WO2009132151A2

WO2009132151A2 - Microfluidic devices and methods of using same

Info

Publication number: WO2009132151A2
Application number: PCT/US2009/041466
Authority: WO
Inventors: Hyongsok Soh; Jonathan D. Adams; Unyoung Kim
Original assignee: The Regents Of The University Of California
Priority date: 2008-04-23
Filing date: 2009-04-22
Publication date: 2009-10-29
Also published as: WO2009132151A3

Abstract

The present disclosure provides microfluidic devices and methods which facilitate high performance separation and/or purification of one or more target species from complex samples with high purity, recovery and throughput. The disclosed microfluidic devices and methods utilize one or more controllable forces to effect the separation and/or purification of the one or more target species.

Description

MlCROFLUIDIC DEVICES AND METHODS OF USING SAME

CROSS REFERENCE TO RELATED APPLICATION [0001] This application claims priority to U.S. Provisional Patent Application No.

61/047,412 filed April 23, 2008, which is incorporated herein by reference in its entirety and for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] The U.S. government has certain rights in this invention, pursuant to grant no.

N00014-08-1-0469 awarded by the Office of Naval Research.

BACKGROUND

[0001] The capability to separate biological targets from complex samples with high purity, recovery and throughput is essential for a wide range of bio technological applications in both diagnostics and therapeutics. Screening methods such as Fluorescence Activated Cell Sorters (FACS) are capable of sorting cells on the basis of multiple parameters with high purity and recovery. However, due to the fact that each target cell must be measured serially, their throughput is severely limited. In contrast, selection techniques such as magnetic activated cell sorting (MACS) allow efficient separation of magnetically-labeled target species with high throughput, but are limited to sorting applications based on a single selection parameter (i.e. magnetization).

SUMMARY OF THE INVENTION

[0002] The present disclosure provides microfluidic devices and methods which facilitate high performance separation and/or purification of one or more target species from complex samples with high purity, recovery and throughput. The disclosed microfluidic devices and methods utilize one or more controllable forces to effect the separation and/or purification of the one or more target species. These and other objects, features and advantages of the present disclosure will become more fully apparent from the following detailed description of the embodiments, the appended claims and the accompanying drawings.

[0003] In a first aspect, the present disclosure provides a microfluidic sorting device including at least one inlet channel configured to provide at least two separate streams. At least one stream defines a sample stream path for a sample suspected of containing one or both of a first target species and a second target species. The sample includes a first magnetic particle having affinity for the first target species, a second magnetic particle having affinity for the second target species, and a non-target species. At least one stream defines a buffer stream path for a buffer, wherein the buffer is substantially free of the sample.

[0004] The microfluidic device of the first aspect also includes a sorting station fluidly coupled to the at least one inlet and located in the sample stream path. The sorting station includes a first array of ferromagnetic elements and a second array of ferromagnetic elements.

[0005] The microfluidic device of the first aspect also includes a magnetic field gradient generator which produces a change in the magnetic field gradient in the sorting station such that the first array deflects the first magnetic particle into the buffer stream path at a first location and out a first outlet channel, such that complexes of the first magnetic particle and the first target species, if present, are deflected out the first outlet channel. As a result of the change in the magnetic field gradient the second array deflects the second magnetic particle into the buffer stream path at a second location and out a second outlet channel, such that complexes of the second magnetic particle and the second target species, if present, are deflected out the second outlet channel.

[0006] The microfluidic device of the first aspect also includes a third outlet channel configured to receive a waste stream from the sample stream path, such that sample that is at least partially depleted of a target species flows through the third outlet channel.

[0007] In one embodiment of the microfluidic device according to the first aspect, the at least one inlet channel includes a first inlet channel for providing at least a portion of the buffer stream and a second inlet channel for providing at least a portion of the sample stream.

[0008] In one embodiment, the ferromagnetic elements are micropatterned nickel elements.

[0009] In one embodiment, the first and second arrays of ferromagnetic elements include ferromagnetic strips. [0010] In one embodiment, the ferromagnetic elements include one or more pins or pegs.

[0011] In one embodiment, the device includes the sample. In one such embodiment, the sample contains at least a first complex including the first target species and the first magnetic particle or at least a second complex including the second target species and the second magnetic particle.

[0012] In a second aspect, the present disclosure provides a method of separating two or more target species in a sample. The method includes introducing a sample into the flow path of a microfluidic sorting device, the microfluidic sorting device including a first array of ferromagnetic elements and a second array of ferromagnetic elements. The sample includes a first target species, a second target species, a first magnetic particle having affinity for the first target species, and a second magnetic particle having affinity for the second target species. The first and second magnetic particles are differentially magnetically separable. [0013] The method according to the second aspect also includes applying an external magnetic field to the microfluidic sorting device, wherein the applying results in deflection of the first target species when present in a complex with the first magnetic particle by the first array to a first outlet of the microfluidic sorting device and deflection of the second target species when present in a complex with the second magnetic particle by the second array to a second outlet of the microfluidic sorting device.

[0014] In one embodiment, prior to said introducing step, the method includes contacting a first target species in a sample with a first magnetic particle having affinity for the first target species to produce a first complex comprising the first target species and the first magnetic particle. The method also includes contacting a second target species in the sample with a second magnetic particle having affinity for the second target species to produce a second complex comprising the second target species and the second magnetic particle. The first and second magnetic particles are differentially magnetically separable.

[0015] In a third aspect, the present disclosure provides a microfluidic sorting device which is configured to sort at least a first target species and a second target species from a sample, wherein members of the first target species include a first controllable force responsive tag and members of the second target species include a second controllable force responsive tag. The device includes at least one inlet; a sorting station in fluid communication with the at least one inlet; a first controllable force generator, which, during operation of the microfluidic sorting device, produces at least a first controllable force in the sorting station, the first controllable force acting on the first controllable force responsive tag, when present, to separate the first target species from the sample. The device according to the third aspect also includes a second controllable force generator, which during operation of the microfluidic sorting device, produces a second controllable force in the sorting station, the second controllable force acting on the second controllable force responsive tag, when present, to separate the second target species from the sample.

[0016] The device according to the third aspect also includes an outlet in fluid communication with the sorting station, wherein the outlet is configured to receive a waste stream at least partially depleted of the first target species and the second target species. [0017] In one embodiment of the microfluidic device according to the third aspect, the first controllable force displaces the first controllable force responsive tag from a first position to a second position in the sorting station to separate the first target species from the sample. [0018] In one embodiment, the second controllable force displaces the second controllable force responsive tag from a first position to a second position in the sorting station to separate the second target species from the sample. In one such embodiment, the second controllable force displaces the second controllable force responsive tag from a first position to a second position in the sorting station to separate the second target species from the first target species.

[0019] In one embodiment, the first controllable force and said second controllable force are different. In one such embodiment, the first controllable force and the second controllable force are selected from the following group: a magnetic field, acoustic radiation, an electric field, and an electromagnetic field.

[0020] In one embodiment, the first controllable force generator and the second controllable force generator are selected from the following group: a magnetic field generator, an acoustic radiation generator, an electric field generator, and an electromagnetic field generator. [0021] In a fourth aspect, the present disclosure provides a microfluidic sorting device configured to sort at least a first target species and a second target species from a sample, wherein members of the first target species include a first controllable force responsive tag and members of the second target species include a second controllable force responsive tag. The device includes at least one inlet, a sorting station in fluid communication with the at least one inlet, a controllable force generator, which, during operation of the microfluidic sorting device, produces a controllable force in the sorting station, the controllable force acting on the first controllable force responsive tag and the second controllable force responsive tag, when present, to separate the first target species and the second target species from the sample. The device according to the forth aspect also includes an outlet in fluid communication with the sorting station, wherein the outlet is configured to receive a waste stream at least partially depleted of the first target species and the second target species.

[0022] In one embodiment of the device according to the fourth aspect, the controllable force displaces the first controllable force responsive tag from a first position to a second position in the sorting station to separate the first target species from the sample. [0023] In one embodiment, the controllable force displaces the second controllable force responsive tag from a first position to a second position in the sorting station to separate the second target species from the sample. In one such embodiment, the controllable force displaces the second controllable force responsive tag from a first position to a second position to separate the second target species from the first target species.

[0024] In one embodiment, the controllable force is selected from the following group: a magnetic field, acoustic radiation, an electric field, and an electromagnetic field. [0025] In one embodiment, the controllable force generator is selected from the following group: a magnetic field generator, an acoustic radiation generator, an electric field generator, and an electromagnetic field generator.

[0026] In a fifth aspect, the present disclosure provides a microfluidic sorting device configured to sort at least a first target species from a sample, wherein members of the first target species include a first controllable force responsive tag and a second controllable force responsive tag. The device includes at least one inlet, a sorting station in fluid communication with the at least one inlet, a first controllable force generator, which, during operation of the microfluidic sorting device, produces a first controllable force in the sorting station, the first controllable force acting on the first controllable force responsive tag, when present, to separate the first target species from the sample. The microfluidic sorting device according to the fifth aspect also includes a second controllable force generator, which during operation of the microfluidic sorting device, produces a second controllable force in the sorting station, the second controllable force acting on said the second controllable force responsive tag, when present, to separate the first target species from the sample. The microfruidic sorting device according to the fifth aspect also includes an outlet in fluid communication with said sorting station, wherein the outlet is configured to receive a waste stream at least partially depleted of said first target species.

[0027] In one embodiment of the fifth aspect, the first controllable force displaces the first controllable force responsive tag from a first position to a second position in the sorting station to separate the first target species from the sample.

[0028] In one embodiment, the second controllable force displaces the second controllable force responsive tag from a first position to a second position in the sorting station to separate the first target species from the sample.

[0029] In one embodiment, the first controllable force and said second controllable force are different. In one such embodiment, the first controllable force and said second controllable force are selected from the following group: magnetic field, acoustic radiation, an electric field, and an electromagnetic field.

[0030] In one embodiment, the first controllable force generator and the second controllable force generator are selected from the following group: a magnetic field generator, an acoustic radiation generator, an electric field generator, and an electromagnetic field generator. [0031] In a sixth aspect, the present disclosure provides a microfruidic sorting device configured to sort at least a first target species from a sample, wherein members of the first target species include a controllable force responsive tag. The device includes at least one inlet, a sorting station in fluid communication with the at least one inlet, a first controllable force generator, which, during operation of the microfruidic sorting device, produces a first controllable force in the sorting station, the first controllable force acting on the controllable force responsive tag, when present, to separate the first target species from the sample. The microfruidic sorting device according to the sixth aspect also includes a second controllable force generator, which during operation of the microfruidic sorting device, produces a second controllable force in the sorting station, the second controllable force acting on the controllable force responsive tag, when present, to separate the first target species from the sample. The microfruidic sorting device according to the sixth aspect also includes an outlet in fluid communication with the sorting station, wherein the outlet is configured to receive a waste stream at least partially depleted of the first target species. [0032] In one embodiment, the first controllable force displaces the controllable force responsive tag from a first position to a second position in the sorting station to separate the first target species from the sample.

[0033] In one embodiment, the second controllable force displaces the controllable force responsive tag from a first position to a second position in the sorting station to separate the first target species from the sample.

[0034] In one embodiment, the first controllable force and said second controllable force are different. In one such embodiment, the first controllable force and said second controllable force are selected from the following group: a magnetic field, acoustic radiation, an electric field, and an electromagnetic field.

[0035] In one embodiment, the first controllable force generator and said second controllable force generator are selected from the following group: a magnetic field generator, an acoustic radiation generator, an electric field generator, and an electromagnetic field generator.

[0036] In a seventh aspect, the present disclosure provides a microfluidic sorting device which includes at least one inlet configured to permit fluidic movement of at least two fluid streams, a sorting station in fluid communication with the at least one inlet, two or more controllable force generators, configured to produce two or more controllable forces in the sorting station, and an outlet in fluid communication with the sorting station.

[0037] In one embodiment of the seventh aspect, the two or more controllable force generators are selected from the following group: a magnetic field generator, an acoustic radiation generator, an electric field generator, and an electromagnetic field generator.

BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 provides an illustration of the MT-MACS separation method and architecture (Panel A), a diagram showing the balance of forces at the microfabricated ferromagnetic strips (MFS) structures (Panel B), and optical micrographs of magnetic tags being separated at two MFS structures.

[0039] Figure 2 shows a simulation of the long-range (Panel A) and short-range (Panel

B) magnetic field gradients in the MT-MACS device.

[0040] Figure 3 provides an illustration of the quantitative measurement of MT-MACS sorting performance for magnetic tags via flow-cytometry. [0041] Figure 4 provides an illustration of the cytometric analysis of simultaneous multi- target bacterial cell sorting via MT-MACS.

[0042] Figure 5 provides an overview of the MT-MACS microfabrication procedure.

[0043] Figure 6 illustrates an exemplary method for the direct attachment of a protein target to a magnetic bead using a carbodiimide coupling method.

[0044] Figure 7 provides an illustration of a combined acoustophoresis-magnetophoresis chip concept (Panel A); a cross-sectional illustration, showing the relative position of the microchannel, microfabricated Ni strips, piezotransducer and external magnets (Panel B); and a photograph of the completed device (Panel C). The sample and buffer fluidic connections are on the device underside.

[0045] Figure 8 provides an illustration showing microfluidic separation based on acoustophoretic response and magnetic content, use of external magnets to produce long range gradient for magnetic settling, and use of microfabricated ferromagnetic structures which allow for magnetic deflection. Figure 8 also provides formulas which describe the acoustophoretic and magnetophoretic responses which occur in the microfluidic device during separation. As used in Figure 8, ( ) refers to time averaged value, F_ac refers to Acoustic radiation force, E refers to

Acoustic energy density, ω refers to Angular frequency, V refers to Particle volume, c_w refers to Speed of sound in fluid, c_p refers to speed of sound in particle; p_w refers to density of fluid, p_p refers to density of particle, k refers to angular wavenumber, x refers to transverse position in channel, η refers to viscosity of fluid, "a" refers to radius of particle, v_ac refers to velocity of movement towards (or away from) acoustic nodes, B refers to magnetic field, τ_s ^max refers to maximum time for magnetic particle to move from top of fluid channel to bottom, h refers to height of fluid channel, v_z refers to velocity of movement in magnetic field, m refers to magnetic moment of particle, p refers to density of magnetic particle, g refers to acceleration due to gravity near earth's surface, F_m refers to magnetic force on magnetic particle, F_HYD refers to hydrodynamic drag force on magnetic particle, f refers to empirical correction factor to drag force due to proximity to channel wall, V_f refers to velocity of fluid, and v_p refers to velocity of magnetic particle.

[0046] Figure 9 illustrates acoustic resonance confinement based on microfluidic channel architecture. Figure 9 also illustrates matching of hydraulic resistance to outlet flow and provides a simulation of flow at an outlet junction. As used in Figure 9, L refers to length of fluid channel; W refers to width of fluid channel; W refers to width of fluid channel in magnetic separation region; f refers to resonance frequency; f refers to resonance frequency in magnetic separation region; Q₁ refers to volumetric flowrate through outlet fraction i; v(x,y) refers to fluid velocity at position x,y; i refers to in integral means integrate over cross sectional area of outlet fraction i; R_HYD,I refers to hydrodynamic resistance for outlet channel i; ΔP refers to pressure drop across outlet channels; const refers to (undefined) constant value; and L₁ refers to length of outlet channel i.

[0047] Figure 10 shows a fabrication scheme and final assembly for an Integrated

Acoustophoretic-Magnetophoretic Separation Device (iAMSD).

[0048] Figure 11 provides a diagram of an experimental setup for the testing of an iAMSD.

[0049] Figure 12 shows close-ups of the microfluidic outlet junction with ultrasound in the off (left panel) and on position (right panel). Figure 12 also shows the results, in graphical form, for a simultaneous separation of a three component mixture using an iAMSD (Panel B).

Finally, Figure 12 shows the results, in graphical form, for a simultaneous separation of a three component mixture using an iAMSD with low initial concentrations of target (Panel C).

[0050] Figure 13 provides a diagram illustrating multi-target bacterial sorting using an

Integrated Dielectrophoretic-Magnetic Activated Cell Sorter (iDMAC). Panel A shows an overview of the experimental scheme and Panel B illustrates the physical movement and separation of differentially labeled particles in the device.

[0051] Figure 14 shows a photograph of a fabricated iDMACS device (Panel A) and optical micrographs (Panel B) of iDMACS operation. The overall size of iDMACS is 7 cm x 1.5 cm, and the device integrates the DEP and Magnetic separation modules. The optical micrographs show DEP tags being deflected and eluted via outlet A (Panel B), and magnetic tags being selectively pulled down at the edges of the ferromagnetic strips (Panel C). Non-target beads are eluted via outlet B.

[0052] Figure 15 shows the results of separation of DEP and magnetic tags in an iDMACS as measured via flow cytometry.

[0053] Figure 16 shows the results of multi-target bacterial cell sorting using an iDMACS. Panel A shows two-color flow cytometry measurement of the initial sample. Panel B shows that after a single round of separation, the outlet A fraction contained almost exclusively target A cells, but no target B cells. Panel C shows that the outlet B fraction contained primarily target B cells and no target A cells. Panel D shows that the waste fraction includes small quantities of target A and target B with much larger quantity of non-target cells. [0054] Figure 17 illustrates a fabrication scheme for the production of an iDMACS.

[0055] Figure 18 illustrates arrangements of ferromagnetic elements for MFGs in accordance with certain embodiments of the invention.

[0056] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0057] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0058] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

[0059] It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a magnetic particle" includes a plurality of such magnetic particles. [0060] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

Targets and Controllable Force Responsive Tags

[0061] The devices and methods disclosed herein provide high performance separation and/or purification of one or more target species from complex samples with high purity, recovery and throughput.

[0062] The homogeneity of the target population may be predetermined using the present devices, for example, in preparation of a substantially homogenous preparation of target species or in a predetermined preparation of defined proportions of a target species.

[0063] The devices use controllable force means for such separation or purification, and, the term "force" as used herein denotes its ordinary usage in a physical sciences context as any external agent that causes a change in the motion of a free body, or that causes stress in a fixed body {see, e.g., "Glossary," Earth Observatory. NASA.

(http://earthobservatory.nasa.gov/Glossary)). As more fully described below, examples of such forces include, but are not limited to magnetic, acoustic, electrophoretic, and mechanical (as, for example, shear incident fluidic movement). As will be illustrated below, the force is controllable with respect to the final outcome: it is applied to achieve the selection, separation or purification result. The force may be controlled so that it is applied in a uniform manner, or may accelerate or decelerate in strength, and may be applied in a continuous or discontinuous fashion (for example in bursts or pulses that may be either cyclical or regular or sporadic in frequency). The force orientation may be altered to position a target in a particular location, or to re-orient the target itself, e.g., cause the target itself to rotate. The force may be controlled in an automated system, or it may be controlled individually by an operator.

[0064] Generally, the controllable forces described herein are produced by one or more controllable force generators as described in greater detail below. As used herein, the term "controllable force generator" refers to a device and/or element or a combination of devices and/or elements which are designed to produce one or more of the controllable forces described herein.

Applications

[0065] The present microfruidic sorting technology has broad application, and the limitation is based largely on the suitability of the target with respect to configuration of the present device and methods. The sample may be a variety of media so long as the target species are capable of migrating in response to a controllable force. In general, samples analyzed should be liquid or at least flowable, although viscosity may be tolerated depending on the configuration of the device. Organic or inorganic moieties, In particular, the present invention has broad applicability in detecting the presence or amount of biological materials as well as small molecules that may be a constituent moiety of a biological - or other - system. [0066] Samples may be from any source, such as an organism, the environment or a food source. For example, samples obtained from a human or animal may be a bodily fluid, such as amniotic fluid surrounding a fetus, aqueous humor, blood and blood plasma, cerumen (earwax), chyme, Cowper's fluid or pre-ejaculatory fluid, interstitial fluid, lymph, breast milk, mucus (including nasal drainage and phlegm), pleural fluid, pus, saliva, sebum (skin oil), semen, sweat, tears, urine, vaginal secretion and vomit. Environmental samples may include samples from air, water, or land, suitably liquefied so that flow properties may operate in a properly configured microfruidic device of the present invention.

[0067] The present devices may be configured to operate under a variety of conditions.

Construction and materials may depend on the application to which the present device will be put, with considerations as described below, such as microfruidic flow. The present devices may be thermoregulated or otherwise configured to account for sample requirements. The devices disclosed herein may have cooling or heating units, and buffers or other materials may be used to adjust pH, viscosity, salinity, etc. as needed depending on the application.

Biological materials

[0068] In general, a suitable target for use in connection with the disclosed devices and methods is a biological material. Biological materials include any biologically-derived materials or materials which, either by accident or design, contain biological agents including bacteria, viruses, micro-organisms, genetically modified organisms / micro-organisms (GMOs, GMMs), prions, or any other biological agents. Biological materials include any virus, whole cell, or cellular component. Exemplary targets include: peptides, proteins, nucleic acids including all types of RNAs, DNAs whether natural or man-made, viruses and constituent moieties including viral coat antigens or proteins displayed through a viral coat; antibodies or fragments thereof (including single chain antibodies, Fabs, and the like), whole cells, cellular components, organic and inorganic small molecules, or combinations thereof.

[0069] Protein targets of interest include, for example, cell surface receptors, signal transduction factors, and hormones. Herein, the term "protein" and "peptide" and "polypeptide" are used synonymously, unless otherwise indicated, and targets may comprise peptidomimetics and other synthetic moieties. One may further have amino acid sequences derivatized to include additional moieties, such as polymers or carbohydrate groups. Aptamers as peptides may also be targets.

[0070] Nucleic acid targets of interest include, e.g., DNA and RNA targets, e.g., aptamers. Nucleic acids may comprise nucleic acid analogs, such as via incorporation of a backbone analog or a base analog. Nucleic acids may be single or double stranded, for example, and may function to interfere with nucleic acid function, e.g., small interfering RNAs. [0071] Cellular targets of interest include any cell prokaryotic or eukaryotic cell or cellular organelle or constituent. Although these are too numerous to mention, particularly contemplated are mammalian (e.g., human) cells including tumor cells and stem cells; bacterial cells, pathogenic organisms, organisms found in air (such as infectious agents, e.g., Mycobacterium tuberculosis; water sources (e.g, algae); and soil (e.g., plant cells or soil microorganisms). Cellular constituents, such as moieties found in cytoplasm, cellular membranes, cell walls, cytoskeletal components, such as microtubules, and organelles may be suitable targets, for example.

[0072] Antibody targets of interest include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single- chain antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein. Targets comprising modified and synthetic antibodies, such as peptibodies are similarly encompassed by the present invention. These antibody targets may be attached to a different moiety, such as a protein, as in, for example, a therapeutic protein having an Fc moiety attached for drug delivery or pharmacokinetic purposes.

Small Molecules

[0073] Small molecule targets of interest include both organic and inorganic small molecules, and one may select from virtually any small molecule capable of having a controllable force responsive tag attached thereto. One may, for example, determine the presence or amount of particular enantiomers within a racemic mix by configuring the tag to be selective for a particular form. One may potentially select enantiomeric forms with no additional controllable force responsive tag where the desired entaniomeric form(s) is (are) disproportionately more reactive with a controllable force, e.g., dielectrophoretic.

Targets and Tags

[0074] More than one type of target may be utilized simultaneously in the devices and methods disclosed herein. For example, two or more different cell types can be separated and/or purified simultaneously using the devices and methods disclosed herein. [0075] Targets which are separated and/or purified in connection with the devices and methods disclosed herein include a tag that responds to a controllable force. A tag that responds to a controllable force, also referred to herein as a "controllable force responsive tag" generally refers to a tag that can be displaced to a second position relative to a first position by the application of the compatible controllable force to the tag. In the devices and methods disclosed herein, the tag is generally selected so as to be compatible with (i.e., responsive to) the particular controllable force that is to be applied to the target comprising the tag or, stated differently, the controllable force used to displace tagged targets is selected so as to be compatible with the tag. For example, where the controllable force to be applied is a magnetic field, a suitable tag is a magnetic particle or bead.

[0076] The tag may be endogenous or intrinsic to a target, e.g., a naturally occurring component of said target which renders the target responsive to a particular controllable force. The tag may also be exogenous (or heterologous) to the target, so that the tag-target combination is not one normally found in nature. Where the tag is either exogenous or heterologous to the target, the tag is positioned with respect to the target such that the application of the controllable force to the tag is effectively applied to the target.

[0077] The tag may be directly bound to the target or the tag may be indirectly bound to the target via an intermediate, e.g., a linker or a bifunctional reagent. In some cases, the target and the tag may be contacted with a bifunctional reagent having one moiety that binds with a target species and another moiety that binds with the surface of the tag. If the tag particles are coated with streptavidin for example, a suitable bifunctional reagent may be a biotinylated antibody specific for the target in the sample. Exemplary tags include, but are not limited to, those that are responsive to controllable forces such as magnetic, electric, condensed-light electromagnetic and acoustic forces.

Magnetically-Responsive Tags

[0078] An exemplary tag-controllable force combination is the use of a magnetically responsive tag and a magnetic field. In such embodiments, a suitable tag is one that responds to a magnetic field, e.g. a magnetic particle. Magnetic particles which may be utilized in the disclosed methods include, for example, magnetic beads or other small objects made from a magnetic material such as a ferromagnetic material, a paramagnetic material, or a superparamagnetic material. The magnetic particles should be chosen to have a size, mass, and susceptibility that allow them to be easily diverted from the direction of fluid flow when exposed to a magnetic field in a microfluidic device (balancing hydrodynamic and magnetic effects). In certain embodiments, the particles do not retain magnetism when the field is removed. In one embodiment, the magnetic particles comprise iron oxide (Fe₂O₃ and/or Fe₃O₄) with diameters ranging from about 10 nanometers to about 100 micrometers. However, embodiments are contemplated in which even larger magnetic particles are used. For example, in certain embodiments, magnetic particles that are large enough to serve as a support medium for culturing cells may be utilized.

[0079] In some instances, the magnetic particles are coated with a material rendering them compatible with the microfluidics environment and allowing coupling to particular targets. Examples of coatings include polymer shells, glasses, ceramics, gels, etc. In certain embodiments, the coatings are themselves coated with a material that facilitates coupling or physical association with a target. For example, a polymer coating on a micromagnetic particle may be coated with an antibody, nucleic acid, avidin, or biotin.

[0080] One class of magnetic particles is the nanoparticles such as those available from

Miltenyi Biotec Corporation of Bergisch Gladbach, Germany. These are relatively small particles made from coated single-domain iron oxide particles, typically in the range of about 10 to about 100 nanometers diameter. They can be coupled to specific antibodies, nucleic acids, proteins, etc.

[0081] Another class of magnetic particles is made from magnetic nanoparticles embedded in a polymer matrix such as polystyrene. These are typically smooth and generally spherical having diameters of about 1 to about 5 micrometers. Suitable beads are available from Invitrogen Corporation, Carlsbad, CA. These beads can also be coupled to specific antibodies, nucleic acids, proteins, etc.

[0082] In some embodiments, the disclosed devices and methods make use of intrinsic magnetic properties of the sample material. In such embodiments, magnetic particles need not be employed. Examples of such materials include erythrocytes, small magnetic particles for industrial applications, etc.

Electrically-responsive tags

[0083] Where the controllable force that is to be applied to the target comprising the tag is an electric field, a suitable tag is an electrically-responsive tag. For example, in one embodiment, a suitable tag is a charged particle which can be immobilized in a flow channel by applying a local electric field. In some embodiments, the disclosed devices and methods make use of an intrinsic electrical charge of the target. In such embodiments, a charged particle need not be employed.

[0084] Suitable electrically-responsive tags are known in the art and include negatively charged polystyrene beads as demonstrated by Jonsson and Lindberg (2006) /. Micromech. Microeng. 16: 2116-2120.

[0085] Another type of suitable electrically-responsive tag is the dielectrophoretic tag.

See for example, Hu et al, "Marker Specific Sorting of Rare Cells Using Dielectrophoresis" Proceedings of the National Academy of Sciences, USA, 102, 44, 15757-15761, (2005). Acoustically-responsive tags

[0086] Where the controllable force that is to be applied to the target is acoustic radiation, a suitable tag is one that responds to the acoustic radiation force. For example, polystyrene and melamine micro beads have been used in the context of acoustic based microfluidic separation. See, for example, Lilliehorn et al. (2005) Sensors and Actuators B: Chemical 106(2): 851-858.

[0087] In some embodiments, the disclosed devices and methods make use of an intrinsic acoustic response of the target. In such embodiments, a separate acoustically-responsive tag need not be employed.

Condensed light-responsive tags

[0088] Where the controllable force that is to be applied to the target is an electromagnetic field in the form of condensed light, e.g., laser light, a suitable tag is a particle or bead responsive to laser light.

[0089] Optical tweezers also referred to as laser tweezers, make use of an electromagnetic field in the form of condensed light. Optical tweezers are known in the art and have been used in the microfluidics context to trap and redirect microspheres as well as live cells. See, for example, Merenda et al. (2007) Opt. Express 15: 6075-6086.

Methods of Attachment

[0090] In some embodiments, the devices and methods disclosed herein make use of a target to which an exogenous controllable force responsive tag has been attached. As indicated above, a tag may be directly bound to the target or the tag may be indirectly bound to the target via an intermediate, e.g., a linker or a bifunctional reagent. A variety of suitable attachment techniques which may be used in connection with the methods disclosed herein is known in the art.

[0091] Figure 6 provides an exemplary method for the direct attachment of a protein target to a magnetic bead using the carbodiimide coupling method. The carboxylic acid groups on magnetic beads are activated by reacting with l-ethyl-3-(3-dimethylaminopropyl)carbodiimde hydrochloride (EDC) to form the active intermediate O-acylisourea. Then this intermediate is immediately reacted with N-hydroxysuccinimide (NHS) to form a less labile active ester. The primary amino groups of the target proteins are then reacted with the NHS ester to form an amide bond with carboxylic acid group on the surface of the magnetic beads. [0092] An example of the indirect attachment of a target molecule to a magnetic bead is provided in U.S. Patent Application No. 11/655,055, filed 1/17/2007, and titled "SCREENING MOLECULAR LIBRARIES USING MICROFLUIDIC DEVICES," which application is incorporated by reference herein. In the referenced application, a biotin tagged target protein is attached to a magnetic microparticle via a steptavidin tag associated with the magnetic microparticle.

[0093] Additional examples include the use of tag particles coated with streptavidin and a bifunctional reagent such as a biotinylated antibody specific for the target in the sample. [0094] The above methods may be readily adapted to the attachment of tags responsive to controllable forces other than magnetic fields. Such as, for example, the attachment of tags responsive to electric fields and/or acoustic radiation.

Controllable Forces to Facilitate Partitioning

[0095] As discussed previously herein, a target to be separated and/or purified using the devices and/or methods disclosed herein comprises a tag that responds to a controllable force. The tag is generally selected so as to be compatible with (i.e., responsive to) the particular controllable force that is to be applied to the target comprising the tag or, stated differently, the controllable force used to displace tagged targets is selected so as to be compatible with the tag. Thus, both the microfluidic device and the controllable force are selected so as to be compatible with the tag and so as to provide appropriate partition efficiency.

Separation and/or Purification of Multiple Target Species - Single Controllable Force Applied

[0096] The devices and methods disclosed herein can be utilized to separate and or purify multiple target species of interest from a complex mixture. In some embodiments, this is accomplished via application of a single controllable force. For example, two or more target species (e.g., two or more types of target cells) can be simultaneously sorted based on differential labeling of the target species with dissimilar magnetic tags and application of a magnetic field to the labeled target species. One may desire to select, for example, a cell with two cell surface receptors. Thus, one may select a first subpopulation containing a first cell surface receptor; and then, select from that first subpopulation a further subpopulation containing both a first cell surface receptor as well as a second cell surface receptor. One could have two populations of selected cells (a first population including cells having a first cell surface receptor and a second population including cells having a first cell surface receptor and a second cell surface receptor). [0097] In some embodiments, the tags need not be dissimilar in order to facilitate simultaneous separation and/or purification of multiple target species. For example, the tags may be designed to bind a molecule or portion thereof, e.g., an antigen, which is present on a first target species at a different level than on a second target species. Similarly, these techniques can be used to separate members of a particular species (e.g., a particular cell type) which have different levels of a particular molecule or antigen which can be labeled and/or which are responsive to a controllable force. This aspect of the present disclosure allows for selection and/or purification of, e.g., cells expressing different levels of a particular antigen. Based on the different levels of antigen, the cells can be labeled with different amounts of tag, e.g., by contacting the cells with biotinylated antibody specific for the antigen and streptavidin coated tag, e.g., a streptavidin coated magnetic bead.

Separation and/or Purification of Single Target Species - Multiple Controllable Forces Applied

[0098] In some embodiments, the devices and methods disclosed herein make use of tags which are responsive to more than one type of controllable force. For example, a particular tag may respond to both a magnetic field and an electric field. When different controllable forces are applied to the same tag, they may be applied either at the same time or alternatively one of the controllable forces may be applied to the tag prior to the application of the other controllable force.

[0099] In some embodiments, each member of a target species is tagged with two or more different tags, wherein each of the different tags is responsive to a different controllable force. In this manner the target species can be separated and/or purified from a complex sample via application of multiple controllable forces. When different controllable forces are applied to different tags present on a member of a target species, they may be applied either at the same time or alternatively one of the controllable forces may be applied to a first tag prior to the application of the other controllable force to a second tag. In this manner, multiple controllable forces can be applied to select and/or purify a particular target species present in a complex sample.

[00100] Such selection using multiple controllable forces may be performed simultaneously or in series, and the present devices may be so configured. If multiple controllable forces are to be used simultaneously, the device should be configured so that the interference between or among controllable forces is taken into account. For example, multiple magnetic fields or multiple acoustic fields may behave differently when presented simultaneously than when alone.

[00101] One may desire to progressively select subpopulation target species. For example, one may first select cells displaying a selected cell surface receptor. From that population, one may then select cells displaying a second cell surface receptor; and so on. Alternatively, one may select sub-cellular moieties, such as mitochondria or nucleic acid species.

[00102] The present devices may be configured in series so that as a first step, a sample is tagged and separated using a first controllable force, and, optionally, as a second step that selected sample has the tag removed (e.g., washed); and as a next step the selected sample is suitably tagged and separated with a second controllable force. The first and second tag/controllable forces may be the same or different.

Separation and/or Purification of Multiple Target Species - Multiple Controllable Forces Applied

[00103] In some embodiments, a complex sample may comprise multiple target species of interest. In such embodiments, a first target species can be labeled with a first tag responsive to a first controllable force, a second target species can be labeled with a second tag responsive to a second controllable force, and so on. In some embodiments, the tags are selected such that they are responsive to different controllable forces and are not responsive to the same controllable force. For example, a first tag on a member of a first target species may respond exclusively to a magnetic field and a second tag on a member of a second target species may respond exclusively to an electric field. When different controllable forces are applied to different tags responsive to different controllable forces, the forces may be applied either at the same time or alternatively one of the controllable forces may be applied to the target species prior to the application of the other controllable force.

Separation and/or Purification Based on Magnitude of Response to Applied Force(s) [00104] Target species selection may be binary (e.g., selecting for the presence or absence of a target having a controllable force responsive tag) or may be based on the magnitude of the response to the application of a controllable force or combination of controllable forces. One may select based on partitioning or fractionating a target species from a sample based on the degree of response, rather than presence of the response alone. For example, one may separate target species into fractions based on a low response to applied force, a medium response to applied force, or a high response to applied force, defined according to desired parameters. The present devices and methods can be configured and performed accordingly. For example, if three fractions are desired, one may configure the subject device with three separate outlet channels for selection into separate collection containers. Devices and methods may be configured in a number of ways for fractionation based on tag response magnitude. [00105] One may desire to select based on the relative response to applied force between two tags or among several tags. (The tags may be any of those as described here, so long as the magnitude of response is detectable). The present disclosure includes a method for using a microfluidic sorting device having a sample flow path to sort target species in a sample, wherein the target species has one or more controllable force responsive tags attached, wherein at least one of the tags produces a response to an applied controllable force, and wherein the target species is sortable based on the magnitude of the response. The method includes introducing a sample into the flow path of a microfluidic sorting device, the microfluidic sorting device including at least one controllable force generator for producing at least one controllable force in the sample flow path; applying the at least one controllable force to the microfluidic sorting device under conditions suitable for generating a response from the controllable force responsive tags; and sorting a target species based on the magnitude of the response generated. In one embodiment, more than one controllable force is applied. In another embodiment, the target species comprises more than one controllable force responsive tag.

[00106] The response magnitude may be absolute, or it may be relative. One may select based only on magnetically-tagged target species responding to relatively weak magnetic forces, indicating that the target species has a particular amount of magnetic tags. One may desire to select a tag response strength that is (for example) one half as strong as a second tag response strength. Measurement of relative tag responses may be so configured in computerized memory storage devices capable of tag response detection.

[00107] For example, one may desire collection of tumor cells from a blood sample, but one may seek to avoid false positives. One may therefore separate only those tumor cells having a response magnitude above a certain threshold.

[00108] Nucleic acids, such as DNAs or RNAs, may be similarly selected. One may determine the frequency of a selected particular nucleic acid sequence in a population of nucleic acids, such as a genome. One may then tag a nucleic acid sample selectively for the selected nucleic acid sequence. One may select target nucleic acid species based on the magnitude of tag response. For example, one may monitor the progression of a nucleic acid containing infectious agent, such as a virus or bacteria. One may similarly monitor the amount of particular cell types, such as tumor or stem cells, where such cell types are tagged for the desired nucleic acid. (One may first prepare suitable conditions for exposing the nucleic acid from the cell populations). [00109] Magnitude of response may be useful in environmental monitoring applications, where amount of moiety is significant. Air, land, and water quality for example typically have threshold amounts of substances below which satisfies public health requirements. One may, for example, use the present devices and methods for monitoring the presence or amount of organisms in open water sources, such as oceans or municipal water sources. [00110] Magnitude of tag response strength may be useful to monitor food quality at a food preparation facility. For example, dairy products may be monitored for the presence of melamine. One may prepare a melamine- selective tag responsive to a controllable force as described herein, and detect quantities above which human or animal health may be affected. [00111] Other examples will be apparent in view of the present disclosure, and combinations of binary (presence/absence) and magnitude detection may be used.

Separation and/or Purification Device Configuration and Methods

[00112] One may sort target species based on any number of desired parameters by performing the present methods on a single sample simultaneously, or in series. [00113] One may separate target species in a single sorting chamber by exposing suitably tagged sample to one or more controllable forces simultaneously. For example, one may contact a sample with suitable controllable force responsive tags under suitable conditions for tagging target species in said sample. One may then apply corresponding controllable forces to said sample. For example, target species are tagged for both magnetic as well as acoustic controllable force separation. A device may be a single input-multi-output configuration, such that the sample is fractionated into (for example) magnetic -only, acoustic-only, magnetic + acoustic, and no label.

[00114] One may separate multiple target species in a series of stages. In a first stage, for example, a tagged sample may first be subject to suitable controllable forces. The target species so selected may then optionally have the tag removed. In a second stage, the target species selected in the first stage is tagged with another tag responsive to a controllable force, and subject to a suitable controllable force. Single or multiple tags may be use in any one stage, and single or multiple controllable forces may be used at any one stage. The tag/controllable forces may be the same or different within and among stages.

[00115] The present microfluidic sorting devices may be so configured to optimize sorting. A device for selecting based on magnitude of tag response strength may have multiple outflow channels for fractionation based on response strength. A device for selection of multiple targets in series may have multiple sorting stations. Fluid communication among any inlet channel, sorting station, and outlet channel may be configured to permit suitable application of controllable force via controllable force generators.

[00116] The present disclosure thus includes a microfluidic sorting device configured to sort at least one target species by using more than one controllable force comprising one or more inlet channels, one or more sorting stations, and one or more outlet channels.

[00117] The present disclosure includes a microfluidic sorting device containing a sample, target species within which are tagged with more than one controllable force responsive tag.

Optionally, the controllable force responsive tags are responsive to different forces, such as dielectrophoretic, acoustic and magnetic.

[00118] A combination of multiple controllable forces and the above methods may be suitable, for example, for sorting virus particles, bacterial cells or mammalian cells. One application may be in the monitoring for the engraftment of stem cells. One may prepare stem cells with a particular cell surface marker to which a controllable force responsive tag is attached, and select for such stem cells based on the presence/absence of the cell surface marker (based on tag response) or amount of cell surface marker (based on magnitude of response). One may further monitor the engraftment of stem cells in a tissue, where the subject stem cells have differentiated into the local tissue type. If the stem cells have a nucleic acid sequence not found in the underlying tissue, one may determine if cells in the tissue derive from the subject stem cell origin. This can be done by preparing a target tissue sample in a fluidic medium, exposing said sample to a controllable force responsive tag under conditions suitable for attachment of said tag to any constituent moieties, and performing a microfluidic separation method as described herein.

Magnetic fields

[00119] In one embodiment of the methods disclosed herein, the controllable force is a magnetic field, the tag is a magnetically responsive tag, and the microfluidic device is a microfluidic device which comprises a magnetic field generating element capable of displacing the tag to a second position relative to a first position by the application of the magnetic field to the tag.

[00120] A variety of microfluidic devices suitable for use in connection with the application of magnetic fields to magnetically responsive targets and/or tags are known in the art. These include those disclosed in U.S. Patent Application No. 11/655,055, filed 1/17/2007, and titled "SCREENING MOLECULAR LIBRARIES USING MICROFLUIDIC DEVICES," incorporated by reference herein; and U.S. Patent Application No. 11/583,989, filed 10/18/06, and titled "MICROFLUIDIC MAGNETOPHORETIC DEVICE AND METHODS FOR USING THE SAME," incorporated by reference herein.

[00121] In some embodiments, the disclosed devices and methods utilize one or more magnetic sorting modules that may be employed in a microfluidics system. In certain embodiments presented below, magnetophoretic sorting modules employ magnetic field gradient generators (MFGs) and employ buffer switching as described in the above patent applications. [00122] As described herein, "magnetic field gradient generators" are elements that generate magnetic field gradients in a manner sufficient to alter the influence of an applied magnetic field on magnetically labeled species or intrinsically magnetic species in the sorting region by increasing or decreasing the field strength and/or changing the direction of the field. As explained more fully elsewhere herein, these magnetic field gradient generators serve to shape the distribution of the magnetic field gradient experienced by the particles traveling through a sorting region of the microfluidic device. In one embodiment, these are nickel strips provided within a flow channel of the sorting region itself. One or more magnets can be used to provide an external magnetic field in the sorting region. In one embodiment, a pair of permanent magnets such as NdFeB magnets is placed on the top and bottom of the sorting region. In other embodiments, one or more electromagnets may be employed to allow precise control of the field shape and homogeneity. The MFG strips interact with the field produced by the magnet to precisely shape and direct the magnetic field gradient within the sorting region.

Magnetic Field Gradient Generating Structures

[00123] The magnetic field gradient is responsible for the magnetic force exerted on magnetic particles in microfluidic devices. In weakly diamagnetic media such as most buffer solutions, the magnetophoretic force on a paramagnetic particle can be approximated as ^_magneti_c ⁼ ^_m ' ^Z' ^ (B² /2μ₀) , where ( μ₀ ) is the permeability of free space, B is the magnetic flux density, Δχ

is the differential magnetic susceptibility of the particle relative to its suspension medium, and V_m, is volume of the paramagnetic particle. Thus, the force depends on the gradient of the square of the flux density B. For many applications such as those described below, where superparamagnetic particles are in the saturation regime, the total volume magnetization (m_p = V_m ■ Aχ • H ) is constant and the equation for the magnetophoretic force on a superparamagnetic particle can be simplified to F_magnetιc = m_ra,|V(β), where m_sat is the saturated magnetization of the particle. Since the direction and magnitude of the force on a superparamagnetic particle are governed by the gradient of the applied field, magnetophoretic separation devices may be designed to accurately control this parameter. [00124] The size and direction of the magnetic field gradient produced via an MFG depends on the applied magnetic field (typically provided by an external magnet proximate the sorting region) as well as the construction of the MFG. Pertinent parameters of MFG construction include the MFG material(s), the size and geometry of the MFG, and the orientation of the MFG with respect to the fluid flow and external magnetic field. [00125] The shape and arrangement or pattern of the elements making up an MFG can be configured to account for the hydrodynamics of the microfluidic device in the sorting channel. In certain embodiments, the direction of the gradient generated by an MFG will be in a direction that promotes buffer switching toward a target collection region. In certain embodiments, the magnetic force exerted in this direction is greater than the component of drag force exerted in the opposite direction. Thus, in some embodiments, F_d-sinθ < F_m, where #is the angle between the direction of flow and the magnetic field gradient generating structures (for linear strips of these elements).

[00126] The material from which an MFG element is made should have a permeability that is significantly different from that of the fluid medium in the device (e.g., the buffer). In certain cases, the MFG element will be made from a ferromagnetic material. Thus, the MFG element may include at least one of iron, cobalt, nickel samarium, dysprosium, gadolinium, or an alloy of other elements that together form a ferromagnetic material. The material may be a pure element (e.g., nickel or cobalt) or it may be a ferromagnetic alloy such as an alloy of copper, manganese and/or tin. Examples of suitable ferromagnetic alloys include Heusler alloys, (e.g., 65% copper, 25% manganese and 10% aluminium), Permalloy (55% iron and 45% nickel), Supermalloy (15.7% iron, 79% nickel, 5% molybdenum and 0.3% manganese) and μ-metal (77% nickel, 16% iron, 5% copper and 2% chromium). Nickel-cobalt alloys may also be used. In some embodiments, non-metallic ferromagnetic materials including ferrites which are mixture of iron and other metal oxides may be used.

[00127] In some embodiments, an MFG is an array of thin nickel stripes micro-patterned on a glass substrate, which becomes magnetized under the influence of an external permanent magnet. Because the nickel possesses much higher permeability than the surrounding material (i.e., the buffer), a strong gradient is created at the interface. Although the magnetic flux density from the MFGs may not be strong compared to the surface of the external magnet, the gradient of the magnetic field is very large within a short distance (e.g., a few microns in some embodiments) of the line edges. As a result, the MFGs allow precise shaping of the field distribution in a reproducible manner inside microfluidic channels. The MFG element may include one or more individual magnetizable elements. The MFG may include a plurality of magnetizable elements, e.g., 2 or more, 4 or more, 5 or more, 10 or more, 15 or more, 25 or more, etc. [00128] In designs where the magnitude of the gradient decreases rapidly with distance from the MFG, the MFG may be formed within or very close to the flow channel where sorting takes place. Therefore, in some microfluidic examples, an MFG should be located within a few micrometers of the sorting region where magnetic particles are to be deflected (e.g., within about 100 micrometers (or in certain embodiments within about 50 micrometers or within about 5 micrometers of the sorting region, such as within about 2 micrometers of the sorting region). However, when large external fields are employed, the MFG design need not be so limited. Generally speaking, the MFG may be located as far away from the sorting region as about 10 millimeters. This may be the case when, for example, the external magnetic field is in the domain of about 1 Tesla or higher. Note that the large gradients afforded by such MFGs allow one to design very high throughput sorting stations with relatively large channels and consequently the capability to support large volumetric flow rates.

[00129] In certain embodiments, the MFG is provided within the sorting region channel; i.e., the fluid contacts the MFG structure. In certain embodiments, some or all of the MFG structure is embedded in channel walls (such as anywhere around the perimeter of the channel (e.g., top, bottom, left, or right for a rectangular channel)). Some embodiments permit MFGs to be formed on top of or beneath the microfluidic cover or substrate. [00130] The pattern of material on or in the microfluidic substrate may take many different forms. In one embodiment it may take the form of a single strip or a collection of parallel strips.

[00131] Examples of suitable dimensions for line-type MFG structures will now be presented. In certain embodiments employing ferromagnetic strips for use in sorting particles in a conventional buffer medium, the strips may be formed to a thickness of between about 1000 Angstroms and 100 micrometers. The widths of such strips may be between about 1 micrometer and 1 millimeter; e.g., between about 5 and 500 micrometers. The length, which depends on the channel dimensions and the angle of the strips with respect flow direction, may be between about 1 micrometer and 5 centimeters; e.g., between about 5 micrometers and 1 centimeter. The spacing between individual strips in such design may be between about 1 micrometer and 5 centimeters. The number of separate strips in the MFG may be between about 1 and 100. The angle of the strips with respect to the direction of flow may be between about -90° and +90°. For fractionation applications, it has been found that angles of between about 2° and 85° work well. One or more dimensions of the MFG pattern may deviate from these ranges as appropriate for particular applications and overall design features.

[00132] In certain embodiments, the pattern of ferromagnetic material may take the form of one or more pins or pegs in the flow channel or on the substrate beside the flow channel or embedded in the substrate adjacent the flow channel. Figure 18, A to E, present arrangements of ferromagnetic elements for MFGs in accordance with certain embodiments of the present disclosure. In each case, the elements are provided within or proximate a flow channel in a magnetophoretic sorting region.

[00133] Figure 18, A and B present two arrangements (rectangular and offset) of pin-type

MFG elements depicted with respect to a direction of flow. The heights and widths of these elements may be in the same ranges as presented for the strip MFG elements presented herein. For comparison, Figure 18, C-E present arrangements of MFG elements taking forms of layers of linear strips (C), layers of curved strips (D), and layers of chevrons (E).

[00134] As indicated an external magnet may provide the magnetic field that is shaped by an MFG to produce a strong magnetic field gradient in a sorting region. Typically the external magnet is a permanent magnet, but it may also be an electromagnet (e.g., a Helmholtz coil). Generally, electromagnets produce smaller magnetic fields (in comparison to permanent magnets), but they may be designed to produce very uniform fields, which may be advantageous. [00135] The position and orientation of the permanent magnet(s) with respect to the sorting region may be determined by the magnetic field strength produced by the permanent magnets, the homogeneity of the field (i.e., the uniformity of the field across the sorting region absent the MFG), the dimensions and shape of the magnet, etc. In some embodiments, it is desirable to have a uniform field produced by the external magnet(s) in the region of the MFG. In some embodiments, two permanent magnets are employed per MFG, one located above the sorting region and the other located below the sorting region. In a specific embodiment, the magnets may be located above and below an MFG. In certain embodiments, two permanent magnets straddle a sorting region (i.e., the permanent magnets are located in the same plane as the sorting region or in a plane parallel to the plane of the sorting region). Certain embodiments employ a single magnet with one pole located above or below the sorting region. Still other embodiments employ generally U-shaped magnets in which poles at the terminal portions of the U straddle the sorting region (e.g., above and below or in the same plane). [00136] In certain embodiments, the permanent magnet provides a field strength of between about 0.01 and 1 T, such as between about 0.1 and 0.5 T. Note that for some applications, it may be appropriate to use stronger magnetic fields such as those produced using superconducting magnets, which may produce magnetic fields in the neighborhood of about 5 T. [00137] Permanent magnets are made from ferromagnetic materials such as nickel, cobalt, iron, alloys of these and alloys of non-ferromagnetic materials that become ferromagnetic when combined as alloys, know as Heusler alloys (e.g., certain alloys of copper, tin, and manganese). Many suitable alloys for permanent magnets are well known and many are commercially available for construction of magnets for use with the present disclosure. A typical such material is a transition metal-metalloid alloy, made from about 80% transition metal (usually Fe, Co, or Ni) and a metalloid component (boron, carbon, silicon, phosphorus, or aluminum) that lowers the melting point. Permanent magnets may be crystalline or amorphous. One example of an amorphous alloy is FeSoB₂O (Metglas 2605).

[00138] In a specific embodiment, the external magnetic field is provided by a pair of 5 millimeter diameter NdFeB magnets (K&J Magnetics, Jamison, PA) attached to the top and bottom sides of a sorting region in a microfluidic device.

Multi-Target Magnetic Activated Cell Sorter (MT-MACS)

[00139] Disclosed herein are the design, fabrication and performance of the Multi-Target

Magnetic Activated Cell Sorter (MT-MACS), which comprises a two-input, multiple output microfluidic device capable of continuously sorting multiple target species into independent, spatially-segregated outputs based on the degree of magnetization of the targets (Figure 1). [00140] In one embodiment, a microfluidic sorting device is disclosed wherein the device comprises at least one inlet channel configured to provide at least two separate streams. A first stream defines a sample stream path for a sample and a second stream defines a buffer stream path for a buffer. The sample is one suspected of containing at least one of two different target species, e.g. two different cell types, which are to be separated from the remaining components of the sample.

[00141] The sample also comprises a first set of magnetic particles having affinity for the first target species and a second set of magnetic particles having affinity for the second target species. These particles can be selected such that they are differentially magnetically separable. Differentially magnetically separable particles include magnetic particles which differ in one or more characteristics which affect their response to a magnetic field. For example, in one embodiment, such particles differ in size and/or magnetization. In additional embodiments, differentially magnetically separable particles comprise different types of magnetic material. In some embodiments, the sample also includes various non-target species.

[00142] The device comprises a sorting station fluidly coupled to the at least one inlet and located in the sample stream path. In one embodiment, the sorting station comprises a first array of ferromagnetic elements and a second array of ferromagnetic elements. In additional embodiments, additional arrays of such elements can also be included. In use, it is generally desirable that the sample be provided under conditions such that complexes between target species and magnetic particles are formed at least prior to entering the sorting station of the device.

[00143] In one embodiment, a magnetic field gradient generator produces a change in the magnetic field gradient in the sorting station such that the first array deflects the first set of magnetic particles into the buffer stream path at a first location and out a first outlet channel and the second array deflects the second set of magnetic particles into the buffer stream path at a second location and out a second outlet channel. In this manner, two distinct species present in a sample can be separated. After separation, the two species can be subjected to various techniques known in the art which allow for the identification and/or quantitation of the two species.

[00144] In some embodiments, the device also comprises a third outlet channel configured to receive a waste stream from the sample stream path, such that sample that is at least partially depleted of a target species flows through said third outlet channel.

[00145] In another embodiment, the microfluidic sorting device comprises at least one inlet channel comprising first inlet channel for providing at least a portion of the buffer stream and a second inlet channel for providing at least a portion of the sample stream.

[00146] The ferromagnetic elements of the microfluidic device can be micropatterned nickel elements. In one embodiment, the first and second arrays of ferromagnetic elements comprise ferromagnetic strips.

[00147] In additional embodiments, the ferromagnetic elements of the microfluidic sorting device comprise one or more pins or pegs. [00148] In specific embodiments, the microfluidic sorting device of comprises a sample, wherein said sample comprises a first target species, a second target species, a first set of magnetic particles having affinity for said first target species, a second set of magnetic particles having affinity for said second target species, and a non-target species. [00149] In one embodiment, a method of separating two or more target species in a sample is disclosed. The method comprises introducing a sample into the flow path of a microfluidic sorting device. The microfluidic sorting device comprises a first array of ferromagnetic elements and a second array of ferromagnetic elements. The sample comprises a first target species, a second target species, a first set of magnetic particles having affinity for said first target species, and a second set of magnetic particles having affinity for said second target species. As discussed above, the first and second magnetic particles are differentially magnetically separable. The method also comprises an applying step in which a magnetic field gradient is applied to said microfluidic sorting device. The application of the magnet field gradient results in deflection of the first target species by said first array to a first outlet of the microfluidic device and deflection of the second target species by said second array to a second outlet of the microfluidic sorting device.

[00150] In one embodiment, prior to said introducing step, the method comprises contacting a first target species in a sample with a first magnetic particle having affinity for said first target species to produce a first complex comprising the first target species and the first magnetic particle; and contacting a second target species in said sample with a second magnetic particle having affinity for said second target species to produce a second complex comprising said second target species and said second magnetic particle, wherein the first and second magnetic particles are differentially magnetically separable.

[00151] It will be appreciated that the device and methods of the present disclosure can be adapted to provide for separation of a plurality of target species, where the device and methods involve use of a plurality of magnetic particles having affinity for a target species, with a plurality of arrays of ferromagnetic elements being adapted for deflection of the magnetic particles. Accordingly, the device and method can be adapted to provide for detection of 1, 2, 3, 4, or more target species. Generally, the "affinity" of the magnetic particle for the target species can be such that a specific complex between the magnetic particle and the target species, e.g., such that a first magnetic particle preferentially binds a first target species relative to non-first target species that may be present in a sample; such that a second magnetic particle preferentially binds a second target species relative to non-second target species that may be present in a sample; and the like.

[00152] In various embodiments, magnetic particles are diverted within the microfluidic device via free flow magnetophoresis. In other words, magnetic particles in a continuous flow are deflected from the direction of flow by a magnetic field or magnetic field gradient. In one example, a microfluidic device includes functionality for generating locally strong magnetic field gradients for influencing the direction of movement of the particles in the device. In certain embodiments, strips or patches or particles of materials are fixed at locations within or proximate the sample flow path. Specific examples are described below.

[00153] The deflection of magnetic particles can be represented as the sum of vectors for magnetically induced flow and hydrodynamic flow. The magnetically induced flow is represented by the ratio of the magnetic force exerted on a particle by the magnetic field (or field gradient) and the viscous drag force. The magnetic force is in turn proportional to the magnetic flux density and its gradient. It is also proportional to the particle volume and the difference in magnetic susceptibility between the particle and fluid. For a given magnetic field gradient and a given viscosity, the magnetic component deflection is dependent on the size and magnetic susceptibility of the particle.

[00154] In certain embodiments, the magnetic flux density applied to a microfluidic channel is between about 0.01 and about 1 T, or in certain embodiments between about 0.1 and about 0.5 T. Note that for some applications, it may be appropriate to use stronger magnetic fields such as those produced using superconducting magnets, which may produce magnetic fields in the neighborhood of about 5 T. In certain embodiments, the magnetic field gradient in regions where magnetic particles are deflected is between about 10 and about 10⁶T/m. In a specific embodiment, the field gradient is approximately 5000 T/m within 1 micrometer from the edge of a magnetic field gradient generator.

[00155] At the point in a microfluidic flow path where separation is to occur, the magnetic field gradient should be oriented in a direction that causes deflection of the particles with respect to the flow. Thus, the magnetic field gradient will be applied in a direction that does not coincide with the direction of flow. In certain embodiments, the direction of the magnetic field gradient is perpendicular to the direction of flow. However, in other embodiments the direction of the magnetic field gradient is not perpendicular to the direction of flow. [00156] Many different magnetic field generating mechanisms may be employed to generate a magnetic field over the displacement region of the microfluidic device. In a simplest case, a single permanent magnet may be employed. It will be positioned with respect to the flow path to provide an appropriate flux density and field gradient. As discussed above, permanent magnets are made from ferromagnetic materials such as nickel, cobalt, iron, alloys of these and alloys of non-ferromagnetic materials that become ferromagnetic when combined as alloys, know as Heusler alloys (e.g., certain alloys of copper, tin, and manganese). In one specific embodiment, the permanent magnet is a cylindrical neodymium-iron-boron magnet. In another example, the magnet is an electromagnet such as a current carrying coil or a coil surrounding a paramagnetic or ferromagnetic core. In some embodiments, a controller is employed to adjust the magnetic field characteristics (the flux density, field gradient, or distribution over space) by modulating the current flowing through the coil and/or the orientation of the magnet with respect to the flowing fluid.

[00157] In some designs, a combination of magnets or magnetic field gradient generating elements is employed to generate a field of appropriate magnitude and direction. For example, one or more permanent magnets may be employed to provide an external magnetic field and current carrying conductive lines may be employed to induce a local field gradient that is superimposed on the external field. In other embodiments, "passive" elements may be employed to shape the field and produce a controlled gradient. Generally, any type of field influencing elements should be located proximate the flow path to tailor the field gradient as appropriate. [00158] In certain embodiments, the magnetic field generating elements are provided within the sorting region channel; i.e., the fluid contacts the magnetic field generating structure. In certain embodiments, some or all of the magnetic field generating structure is embedded in channel walls (such as anywhere around the perimeter of the channel (e.g., top, bottom, left, or right for a rectangular channel)). Some embodiments permit magnetic field generating elements to be formed on top of or beneath the microfluidic cover or substrate. [00159] The magnetophoretic separation and/or purification methods and devices described herein may be used in combination with one or more additional separation and/or purification methods and/or devices described herein in a single integrated microfluidic device. For example, a single device can include some combination of magnetophoretic, acoustophoretic, electrophoretic (e.g., dielectrophoretic), and electromagnetophoretic (e.g., laser light induced) separation and/or purification as described herein.

Electric fields

[00160] In one embodiment of the methods disclosed herein, the controllable force is an electric field, the tag is an electric field responsive tag, and the microfluidic device is a microfluidic device which comprises an electric field generating element capable of displacing the tag to a second position relative to a first position by the application of the electric field to the tag.

[00161] In one embodiment, a planer microfluidic electrocapture device can be utilized which captures and concentrates beads by a local electrical field. For example, negatively charged polystyrene beads may be captured and released at an applied potential of 300V in

25min.

[00162] In one embodiment, members of a target species which include an electric field responsive tag are passed through a dielectrophoretic separation module in a microfluidic device, where they are subjected to dielectrophoretic forces (F_DEP)- These forces can be created, for example, by the non-uniform electric field generated by a set of electrodes as described in greater detail in the examples below. The device can be configured such that tagged cells undergo selective deflection into a buffer stream upon being subjected to the dielectrophoretic forces.

Acoustic radiation

[00163] In one embodiment, the controllable force is acoustic radiation, the tag is an acoustic radiation responsive tag, and the microfluidic device is a microfluidic device which comprises an acoustic radiation generating element capable of displacing the tag to a second position relative to a first position by the application of the acoustic radiation to the tag. [00164] In one embodiment, a suitable microfluidic device separates tagged targets based on ultrasonic trapping of the tagged targets using acoustic forces in standing waves. In specific embodiments, piezoelectric micro tranducers may be integrated into a microfluidic channel to produce the appropriate acoustic forces. Specific microfluidic devices capable of producing the appropriate acoustic forces are described in greater detail in the examples below. [00165] Generally, the acoustic force applied to a particle is proportional to the volume of the particle. As such, a particular acoustic tag can be selected in accordance with the volume of tag, the force to be applied and the deflection and/or trapping architecture of the microfluidic device. Upon ultrasonic actuation, e.g., using a piezoelectric micro tranducers, particles which are present in the microfluidic channel and responsive to an acoustic force will experience an acoustic force towards an acoustic node generated by the ultrasonic actuation. In this manner, targets of interest can be deflected and/or trapped as desired via application of an acoustic force.

Optical tweezers

[00166] In one embodiment of the methods disclosed herein, the controllable force is an electromagnetic field in the form of condensed light, e.g., laser light, the tag is a particle or bead responsive to laser light and the microfluidic device is a microfluidic device which comprises a laser light generating element capable of displacing the tag to a second position relative to a first position by the application of the laser light to the tag.

[00167] Optical tweezers, also referred to as laser tweezers, make use of an electromagnetic field in the form of condensed light. Optical tweezers are known in the art and have been used in the microfluidics context to trap and redirect microspheres as well as live cells. See, for example, Merenda et al. (2007) Opt. Express 15: 6075-6086.

Flow Systems and Hydrodynamics

[00168] Generally, in the context of the devices and methods disclosed herein the physical separation between the tagged and untagged sample components occurs through the balance between hydrodynamic forces and the application of one or more controllable forces as described herein. For example, in the context of magnetophoretic separation, as the magnetically labeled components travel within the microfluidic channel, there exists a hydrodynamic drag force (F_d). Assuming that the magnetically labeled component is spherical, then the force can be approximated as F_d = 6πη-R_p-V, where η is the viscosity of the medium, R_p is the particle radius and υ is the velocity. When the labeled component is exposed to an MFGs positioned at an angle, the MFG imposes an attractive magnetophoretic force (F _m). In this case, if the component of F_d perpendicular to the MFG is less than the value of F_m (i.e., F_d-sinθ < F_m), then the velocity vector of the labeled cell will be significantly modified in the direction parallel to the MFG pattern.

[00169] In view of the above, the linear fluid velocity in the sorting regions can be controlled to provide proper balance with the controllable force to be applied, e.g., the linear fluid velocity can be controlled to provide proper balance with the magnetic gradient size to ensure that efficient sorting can be accomplished. For example, in some embodiments a fluid velocity of approximately 3 mm/s has been found to be appropriate. At this velocity, viscous drag forces on Dynal M280™ 2.8 μm microbeads are expected to be -160 pN (with η -0.002 kg-m^-s^"1 for the suspension medium). For a MFG oriented at approximately 28° with respect to the fluid direction and producing a field gradient of approximately 5000 T/m in the sorting region, a 3 mm/s allows efficient sorting.

[00170] Turning now to configurations of the sorting station, certain buffer switching designs will now be described. Although these buffer switching designs are described in the context of magnetophoretic separation and/or purification, it should be noted that these designs can be readily adapted to accommodate separation via application of any of the controllable forces described herein. Buffer switching employs separate streams of buffer and sample that are delivered to a sorting region. The force exerted on, e.g., magnetic particles in the sorting region guides those particles out of the sample stream and into the buffer stream. Due to the low Reynolds number, the flow streams within the sorting region are generally uniaxial and laminar (e.g., Re is approximately 2000 or less). Buffer switching regions may be designed such that in the absence of an external magnetic field, only the buffer medium arrives at a collection channel. When an external field is applied, the MFG elements become magnetized and the magnetically labeled components are selectively transported across the stream boundary, from the inlet stream into the buffer medium, resulting in a high purity of the target species in the collection channel. On the other hand, the unlabeled components (typically the majority of the components in the sample) are not deflected by the magnetic field and will enter the waste channels. [00171] In buffer switching devices, the separate buffer and sample streams may be provided to the sorting region via one, two or more separate channels. Typically, though not necessarily, a sorting region will have at least one inlet channel dedicated to delivering buffer and another channel dedicated to delivering sample. In certain embodiments, at least one inlet channel to the separation region provides both the sample stream and the buffer stream. Because these streams are provided as laminar flows, they can be combined into an inlet channel upstream from the sorting region. They will then flow into the sorting region separated from one another as separate streams.

[00172] In some examples, the buffer and sample streams may be stacked vertically within a channel or sorting region. Devices normally operated in horizontal arrangement can be turned by 90 degrees to a vertical orientation. However, a device may also be designed such that when the substrate lies flat on a surface, the buffer and sample streams flow within the sorting region on top of one another.

[00173] Many other buffer switching structures are within the scope of the present disclosure. In one example, a multi-layer network or flow channels is provided with the buffer channels being provided at one layer of a device and the sample channels being provided at a different layer of the device. In this manner, the paths of the various flow lines can cross over one another without actually intersecting (analogous to multiple layers of metallization in an integrated circuit design). In some embodiments, the buffer and sample lines come together on the same level only as necessary to implement sorting modules. Such designs permit single entry ports for sample and buffer (as well as single outlet ports for waste and target collection) while providing parallel processing for high throughput.

[00174] The above embodiments contemplate that target species including controllable force responsive tags (e.g., magnetically labeled target species) move through the sorting station during a sorting a process. While this movement is typically envisioned to be continuous, that is not necessarily the case. In some cases, during their transport the tagged species may become temporarily suspended against the flow of sample and/or buffer mediums. In the case of magnetophoretic separation, this situation becomes increasingly likely as the force exerted on the magnetic particles by the MFGs increases relative to the force exerted by the flow field. [00175] In some embodiments, a sorting device is designed to temporarily hold members of a tagged target species in place within the sorting station. Later, they are released and collected. In such embodiments, the tagged members of the target species stop moving through the sorting station while the other sample components (e.g., non-magnetic components) flow through and out of the station, thereby purifying the tagged target species. In one embodiment, the magnetic components are released and separately collected at an outlet of the sorting station only after the non-magnetic sample components have flowed out of the sorting chamber. [00176] Generally, the buffer entering the sorting region should contain little if any sample. It should provide a medium for collecting relatively pure target material from the sample, as carried by the tag particles. Therefore it preferably should contain relatively little sample material that might interfere with subsequent detection and/or treatment of the target material. Further, the buffer should be compatible with both the target and the tag particles that carry the target. Thus, the buffer may be aqueous or non-aqueous depending on the sample being analyzed. For some applications, the buffer should have a density and composition that maintains magnetic particles and/or the sample materials in suspension. In certain embodiments, the density of buffer is between about 1 and 1.2 g/ml.

[00177] Some commonly used sorting buffers include phosphate buffered saline, deionized water, etc. The actual buffer composition can vary depending on the application and the nature of the sample and target. In a specific embodiment used to sort bacteria, the buffer comprises IX PBS (phosphate buffered saline)/20% glycerol/1% BSA (bovine serum albumin) (all by volume) and has a density of 1.06 g/ml.

[00178] In certain embodiments, a sorting stage operates in constant flow processes to effect sorting. This does not mean that certain sorting operations cannot be performed without interruption of fluid flow. For example, in certain embodiments it may be necessary to intermittently pause the flow for process tuning or for certain designated operations such as detection, amplification, and/or lysis.

[00179] During constant flow conditions, the overall flow rate within the sorting region of a microfluidic device will depend upon throughput goals as well as the total area of the channels and the resistance of the channels within the device. In certain embodiments, the process is performed with a volumetric sample flow rate of between about 10 μL/hour and 500 ml/hour. Typically, the high end of this range is attained with a multi-station parallel flow device or system. For a single sorting station, the sample flow rate may be between about 10 and 5000 μL/hour (preferably between about 50-1000 μL/hour), and the buffer flow rate of about 1-10 times that of the sample flow (preferably 2-4 times the sample flow). In one example, the fluid velocity in the sorting region is between about 100 μm/s and 50 cm/s, typically in the range of about 1-10 mm/s (e.g., approximately 2-5 mm/s).

[00180] Generally, sorting stages should be designed so that little if any unlabeled components cross the stream boundaries by diffusion. This may be accomplished by designing the device to have a relatively fast flow rate in the sorting region, and/or a relatively large distance for sample to traverse from a sample stream to a collection outlet channel. As an example, the typical diffusivity of a 1 μm-sized cell in an aqueous buffer at room temperature is D = 0.2 μm²/s. At a velocity of ~ few mm/s, the dwelling time of each cell in the channel is typically less than a second, during which the cell can diffuse by only a few microns. If the device is operated such that a portion of the buffer stream is bled into the waste channel with a width > 10 μm, which ensures that non-target cells that are able to cross the stream boundary through diffusion are unable to enter the collection channel.

[00181] The channels, inlets, vias, pumps, etc. required for a microfluidic sorting station of this disclosure may be fabricated using well know fabrication techniques (e.g., various microfabrication procedures) or purchased as necessary. In a specific example, borosilicate glass wafers may be affixed to PDMS replicas of a silicon master mold fabricated by applying a precursor to the silicon master, followed by curing. A binding agent such as epoxy may be used to bond the glass and PDMS layers.

[00182] Fluid flow may be either pressure-driven or electrokinetic-driven. Pressure-driven flows are created by pumps (e.g., peristaltic pumps), syringes, etc. that are readily available for small volume microfluidics applications. In a specific embodiment, a dual-track programmable syringe pump (Harvard Apparatus Ph.D. 2000, Holliston, MA) is employed to deliver both the sample mixture and the sorting buffer into the device at constant flow rates. [00183] The flow of sample in the microchannel may be monitored through a suitable detector such as a bright-field microscope (e.g., the DM 4000, LEICA Microsystems AG, Wetzlar, Germany) and a cooled CCD camera (e.g., the ORCA-AG, Hamamatsu Corporation, Bridgewater, NJ).

[00184] The sorting modules of this disclosure may be integrated with various other types of on-chip modules (e.g., multiplexed on-chip detection modules or methods). For example the sorting modules could serve as an initial stage in this regard.

EXAMPLES

[00185] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Standard abbreviations may be used, e.g., bp, base pair(s); kb, kilobase(s); pi, picoliter(s); s or sec, second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb, kilobase(s); bp, base pair(s); nt, nucleotide(s); Lm., intramuscular(ly); Lp., intraperitoneal(ly); s.c, subcutaneous (Iy); and the like.

Example 1: Fabrication and Performance of a Multi-Target Magnetic Activated Cell Sorter (MT-MACS)

[00186] Example 1 describes the fabrication and performance of a Multi-Target Magnetic

Activated Cell Sorter (MT-MACS), which comprises a two-input, multiple output microfluidic device capable of continuously sorting multiple target species into independent, spatially- segregated outputs based on the degree of magnetization of the targets (Figure 1). [00187] As a test of the efficiency and accuracy of MT-MACS sorting, two rare bacterial targets were simultaneously enriched from a mixture dominated by non-target cells, demonstrating high purity (>90%) and high throughput (10⁹ cells/hr) multi-target separation in a single pass.

Device Fabrication and Architecture

[00188] The MT-MACS chips were fabricated using a glass-PDMS-glass architecture.

(Figure 5) The assembled chip has a width, length and thickness of 15.7 x 64 x 1.5 mm (excluding external magnets and fluidic connections) (Figure 1, Panel A). The microchannel at the main flow path is 50 μm high and 500 μm wide, and contains two consecutive arrays of microfabricated ferromagnetic strips (MFS). Each set incorporates twenty 200-nm-thick nickel deflector strips, which are 10 μm wide at a pitch of 20 μm. Each MFS region is connected to corresponding outlets for the collection of deflected target cells; these channels are 50 μm tall and 300 μm wide.

[00189] An exemplary microfabrication procedure for a MT-MACS device is provided in

Figure 5. Panel (A) The MFS regions were defined by electron beam physical vapor deposition of 20 nm Ti and 200 nm Ni on a glass wafer, using standard metal liftoff techniques to define the desired pattern. 100 nm of SiO₂ were subsequently deposited onto the wafer by plasma enhanced chemical vapor deposition as a passivation layer. Inlet and outlet holes were drilled using a CNC mill with a diamond drill bit. Panel (B) The fluidic channels were fabricated with Polydimethylsiloxane (PDMS), using a Si mold defined with deep reactive ion etching. After curing, the PDMS was removed from the mold and placed on a glass backing wafer. Panel (C) Alignment and assembly of the device layers was performed via flip-chip bonding. Microfluidic inlet and outlet connections were made with a brass eyelet for the buffer inlet, a microfluidic connector port (Labsmith, Livermore, CA) for the sample inlet, and Teflon tubing for the outlets. All connections were bonded in place with epoxy.

[00190] Prior to MT-MACS sorting, each class of target cell was labeled with corresponding superparamagnetic tags coupled to affinity reagents (e.g., antibodies) that recognize target-specific cell surface markers (Figure 1, Panel A, Step A). The magnetic tags were chosen such that each type has a distinct magnetization (M) and radius (r). The sample mixture and running buffer were volumetrically pumped into the device by two independently controlled syringe pumps (Figure 1, Panel A, Step B). Collected fractions were subsequently analyzed via flow cytometry to quantitatively measure purity and enrichment (Figure 1, Panel A, Step C).

[00191] Assuming a spherical geometry, the fluidic drag ( Fd ) on a labeled cell in the device can be approximated with Stokes' equation to be

where η is the fluid viscosity, v_f the velocity of the fluid, and v_p the velocity of the particle. Within the microchannel, the two MFS regions generate high magnetic field gradients, such that superparamagnetically-labeled cells traveling through these regions will experience a magnetophoretic force F_1n . This can be approximately calculated as ζ = v(m- 5) = mV5 =— Mr³VB (₂) where m is the magnetic moment of the tag, and B is the magnetic field. The MFS arrays are aligned at two different angles (θj =15° & 6^=5°) with respect to the flow direction (Figure 1, Panel B). In this way, the net amplitude and direction of the force on the labeled cells are governed by the sum of the two forces. Selective deflection of the species occurs when the magnitude of F_m attracting the labeled cell towards the ferromagnetic strip exceeds the component of F_d that pulls the object away from the strip (i.e. F_1n > F_d sin (θ) ).

[00192] The simultaneous sorting of multiple target cells is achieved by exploiting the fact that the balance of the two forces has a non-linear dependence on the radius; F_d has a linear dependence on r (Eq 1) whereas m (and hence F_m) has a cubic dependence (Eq 2), and MT- MACS separates the target cells according to the ratio of F_m/F_d . Thus, cells labeled with tag 1, which is larger in terms of magnetization and radius, are deflected by the first set of MFS (θj =15°) (Figure 1, Panel B, left) and glide along the strips to elute through outlet 1 (Figure 1, Panel C, left). Cells labeled with the smaller tag 2 beads are not deflected here because F_m/F_d is insufficient to cause deflection. Instead, they are deflected by the second set of MFS (θj =5°) (Figure 1, Panel B, right) and elute through outlet 2 (Figure 1, Panel C, right). Unlabeled non- target cells are not deflected by either sets of MFS, and elute through the waste outlet. During the selective deflection process, a running buffer stream is introduced alongside the sample flow to act as a sheath, preventing non-target objects from entering either of the target outlets.

High Precision Magnetophoresis

[00193] Multi-target separation via MT-MACS as described herein can precisely and reproducibly generate magnetophoretic forces. In a specific embodiment, a long-range magnetic field gradient is created with a custom magnetic fixture containing neodymium-iron-boron (NeFeB) permanent magnets (Figure 2, Panel A), which are placed underneath the chip. This gradient is designed to attract the labeled cells to the bottom plane of the microchannel in the MT-MACS device. In addition, a short-range, high-precision, strong magnetic field gradient is created within the microchannel by the MFS regions. When the long-range magnetic field is applied, a large gradient is automatically generated at the interface between the MFS and the buffer due to the fact that nickel possesses significantly higher magnetic permeability compared to aqueous buffer

200, μ_r,buffer~l), thereby creating a precise and reproducible F_m. [00194] The magnetic field gradients within the MT-MACS were numerically calculated using finite element analysis software (Comsol Multiphysics 3.2, Comsol, Los Angeles, CA). In the calculations, relative permittivity μ _^=I.05 and remnant field strength B,=1.27 T for the NeFeB magnets was assumed. These calculations revealed that the strength of the magnetic field within the microchannel was approximately 0.5 T (Figure 2, Panel A), indicating that the superparamagnetic tags are saturated. The downward component (-y direction) of the long-range magnetic field gradient was approximately 200 T/m in the microchannel; assuming a volumetric throughput of -50 ml/hr, this gradient is sufficient to draw all magnetic tags to the bottom plane of the microchannel within 1 cm of the device under operating conditions. The calculations also showed that the short-range gradient from the MFS had a magnitude of ~10⁴ T/m within an approximate distance of 8 μm from the features, with a peak gradient of ~10⁷ T/m (Figure 2, Panel B). These results indicate that under typical MT-MACS operation conditions, in which the volumetric throughput is -50 ml/hr, both F_d and F_m are on the order of ~ tens of nN, confirming the validity of this approach to device design and the experimental observations. [00195] Figure 2 shows a simulation of the long-range and short-range magnetic field gradients. The arrows provide approximate positions of values associated with color scale on y- axis. Panel (A) The long-range magnetic field gradient is generated by the external permanent magnets. The magnitude of the downward (-Y direction) gradient (>200 T/m) extends over the full length of the main fluidic channel (inset). Panel (B) An abrupt change in relative permittivity (μ_r) between the microfabricated nickel features (μ_τ -200) and the surrounding material (μ_τ -1) creates large, short-range magnetic field gradients in the vicinity of the MFS. The magnitude of this gradient is high (>10⁴ T/m) and it extends about - 8 μm from the MFS. The large magnetophoretic forces for target deflection are generated by these short-range gradients. The height of the image corresponds to the height of the fluidic channel.

Magnetic Tag Separation Performance

[00196] The performance of the MT-MACS device was first characterized by simultaneously sorting multiple magnetic tags in the presence of excess non-target polystyrene beads. The device and the magnetic module were mounted under the objective of an epifluorescence microscope (DM4000B, Leica, Bannockburn, IL). Two dual-track programmable syringe pumps (PhD 2000, Harvard Apparatus, Holliston, MA) were used to deliver the sample mixture (flow rate = 5 ml/hr) and buffer (flow rate = 42 ml/hr) into the device. The ternary sample mixture (Figure. 3, Panel A), which was composed of 0.020% tag 1 (r=2.25 μm, color=low green, low red), 0.006% tag 2 (r=1.4 μm, color=low green, high red) and 99.974% non-target particles (polystyrene, r=5 μm, color=high green, high red), was sorted at an overall throughput of 10 particles/hr. The sorted fractions from each outlet were then quantitatively analyzed via flow cytometry.

[00197] After a single round of purification, the flow cytometry results revealed several thousand-fold enrichment of the individual tags. At outlet 1, the population of tag 1 was enriched from 0.020% to 95.876% of the total population, an approximately 5000-fold enrichment (Figure 3, Panel B). This fraction also contained 2.974% of tag 2, and 1.150% of non-target beads. Similarly, the population of tag 2 in outlet 2 was enriched approximately 15000-fold from 0.006% to 90.517% (Figure 3, Panel C); the remainder of the output consisted of 3.125% target 1 and 6.358% non-target beads. Waste output consisted almost entirely of non-target beads (99.997%), as both tags were efficiently depleted by magnetophoretic separation (Figure 3, Panel D), and the waste elute contained only 0.002% of tag 1 and 0.001% of tag 2.

Multi-Target Bacterial Cell Sorting Performance

[00198] The separation performance of MT-MACS for sorting multiple bacterial strains was characterized with three distinct E. coli MC 1061 cell types. Target 1 cells, which inducibly express the T7»tag peptide sequence (MASMTGGQQMG) on their surface, were labeled using anti-T7 mAb-functionalized tag 1 beads. Target 2 cells, which inducibly express a streptavidin- binding peptide (SAl) on their outer membrane, were labeled with streptavidin-functionalized tag 2 beads. Target 2 cells also express green fluorescent protein (GFP), to facilitate visualization. E. coli cells that express blue fluorescent protein (BFP), but no surface marker, were employed as non-target cells. A sample mixture containing low concentrations of labeled target 1 (0.175%) and target 2 (0.383%) cells doped into an excess of non-target cells (99.442%) was pumped into the device at a sample flow rate of 5 ml/hr, with a buffer flow rate of 37 ml/hr (Figure 4, Panel A). This corresponds to a total cell throughput of ~10⁹ cells/hr. Compared to the tag sorting experiment, a slightly lower buffer flow rate was used to account for an increased fluidic drag force on the target cell-tag complex compared to the tag alone. After MT-MACS separation, the cells collected at each of the three outlets were cultured overnight in the absence of induction for subsequent quantification via flow cytometry. Since all the cells are derived from the same host strain (E. coli MC1061) there was no growth bias among the cell types (data not shown). [00199] After a single pass through the MT-MACS device, flow cytometry analysis revealed that the targets were each enriched to over 90% purity (Figure 4). The percentage of target 1 cells in outlet 1 increased from an initial population of 0.175% (Figure 4, Panel A) to 91.575% (Figure. 4, Panel B), a 523-fold enrichment. A small amount of cross-over of target 2 cells (8.393%) and non-target cells (0.032%) was observed in outlet 1. In outlet 2, the population of target 2 cells was enriched from 0.383% to 93.865% (Figure 4, Panel C), corresponding to a 245 -fold enrichment. Outlet 2 also contained some target 1 cells (6.123%) and non-target cells (0.012%). The overwhelming majority of the cell population recovered via the waste outlet were non-target cells (99.621%), with a small amount of target 1 (0.102%) and target 2 cells (0.277%) (Figure 4, Panel D). Since so few magnetic beads passed through the waste outlet under similar experimental conditions, it is likely that these are target cells that detached from their magnetic tags during the separation process.

[00200] As discussed previously herein, certain embodiments pertain to simultaneous sorting of two or more target species (e.g., target cells) via differential labeling of the target species with dissimilar magnetic tags. Device performance has been demonstrated by sorting both target- free magnetic tags and target- tag complexes. In both cases, the results demonstrate simultaneous enrichment of two targets, from a large background of non-targets to over 90% purity in a single round of sorting, at a throughput of order of 10⁹ objects/hr. [00201] The microfluidic, microfabricated architecture of the MT-MACS device affords many advantages. Microfabricating the MTS regions allows for accurate and reproducible generation of very large magnetic gradients within the fluidic channel. The microscale fluidic channel dimensions permit consistent, laminar flow at high velocities (v_f ~ 0.5 m/s, Re~50), enabling well-formed streamlines with large fluidic drag forces. These large magnetic and fluidic forces couple to permit high throughput compared with reported values not only for other microfluidic, magnetophoretic cell sorting devices, but also for flow cytometry. The laminar flow condition additionally helps improve output purity by maintaining segregation of deflected and undeflected objects, and the high fluid velocity helps increase recovery in the device. Although some loss is to be expected at the fluidic connections, only negligible sticking of magnetic tags in the fluidic channels or on the MFS regions was observed. [00202] The MT-MACS concept is scalable and there is no fundamental limitation to only two targets with the outlined methodology. Judicious choice of magnetic beads and MFS angles could extend this number to three targets or more, further extending the versatility and potential applications of this technique. The device outlined here can be scaled to achieve much higher throughputs (e.g., throughput of order several hundred ml/hr) with a widened device and/or parallel implementation of multiple MT-MACS units. Furthermore, additional gains in purity can be achieved with the use of multiple sorting stages in series.

[00203] The sorting performance is ultimately limited by the introduction of undesired cells into a target outlet and by the efficiency of the labeling protocol. One source of impurities in the collected fractions may arise from crossover of targets between the target outlets. Because optimal device performance necessitates a clear distinction between the magnetic moments of the two magnetic tags, one possible source of this crossover may come from variation in the magnetic properties of the tags. Although the enrichment for the target- free tags was about an order of magnitude higher than that of the target-tag complexes (-15000 fold enrichment versus -500), the output purity was roughly equal in each case.

Materials and Methods for Example 1

Magnetic Tags, Cells and Reagents

[00204] As labeling tags, 4.5 μm diameter epoxy-coated M-450 magnetic beads (tag 1) and 2.8 μm diameter streptavidin-coated M-280 magnetic beads (tag 2; Invitrogen, Carlsbad, CA) were used. The tag 1 beads were labeled with antibody for cell separations, and were fluorescently labeled for bead-only separations. A similar labeling protocol was used for both preparations. First, the beads were washed twice and resuspended in a solution of 0.1 M sodium phosphate, pH 7.8. Next, 1 μM BSA-conjugated Alexa Fluor 488 (Invitrogen, Carlsbad, CA) or 300 nM T7»tag monoclonal antibody (EMD Biosciences, San Diego, CA) were added for fluorescent or antibody labeling respectively. After 15 minutes of incubation at room temperature, BSA was added to 0.1% w/v, and the beads were incubated at room temperature for an additional 24 hours. The beads were then washed three times in wash buffer (IX PBS, 0.1% BSA, pH 7.4). The tag 2 beads were used as-is (i.e. streptavidin coated) for cell sorting, but were fluorescently labeled for bead-only separations. For the latter preparation, beads were first washed twice in binding and washing (BW) buffer (5 rnM Tris-HCl, pH 7.5, 0.5 rnM EDTA, 1.0 M NaCl). Next, 1.7 μM biotin-conjugated r-phycoerythrin (Invitrogen, Carlsbad, CA) was added, and the mixture was incubated at room temperature for 30 minutes. The beads were then washed three times in BW buffer.

[00205] Three different E. coli MC 1061 cell types were used, each engineered to express transgenes under the control of an arabinose-inducible promoter. Target 1 cells expressed the peptide sequence (MAS MTGGQQMG), which is recognized by the anti-T7»tag monoclonal antibody. Target 2 cells expressed the outer membrane peptide CPX-SAl, which binds to streptavidin; these cells were also engineered to express green fluorescent protein (GFP). Non- target cells expressed Azurite, a blue fluorescent protein (BFP).

Tag separation protocol

[00206] Prior to sorting, the tubing for the device, sample and buffer inlets was washed extensively with ddH₂0. The device was connected to the tubing, placed under the viewfield of a microscope (Leica Microsystems GmbH, Wetzlar, Germany), and was then washed for at least 30 minutes with 4 ⁰C running buffer (IX PBS, 0.1% Tween-20). The fluorescently-labeled tags were combined with 5 μm green fluorescent polystyrene non-target beads (Duke Scientific, Fremont, CA), volume adjusted to 600 μl with PBS, mixed by pipette, and loaded into the sample inlet tubing. Buffer and sample flow were delivered using two dual-track programmable syringe pumps (Harvard Apparatus, Holliston, MA), and device output was collected in microcentrifuge tubes. Following sorting, the device and tubing were thoroughly cleaned with running buffer, followed by ddH₂O, and then allowed to dry. The remaining initial mixture and each of the outlet collections were subsequently analyzed via FACS (FACSAria, BD Biosciences, San Jose, CA).

Bacterial Cell Sorting Protocol

[00207] Target cells were grown in Luria-Bertani (LB) medium with 34 μg/ml chloramphenicol (CM) for 4 hours at 37 ⁰C. The cells were then subcultured at a 1:50 dilution for 2 hrs at 37 ⁰C. Protein expression was induced with the addition of 0.02% w/v L-arabinose for 3 hrs. Tag 1 and 2 beads were washed thrice with wash buffer. Next, a small number of target cells were added to a 10-fold excess of washed beads (target 1 cells to tag 1 beads, target 2 cells to tag 2 beads) and IX PBS added such that the total volume of each mixture was -100 μl. These mixtures were incubated overnight at 4 ⁰C. The following morning, non-target cells were grown in LB with CM for 6-7 hrs. Prior to sorting, the device and tubing were prepared as described above. The non-target cells were pelleted via centrifuge at 5000 rpm for 5 mins and resuspended in IX PBS. The non-target cells were then combined with small volumes of each of the labeled target cell samples; these were mixed by pipette and then loaded into the sample inlet. Cold running buffer was loaded via the buffer inlet, and the sorted fractions were collected from each outlet in microcentrifuge tubes. Following sorting, the device and tubing were cleaned as described above. Sample volumes from the initial mixture and each of the collected outlets were added to LB with CM and 0.2% D-glucose. These were incubated overnight at 37 ⁰C. The following morning, the cells were subcultured and induced as above. Next, 10 μl of subcultured cells from each collection sample were added to 200 μl PBS with 20 nM streptavidin-conjugated phycoerythrin (SAPE) (Invitrogen, Carlsbad, CA), and incubated at 4 ⁰C for 1 hr. The cells were pelleted via centrifuge at 5000 rpm for 5 mins, resuspended in 200 μl PBS, and analyzed immediately with FACS.

Example 2: Fabrication and performance of an Integrated Acoustophoretic- Magnetophoretic Separation Device (iAMSD)

[00208] Example 2 describes the fabrication and performance of an Integrated

Acoustophoretic- Magnetophoretic Separation Device (iAMSD) which provides rapid mulit- target cell sorting.

Fabrication of the iAMSD

[00209] As shown in Figure 10, the device is constructed by initially etching a microchannel in silicon via deep reactive ion etching. The microchannel is capped by anodically bonding a glass wafer to the Si. Microfabricated nickel strips on the glass wafer provide the magnetic deflection force to transfer the magnetic target beads. The device is completed by bonding a piezotransducer to the Si side and external magnets to the glass side. A cross sectional schematic (Panel A) and photograph of the completed device (Panel C) are shown in Figure 7. Sorting Protocol

[00210] The iAMSD is designed to sort acoustic targets, magnetic targets and non-targets into independent, spatially segregated outlets in a continuous flow manner. A diagram of the experimental setup is provided in Figure 11. First, the buffer and sample mixture containing the three different types of targets are injected into the device. Due to the ultrasonic actuation, all three types of particles experience an acoustic force towards the first acoustic node as shown in Figures 7 and 8. Due to the fact that the acoustic force is proportional to the volume of the particles, only the acoustic and magnetic targets are effectively focused into the magnetic separation region, and the non-target particles elute through the waste outlet (Figures 7 and 8). At the magnetic separation region, the acoustic and magnetic targets are further separated as the magnetic targets are selectively deflected along the microfabricated Ni strips (Figures 7 and 8). As shown in Figure 9, a small indent in the microchannel sidewall in the magnetic separation region prevents acoustic resonance in this area, facilitating magnetic deflection.

Device Physics

[00211] The combined acoustic-magnetic separation device is designed to sort acoustic targets, magnetic targets and non-targets into independent outlets in a continuous flow manner. First, the buffer and sample mixture containing the three different types of targets are injected into the device. Due to the ultrasonic actuation, all three types of particles experience an acoustic force towards the first acoustic node, and move towards the acoustic node at velocity v_ac = (F_ac μβπηa , where F_ac is the acoustic radiation force, η is the fluid viscosity and a is the particle radius. The acoustic radiation force is described by

where E is the acoustic energy density, ω the angular frequency, V the particle volume, k the angular wavenumber, x the transverse position in the channel, a = p_p I p_w where p_p and p_w are the particle and fluid densities, and β = c_p /c_w where c_p and c_w are the speeds of sound in the particle and fluid. The separation velocities of the acoustic and magnetic targets particles are on the order of 10-30 times higher than the nontarget particles; thus, only the acoustic and magnetic targets are effectively focused into the magnetic separation region, and the non-target particles elute through the waste outlet.

[00212] Subsequently, the particles enter a magnetic separation region. Here, a small indent in the microchannel sidewall in the magnetic separation region precludes any acoustic resonance in this area, facilitating magnetic deflection (detailed in Figure 9). A set of 3 permanent magnets serve to draw all magnetic objects down towards the lower plane of the fluid channel and also to magnetize a series of micro fabricated Ni strips. It is estimated that the maximum time needed to draw all magnetic objects to the bottom of the channel is about 42 ms, or 1/7 the average total time the particles are present in the fluid channel, assuming an average flow velocity of 0.1 m/s. The microfabricated Ni strips generate large and reproducible magnetic gradients, which serve to selectively deflect the magnetic targets into a new flow stream. Nontarget particles and acoustic target particles do not experience any magnetic force, and remain undeflected in this region of the device. Magnetic separation occurs if the magnetic force is greater than the component of the hydrodynamic drag force perpendicular to the Ni strip, or F_m > F_HYD± . From numerical simulation, it is estimated that the magnetic field gradient is 1000

T/m within ~5 μm of the Ni strip, which translates to an estimated magnetic force of F_m ~ 1.4 nN on the magnetic target. Correspondingly, at 0.1 m/s flow rate, calculation of an upper-end estimate of the perpendicular component of the hydrodynamic drag force on the magnetic target gives F_HYD-¹- ~ 0.4 nN, confirming the observation that the magnetic targets are deflected along the Ni line.

Performance of the iAMSD

[00213] The performance of the iAMSD was measured by simultaneously sorting multiple bead types. The sample mixture consisted of 19.3 % acoustic target particles (5 μm diameter polystyrene, Duke Scientific), 33.5 % magnetic target particles, (4.5 μm diameter M450, Dynal Invitrogen) and 47.2% non-target particles (1 μm diameter, MyOne™ Streptavidin T, Invitrogen) (Figure 12). The separation was performed at a sample flow rate of 0.5 ml/hr and buffer flow rate of 20 ml/hr, corresponding to a throughput of ~10⁷ particles/hr, and the collected fractions from each outlet were analyzed via flow cytometry (FACS Aria™, BD Biosciences). Significant enrichment of multiple targets was observed after a single pass through the device; the Acoustic outlet contained 97.5 % acoustic targets, 0.1% magnetic targets, and 2.4% non-targets. The Magnetic outlet contained 78.4 % magnetic targets, 4.6 % acoustic targets and 17.0 % non- targets. The waste outlet contained primarily non-target particles. These results are depicted in Figure 12, Panel B.

[00214] An additional separation was performed to show device performance given initial low target purities. A sample mixture having initial target purities of < 1% was provided to the device. The separation was performed at a sample flow rate of 0.5 ml/hr and buffer flow rate of 20 ml/hr, corresponding to a throughput of ~10⁸particles/hr, and the collected fractions from each outlet were analyzed via flow cytometry (FACSAria™, BD Biosciences). Target purities of 89% for the acoustic target and 95% for the magnetic target were obtained, with 91% of all targets being separated. These results are depicted in Figure 12, Panel C.

Example 3: Fabrication and Performance of an Integrated Dielectrophoretic-Magnetic Activated Cell Sorter (iDMACS)

[00215] Example 3 describes the fabrication and performance of an Integrated

Dielectrophoretic-Magnetic Activated Cell Sorter (iDMACS). The iDMACS is a two-input, multiple-output device. The inputs introduce the running buffer and the sample into the system; the sample in this experiment included two types of target cells, labeled with either micrometer- scale dielectrophoretic or nanometer-scale magnetic tags, within a large excess of unlabeled non- target cells. After a single pass through the device, both target cell types were purified and eluted through independent, spatially-segregated outlets.

Buffer conditions and DEP and Magnetic Tags

[00216] Green fluorescent polystyrene beads (5.0 μm diameter, Duke Scientific, Fremont,

CA) were used as Dielectrophoretic (DEP) tags, and red fluorescent magnetic beads (1.0 μm diameter, Invitrogen, Carlsbad, CA) were used as magnetic tags. For tag separation experiments, blue fluorescent polystyrene beads from Duke Scientific (2.0 μm diameter), were used as the non-target background particles. Bead separations were performed using concentrations of 3.6 x 10³ beads/ml for DEP tags, 3.5 x 10³ beads/ml for magnetic tags and 1 x 10⁸ beads/ml for non- target particles. The bead mixture was suspended in O.lx PBS with 1% BSA (Fraction V, Sigma, St. Louis, MO). In order to prevent settling of the beads during the sorting experiments, the density of the solution was adjusted to that of polystyrene beads (1.06 g/ml) by addition of glycerol to a final concentration of 20% (vol/vol).

Cells and Reagents

[00217] Cell sorting experiments were performed with E. coli strain MC1061 [F- araD139

Δ(ara-leu)7696 galE15 galK16 Δ (lac)X74 rpsL(StrR) hsdR2 (rK- mK+) mcrA mcrBl]. Genes coding for the surface and fluorescent proteins were expressed using the plasmid pBAD33. The surface peptides were expressed as N-terminal fusions to CPX for T7»tag peptides or eCPX for streptavidin-binding peptides. Cells were cultured overnight at 37 ⁰C in Luria-Bertani (LB) medium with 34 μg/mL chloramphenicol (Sigma, St. Louis, MO), and subcultured at a 1:50 dilution for 2 h at 37 ⁰C. The expression of the binding peptides and fluorescent proteins were induced by adding L-arabinose (0.02% w/v) to the culture media for 45 min at 37 ⁰C. Roughly 10⁴ target A cells (expressing T7»tag) and target B cells (expressing streptavidin-binding peptide) were centrifuged (265Og, 5 min) and resuspended in 10 μL 0.25x PBS, 0.5% BSA (PBSB). 9.6-μm polystyrene beads (Bangs Lab, Fishers, IN) were used as DEP tags. Anti-T7»tag mAb (EMD Biosciences, La Jolla, CA) were conjugated to ~10⁶ carboxylic acid-coated DEP tags via standard EDC-NHS covalent coupling. The tags were subsequently used to label approximately 10⁴ target A cells in an inversion shaker at 4 ⁰C overnight. 0.2 μL of streptavidin- coated, 50-nm MicroBeads (Miltenyi, Auburn, CA) were used as magnetic tags, and were used to label approximately 10⁴ target B cells overnight in an inversion shaker at 4 ⁰C. Approximately 10⁷ non-target cells expressing blue fluorescent protein (BFP) were washed in 500 μL PBSB and resuspended in the cell/bead mixture. The mixture was washed once and resuspended in 100 μL sorting buffer (O.lx PBS, 1% BSA, 20% glycerol), and finally passed through a CellTrics® 20-μm mesh filter (Partec, Mϋnster, Germany).

iPMACS Design and Fabrication

[00218] The iDMACS device was fabricated using a glass-polyimide-glass sandwich architecture, and contains titanium/gold electrodes for DEP sorting and titanium/nickel structures for magnetic traps. The microfabrication process flow is shown in Figure 17. Step a) Ni strips were patterned with 20 nm of titanium and 200 nm of nickel via a standard lift-off process (Temescal, Berkeley, CA) on 4-inch glass wafers (Pyrex 7740 borosilicate glass; Corning, Corning, NY) as bottom substrate. Step b) A 100-nm-thick passivation layer of SiO₂ was deposited by plasma-enhanced chemical vapor deposition (Plasma-Therm, Prosper, TX). Step c) Top and bottom DEP electrodes were patterned with 20 nm of titanium and 200 nm of gold (Temescal). Step d) The microchannels were formed with photosensitive polyimide (HD4010; HD MicroSystems, Parlin, NJ) on the top substrate, which served as the spacer between the two glass substrates. It was spun on the top and bottom substrates at 1,000 rpm for 45 sec, which results in a 20-μm-thick film after curing and bonding. Channels were defined on this layer by photolithography using a standard photolithographic tool (SUSS MicroTec, Garching, Germany; 350-nm wavelength, 1-min exposure) and development process (2 min in 100% developer, 1 min in 50% developer and 50% rinser, and 30 sec in 100% rinser). Step e) Microfluidic vias on the top substrate were drilled with a computer-controlled milling machine (Flashcut CNC, Menlo Park, CA), and both substrates were diced. Step f) The two substrates were aligned and bonded at 300 ⁰C for 2 min using a Flip-Chip aligner bonder (Research Devices, Piscataway, NJ). To complete the bonding process, a wafer bonder (SB-6; SUSS MicroTec) was used to cure the polyimide layer at 375 ⁰C for 40 min and then bond for 10 min. Microfluidic inlets and outlets were manually fixed on the drilled vias of the device using epoxy.

Experimental Setup

[00219] The iDMACS was mounted beneath the objective of an epifluorescence microscope (DM4000B, Leica, Bannockburn, IL) in order to monitor the separation process. Buffer and the sample were delivered into the device using two dual-track programmable syringe pumps (PhD 2000, Harvard Apparatus, Holliston, MA). For the DEP separation module, the electrodes were powered using a function generator (AFG320, Tektronix, Beaverton, OR), and the frequency and magnitude of applied voltages were measured with a digital oscilloscope (54622A, Agilent Technologies, Palo Alto, CA). The magnetic separation module made use of two 1/2" x 1/4" x 1/16" thick neodymium external magnets (grade N42, K&J Magnetics, Jamison, PA). A separate permanent magnet of the same type was placed on top of the chip to fix the position of the magnets. During the sorting experiment, all cells were kept on ice, and the sorting experiments were performed within ~ 1 hour. Cytometry Analysis

[00220] After the iDMACS separation, the eluted cells from A, B and waste outlets were grown overnight in LB medium, with 0.2% glucose to repress pBAD33 gene expression; this ensured that there were no growth biases among the various cell types. The cells were then subcultured at a 1:50 dilution for 2 h at 37 ⁰C, after which L-arabinose (0.02% w/v) was added to the culture media for 3 h at 37 ⁰C in order to induce expression of cell surface peptides and fluorescent proteins. 5 μL of cells from each outlet-derived subuculture were incubated in 100 μL Ix PBS, 0.5% BSA (PBSB) containing 20 nM streptavidin-phycoerythrin (SAPE) for 1 h on ice, followed by centrifugation and removal of the supernatant. Finally, the cells were resuspended in cold PBSB at ~10⁶ cells/ml and immediately analyzed by FACS (FACSAria™, BD Biosciences, San Jose, CA).

Device Physics

[00221] Prior to separation, each class of target cells was labeled with either DEP or magnetic tags via their surface markers (Figure 13, Panel A, Step A). Polystyrene beads (PSB) were used as DEP tags to label target A cells because their low surface conductivity (σ) leads to complex permittivities that differ significantly from those of bacterial cells. Target B cells were labeled with superparamagnetic tags. After labeling, the sample mixture and running buffer was pumped into separate inlets of the iDMACS using two independently controlled syringe pumps (Figure 13, Panel A, Step B). Due to the low Reynolds number of the flow (Re ~ 0.1), the buffer and sample streams are laminar and remain segregated during the separation. All cells entering the device are subject to a hydrodynamic force, F_HD = 6ττμa(v_f - v_p ) , where μ is the viscosity of the fluid, a is the diameter of the cell, v_f is the velocity of the fluid, and v_p is the velocity of the cell.

[00222] Cells subsequently enter the dielectrophoretic separation module, where they are subjected to dielectrophoretic forces (F_DEP) created by the non-uniform electric field generated by a set of titanium/gold electrodes (Figure. 13, Panel B). Only the DEP tag-labeled cells undergo selective deflection into the buffer stream by the angled electrodes, because they experience F_DEP (~ 2 nN) which exceeds the F_HD (-0.4 nN) in the direction perpendicular to the electrodes (i.e. F_DEP > F_HD sin θ ). The magnetically labeled (target B) and unlabeled (non- target) cells in the sample are not deflected here because F_HD sin θ > F_DEP . In this way, the DEP tag-labeled target A cells elute through outlet A, while target B and non-target cells proceed to the magnetic separation module.

[00223] The magnetic separation module includes an array of microfabricated nickel ferromagnetic strips that efficiently trap magnetically-labeled cells with extremely high localized magnetic field gradients (Figure 13, Panel B). The application of an external magnetic field (B_ext) to the ferromagnetic strips creates large magnetic field gradients ( VB ) due to the mismatch in magnetic permeabilities between the buffer medium and nickel structures (//_r,buffer~l,

200). From numerical simulations, the magnetic field gradient near the Ni stripes is estimated to be greater than 10⁴ T/m¹², and so it is estimated that the magnetophoretic force (F_MAG) on the magnetically labeled cells is ~ 0.3 nN. On the other hand, F_HD on the magnetically labeled cells is ~ 0.07 nN under the experimental operating conditions ( v_f = ~ 2 mm/s), confirming that F_MAG > F_HD, which allows for effective trapping.

Particle Separation Performance

[00224] The performance of the iDMACS chip (shown in Figure 14, Panel A) was initially characterized with a mixture of DEP and magnetic tags in order to optimize the operating conditions of the device. A ternary mixture of DEP tags (5-μm-diameter polystyrene beads), magnetic tags (1.0-μm-diameter superparamagnetic beads) and non-target particles (2.0-μm- diameter polystyrene beads) were suspended in sorting buffer (O.lx PBS, 1% BSA, 20% glycerol). Two dual-track programmable syringe pumps delivered the sample mixture and buffer into the device at flow rates of 250 μL/h and 500 μL/h, respectively. This flow rate corresponds to a throughput of ~5 X 10⁷ particles/hour/microchannel. A sinusoidal voltage of 20 V_{peak to peak} was applied to the DEP electrodes at 500 kHz.

[00225] High-speed videos of the separation process revealed that DEP tags were efficiently separated at the DEP separation module and eluted into outlet A (Figure 14, Panel B, top). As expected, the magnetic tags and non-target beads were not deflected by dielectrophoresis (Figure 14, Panel B, bottom). However, the magnetic tags were efficiently captured in the magnetic separation module at the edges of the nickel patterns (Figure 14, Panel C), where the magnetic field gradient is highest, while non-target particles were continuously eluted through outlet B. The magnetic tags were retrieved by removing the external magnet after the entire sample had been processed. Although some particles were lost in the tubing and other fluidic interconnections, no sticking of the particles to the device was observed, and all particles that entered the device were successfully recovered.

[00226] In order to quantify the purity of the separation, the eluted fractions from outlet A and outlet B were analyzed via flow cytometry (FACSAria™, BD Biosciences, San Jose, CA). The initial sample consisted of 0.036% DEP tags, 0.035% magnetic tags, and 99.929% non- target beads (Figure 15, column 1). After a single pass through the iDMACS device, the eluted fraction at outlet A contained 98.9% DEP tags, 0% magnetic tags and 1.1% non-target beads (Figure 15, column 2), which translates to a -2,700-fold enrichment of DEP tags. Likewise, the outlet B fraction consisted of 98.5% magnetic tags, 0% DEP tags and 1.5% non-target beads, corresponding to a -2,800-fold enrichment (Figure 15, column 3). The waste fraction was comprised purely of non-target beads, with no detectable DEP or magnetic tags (Figure 15, column 4).

Multi-target Bacterial Cell Separation

[00227] Three distinct bacterial clones of E.coli MC 1061 strain were generated for use in cell separation experiments. Target A cells expressed the T7»tag peptide sequence (MASMTGGQQMG) on their surface, and were labeled with anti-T7 mAb-functionalized DEP tags. These cells also expressed green fluorescent protein (GFP) allowing facile visualization and sorting via flow cytometry. Target B cells inducibly expressed a streptavidin-binding peptide (SAECHPQGPPCIEGR) on their outer membrane, and these were labeled with streptavidin- coated magnetic tags. Finally, bacteria expressing Azurite, a blue fluorescent protein (BFP), were employed as non-target cells; these cells did not express any particular surface markers for labeling.

[00228] The initial sample contained low concentrations of target A (0.32%) and target B

(0.11%) cells doped into a large excess of non-target cells (99.57%) (Figure 16, Panel A). The parameters of iDMACS operation (i.e., flow rates, DEP voltage amplitude and frequency, and magnetic actuation) were the same as those used in the tag separation experiment described above, corresponding to a throughput of -2.5 X 10⁷ cells/hour. After the separation, the cells eluted at each outlet were cultured overnight and quantified using flow cytometry. [00229] Flow cytometry analysis revealed that, after a single pass through the iDMACS device, the population of target A cells in outlet A increased from 0.32% to 98.6%, corresponding to a 310-fold enrichment (Figure 16, Panel B). Similarly, in outlet B, the fraction of target B cells increased 870-fold, from 0.11% to 95.6%, (Figure 16, Panel C). It is noteworthy that there was no cross-contamination between target A and target B in either outlet (i.e., no target A cells in outlet B, and no target B cells in outlet A). As expected, the waste fraction consisted mostly of non-target cells (99.74%) with 0.17% target A and 0.09% target B cells (Figure 16, Panel D).

[00230] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims.

Claims

What is claimed is:

A microfluidic sorting device comprising:

(a) at least one inlet channel configured to provide at least two separate streams, wherein

(i) at least one stream defines a sample stream path for a sample suspected of containing one or both of a first target species and a second target species, wherein said sample comprises a first magnetic particle having affinity for said first target species, a second magnetic particle having affinity for said second target species, and a non-target species, and

(ii) at least one stream defines a buffer stream path for a buffer, wherein the buffer is substantially free of the sample;

(b) a sorting station fluidly coupled to said at least one inlet and located in the sample stream path, wherein said sorting station comprises a first array of ferromagnetic elements and a second array of ferromagnetic elements;

(c) a magnetic field gradient generator which produces a change in the magnetic field gradient in the sorting station such that said first array deflects said first magnetic particle into the buffer stream path at a first location and out a first outlet channel, such that complexes of said first magnetic particle and said first target species, if present, are deflected out said first outlet channel; and said second array deflects said second magnetic particle into the buffer stream path at a second location and out a second outlet channel, such that complexes of said second magnetic particle and said second target species, if present, are deflected out said second outlet channel; and

(d) a third outlet channel configured to receive a waste stream from said sample stream path, such that sample that is at least partially depleted of a target species flows through said third outlet channel.

2. The microfluidic sorting device of claim 1, wherein the at least one inlet channel comprises a first inlet channel for providing at least a portion of the buffer stream and a second inlet channel for providing at least a portion of the sample stream.

3. The microfluidic sorting device of claim 1, wherein the ferromagnetic elements are micropatterned nickel elements.

4. The microfluidic sorting device of claim 1, wherein the first and second arrays of ferromagnetic elements comprises ferromagnetic strips.

5. The microfluidic sorting device of claim 1, wherein the ferromagnetic elements comprises one or more pins or pegs.

6. The microfluidic sorting device of claim 1, wherein the device comprises the sample.

7. The microfluidic sorting device of claim 6, wherein the sample contains at least a first complex comprising the first target species and the first magnetic particle or at least a second complex comprising the second target species and the second magnetic particle.

8. A method of separating two or more target species in a sample, said method comprising: introducing a sample into the flow path of a microfluidic sorting device, said microfluidic sorting device comprising a first array of ferromagnetic elements and a second array of ferromagnetic elements, wherein said sample comprises a first target species, a second target species, a first magnetic particle having affinity for said first target species, and a second magnetic particle having affinity for said second target species, and wherein the first and second magnetic particles are differentially magnetically separable; and applying an external magnetic field to said microfluidic sorting device, wherein said applying results in deflection of the first target species when present in a complex with said first magnetic particle by said first array to a first outlet of the microfluidic sorting device and deflection of the second target species when present in a complex with said second magnetic particle by said second array to a second outlet of the microfluidic sorting device.

9. The method of claim 8 wherein, prior to said introducing step, the method comprises: contacting a first target species in a sample with a first magnetic particle having affinity for said first target species to produce a first complex comprising the first target species and the first magnetic particle; and contacting a second target species in said sample with a second magnetic particle having affinity for said second target species to produce a second complex comprising said second target species and said second magnetic particle; wherein the first and second magnetic particles are differentially magnetically separable.

10. A microfluidic sorting device configured to sort at least a first target species and a second target species from a sample, wherein members of said first target species comprise a first controllable force responsive tag and members of said second target species comprise a second controllable force responsive tag, said device comprising:

(a) at least one inlet;

(b) a sorting station in fluid communication with said at least one inlet;

(c) a first controllable force generator, which, during operation of the microfluidic sorting device, produces at least a first controllable force in said sorting station, said first controllable force acting on said first controllable force responsive tag, when present, to separate said first target species from said sample;

(d) a second controllable force generator, which during operation of the microfluidic sorting device, produces a second controllable force in said sorting station, said second controllable force acting on said second controllable force responsive tag, when present, to separate said second target species from said sample; and

(e) an outlet in fluid communication with said sorting station, wherein said outlet is configured to receive a waste stream at least partially depleted of said first target species and said second target species.

11. The microfluidic device of claim 10, wherein said first controllable force displaces said first controllable force responsive tag from a first position to a second position in said sorting station to separate said first target species from said sample.

12. The microfluidic device of claim 10, wherein said second controllable force displaces said second controllable force responsive tag from a first position to a second position in said sorting station to separate said second target species from said sample.

13. The microfluidic device of claim 12, wherein said second controllable force displaces said second controllable force responsive tag from a first position to a second position in said sorting station to separate said second target species from said first target species.

14. The microfluidic device of claim 10, wherein said first controllable force and said second controllable force are different.

15. The microfluidic device of claim 14, wherein said first controllable force and said second controllable force are selected from the group consisting of a magnetic field, acoustic radiation, an electric field, and an electromagnetic field.

16. The microfluidic sorting device of claim 10, wherein said first controllable force generator and said second controllable force generator are selected from the group consisting of a magnetic field generator, an acoustic radiation generator, an electric field generator, and an electromagnetic field generator.

17. A microfluidic sorting device configured to sort at least a first target species and a second target species from a sample, wherein members of said first target species comprise a first controllable force responsive tag and members of said second target species comprise a second controllable force responsive tag, said device comprising:

(a) at least one inlet;

(b) a sorting station in fluid communication with said at least one inlet; (c) a controllable force generator, which, during operation of the microfluidic sorting device, produces a controllable force in said sorting station, said controllable force acting on said first controllable force responsive tag and said second controllable force responsive tag, when present, to separate said first target species and said second target species from said sample; and

18. The microfluidic device of claim 17, wherein said controllable force displaces said first controllable force responsive tag from a first position to a second position in said sorting station to separate said first target species from said sample.

19. The microfluidic device of claim 17, wherein said controllable force displaces said second controllable force responsive tag from a first position to a second position in said sorting station to separate said second target species from said sample.

20. The microfluidic device of claim 19, wherein said controllable force displaces said second controllable force responsive tag from a first position to a second position to separate said second target species from said first target species.

21. The microfluidic device of claim 17, wherein said controllable force is selected from the group consisting of a magnetic field, acoustic radiation, an electric field, and an electromagnetic field.

22. The microfluidic sorting device of claim 17, wherein said controllable force generator is selected from the group consisting of a magnetic field generator, an acoustic radiation generator, an electric field generator, and an electromagnetic field generator.

23. A microfluidic sorting device configured to sort at least a first target species from a sample, wherein members of said first target species comprise a first controllable force responsive tag and a second controllable force responsive tag, said device comprising:

(a) at least one inlet;

(b) a sorting station in fluid communication with said at least one inlet;

(c) a first controllable force generator, which, during operation of the microfluidic sorting device, produces a first controllable force in said sorting station, said first controllable force acting on said first controllable force responsive tag, when present, to separate said first target species from said sample;

(d) a second controllable force generator, which during operation of the microfluidic sorting device, produces a second controllable force in said sorting station, said second controllable force acting on said second controllable force responsive tag, when present, to separate said first target species from said sample; and

(e) an outlet in fluid communication with said sorting station, wherein said outlet is configured to receive a waste stream at least partially depleted of said first target species.

24. The microfluidic device of claim 23, wherein said first controllable force displaces said first controllable force responsive tag from a first position to a second position in said sorting station to separate said first target species from said sample.

25. The microfluidic device of claim 23, wherein said second controllable force displaces said second controllable force responsive tag from a first position to a second position in said sorting station to separate said first target species from said sample.

26. The microfluidic device of claim 23, wherein said first controllable force and said second controllable force are different.

27. The microfluidic device of claim 26, wherein said first controllable force and said second controllable force are selected from the group consisting of a magnetic field, acoustic radiation, an electric field, and an electromagnetic field.

28. The microfluidic sorting device of claim 23, wherein said first controllable force generator and said second controllable force generator are selected from the group consisting of a magnetic field generator, an acoustic radiation generator, an electric field generator, and an electromagnetic field generator.

29. A microfluidic sorting device configured to sort at least a first target species from a sample, wherein members of said first target species comprise a controllable force responsive tag, said device comprising:

(a) at least one inlet;

(b) a sorting station in fluid communication with said at least one inlet;

(c) a first controllable force generator, which, during operation of the microfluidic sorting device, produces a first controllable force in said sorting station, said first controllable force acting on said controllable force responsive tag, when present, to separate said first target species from said sample;

(d) a second controllable force generator, which during operation of the microfluidic sorting device, produces a second controllable force in said sorting station, said second controllable force acting on said controllable force responsive tag, when present, to separate said first target species from said sample; and

30. The microfluidic device of claim 29, wherein said first controllable force displaces said controllable force responsive tag from a first position to a second position in said sorting station to separate said first target species from said sample.

31. The microfluidic device of claim 29, wherein said second controllable force displaces said controllable force responsive tag from a first position to a second position in said sorting station to separate said first target species from said sample.

32. The microfluidic device of claim 29, wherein said first controllable force and said second controllable force are different.

33. The microfluidic device of claim 32, wherein said first controllable force and said second controllable force are selected from the group consisting of a magnetic field, acoustic radiation, an electric field, and an electromagnetic field.

34. The microfluidic sorting device of claim 29, wherein said first controllable force generator and said second controllable force generator are selected from the group consisting of a magnetic field generator, an acoustic radiation generator, an electric field generator, and an electromagnetic field generator.

35. A microfluidic sorting device comprising:

(a) at least one inlet configured to permit fluidic movement of at least two fluid streams;

(b) a sorting station in fluid communication with said at least one inlet;

(c) two or more controllable force generators, configured to produce two or more controllable forces in said sorting station; and

(d) an outlet in fluid communication with said sorting station.

36. The microfluidic sorting device of claim 35, wherein said two or more controllable force generators are selected from the group consisting of a magnetic field generator, an acoustic radiation generator, an electric field generator, and an electromagnetic field generator.

37. A method for using a microfluidic sorting device having a sample flow path to sort target species in a sample, said target species having one or more controllable force responsive tags attached, wherein at least one of said tags produces a response to an applied controllable force, wherein the target species is sortable based on the magnitude of the response, comprising:

(a) introducing a sample into the flow path of a microfluidic sorting device, said microfluidic sorting device comprising at least one controllable force generator for producing at least one controllable force in the sample flow path;

(b) applying said at least one controllable force to said microfluidic sorting device under conditions suitable for generating a response from the controllable force responsive tags; and

(c) sorting a target species based on the magnitude of the response generated.

38. The method of claim 37, wherein more than one controllable force is applied.

39. The method of claim 37, wherein the target species comprises more than one controllable force responsive tag.