MICROFLUIDIC APPARATUS FOR SEPARATION OF PARTICULATES IN A FLUID

Abstract

An apparatus for separation of cells in a liquid into subsets of cells comprises a microfluidic chip comprising: a microfluidic channel having a liquid inlet for receipt of a stream of cell containing fluid; a detection zone disposed in the microfluidic channel and comprising a sensor configured to detect changes in the microfluidic channel corresponding to cells passing the sensor; and a separation zone distal of the detection zone in which the microfluidic channel divides into at least two secondary microfluidic channels, the separation zone comprising two or more separation electrodes, wherein the two or more separation electrodes include at least one separation electrode disposed in electrical contact with an interior of the microfluidic channel and at least one further separation electrode disposed in electrical contact with an interior of the microfluidic channel or one of the secondary microfluidic channels.

Claims

1. An apparatus for separation of cells in a liquid into subsets of cells, the apparatus comprising a microfluidic chip comprising: a microfluidic channel having a liquid inlet for receipt of a stream of cell containing fluid; a detection zone disposed in the microfluidic channel and comprising a sensor configured to detect changes in the microfluidic channel corresponding to cells passing the sensor; and a separation zone distal of the detection zone in which the microfluidic channel divides into at least two secondary microfluidic channels, the separation zone comprising two or more separation electrodes, characterised in that the two or more separation electrodes include at least one separation electrode disposed in electrical contact with an interior of the microfluidic channel and at least one further separation electrode disposed in electrical contact with an interior of the microfluidic channel or one of the secondary microfluidic channels, and wherein the pair of separation electrodes are configured during use to pass a pulse of direct current through the microfluidic channel.

2-36. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0088] FIG. 1 is a plan view of a microfluidic chip forming part of an apparatus of the invention showing the microfluidic channel, detection zone and separation zone having one pair of separation electrodes that span the separation zone in a longitudinal and transverse direction.

[0089] FIG. 2 is a plan view of a microfluidic chip forming part of an apparatus according to an alternative embodiment of the invention showing the microfluidic channel and separation zone having two pairs of separation electrodes.

[0090] FIG. 3 is a plan view of a microfluidic chip forming part of an apparatus according to an alternative embodiment of the invention showing the microfluidic channel and separation zone having two pairs of separation electrodes.

[0091] FIG. 4 is a plan view of a microfluidic chip forming part of an apparatus according to an alternative embodiment of the invention showing the microfluidic channel and separation zone having one pair of separation electrodes that span the separation zone in a longitudinal direction.

[0092] FIG. 5 is a plan view of a microfluidic chip forming part of an apparatus according to an alternative embodiment of the invention showing the microfluidic channel and separation zone having one pair of separation electrodes that are disposed proximal to the bifurcation of the microfluidic channel and in use generate an electrical field that is orthogonal to a direction of flow of sample fluid in the microfluidic channel.

[0093] FIG. 6 is a plan view of an apparatus of the invention showing the detection zone with lock-in amplifier, separation zone voltage/current generator.

[0094] FIG. 7 is a sectional view taken along the line A-A of FIG. 6 showing the detection electrode having an energising electrode below the microfluidic channel and a signal electrode disposed above the microfluidic channel.

[0095] FIG. 8 is graph of voltage V vs time of the output signal obtained from the comparator of FIG. 6.

[0096] FIG. 9 is a plan view of a microfluidic chip forming part of an apparatus according to an alternative embodiment of the invention showing the microfluidic channel which bifurcates into three channels and separation zone having two pairs of separation electrodes.

[0097] FIGS. 10a,10b, 11 and 12 are illustrations of microfluidic channels having secondary channels of different dimensions and architecture.

DETAILED DESCRIPTION OF THE INVENTION

[0098] All publications, patents, patent applications and other references mentioned herein are hereby incorporated by reference in their entireties for all purposes as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference and the content thereof recited in full.

Definitions and General Preferences

[0099] Where used herein and unless specifically indicated otherwise, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms might enjoy in the art:

[0100] This invention deals with instruments for counting, identification and separation of particles in particulate-containing fluids such as e.g. flow cytometry or particle flow analyser. For many common envisaged applications in this invention, the particles in the fluid are cells, and particulate containing fluid could be a suspension of mammalian cells, semen, blood cells, viruses, bacteria or indeed any other type of isolated particles. However, the technology could also be used with numerous other types of particles: organic, inorganic, ceramic, composite, nanoparticles, etc., both solid, semi-solid or liquid (e.g. protein particles, fatty particles, blends of soft particles and their compositions, drops of one liquid in the stream of another one, etc.). The solid particles could be particles of metals, oxides, nitrides, sulphides, polymer particles and particles of numerous other inorganic and organics materials, also mixed particles containing blends and composites of materials within individual particles and various nano- and micro-particles and clusters. We shall use the term “particle” or “cell” to cover any and all of these. We shall also use the same to describe clusters of particles/cells thus forming larger particle/cell. We shall call the liquid/fluid carrying particles in microfluidic channel as sample fluid, or particle containing fluid or particulate containing fluid.

[0101] The emphasis in the document is on technologies that can perform counting and identification of particles and/or cells in a microfluidic chip format. The said microfluidic chip has at least one microfluidic channel where such counting and identification takes place in the detection area (zone) of the channel. The preferred embodiment deals with the method of counting and/or identification of particles/cells based on AC electrical impedance spectroscopy. For each passing particle/cell, such counting and/or identification is normally done before the separation step.

[0102] The term “separation” of particles/cells is used to describe the process of physical separation of these, e.g. moving different subsets of the entire set towards different destination points. We will use the term “identification” of particles/cells to describe the process of attaining the information on the sub-set to which the particle/cell belongs to.

[0103] Unless otherwise required by context, the use herein of the singular is to be read to include the plural and vice versa. The term “a” or “an” used in relation to an entity is to be read to refer to one or more of that entity. As such, the terms “a” (or “an”), “one or more,” and “at least one” are used interchangeably herein.

[0104] As used herein, the term “comprise,” or variations thereof such as “comprises” or “comprising,” are to be read to indicate the inclusion of any recited integer (e.g. a feature, element, characteristic, property, method/process step or limitation) or group of integers (e.g. features, element, characteristics, properties, method/process steps or limitations) but not the exclusion of any other integer or group of integers. Thus, as used herein the term “comprising” is inclusive or open-ended and does not exclude additional, unrecited integers or method/process steps.

[0105] In the context of treatment and effective amounts as defined above, the term subject (which is to be read to include “individual”, “animal”, “patient” or “mammal” where context permits) defines any subject, particularly a mammalian subject, for whom treatment is indicated. Mammalian subjects include, but are not limited to, humans, domestic animals, farm animals, zoo animals, sport animals, pet animals such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows; primates such as apes, monkeys, orangutans, and chimpanzees; canids such as dogs and wolves; felids such as cats, lions, and tigers; equids such as horses, donkeys, and zebras; food animals such as cows, pigs, and sheep; ungulates such as deer and giraffes; and rodents such as mice, rats, hamsters and guinea pigs. In preferred embodiments, the subject is a human.

[0106] “Particulate” as applied to a particulate containing fluid means a solid body in the fluid or a semi-solid, i.e. a body with properties different to that of the fluid. Examples include particles of metals, oxides, nitrides, sulphides, polymer particles, particles of inorganic or organic materials, particles of gel, also composite particles, and mixed particles, nano-particles, microparticles, particulate complexes, cells, clusters of cells, bacteria, fungi, virus, clusters of viruses, particles of proteins. Likewise, “particulate containing fluid” means a fluid containing particulates. Examples include cell containing fluids, such as sperm containing fluid.

[0107] “Analysis” means determining a qualitative or quantitative characteristic of the particulates in the fluid, for example determining whether the particulates are a homogenous population or a heterogenous population, determining the amount or concentration of particulates, or differentiating or sorting the particulates based on differences. Thus, the term broadly covers analysis of the particulates (i.e. cells) qualitatively or quantitatively, or differentiation or sorting of the particulates based on detected impedance response differences.

[0108] “Cells” means any type of cell, including mammalian cells such as sperm, white blood cells, red blood cells, bone marrow cells, immune cells, epithelial cells, nerve cells, pulmonary cells, vascular cells, hepatic cells, kidney cells, skin cells, stem cells, or bacterial and fungal cells and hybridomas, yeast cells, cancerous cells. Generally, the particulate containing fluid contains at least two different types of particulates, for example different cell types, sperm of different sex, sub-populations of the same cell types, the same cell type having different phenotypes, dead and living cells, diseased and non-diseased cells, immature and mature cells of the same kind. The apparatus and methods of the invention may be employed to analyse and/or differentiate and/or separate these different types or phenotype of particulates/cells.

[0109] “Different phenotypes” as applied to cells means different populations of cells (i.e. hepatic cells and vascular cells), different sub-populations of the same cell type (i.e. different types of cartilage cells), different phenotypes of the same cell type (i.e. cell expressing different markers, diseased and healthy cells, transgenic and wild-type cells, stem cells at different stages of differentiation).

[0110] “X and Y population” as applied to sperm cells means male sperm and female sperm cells.

[0111] “Focused stream of particulate containing fluid” means a fluid containing particulates in the form of a focused beam of particulates positioned within a guidance stream. In one embodiment the particulates in the focused beam are focused into a single cell stream arrangement. In one embodiment, in which the particulates have an anisotropic shape, particulates in the focused beam are aligned in the same direction.

[0112] “Microfluidic chip” means a chip having at least one microfluidic channel having a cross-sectional area of less than 1 mm.sup.2 and a length of at least 1 mm. In one embodiment, the microfluidic chip has at least one microfluidic channel having a cross-sectional area of less than 0.25 mm.sup.2. In one embodiment, the microfluidic chip has at least one microfluidic channel having a cross-sectional area of less than 0.01 mm.sup.2. In one embodiment, the microfluidic chip has at least one microfluidic channel having a cross-sectional area of less than 0.0025 mm.sup.2. In one embodiment, the microfluidic chip has a plurality of microfluidic channels, for example at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 microfluidic channels. In one embodiment, the microfluidic chip has at least one microfluidic channel having a length of at least 1.5 mm. In one embodiment, the microfluidic chip has at least one microfluidic channel has a length of at least 2 mm. In one embodiment, the microfluidic chip has a length of at least 3 mm. In one embodiment, the microfluidic chip comprises a plurality of layers, for example at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 layers.

[0113] “AC impedance changes” should be understood to mean changes in impedance detected between the detection electrodes. The changes may include changes in amplitude, phase, or amplitude and phase of the signal.

[0114] “In electrical communication with the microfluidic channel” as applied to the electrodes means that the electrodes are in direct contact with the fluids analysed in the microfluidic channel.

[0115] “Separation zone” is a part of the microfluidic chip, distal of the detection zone, where particulates in the fluid can be separated in accordance with the results of the characterization of the particulates in the detection zone. The separation zone comprises at least one pair of electrodes, optionally two pairs of electrodes. One or more Voltage/current generators are provided and electrically coupled to the separation electrodes. The or each voltage/current generator is configured to provide a pulse of current across the microfluidic channel. Generally, at least one separation electrode is disposed in the separation zone proximal (upstream) of the bifurcation (splitting) of the microfluidic channel, and at least one separation electrode is disposed either proximal (upstream) of the bifurcation (splitting) of the microfluidic channel or distal of the bifurcation (i.e. in one of the secondary channels. Thus, the separation electrodes may be disposed on opposite sides of the microfluidic channel proximal of the bifurcation (i.e. FIG. 5). The separation electrodes may be disposed proximally and distally of the bifurcation point (FIG. 1, 2, 3, 4). The separation electrodes may be disposed on the same side of the microfluidic channel and proximally and distally of the microfluidic channel (FIGS. 3, 4). The separation electrodes may be disposed on the opposite side of the microfluidic channel and proximally and distally of the microfluidic channel (FIG. 2). One of the separation electrodes may be disposed on a side of the microfluidic channel proximal of the bifurcation and one may be disposed on a side |(inner side wall (FIG. 2) or outer sidewall (FIG. 3)) of a separation channel distal the bifurcation.

[0116] The term “anisotropic” refers to being not spherical in overall symmetry of particle's shape or its response to the stimulus used in the apparatus. In the simplest case, this refers to overall shape of the particle (cell). For example, if the particle is elongated, ellipsoidal, bar-shaped or disk-shaped, discoid, this is then described as anisotropic in contrast to a spherical shape particle that is being described as isotropic. However, the overall shape in its own right is insufficient to distinguish between anisotropic and isotropic particles (cells). For example, if a conducting rod (segment of wire) is embedded into an insulating sphere, this forms an anisotropic particle even if the overall shape of the particle is spherical, i.e. isotropic. The reason is that such a particle has different response to the Radio Frequency (RF) electromagnetic field depending on whether it is directed with the length of the rod along the field or perpendicular to the field. The main response to the RF field will be in this case from the metallic rod, this response will be highly anisotropic, the insulating spherical envelope will have little effect on the situation. The same applies to optical response: it will be different depending on the direction of the light incidence and the polarization with respect to the long axis of the rod, again the effect of the isotropic dielectric envelope on the optical response will not alter anisotropic response from the conducting rod. The same applies to the cells. The main contribution to RF signal response from a cell may not come from the exterior periphery of the cell but from its interior features. This depends on the structure of the cell and the RF frequency.

[0117] When referring to laminar flow regime, we shall imply the flow conditions that fall under the Stokes regime (−1<Re<˜1000). Re is the Reynolds number defined as Re=ρUH/μ, where μ, U and μ, are the fluid density, the average velocity and dynamic viscosity respectively and H is the characteristic channel dimension. In some cases the effect of particle focusing may still be achieved when Reynolds number is below 1 and therefore the invention is not restricted to the situation of <Re<˜1000. Generally the range of Re values at which the focusing is achieved, also depends on the difference between the density of the liquid and the density of the particles. The greater is the difference, e.g. the heavier are the particles compared to the liquid, the greater is the effective gravity force (difference between the gravity force and the buoyance force) pulling the particles down from the locations defined by the hydrodynamic forces. Therefore, the greater is the difference between the densities, the greater should be the force acting on the particles to achieve effective focusing of the particle's trajectories.

[0118] The term “separation of particulates in a fluid into subsets of particulates” should be understood to mean separating a type of particulate within a fluid. The fluid may contain just that particulate or more probably contains at least two subpopulations of particulates. The particulates may be cells, and may be separated into subpopulations based on phenotype, for example cell type, cell health (diseased, normal), viability (dead or alive), sex (X and Y subpopulations), level of differentiation (stem cells). The method employs an electric current to deflect specific cells, provided as a pulse. In one embodiment, the method of the invention employs at least two different current pulses, each configured to deflect a different subpopulation of particulate.

Exemplification

[0119] The invention will now be described with reference to specific Examples. These are merely exemplary and for illustrative purposes only: they are not intended to be limiting in any way to the scope of the monopoly claimed or to the invention described. These examples constitute the best mode currently contemplated for practicing the invention.

[0120] FIG. 1 describes a section of an embodiment of a microfluidic chip comprising the common microfluidic channel 2, detection area 5 and the separation area 16. There can be other optional elements of the microfluidic chip such as area where a number of channels containing sample fluid 3a and the guidance (sheath) fluid 4a merge into the single common microfluidic channel 2. Again, this arrangement is optional. One may operate microfluidic chip without any sheath fluid 4a, for example by filling the entire cross-section of the microfluidic channel with sample fluid 3a alone. Such embodiment is also covered by the present invention. The optional area where multiple channels, sample channel and guidance microfluidic channel, merge into a common microfluidic channel, is not shown in FIG. 1 and most other figures of this document for brevity. This optional area is located upstream from the detection area 5. In this embodiment, the common microfluidic channel 2 has a rectangular cross-section with the dimensions in the following range: width w 20 to 3000 micrometres, more preferably 20 to 500 micrometres and the height h in the range of 10 to 3000 micrometres, more preferably 20 to 500 micrometres. The width w is the dimension along the X axis and it is shown in FIG. 1 and the height h is the dimension along the Z-axis and it is not shown in FIG. 1. To simplify understanding of the invention it is best thinking in terms of a channel with rectangular cross-section. The cross-section of the common microfluidic channel 2 does not need to be square or rectangular, and channels with other cross-sections can also be deployed. The microfluidic chip could be also equipped with means for aligning anisotropic cells along a given direction. These means are not described here in further details. The chip may be equipped with a detection area where the particles/cell are identified. The detection area 5 is shown in FIG. 1 but it is not shown in some other figures. The detection area is normally located upstream from the separation area 16. The detection area 5 is equipped with detection electrodes 5a, 5b, 5c, 5d and optional shielding electrodes 8a, 8b, 8c, 8d to reduce the noise induced in the detection electrodes and improve signal detection. Such noise is manifested as voltage or current induced in the detection electrodes from various other electrodes of the microfluidic chip and various other sources of noise. The shielding electrodes 8a, 8b, 8c, 8d are connected to a fixed potential, e.g. a ground potential. The shielding electrodes are preferably in electric contact with the interior of the common microfluidic channel 2. One could also devise additional shielding electrodes on the microfluidic chip that are not in direct contact with the interior of the microfluidic channel. These mainly serve to shield the detection electrodes from the outside interference and induced voltage and noise, and are not shown in the figures of this document.

[0121] The detection electrodes normally have electric contact with the interior of the common channel but for some embodiments, especially operating at higher end of frequency range described in this document, the detection electrodes may also be electrically insulated from the interior of the common microfluidic channel. The detection electrodes 5a, 5b, 5c, 5d are connected to the detection circuit. Some detection electrodes are connected to AC voltage or current generators and others are connected to the AC voltage or current detectors. These are not shown in FIG. 1. We shall stress that some of the detection electrodes are connected to excitation AC voltage or current sources even though we attribute them to the set of detection electrodes; and other detection electrodes are connected to AC voltage or current meters/analysers. To differentiate between the two subsets of the detection electrodes, we shall call the detection electrodes connected to the AC voltage or current generator, the excitation electrodes; and the detection electrodes connected to the AC voltage current detector/meter, the signal electrodes. In the embodiment shown in FIG. 1, there are two excitation electrodes and two signal electrodes. They have the same dimensions and positioned just one under the other, one along the upper wall of the channel and the other one along the lower wall. The pair of electrodes which comprises one signal electrode and one excitation electrode is labelled a detection electrode.

[0122] The separation area 16 shown in FIG. 1 has the common channel 2 splitting into two secondary channels. The secondary channels 17a and 17b are terminated with two sample collection reservoirs. This splitting of the common microfluidic channel into secondary channels is also called bifurcation. There are two separation electrodes 16a and 16b along the walls of the common microfluidic channel and secondary channels: one of these is located along the wall of the common channel and the other one is located along the walls of one of the secondary channels. The two separation electrodes are connected to two voltage/current generators. The voltage/current generators are capable of supplying voltage/current pulse with the duration in the range from 1 microsecond to some 100 milliseconds and the amplitude in the range of 1 mV to 100 V. The repetition rate of the pulses is variable. Under normal operation one pulse is supplied per one cell guided through the separation area. However, this does not need to be always the case. One could supply a chain of shorter pulses of duration say 10 microseconds instead of a single pulse of duration of 1 millisecond. The timing of the pulses is coordinated with signals from the detection area 5. The electrical connection from the control circuit of the detection area to the voltage/current generators of the separation area, is not shown in FIG. 1. How this is done, will be well understood by those skilled in the art of electronics. We shall discuss the issue of the timing of triggering of pulses supplied to the separation electrodes, below. Preferably the particles/cells are travelling in the middle of the common microfluidic channel 2 and in the absence of any voltages applied to the electrodes, they could equally well go into the secondary channel 17a or 17b. The flow of particles/cells in the common microfluidic channel is shown as sample fluid 3a. The sample fluid flow is positioned in the middle of the common microfluidic channel 2 and it is flanked by the sheath fluid 4a on both sides. This is known as hydrodynamic focusing, a phenomenon well known to those skilled in the art of microfluidics. As will be appreciated by those skilled in the art of microfluidics, the flow of sample fluid from the common microfluidic channel into the secondary channels is defined by the flow impedance of the secondary channels 17a and 17b from the point of bifurcation of the common channel into the secondary channels to the terminal point of the secondary channels (i.e. sample collection reservoirs). In the case of symmetric split and with the particles/cells positioned in the middle of the common microfluidic channel as shown in FIG. 1, equal splitting of the flow between the two secondary channels is achieved when the two secondary channels have equal fluidic impedance.

[0123] We teach in this patent application that such a symmetric situation will be altered if there is a voltage/current pulse applied between the electrodes. When the voltage is applied between the separation electrodes 16a and 16b, there is current tangential to the flow direction that will introduce non-equivalence into the entrances from the common microfluidic channel and the secondary channel 17a and the same for the secondary channel 17b. This can be considered as additional fluidic impedance or pressure source inserted between the common microfluidic channel and one of the secondary channels. The non-equivalence is results from the following key contributions. Firstly, there is electroosmotic pressure arising in the channel and this pressure will alter the difference between the distribution of flows of liquid into the secondary channels 17a and 17b. Secondly, there is electrophoretic force acting on the particles/cells. This force arises from the ionisation on the particles/cells that results from their interaction with the liquid in which they are immersed. The ionised particle/cell interacts with the electric field between the electrodes 16a and 16b and this produces the force acting on the particle/cell moving predominantly into the secondary channels 17a and 17b. Thirdly, there is dielectrophoretic force. This force results from the fact that the electric field between the electrodes 16a and 16b is not homogeneous and there is a region of gradient electric field. The dielectric permittivity of the particle/cells differs from that the liquid surrounding it. Therefore, there is force acting on the particle/cell that is proportional to the gradient of the electric field and the difference in the dielectric constants of the particle/cell and that of the liquid around it. Depending on the electric permittivity of the cell and the liquid carrying the particles/cells and also on the polarity of the voltage applied between the electrodes 16a and 16b, the particles/cells will be pulled towards the secondary channel 17a or 17b.

[0124] The quantitative effects of these three contributions to the force are difficult to describe theoretically. They depend on properties of the liquid, its pH, electric properties of the particle/cell, configuration of the electrodes.

[0125] The exact balance of the forces contributing to the overall force should preferably be determined experimentally and can be performed by a person skilled in the art. However, it is important that in any event, the introduction of the electric current between the electrodes as shown in FIG. 1 starts diverting cells into one of the two secondary channels, at the expense of the second secondary channel. Since all these contributions for the force described above scale up with value of the voltage applied between the separation electrodes, and in most cases they are proportional to the voltage, the net force also scales up with the voltage. The direction of the force is reversed once the polarity of the voltage is altered sending the particles/cells into the other secondary channel. Lines of the electric fields are also shown schematically.

[0126] The voltage/current pulse should be short enough and coordinate with the time of arrival of a specific cell that has been measured in the detection area 5, to the separation area 16.

[0127] FIG. 2 shows embodiment with four separation electrodes 16a, 16b, 16c and 16d. In this case one could apply voltage between the two sets of separation electrodes simultaneously of opposite polarities to enhance the force separating the particles/cells from the common microfluidic channel into one of the secondary channels. The two polarities of the voltage that favour the movement of particles/cells into the separation channels 17a and 17b are shown in FIG. 2. The polarities are shown schematically. As explained in relation to FIG. 1, the exact polarity depends on the properties of the particles/cells and those of the liquid around the particles/cells. Preferably the correct polarity needs to be established/confirmed experimentally. This is straightforward experimental procedure that needs to be carried out once for each new untested set of particles/cells. However, it is preferable in this embodiment that the potential difference from the electrode 16a to electrode 16d is opposite to the one from the electrode 16c to the electrode 16b. It is also important that by reversing the polarity as shown in FIG. 2, we can change the choice of the secondary channel receiving the particles/cells from the common microfluidic channel.

[0128] One example of sets of voltages applied to the four separation electrodes is now described: electrodes 16a and 16c connected to ground potential, electrode 16d connected to positive potential +V and electrode 16b is connected to negative potential −V. Electric fields produced by this set of voltages are shown schematically in FIG. 2. To reverse the selection of flow between the secondary channels 17a and 17b, one can maintain the potentials of separation electrodes 16a and 16c fixed, i.e. still connected to the ground potential and reverse the potentials applied to the electrodes 16b and 16d connecting electrode 16d to a negative potential −V and electrode 16b to a positive potential +V. It is important to note that the voltages applied to the separation electrodes 16b and 16d do not need to be of the same value. For example, one could operate the separation device with the voltage of +10 V applied to the separation electrode 16b and voltage −5 V applied to the separation electrode 16d. In this case the separation force acting on the particles/cell will be smaller than in the case of identical voltage values: +10 V to the electrode 16b and −10 V to the electrode 16d. The electrodes 16a and 16c in FIG. 2 are shown connected to the ground potential. This does not need to be the case. These could be connected to another potential as will be appreciated by those skilled in the art of mapping electric field generated by metallic electrodes. They could also be connected to different potentials.

[0129] FIG. 3 shows embodiment with four separation electrodes positioned in a somewhat different way around the channel bifurcation. These are connected to another set of potentials to achieve broadly similar effect of the forces to the one in FIG. 2. In this case the electrodes 16a and 16c are connected to the same potential V. To achieve transfer of particles/cells into one of the secondary channels, the electrode 16d is connected to the ground potential and the electrode 16b is connected to the same voltage V as the electrodes 16a and 16c. Those skilled in the art will appreciate that the voltage connected to the electrode 16b does not need to be the same as the one of electrodes 16a and 16c. This is given as a particularly simple specific example of energising the electrodes. To send the particles/cells into the alternative secondary channel, we reverse the connections of the electrodes: the electrode 16b is connected to the ground potential and the electrode 16d is connected to the voltage V.

[0130] FIG. 4 shows another embodiment of microfluidic chip, more specifically section of the chip at the point of bifurcation. Other sections of the microfluidic chip are not shown. This embodiment has just two separation electrodes 16a and 16d. The voltage of one of the electrodes is fixed. For example, separation electrode 16a could be connected to the ground potential with the second separation electrode 16d is connected to potential +V or −V for sending the particles/cells to one secondary channel or the other.

[0131] What is common is all these configurations is that all of them create electric field and current that is significantly non-collinear with the direction of the fluid flow in the common microfluidic channels in proximity of the point of bifurcation of the common microfluidic channel into a number of secondary channels.

[0132] The voltage values applied to all these electrodes do not need to be constant in time. In a typical operation the voltages applied to the electrodes switch on and off or alter between the set values in order to achieve switching of the flow between different secondary channels. This is explained in further detail using FIGS. 6, 7, 8.

[0133] FIG. 5 shows another embodiment having two separation electrodes 16a and 16c upstream of and in close proximity to the point of bifurcation. The current between the separation electrodes is perpendicular to the flow direction in the common microfluidic channel. This is shown schematically using lines of the electric field. To displace the particles/cells for subsequent migration into the selected secondary channel, one of the separation electrodes needs to be connected to a positive potential with respect to the other one and vice versa. The direction of the electric field in FIG. 5 is given for the case when the top electrode has higher potential than the bottom electrode.

[0134] The voltage applied to the electrodes can be switched on and off on demand. In most preferred embodiments the voltage to the electrodes is supplied in the form of pulses of fixed shape and amplitude: one pulse or one fixed train of pulses is supplied to direct a single particle/cell. In the typical embodiment, the generator/generators supplying the voltages to electrodes are controlled from the output of the circuit detecting the particles/cells. This is shown schematically in FIG. 6. The microfluidic chip shown in FIG. 6 has detection area 5 and separation area 16. The separation area 16 is similar to the one described in relation to FIG. 5 and it is located downstream of the detection area 5. There are two pairs of detection electrodes shown in the detection area. Each of these detection electrodes is composed of one excitation electrode and one signal electrode. The two excitation electrodes and the two signal electrodes in each pair of detection electrodes are positioned at opposite sides of the microfluidic channel and are electrically coupled with each other via the fluid contained in the channel. This coupling can be of resistive- or capacitive nature but more generally, with AC coupling it has a combined resistive-capacitive character. In the embodiment of FIG. 6, the two excitation electrodes 5a and 5c belonging to the group of detection electrodes, are connected to the same AC potential: voltage sources generating voltage in the range of 0.001-50 V and the frequency of 10 kHz-200 MHz.

[0135] The positions of the electrodes are more clearly shown in FIG. 7 that is a cross-section of FIG. 6 through the line A-A′ along a vertical plane perpendicular to the flow direction, i.e. in the ZX plane of FIG. 6. The other two detection electrodes, signal electrodes 5b and 5d, are electrically connected to two pre-amplifiers: pre-amplifier 13 and pre-amplifier 13′. In the simplest configuration, the two signal electrodes have identical dimensions, identical areas of contact with the interior of the common microfluidic channel, and are positioned at the same distance from the flow of the sample fluid 3a within the cross-section of the microfluidic channel, and the gains of the two pre-amplifiers are also identical. The signals from the preamplifiers are sent to ADCs and then digitised signals are processed using phase-sensitive detector/demodulator/lock-in amplifier.

[0136] The detection electrodes 5a and 5b detect the change in AC complex impedance between them (between the excitation electrode and signal electrode) resulting from the particles/cells passing through the detection area in between the excitation electrode/electrodes and the signal electrode/electrodes.

[0137] Once a particle/cell passes in between a pair of detection electrodes, it obstructs the electric coupling between the excitation electrode and the signal electrode. This changes the voltage induced in the signal electrode. The amplitude and the phase of the voltage induced in the signal electrodes depends on the properties of particle/cell and therefore can be used as a key indicator in identifying the particle (cell) or the subset to which the particle/cell belongs. The detection of this signal is done using the method of demodulation with the help of the lock-in or phase sensitive detection as described in the state of the art section of this document.

[0138] The identification of the particle/cell position and alignment should be done in real time within the same time interval as the measurements using the demodulation of the AC impedance change described above.

[0139] In order to improve signal-to-noise ratio of the signals induced in the signal electrodes, it is beneficial to use differential amplifiers. For this, a single signal electrode is replaced by two electrodes: signal electrodes 5b and 5d. These are connected to inputs of pre-amplifiers: pre-amplifiers 13 and 13′. The outputs of the pre-amplifiers are connected to a comparator/differential amplifier 14 as shown in FIG. 6. The excitation electrode is also separated into two electrodes 5a and 5c; and these are positioned opposite their respective signal electrodes 5b, 5d. In this case the signals from the pre-amplifiers 13 and 13′ are introduced as inputs into the differential amplifier/comparator 14. The output from the pre-amplifiers is digitised by analogue-to-digital converter ADC1.

[0140] The comparator/differential amplifier 14 amplifies the difference between two inputs which are the outputs of the pre-amplifiers 13 and 13′. The output from the comparator is coupled to a lock-in amplifier or demodulator/phase sensitive detector capable of measuring the signal at the frequency ω of the AC voltage source connected to the two excitation electrodes. The generator connected to the excitation electrodes is not shown in FIG. 6. Let us consider the operation by referring to one pair of excitation electrodes (5a and 5c) and one pair of signal electrodes (5b and 5d). In the absence of any particles/cells in the microfluidic channel, the output from the comparator is set to zero if the outputs of the pre-amplifiers 13 and 13′ are set accurately to compensate for each other.

[0141] Once the particle/cell passes in between the excitation electrode and the signal electrode, it will induce signal in the comparator at the frequency ω of the generator connected to the excitation electrodes. The typical amplitude of the output signal from the comparator is shown in FIG. 8. The signal will appear as a wave of one polarity followed by the wave of the opposite polarity and the same magnitude. This shape of the signal is due to the cell/particle passing first in front of the signal electrode connected to the pre-amplifier 13′ and inducing signal of amplitude e.g. V1 that is introduced into the comparator with the positive polarity and then passing in front of the second signal electrode connected to the pre-amplifier 13 inducing rather the same signal V1 that is introduced into the comparator with a negative polarity. The time delay Δt between the maxima/minima of first hump (peak) and the second one is indicative of the flow velocity in the channel as it is equal to the time required for the same cell/particle to travel from the position in front of the first sensing electrode to the same position in front of the second one. The detection of this signal is done using the method of demodulation with the help of the lock-in amplifier or phase sensitive detection. The amplitude and the phase of the signal V1 are indicative of the characteristics of the particle (cell) and therefore can be used as a key indicator in identifying the particle (cell) or the subset to which the particle (cell) belongs. FIG. 6 does not clearly show the difference between the excitation electrodes 5a and 5c on one hand and the signal electrodes 5b and 5d on the other hand. The excitation electrodes and the signal electrodes have the same size and positioned one underneath the other and therefore are not distinguishable from a top view as shown in FIG. 6.

[0142] Once the particle/cell is identified/measured in the detection area, it is possible to determine the time required for it to travel from the detection area to the separation area. In FIG. 6, this distance is marked as L. Exact definition of the distance L is embodiment-dependent. In FIG. 6 this is the distance from the centre of the first detection electrode to the beginning of the separation electrode. The flow velocity is normally well known in the in the microfluidic chip. There are several methods of detecting it and one of these methods is described above in relation to FIG. 8. The flow velocity V.sub.flow is equal to V.sub.flow=I1/Δt where Δt is the time interval between the two maxima/minima of the peaks of FIGS. 8, and 11 is the distance between the centres of the two subsequent detection electrodes (FIG. 6). Therefore, the control circuit can establish the time required for the particle/cell to travel from the detection area to the separation area. With reference to FIG. 6, if the flow velocity is V.sub.flow, then the time required for the particle/cell to arrive to the separation area is Δt=L/V.sub.flow measured from the moment of the first peak shown in FIG. 8. Therefore, in order to direct the identified particle/cell into the correct secondary channel, we need to apply a pulse of voltage to the separation electrodes 16a and 16c at time interval Δt after detection of the first peak in the detection area. There may be some corrections to be introduced to take into the account the time processing the signal in the detection circuit. We will not expand this in further details as this is clear to those skilled in the art of signal processing. In FIG. 6 the separation voltage pulse is shown applied to the separation electrode 16a while the separation electrode 16c is shown connected to the ground potential. We discussed earlier with reference to the previous figures that there are many other options of such a connection.

[0143] As will be appreciated by those skilled in the art of electronics, the supply of the signal from the AC voltage generator into demodulator, lock-in amplifier/phase sensitive detector as the frequency reference, is important. This is not shown for clarity of the figures.

[0144] In a typical embodiment the width of the excitation and signal electrodes is in the range of 0.05 mm to 50 mm. The typical width of the separation electrodes is also in the same range of dimensions.

[0145] The detection circuit given in FIG. 6 is just one possible embodiment. This invention can be practiced regardless of the detection method. This can be practiced with numerous other varieties of the detection based on AC impedance measurements. This can also be practiced in conjunction with optical detection, based on scattering of light by the particles/cells, optical fluorescence detection and indeed other methods of detection. The time interval from the moment of detecting/identifying the particle/cell to the moment of actuating the separation electrodes should preferably be calculated using the known distance from the detection element to the separation area and the using the known flow velocity. If required, one may have to add correction to take into account the delay time for the operation of the detection circuit.

[0146] In relation to the frequency of the voltage source/sources, the signal induced in the signal electrodes by cells/particles passing in front of the electrodes is due to conductance change in the space between the excitations and sensing electrodes and also due to permittivity change in the same space. It should be appreciated that the dielectric permittivity ε(f) of a typical cell is frequency f dependent, and its conductance properties are also frequency dependent. These properties may also depend on other external factors such as pH liquid or temperature as the internal properties of the cells are affected by the conditions outside the cell. The conductance of the liquid bi-layer along the cell surface also depends on the pH of the liquid carrying the cells and its internal structure. It is preferable to choose the AC frequency f of the voltage energising the excitation electrodes such that the dielectric permittivity E at this frequency is significantly different from that of the liquid where the cells are placed. The dielectric permittivity is a complex function that has two components: the real Re ε(f) and imaginary Im ε(f) ones, both of these being functions of frequency. The same applies to conductance. It is preferable to operate the excitation electrodes at the frequency where there is significant difference between the conductance of the particles/cells and that of the liquid carrying these. Such a difference is responsible for the magnitude of the signal induced in the signal electrodes. Since cells have internal fine structure with elements of the structure having their own different dependencies of permittivity, there is generally a rather broad window of frequencies where the signal from the cells/particles can be readily detected. The frequency ω is usually selected in such a window by tuning the frequency to optimise the value of the signal induced in the signal electrodes.

[0147] The walls of the microfluidic common microfluidic channel could be made of plastic, glass or other materials, e.g. silicon. The walls of the common microfluidic channel are indicated on FIG. 6 and FIG. 7 with numeral 2. The detection and the separation electrodes are made of metal, these could be e.g. gold, copper, silver electrodes or these could also be made out of other metals that have special bio-compatibility properties and enhanced resistance to corrosion such as palladium, platinum, tantalum, titanium, etc. The thickness of the electrodes is in the range 0.1-10 micrometers. The electrodes could be also be composed of several layers, for example a sub-layer of metal A serving the purpose of enhanced adhesion to the walls of the channel followed by a layer B deposited on the surface of the layer A. These details are well appreciated by those skilled in the art of making film electrodes for electronic and electrical applications. The detection electrodes are usually open to the interior of the common channel 2 across their width of the electrodes to make sound electric contact with the fluid inside the channel. The shielding electrodes 8a, 8b, 8c and 8d are shown in FIG. 6 but not shown in most other figures of the document for brevity. They also make electric contact with the interior of the common microfluidic channel. The configuration of the shielding electrodes and their positions are important as they change the distribution of the electric potential along the microfluidic channel and the pattern of current distribution between the excitation electrodes and the signal electrodes. This pattern is defined by the distribution of potentials in between the excitation and the signal electrodes and therefore, once the distribution of potential changes, the distribution of currents between the excitation electrode and the signal electrode, will also change.

[0148] As a brief note of the AC impedance detection, the signals received at each signal electrode in general are complex signals. They are characterised by amplitude and phase or otherwise by real and imaginary parts of the signal. Such complex response arises from a complex circuit in between the excitation and the signal electrode: there is resistive and capacitive coupling between the electrodes. The cells are also characterised by a dielectric constant that has real and imaginary parts. The imaginary part is responsible for the losses in the cell under the influence of the AC electric field. The liquid carrying particles/cells also has real and imaginary dielectric constant. For a given configuration of the electrodes and given characteristic of the liquid and the particles/cells, the importance of the phase contained in the signal is dependent on the excitation frequency ω. There can be a window of frequencies where the phase characteristics of the signal collected from the signal electrode should not be neglected as it contains valuable information helping to identify the particles/cells. This information can be readily collected if the signal detection is done using phase-sensitive detector or lock-in amplifier.

[0149] The microfluidic device may also include means for sustaining and control of the fluid flow in the microfluidic channel, mixing the flows of the sample fluid and the sheath fluid, regulating the flows. The flow of fluids in the channels is sustained by a pump or multiple pumps or a pressure source/sources. This could be e.g. one or several UniGo pressure driven pumps from Cellix Limited, Dublin, Ireland. The pump/pumps are not shown in figures of this document for shortness as those skilled in the art of microfluidics are familiar with this aspect. There is a flow of the particle containing fluid 3a enveloped in the flow of the guidance fluid 4a. For shortness, we may call the particles-containing fluid also the sample fluid. We shall also refer to it as the liquid carrying particles/cells. Therefore, it is understood that the liquid carrying particles/cells and the particles/cells themselves form sample fluid. For example, this could be For example, this could be TRISA based buffer which is commonly used for holding sperm cells which might contain concentration of sperm cell from 0.1-100 million sperm cells per millilitre. The particle containing fluid is the fluid that contains the particles of interest. These could be organic or inorganic particles. These could also be alive or dead cells including mammalian cells, sperm cells, yeast cells, particles of biological and non-biological origin etc. In this embodiment, the particles-containing fluid stream (sample fluid) is located at the centre of the guidance fluid 4a flow, i.e. at the centre of the cross-section of the common microfluidic channel 2. Therefore, the guidance fluid 4a performs the function of the sheath fluid focusing the flow of the particle containing fluid into a tighter flow of reduced cross-section. One could construct other embodiments where the sample fluid is guided by the guidance fluid to be positioned not at the centre of the common microfluidic channel but e.g. at a corner of rectangular cross-section of the common microfluidic channel or along its wall and there are certain benefits of such a positioning. The guidance fluid also does not need to envelope the sample fluid all around but could be enveloped e.g. on two or three sides. All these embodiments are included in the present document. To obtain the focusing of the particles-containing fluid 3a by means of the guidance fluid, the microfluidic chip 1 comprises sample microfluidic channel 3 sustaining a flow of particles containing fluid and a guidance microfluidic channel or channels 4 sustaining a flow of guidance (sheath) fluid. Typically the flow rate of the guidance fluid is substantially greater than the flow rate of the sample fluid, i.e. greater by a factor of 2 to 100.

[0150] It will be appreciated by those familiar with hydrodynamic focusing that there is no sharp physical boundary between the sample fluid flow and the guidance fluid in the common channel. These two liquids gradually intermix by diffusion and under influence of other forces, along the common microfluidic channel as they move from the point of their mergence downstream. However, over the distance of 0.1-10 millimeters one could readily consider the flow as the flow of two fluids: the sample fluid enveloped by a guidance fluid. The guidance fluid 4a may be interchangeably called the sheath fluid in this document. The linear velocity of the liquid in the sample microfluidic channel and the guidance channel is in the range 0.01 meters/sec to 10 meters/sec. Those skilled in the art appreciate that the linear velocity varies strongly across the channel and normally is greatest at the centre of the channel at least for pressure-driven flows. This compression of the sample fluid by the sheath fluid through the laminar mixing of the streams in one common microfluidic channel, is known as hydrodynamic focusing. The focused flow of the particles containing fluid gradually becomes defocused due to the diffusive movement of the particles perpendicular to the flow direction. The detection area is located at short enough distance away from the point where the sample microfluidic channel merges with the guidance channels, at such a distance where the particles containing fluid still remains focused at the centre of the common microfluidic channel 2. This distance could be in the range 20 to 2000 micrometers.

[0151] We refer to particulates/particles throughout this document. This also includes clusters of particulatesparticles. The term cluster describes a small group of particles, for example 2-20 particles linked together by binding forces. For the purpose of this document we shall treat such clusters in the same way as single particles.

[0152] Figures of this document show excitation electrodes and signal electrodes making direct contact with the interior of the common microfluidic channel. This does not have to be always the case and one could construct embodiment where some or all of these electrodes are electrically insulated from the interior of the common microfluidic channel. This embodiment could more practical for operation at very high excitation frequencies, e.g. above 10 MHz.

[0153] The cross-section of the channel does not need to be rectangular or square. One could have circular, triangular, elliptical channel or indeed channels of various other cross-sections.

[0154] It is understood that cells addressed by the above description could be any live or dead cells, and non-mammalian mammalian cells, sperm cells, etc.

[0155] The use of hydrodynamic focusing is entirely optional. One may arrange the flow of sample fluid through the common microfluidic channel without any use of the sheath fluid.

[0156] All the embodiments shown in this document have separation electrodes positioned on two vertical walls. This is done so only for the clarity of the drawings. For example, with reference to FIG. 1, one or both of the separation electrodes could be positioned on the top of bottom wall of the common microfluidic channel or secondary channel, i.e. parallel to the plane YX. One could construct embodiment where some of the separation electrodes are positioned on the vertical wall (e.g. plane ZY of FIG. 1) and some other ones are positioned on the horizontal wall (e.g. plane YX of FIG. 1). The separation electrode does not need to cover the entire width or height of the wall of the channel. It could cover a fraction of the wall's width or height. It could also cover corner of two adjacent walls covered by separation electrodes across their entire width or height or partially.

EQUIVALENTS

[0157] The foregoing description details presently preferred embodiments of the present invention. Numerous modifications and variations in practice thereof are expected to occur to those skilled in the art upon consideration of these descriptions. Those modifications and variations are intended to be encompassed within the claims appended hereto.