a speed (VTW) equal to the distance between the electrodes with the same phase divided by the period of the single sine voltage. The phase-shift between the polarization induced on the particle and the electric field that generates it causes a translation of the particle, at a speed of orders of magnitude less than VTW, and asynchronous with it. Accordingly it is not possible to maintain an accurate control on the position of the particles after separation, which may be desirable in many applications, for instance if it is wished to determine the quantity of separated particles by placing them above sensors.
The limitations of the known art are superseded by this invention that permits separating particles even if they are subject to the same force (e.g. NDEP), it requires no flow of liquid, and it permits keeping a control on the position of the particles during and after separation or measurement.
The invention pertains to a method and apparatus to separate particles using time-variable, non-uniform force fields. The force fields can be for dielectrophoresis (positive or negative), electrophoresis or electrohydrodynamic motion, characterized by a set of stable-equilibrium points for the particles.
The physical properties of the particles determine the speed with which they move, subject to the action of the force field. A first objective is to isolate the particles that are most affected by the force field, in other words the fastest particles. A second objective is to isolate the particles that are less affected by the force field, in other words the slower particles. A third objective is to separate in one direction the ones that are most affected by the force field and in the opposite direction the ones that are less affected. An additional objective is to indirectly quantify groups of particles of one type determining the response speed of groups of them or the size of single particles.
In one aspect of the method, the stable-equilibrium points of the field are translated in space at a speed substantially comparable to the speed of translation of the fastest particles in the sample so that only these follow by changing position, while the slowest particles are not affected.
According to the invention the translation of these points of equilibrium can also occur with varying speed, which is especially useful when this happens with periodic law on a field with spatial periodicity. In one aspect of the method, the points of equilibrium of a force field with spatial periodicity are translated in a first direction at high speed, for such a period of time as to cause a movement equal to the spatial period of the field, and at low speed in a second direction, opposite to the first one, for such a period of time as to cancel the overall movement of the field, causing the translation of the slowest particles in the second direction and no movement of the fastest particles.
In an additional aspect of the method, the stable-equilibrium points of the field are translated in a first direction at high speed, for such a period of time as to cause a movement equal to the spatial period of the field minus a quantity corresponding to the period of the electrodes generating it, causing the translation of the fastest particles in the first direction and of the slowest particles in the opposite direction.
In another aspect of the method the particles, subject to the action of the force of gravity, suffer a different attenuation of the effects of the forces applied such as to induce a variation on the speed of movement, therefore determining the separation of the particles.
In another aspect of the method, the particles are grouped by the force of dielectrophoresis and their different response speed to translations of the field is utilized to indirectly determine the quantity of particles in the group or the size of the particles.
Another aspect of the invention is a device that permits advantageously producing some of the aforesaid methods, composed of just two array of electrodes to which different varying-time potentials are applied.
Hereinafter, the term particles will be used to indicate micrometric or nanometric, natural or artificial entities, such as cells, subcellular components, viruses, liposomes, niosomes, microbeads and nanobeads, or even smaller entities such as macro-molecules, proteins, DNA, RNA, etc, as well as drops of liquid immiscible in the suspension medium, for example oil in water or water in oil.
The purpose of the invention is to create a method and an apparatus for the separation of particles according to their physical properties or their quantification or the determination of their size. The method is based on using a non-uniform force field (F). This field can for instance be a field of dielectrophoresis (DEP) negative (NDEP) or positive (PDEP), an electrophoretic field (EF) or a field of electrohydrodynamic motion (EHD).
Generation of the Force Field
The field can have a spatial periodicity (CPIT) that is a multiple of the periodicity of the electrodes (EPIT), in the cases in which this can be defined.
Property of Separation
When the points of stable equilibrium of the force field (F) are moved by a step (EPIT) changing the configuration of the voltages (+, −) applied to the electrodes (EL), the particles converge toward the new point of equilibrium with a time that substantially depends on the ratio between the force of viscous friction and the force of the field (F).
It is moreover evident that the method is also effectively applied in cases in which the field acts with the same force on all the particles, but the viscous friction is different due, e.g. to the different material or shape of the particles.
The principle of the separation exploits the balancing between a force generated by the field (F), not uniform in the space and that moves in a controlled manner, and a force that acts uniformly. This can be the viscous friction, as described above, but also, for example, the force of gravity. For example, two particles on which the field (F) acts with the same force and that have the same viscous friction (for instance having the same diameter) would nevertheless be able to have different mass. In this case, tilting the device so that the force of gravity has a non-null component in the direction of movement of the force field (F) will cause the particles to have a different speed, so that it is anyhow possible to apply the method.
In general, one different physical property between the particles is enough to permit separation. The physical properties according to which the particles are discriminated can for instance be the diameter, shape, material of which they are made or the relative proportion and the arrangement of the component materials, or even the surface properties. In other cases they can include the electric charge. They can obviously also be combinations of the aforesaid properties. By way of example, but without restricting the purpose of the invention we can mention the separation of:
cells of the same type but of different dimensions;
cells of the same type but with different functional properties;
cells of the same dimensions but with a different composition of the cytoplasm;
apoptotic cells from vital cells;
cells infected with viruses or intracellular parasites from non-infected cells;
cells decorated with antibodies from non-decorated cells;
cells of different types:
liposomes of different dimensions but with the same content;
microbeads of different dimensions;
microbeads of the same dimensions but with different surface functionalities:
microbeads of the same dimensions but with different surface charge;
positively charged microbeads from neutral microbeads;
cells from viruses
viruses of different types
fragments of DNA of different molecular weight;
proteins with a different ratio between mass and charge.
Likewise to the above, separation can be done with the speed of translation of the point of attraction increased over time. In this case, illustrated for instance in
It is clear that the method also applies when the starting force field does not have a single point of equilibrium but a multiplicity of points.
The same technique of separation is practicable using continuous or discontinuous or piece-wise movements of the field of forces in space.
The speed of translation of the force field (F) may also be periodic. This is especially useful when the force field has spatial periodicity (CPIT). For simplicity we define the number pitch=CPIT/EPIT representing the period of repetition of the field expressed in the number of electrodes. Some aspects of the method are listed hereunder.
Translation of Fast Particles
In terms of time, a conventional critical time can be defined
The particles that take longer than this time to complete a step will return to the position of departure. In reality, the force along the X axis is normally not constant. Therefore also the limiting speed that is obtained when there is equilibrium with the viscous friction is not constant between WS and DEPC. Accordingly, the above formula is indicative and it must be empirically corrected or determined by means of numerical simulations to have a more precise value. The waiting time (tw), in which the field remains stationary to allow settling, in the original position for the slower particles (SP), in the new position for the fastest particles (FP), is indicatively equal to at least tw=αw(pitch−2)·tf, where the factor αw≧1 takes account of the fact that the speed of settling is typically lower than that during translation. Indeed, during translation the particles are for most of the time in regions of the field where the force is more intense, and they are therefore moving faster.
The total period is therefore tp=[pitch+αF·(pitch−2)]·tf. We are able from this to define the average speed of separation νsep=CPIT/tp=VF/[pitch+αF·(pitch−2)], at which the fast particles (FP) move in the period, that is those for which ν≧νws.
Translation of Slow Particles
Translation of Slow and Fast Particles in Opposite Directions
For a first time period (pitch−1)·tf the field translates in a first direction at a constant average-speed in the spatial period EPIT equal to VF=EPIT/tf, such as to cause total translation of the field equal to (pitch−1)·EPIT. For a second time period (tw) the field remains immobile, allowing the particles to reach a new point of equilibrium, varying its coordinate, with reference to the original position and the direction of translation of the field, of −EPIT for the slower particles (SP) and CPIT−EPIT for the fastest particles (FP),
In this case the critical speed that is distinguished is:
And the conventional critical time is tws=EPIT/νws=2·tf. The particles that take longer than this time to complete a step will return to the category of slow particles (SP), the ones that take less to the category of fast particles (FP).
The second time period in which the field remains immobile to allow the slow particles to reach the new point of equilibrium must be greater than tw=αw(pitch−1)·tf, where the factor αw≧1 takes account of the fact that the settling-time to reach the new point of equilibrium starting from the coordinate WS is typically greater than the time required from a point of equilibrium to WS, as discussed above. In this case, too, the real value of tw determines a further limit. Particles that are excessively slow that are not able in this time to recover the point of equilibrium at −EPIT lose their synchronism.
Since the above described translation should be made a number of times equal to pitch to complete a translation equal to the period of the field, the period of the cycle of separation can be defined as
tp=pitch·[(pitch−1)·tf+tw]=pitch·[(1+αw)(pitch−1)·tf]
In this period the slow particles translate by DXSP=−pitch·EPIT and the fast ones by DXFP=pitch·(pitch−1)·EPIT. The speed of separation is
and for the fast particles (FP) and
for the slow particles (SP).
It is evident to experts in the sector with ordinary capacities that other methods can be realized, according to the invention, as a combination of the above methods. For instance the periodic translation of fast particles in which, in the period, the field translates by a multiple of the spatial period of the field. Or the repetition in sequence of the separation with periodic speed of fast particles, but for speed (VF) of the force field decreasing, so as to segregate the particles of the initial population in a series of groups, where each group is characterized by a speed interval.
It is moreover evident that it is also easy to generalize, as concerns the case of NDEP, the above described methods to cases in which the dimensions of the cage are multiples of a single electrode (e.g. the cage is generated by two adjacent electrodes in phase (−) with the cover, and surrounded by electrodes in the opposite phase (+), instead of by a single electrode).
It is lastly evident that the above described case relating to a translation of the force field (F) can easily be generalized to other types of movement, such as for instance a rotation, as could be useful for symmetrical array of electrodes in relation to a point (instead of an axis). For simplicity nevertheless reference will be made, also in the continuation of the description, to translations.
Method of Separation with Non-Uniform Voltages
In addition, it is possible to vary the intensity of the voltages applied to the electrodes during the translation. This generally involves a variation in the intensity of the field, that nevertheless maintains the same configuration for the force lines. For instance, it is possible to translate the field (F) at a constant speed to separate the fast particles, but to decrease its intensity during the time, so that only the more responsive particles (FP) follow the field, while those (SP) less affected by the field, in relation to the force of viscous friction, remain behind and lose their step. One example is given in
Method of Separation with Non-Uniform Array of Electrodes
In the above examples we have considered a regular array of electrodes, of equal width, with spatial periodicity EPIT. Another aspect of the method according to the invention covers the case in which the array of electrodes is irregular. For example, it is possible to use electrodes of fixed dimensions but with a variable distance between the electrodes, or electrodes of varying dimensions with the same distance between the electrodes or variable dimensions both in their width and for the distance between the electrodes.
In these cases, besides varying the position of the points of equilibrium of the force field, its intensity varies during the translation, and in general the shape of the field also varies, that is the lines of force are not only translated but distorted too.
Likewise to the descriptions for the preceding methods it is possible to separate the particles that substantially follow the translation of the field from those that are not able to keep step, or, by exploiting the non-homogeneity of the field, particles of different diameter can be separated by pushing them both toward the same direction until the field deforms so much as to no longer be able to transport just one of the two types of particles. It is moreover evident that the same method of separation can be applied to the generic case of the separation of more than two types of particles.
Method of Separation with Only Two Field Configurations
In the above examples we have considered a succession of configurations of forces in the space; the minimum number of configurations of forces, necessary to permit the movement of particles in a specific direction and therefore their separation, is two. Indeed, as shown in
According to this method it is possible to separate particles of at least two types (FP, SP), so as to obtain a net movement of only said particles of a first type (FP) by changing the configuration of the field of forces at a frequency substantially comparable to the inverse of the settling-time of the particles of a first type (FP) at the new point of equilibrium, but greater than the inverse of the settling-time of the particles of at least a second type (SP).
According to this method it is also possible to separate particles of at least two sizes (BP, LP). In fact, when a particle (BP) is much bigger than the basin of attraction of a stable equilibrium point, so that it is subject to the attraction of more than one point of equilibrium, the particle does not move, since the resultant of forces is not enough to propel the particle (BP) toward either of the equilibrium points. Hence, to carry out the separation of small particles (LP) one can generate a first distribution in the space of a force field (F1) characterized by a first set of points of stable equilibrium (GSEP1) for said particles (BP, LP); and at least a second distribution in the space of a force field (F2) characterized by a second set of points of stable equilibrium (GSEP2) for said particles (BP, LP); wherein said two distributions of forces (F1, F2) are such that any first point of equilibrium (SEP1, SEP2) of one of said groups (GSEP1, GSEP2) is unequivocally included inside the basin of attraction of a single second point of stable equilibrium (SEP2, SEP1) of the other group (GSEP2, GSEP1), and that said second point in its turn is not contained in the basin of attraction of said first point (SEP1, SEP2); and that the size of the basin of attraction of each first point (SEP1, SEP2) is greater or less than the size of the basin of attraction of said single second point of stable equilibrium (SEP2, SEP1) containing it; and then can alternate the distributions (F1, F2) of said force field at a frequency comparable to the inverse of the settling-time of both types of particles (BP, LP), so as to move said particles (BP, LP) from one new point of equilibrium to the next one until the size of the particles is compatible with that of the basin of attraction. If the size of the basins of attraction gradually reduces, the big particles (BP) will in fact stop moving earlier than the small particles (LP), so as to obtain a net overall movement of the small particles (LP) greater than the net overall movement of the large particles (BP).
In the above methods it is an advantage that the density of the particles subject to the field is not such as to substantially affect the speed of translation of the single particles because of their mutual interactions. In fact, when grouped together, even small particles behave as larger particles, approximately with a volume equivalent to the sum of the volumes of the single particles, as shown in
According to a further method of the invention, illustrated in
Starting from a homogeneous sample with an unknown concentration of particles of known speed, the position of a group of particles is determined.
Then the point of stable equilibrium between two positions is moved, at known speed (Vfh), verifying whether the group responds to this change in the field with a corresponding movement of the rest position. If this is not so, the critical speed of the group of particles is evidently lower than the speed of movement of the point of stable equilibrium (Vfh). After a waiting period (tw), to allow the particles to settle at their initial point, the movement of the field is repeated at a lower speed (Vfl), until the particles are able to follow the movement of the rest position, which indicates that the critical speed of the particles is higher. Repeating the measurement so as to identify at least one speed of movement of the field greater than the critical speed and one lower, an interval is determined inside which the critical speed of the group of particles must necessarily be included, which corresponds to a specific range of quantity of particles. In this way, knowing the range in which the critical speed νws falls we can calculate the total volume of the particles in the group (with a certain approximation related to the width of the range) by using the ratio between volume and speed that is ideally linear. If this ratio is not linear, the ratio can be determined experimentally, producing a pre-characterization of the parameter subject of the measurement, that is the quantity of particles in the sample. The search for the critical speed can be made in various ways, for example with binary or linear searching.
The position can be measured with integrated or external sensors of the optical type, impedentiometric, etc., in accordance with the known art.
If the sensitivity of these sensors were not suitable to detect the presence of the initial quantity of the particles on an electrode, according to the invention the quantity should be increased, concentrating the content of several cages, to allow detection and exploiting the linear relationship or the pre-characterization to determine the measured quantity.
The above-described method for the quantification of particles can be applied in a similar manner to the problem of determining the size of particles. For this purpose, we act on groups of non-interactive particles or ones with negligible interaction between each other. In this case, as illustrated in
Apparatus for the Separation of Particles
The subject of this invention is also an apparatus to make appropriate field configurations that are necessary for the selective movement of particles; this device, in one of its typical implementations, has two array of electrodes (EL1, EL2) and, if necessary, a cover (LID); means to apply an electric potential (V1) to said first array of electrodes (EL1) and at least a second potential (V2) to said at least a second array of electrodes (EL2) are also included even if not shown because substantially of know kind; said electrodes and said means to apply an electric potential to said electrodes are operatively associated such that on the electrodes (EL1, EL2) appropriate stimuli (V1, V2) are applied to make at least two different spatial distributions of forces (F1, F2), each characterized by a group of points of stable equilibrium (SEP1, SEP2). A characteristic of these points of stable equilibrium is that each one is unequivocally included in just one of the basins of attraction of the stable-equilibrium points of the other configuration of forces. In this way the device subject of the invention permits trapping particles and moving them in a direction at a speed that depends on the speed with which the two configurations of forces are alternated and on the responsiveness of the particles. These distributions of forces can be made, for example, by utilizing electrodes of increasing or decreasing width and constant inter-electrode distance (
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| Number | Date | Country | |
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| Parent | 11651313 | Jan 2007 | US |
| Child | 11724697 | US | |
| Parent | PCT/EP2005/053235 | Jul 2005 | US |
| Child | 11651313 | US |
