WO2024073585A2

WO2024073585A2 - Systems and methods for single-cell trapping via dielectrophoresis

Info

Publication number: WO2024073585A2
Application number: PCT/US2023/075399
Authority: WO
Inventors: Marc S. CHOOLJIAN; Amir TAHMASEBIPOUR; Yu Ting Chow
Original assignee: Mekonos, Inc.
Priority date: 2022-09-30
Filing date: 2023-09-28
Publication date: 2024-04-04
Also published as: WO2024073585A3

Abstract

A method of capturing a cell by flowing a fluid having a plurality of cells at a first flow rate into a fluidic channel of an apparatus, generating a set of capture sites comprising one or more captured cells by applying a signal at a first signal strength to one or more electrodes disposed in the fluidic channel of the apparatus, thereby generating a non-uniform electric field at or in a vicinity of the one or more electrodes, wherein the fluidic channel comprises a capture site disposed approximate the one or more electrodes; and increasing the flow rate of the fluid to a second flow rate and/or changing the signal to a second signal strength to remove all but one captured cell at one or more of the capture sites in the set of capture sites.

Description

DESCRIPTION

SYSTEMS AND METHODS FOR SINGLE-CELL TRAPPING VIA DIELECTROPHORESIS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63/377,965 filed September 30, 2022, the contents of which are incorporated into the present application by reference in its entirety.

BACKGROUND

[0002] The embodiments disclosed herein are generally directed towards systems, processes and methods for single-cell trapping via dielectrophoresis.

[0003] Dielectrophoresis (DEP) is an electro-physical phenomenon that occurs when an electrically neutral, but polarizable, substance, such as a biological molecule or a cell, in a nonlinear and/or non-uniform electric field experiences a force in the electric field gradient. This occurs because one side of the particle experiences a larger dipole force than the other due to the variation in electric field across the particle. The DEP force is nominally given by the following equation:

F_DEP = ne_mr³Re f_CM)V\E\² where r is the radius of the particle, e_m is the permittivity of the fluid, E is the electric field, and fCM is the Clausius-Mossotti factor, a complex value that depends on the difference in permittivity between the fluid and the particle, and which determines if the DEP force will be positive or negative.

[0004] Based on the ability to trap and sort neutral particles or biological molecules in fluidic environments, DEP can be exploited, for example, for single-cell analyses in microfluidic-based applications. Using DEP in standard biochemical assays, for example, by applying DEP to isolate cells for impedance or fluorescence characterization (or any non-contact evaluation technique) has been demonstrated in fluidic environments. However, using DEP to isolate single cells for direct manipulation of the cells presents additional challenges due to, for example and not limited to, multiple cells localizing at a capture point in the DEP apparatus. Existing single-cell dielectrophoretic traps generally use negative DEP (nDEP) and exclude additional cells by using a “cage” of repulsive force (see Jang et al, DOI: 10.1016/j.bios.2009.05.027) or make use of additional fluidic features such as chambers or microwells to confine cells (see Qin & Wu et al, DOI: 10.1002/anie.201807314). These require larger trapping sites and therefore lower trapping densities. Therefore, there is a need in the art to remove unwanted cells from the capture points in a DEP apparatus.

BRIEF SUMMARY

[0005] In accordance with various embodiments, a method of capturing a cell is provided. The method can include any of the following steps: flowing a fluid at a first flow rate into a fluidic channel of an apparatus; generating a set of capture sites comprising one or more captured cells by applying a signal at a first signal strength to one or more electrodes disposed in the fluidic channel of the apparatus, thereby generating a non-linear and/or non-uniform electric field at or in a vicinity of the one or more electrodes; increasing the flow rate of the fluid to a second flow rate; and flowing a buffer solution for passivating a surface of the fluidic channel. In accordance with various embodiments, the fluid comprises a plurality of cells. In accordance with various embodiments, the fluidic channel comprises a capture site disposed approximate the one or more electrodes. In accordance with various embodiments, increasing the flow rate and/or changing the signal is done to remove all but one captured cell at one or more of the capture sites in the set of capture sites.

[0006] In accordance with various embodiments, the method can include any of the following steps: flowing a fluid at a first flow rate into a fluidic channel of an apparatus; applying a signal at a first signal strength to one or more electrodes disposed in the fluidic channel of the apparatus; generating a non-linear and/or non-uniform electric field across the one or more electrodes based on the applied signal to immobilize one or more cells of the plurality of cells at one or more capture site; adjusting the flowing of the fluid to a second flow rate; adjusting the signal to a second signal strength; cleaning the fluidic channel by removing any cell that is not immobilized at the capture site; and flowing a buffer solution for passivating a surface of the fluidic channel. In accordance with various embodiments, the fluid comprises a plurality of cells. In accordance with various embodiments, the fluidic channel comprises a capture site disposed approximate the one or more electrodes. In accordance with various embodiments, adjusting the flowing of the fluid and/or adjusting the signal is sufficient to remove all but one immobilized cell at the one or more capture sites. In accordance with various embodiments, cleaning the fluidic channel is done prior to adjusting the flowing of the fluid.

[0007] In accordance with various embodiments, a method for capturing a single cell at a capture point in a fluidic channel is provided. In accordance with various embodiments, the method can include any of the following steps: flowing a fluid into the fluidic channel at a first flow rate; applying a di electrophoretic force at a first strength on the plurality of cells; changing the dielectrophoretic force to a second strength; flowing the fluid into the fluidic channel at a second flow rate; and flowing a buffer solution for passivating a surface of the fluidic channel. In accordance with various embodiments, the fluid comprises a plurality of cells. In accordance with various embodiments, the fluidic channel comprises a plurality of capture points. In accordance with various embodiments, the first flow rate and the first dielectrophoretic force are balanced to generate one or more captured cells from the plurality of cells at one or more capture points of the plurality of capture points. In accordance with various embodiments, changing the dielectrophoretic force and/or flowing the fluid at a second flow rate dislodges all but one cell of the captured cells.

[0008] In accordance with various embodiments, an apparatus configured for immobilization of a particle is provided. The apparatus includes a membrane for separating a fluid from a compartment; one or more electrodes disposed proximate to the membrane; a counter-electrode, wherein the one or more electrodes and the counter-electrode are configured to generate a nonlinear electric field across the one or more electrodes and the counter-electrode; and a power source for providing an alternating current (AC) across the one or more electrodes and the counterelectrode, thereby generating an oscillating non-linear electric field for immobilizing a particle suspended in the fluid that flows between the one or more electrodes and the counter-electrode.

[0009] In accordance with various embodiments, a method for operating an apparatus for immobilization of a particle is provided. The method includes providing a power source; providing a membrane configured for separating a fluid from a compartment; providing one or more electrodes disposed proximate to the membrane; providing a counter-electrode, wherein the one or more electrodes and the counter-electrode are configured to generate a non-linear electric field across the one or more electrodes and the counter-electrode; supplying, via the power source, an alternating current (AC) across the one or more electrodes and the counter-electrode, thereby generating an oscillating non-linear electric field; and immobilizing, via a dielectrophoretic force generated by the oscillating non-linear electric field, a particle suspended in the fluid that flows between the one or more electrodes and the counter-electrode.

[0010] In accordance with various embodiments, an apparatus configured for immobilization of a particle is provided. The apparatus includes one or more electrodes and a counter-electrode configured for generating a non-linear electric field for immobilizing a particle suspended in a fluid that flows between the one or more electrodes and the counter-electrode; and a membrane disposed proximate a surface of the one or more electrodes, the surface of the one or more electrodes distal the counter-electrode, wherein the membrane is configured for separating the fluid from a compartment, and has an opening configured to allow for insertion of a sharp member disposed in the compartment.

[0011] In accordance with various embodiments, a method for operating an apparatus for immobilization of a particle is provided. The method includes providing a power source; providing one or more electrodes and a counter-electrode configured for generating a non-linear electric field for immobilizing a particle suspended in a fluid that flows between the one or more electrodes and the counter-electrode; providing a membrane disposed proximate a surface of the one or more electrodes, the surface of the one or more electrodes distal the counter-electrode, wherein the membrane is configured for separating the fluid from a compartment, and has an opening configured to allow for insertion of a sharp member disposed in the compartment; supplying, via the power source, an alternating current (AC) across the one or more electrodes and the counterelectrode, thereby generating an oscillating non-linear electric field; and immobilizing, via a di electrophoretic force generated by the oscillating non-linear electric field, a particle suspended in the fluid.

[0012] In accordance with various embodiments, a method for operating an apparatus for immobilization of a particle is provided. The method includes providing a power source; providing a membrane configured for separating a fluid from a compartment; providing a pair of electrodes disposed proximate a surface of the membrane, wherein the pair of electrodes is configured to generate a non-linear electric field across the electrodes; supplying, via the power source, an alternating current (AC) across the electrodes, thereby generating an oscillating non-linear electric field; and immobilizing, via a di electrophoretic force generated by the oscillating non-linear electric field, a particle suspended in the fluid that flows between the electrodes. The method also includes providing a counter-electrode. The method also includes providing a third electrode disposed proximate the surface of the membrane.

[0013] In accordance with various embodiments, a system configured to perform the method of any of the methods disclosed herein is provided.

[0014] These and other aspects and embodiments are discussed in detail below. The foregoing information and the following detailed description include illustrative examples of various aspects and embodiments, and provide an overview or framework for understanding the nature and character of the claimed aspects and embodiments. The drawings provide illustration and a further understanding of the various aspects and embodiments, and are incorporated in and constitute a part of this specification.

BRIEF DESCRIPTION OF THE DRAWINGS

[0015] The accompanying drawings are not intended to be drawn to scale. Like reference numbers and designations in the various drawings indicate like elements. For purposes of clarity, not every component may be labeled in every drawing. In the drawings:

[0016] Figures 1 A-1D show schematic views of an apparatus configured for immobilization of a particle, in accordance with various embodiments.

[0017] Figures 2A-2B show schematic views of an apparatus capture site in a dielectrophoretic field.

[0018] Figures 3A-3H show simulation and experimental results using methods and apparatuses according to various embodiments. (3A) Simulated capture volume corresponding to an electric potential of <p^el = 4[F] at frequency f = l[MHz] and a flow velocity Uf^l = 1 mm/s. (3B) Heatmap of capture radius vs. Uf^l and <p^el . (3C-3H) Stills from video showing capture of multiple cells at capture site followed by release of surplus cells.

DETAILED DESCRIPTION

[0019] As described herein, the term “particle” refers to an object or a group of objects that individually or together have a physical property. The particle has a composition that can include mixtures, including, but not limited to living cells, viruses, oil droplets, liposomes, micelles, reverse micelles, protein aggregates, polymers, surfactant assemblies or their combination. The particle can be an individual, or a plurality of, cell (or cells), virus (or viruses), bacterium or bacteria, or any organism(s), alive or dead. The particle can be free floating in a fluid, e.g., suspended in the fluid, can be adherent, can change shape, can merge, can split apart, etc.

[0020] Unless otherwise defined, scientific and technical terms used in connection with the present teachings described herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0021] The disclosure generally relates to an apparatus, and methods using the apparatus, for isolating a single cell at a plurality of capture points in the apparatus. For example, the disclosure at least partially relates to balancing a dielectrophoretic force within the apparatus with a drag force felt by a plurality of cells that are flowing through the apparatus such that one or more of the plurality of cells are captured at one or more of the plurality of capture points. According to various embodiments, the dielectrophoretic force and the drag force are rebalanced in order to remove all but one of the captured cells from each of (or a majority of, or a plurality of, or at least one of) the one or more capture points.

[0022] Accordingly, systems, processes and methods for single-cell trapping via dielectrophoresis are disclosed. In various embodiments, a single-cell capture array using dielectrophoresis is disclosed. In various embodiments, systems, processes and methods using a positive dielectrophoresis (pDEP) are disclosed and are integration-compatible with other MEMS and microfluidic components. A pDEP array of this type enables active control of cell trapping at each site, allowing for programmatic capture and release of cells and single-cell transfection in the context of the disclosed MEMS nanoinjection platform. The disclosure includes architectures and workflows developed using numerical simulations and experimental methods which take advantage of the non-linear scaling of capture volume with electric potential and flow rate to achieve single-cell capture. In various embodiments, the disclosure is related to dielectrophoresis, cell capture, and/or nanoinjection.

[0023] In various embodiments, the disclosure relates to a technique for large-scale (thousands to millions of cells), addressable, single-cell trapping using positive dielectrophoresis (pDEP). The disclosed method includes a set of electrode architectures that allow for a large-scale addressable DEP array compatible with other device architectures. However, without adjustments to the control systems and workflow, this architecture may trap multiple cells per site if cells are loaded into the device at sufficient density to trap cells at the majority of sites (i.e., the cell number at each site will follow a Poisson distribution). The disclosure also includes a workflow that may achieve sub-Poisson statistics - ideally, exactly one cell per site - using positive DEP trapping.

I, Methods For Isolating Single Cells According to Various Embodiments

[0024] Disclosed herein, according to various embodiments, are methods of isolating a single cell at a plurality of capture points in an apparatus.

[0025] According to various aspects, steps for loading and initial capturing of one or more cells at capture sites are provided. Fluidic channels, which can including tubing, can be initially filled with a fluid, such as low-conductivity DEP buffer for example, at a comparatively high flow rate, referred to as the loading flow rate. The loading flow rate can be, in various embodiments, on the order of at least, at most, approximately, or exactly 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, pL/min, or any range derivable therein. According to various embodiments, the flow rate is then slowed to a first trapping flow rate, on the order of at least, at most, approximately, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 pL/min, or any range derivable therein. According to various embodiments, the flow rate can have an average fluid velocity of at least, at most, approximately, or exactly 10,

I I, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,

37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62,

63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88,

89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110,

I I I, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129,

130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,

149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167,

168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186,

187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205,

206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224,

225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243,

244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,

263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300,

301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319,

320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338,

339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357,

358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376,

377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391, 392, 393, 394, 395,

396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410, 411, 412, 413, 414,

415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 433,

434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452,

453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471,

472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490,

491, 492, 493, 494, 495, 496, 497, 498, 499, 500 pm/s, or any range derivable therein. According to various embodiments, a DEP signal is turned on, which can comprise a voltage at least, at most, approximately, or exactly 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10V and/or a frequency of at least, at most, approximately, or exactly 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 MHz, or any range derivable therein. According to various embodiments, this results in multiple cells being trapped at each site, as shown in Figure 3F.

[0026] According to various embodiments, a procedure for single-cell trapping using flow rate modulation is provided. In various embodiments, the flow rate is increased to a second flow rate, which can be faster (including substantially faster) than the first flow rate. In various embodiments, the signal is changed to create a different dielectrophoretic force on the cells. In various embodiments, the flow rate is modulated and the signal is changed. In various embodiments, the flow rate is not modulated. In various embodiments, the signal is not changed. In various embodiments, the second flow rate is at least, at most, approximately, or exactly 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125,

126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144,

145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163,

164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201 ,

202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220,

221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239,

240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258,

259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277,

278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296,

297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315,

316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334,

335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353,

354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372,

373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, 386, 387, 388, 389, 390, 391,

392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 409, 410,

411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 421, 422, 423, 424, 425, 426, 427, 428, 429,

430, 431, 432, 433, 434, 435, 436, 437, 438, 439, 440, 441, 442, 443, 444, 445, 446, 447, 448,

449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463, 464, 465, 466, 467,

468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486,

487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500 pL/min, or any range derivable therein. According to various embodiments, the second flow rate has an average fluid velocity of at least, at most, approximately, or exactly 0.8, 0.9, 1, 2, 3, 4, 5 mm/s, or any range derivable therein. According to various embodiments, the signal is changed by decreasing the voltage and/or frequency. According to various embodiments, the signal is changed by increasing the voltage and/or frequency. According to various embodiments, the signal can be changed to a frequency of at least, at most, approximately, or exactly 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 MHz, or any range derivable therein. According to various embodiments, the voltage can be changed to at least, at most, approximately, or exactly 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4V, or any range derivable therein. According to various embodiments, one or more of the changes in flow rate, fluid velocity, voltage, and/or frequency results in the release of weakly adhered cells which are not at the exact center of the trapping site (see Figures 3G and 3H). According to various embodiments, only a single cell is left at the center of each trap (which may be referred to as a capture site). According to various embodiments, only a single cells is left at the center of one or more, a majority of, or substantially all of the capture sites in the apparatus. According to various embodiments, the capture sites are proximal to the tip of the electrode. According to various embodiments, the capture site is offset by half of the separation distance between the electrodes.

[0027] According to various embodiments, a blocking step is performed prior to flowing the plurality of cells into the fluidic channel. In various embodiments, the blocking step comprises flowing a solution comprising a blocking agent into the fluidic channel. The blocking agent can be any agent that sufficiently prevents non-specific interactions, such as non-specific binding, of the cells with the fluidic channel and/or reduce nonspecific cell adhesion to the surface of the fluidic channel. Blocking agents can be proteinaceous compositions. The blocking agents can comprise an albumin, such as a bovine serum albumin. The blocking agent can be a polymer composition. The blocking agent can comprise a pluronic triblock copolymer, or a similar molecule. According to various embodiments, the blocking agent is loaded into and/or flown through the fluidic channel and allowed to incubate briefly before being replaced with a separate fluid, such as a fluid comprising the plurality of cells.

II. Apparatuses According to Various Embodiments

[0028] Disclosed according to various embodiments herein is an apparatus for local manipulation of neutral particles or biological molecules in a fluidic and non-linear and/or non- uniform electric field environment and various (e.g., microfluidics) applications thereof. In particular, various embodiments relate to an apparatus for dielectrophoresis-based (DEP -based) immobilization of biological objects, single cells or groups of cells in proximity to a compartment (or cavity) for local manipulation of the molecules or cells. In various embodiments, the compartment or cavity can be filled with one of an aqueous fluid, an aqueous buffer, an organic solvent, a hydrophobic fluid, or a gas. In various embodiments, the compartment can contain a fluid within the compartment that is immiscible with a fluid outside the compartment. In various embodiments, the compartment can contain a non-aqueous fluid or microelectronics incompatible with an aqueous environment. [0029] In particular, the technology described herein relates to a high-throughput, DEP -based particle immobilization (trapping) apparatus that pins and immobilizes one or more particles in a fluid that flows adjacent to a membrane that separates the fluid from a compartment (isolated compartment or cavity) that contains electronic components. As described herein, in order to provide access from the cavity into the fluidic environment, one or more membrane openings (also refer to herein as “pores” or “micropores”) through the membrane can be used. As described herein, the membrane can also be designed to maintain a stable liquid/gas interface or liquid-liquid interface between two immiscible fluids using fluid dynamic strategies that include, but are not limited to, surface patterning via hydrophobic or hydrophilic coatings, and/or pressure control of both fluid media on either side of the membrane. This interface can also be controlled to intentionally move fluid into or out of the cavity via modulation of surface energy via electrostatics, by pressurizing or depressurizing the cavity, or by changing the size or shape of the pore (e g. by inserting a hollow microneedle into the pore to decrease the effective capillary radius).

[0030] By providing a platform to interface the DEP-mediated particle immobilization technique (e.g., a trapping technique) with highly local manipulation of individual biological molecules or cells at the single-cell level (e.g., single cell resolution), a highly controllable approach to extraction of genetic materials and/or delivery of drug molecules into individual cells, for example, can be achieved, not just for single cells, but in a high-throughput, reliable, and reproducible fashion.

[0031] As described herein, various embodiments of an apparatus for immobilizing a particle in fluid is described. In various embodiments, the apparatus includes a membrane for separating a fluid, for example, in a microfluidic channel, from a compartment. In various embodiments, the apparatus also includes one or more electrodes disposed on the membrane away from the compartment and a counter-electrode having a dissimilar surface area than the one or more electrodes. In various embodiments, the one or more electrodes and the counter-electrode (also referred to herein as “DEP electrodes”) are configured to generate a non-linear electric field across the one or more electrodes and the counter-electrode. In various embodiments, the one or more electrodes and the counter-electrode (also referred to herein as “DEP electrodes”) are configured to generate a non-uniform electric field across the one or more electrodes and the counterelectrode. In various embodiments, the one or more electrodes and the counter-electrode (also referred to herein as “DEP electrodes”) are configured to generate a non-linear and non-uniform electric field across the one or more electrodes and the counter-electrode. In various embodiments, the apparatus also includes an electrical input and output source for providing and sensing a signal across the one or more electrodes and/or the counter-electrode. In various embodiments, the signal is an AC voltage for generating an oscillating non-linear electric field for immobilizing a particle suspended in the fluid that flows between the one or more electrodes and the counter-electrode. In various embodiments, the signal is an AC voltage for generating an oscillating non-uniform electric field for immobilizing a particle suspended in the fluid that flows between the one or more electrodes and the counter-electrode. In various embodiments, the signal is an AC voltage for generating an oscillating non-linear and non-uniform electric field for immobilizing a particle suspended in the fluid that flows between the one or more electrodes and the counter-electrode.

[0032] In various embodiments, the apparatus includes an array of electrodes (or the array of one or more electrodes, e.g., a pair of electrodes, a set of three electrodes, a set of four electrodes, and so on) co-localized with pores (e.g., opening 125, 225a-d, etc.), allowing access to trapped particles from a cavity. In various embodiments, the pores are made to be hydrophobic by a chemical treatment coating the interior walls of the pores. In various embodiments, the edge surface of the pores on either side of the membrane and/or pore interior are coated/chemically functionalized with a range of material classes including, for example, any small molecule, proteins, peptides, peptoids, polymers, or inorganic materials listed above in any suitable combination. Some examples of surface chemistries and their functionalities are included herein. In accordance with various embodiments, coatings of the interior of the pore and/or one side of the membrane can include a hydrophobic material, such as a hydrophobic organosilane, e.g. a fluorosilane, in order to prevent leakage of aqueous solution through the pore. In accordance with various embodiments, a surface can be coated to discourage cell adhesion, using a chemical such as for example, but not limited to, a poloxamer or poly(2-hydroxyethyl methacrylate) or any suitable protein blocking solution, such as for example, bovine serum albumin, in order to prevent nonspecific cell adhesion away from trapping sites, for example, approximate the opening or pore. Some examples of surface coatings may include, for example, biological or organic materials, such as proteins, peptides, polymers, hydrocarbon chains of varying lengths, any combination of which can be used for preventing cell adhesion as well as payload/analyte adhesion prevention. In accordance with various embodiments, such surface coatings may be used for preventing molecular payload adhesion, particularly with respect to molecular payload that is disposed on a sharp member or needle. In accordance with various embodiments, a coating on one side of the membrane with a hydrophilic material such as hyaluronic acid, titanium oxide, polyethylene glycol, etc. in order to ensure efficient wetting of those surfaces and prevent outflow flow of hydrophobic material from the opening. In accordance with various embodiments, any combination of the aforementioned approaches can be employed in order to separate hydrophobic and hydrophilic fluids in separate openings, pores, or cavities.

[0033] The various embodiments disclosed herein represent a unique capability for high- volume trapping of biological objects and/or cells for characterization, sampling, payload delivery, or modification. As described herein, physical and material properties and parameters, such as, for example, the size and hydrophobicity of the pore (or opening), size of the electrode, conductivity of the fluid medium, and operating frequency of the electrodes can be optimized based on the application and the biological objects or cells to be interrogated. In various embodiments of the technology described herein, the apparatus can be configured for selective release of cells after trapping/capture, and probing/interrogation/manipulation.

[0034] Moreover, according the various embodiments as described herein, the apparatus can also be optimized by exploiting the di electrophoretic (DEP) force. For example, since the DEP force generated is proportional to the square of the field gradient according to the DEP equation described above, a highly non-linear and/or non-uniform electric field can be generated across the one or more electrodes and the counter-electrode. In various embodiments, by applying an alternating current (AC) between one or more electrodes with a geometry that creates a large electric field gradient via size difference and/or proximity, a confined highly non-linear and/or non-uniform electric field can be generated to act on a biological object or cell, and immobilize it in the trapping area. For example, if the one or more electrodes, e.g., a pair of electrodes, are arranged around an opening, the DEP force can be tuned to trap the object between the electrodes at the opening. In addition, if the wall of the opening in the electrode is coated with a hydrophobic material, the contact angle of the coated inner wall of the opening can relate to the capillary pressure of the fluid via the following equation:

(00351 Ap = - 22^

[0036] where r is the radius of the opening, y is the surface tension (approximately 72.75 mN/m for water and air) and 0 is the contact angle. Conventionally, a contact angle 0 of above 90 represents a hydrophobic material while a contact angle below 90 represents a hydrophilic material. By increasing the contact angle 6 to around 130 degrees by applying, for example, a hydrophobic silane coating, the capillary pressure for an air- water interface reaches 40-60 kPa with a relatively large opening of about 4 pm or 5 pm. As described herein, a hydrophobic coating on the inner wall of the opening can prevent fluid from flowing through the opening from the aqueous side into an air-fdled compartment that can other electronic components. The same principle holds for other types of fluid phase separation across the membrane, depending on whether the aqueous or non-aqueous side of the membrane is at higher or lower pressure the pore can be patterned with a hydrophobic or hydrophilic surface treatment respectively. Therefore, the apparatus having one or more electrodes and a counter-electrode arranged in such a way to produce a nonlinear electric field can be configured to trap, immobilize or confine a biological object or a cell in a fluid without compromising any fluid exposure to the sensitive electronic components. In various embodiments, the apparatus has one or more electrodes and a counter-electrode that are of the same or substantially similar size can be configured to generate a highly non-linear and/or non-uniform electric field in order to trap, immobilize or confine a biological object or a cell in a fluid without compromising any fluid exposure to the sensitive electronic components. In various embodiments, each of the trapping sites e.g., an opening or a pore, can include an electrode, two electrodes, three electrodes, four electrodes, and so on. In various embodiments, additional electrodes can be configured for impedance sensing in the presence of an object, for example, a particle or a cell.

[0037] Figures 1A-1D show schematic views of an apparatus for immobilization of a particle, according to various embodiments as disclosed herein. Figure 1A shows a schematic top view of an example apparatus 100, in accordance with various embodiments. As shown in Figure 1A, the apparatus 100 includes an opening 125 (also referred to herein as “pore”), a plurality of electrodes 120 and one or more interconnects 130. For example, the plurality of electrodes 120, as illustrated, can include a plurality of individual disparate electrode surface areas formed in an array or a grid. Although the electrode 120 is illustrated as a ring or circular electrode, the electrode 120 can be a pair of electrodes, or any number of sets of electrodes disposed proximate the opening 125, in accordance with various embodiments. Accordingly, the physical, chemical, material parameters as described further below with respect to the electrode 120 can be applicable to any of the pair of electrodes. [0038] In various embodiments, the electrode 120 has a thickness between about 1 nm to about 50 pm. In various embodiments, the electrode 120 has a thickness between about 10 nm to about 5 pm, about 10 nm to about 10 pm, about 10 nm to about 5 pm, about 100 nm to about 4 pm, about 300 nm to about 3 pm, about 400 nm to about 5 pm, about 500 nm to about 5 jam, inclusive of any thickness ranges therebetween.

[0039] In various embodiments, the electrode 120 includes at least one of a transparent conducting material or a doped semiconducting material with sufficient electrochemical stability. In various embodiments, the transparent conducting material includes indium tin oxide, graphene, doped graphene, a conducting polymer, or a thin metal layer.

[0040] As shown in Figure 1 A, in various embodiments, each of the plurality of electrodes 120 (referring to an array of electrodes 120) has an opening 125. In various embodiments, some of the plurality of electrodes 120 have an opening 125 and some electrodes 120 do not have an opening 125. In various embodiments, the electrodes 120 that have an opening 125 and the electrodes 120 that do not have an opening 125 are strategically arranged based on the application of the apparatus 100.

[0041] In various embodiments, the opening 125 has a size (also referred to herein as a diameter if circular or a lateral dimension if any non-circular geometry) between about 0.1 nm to about 1 mm. In various embodiments, the opening 125 has a size between about 1 nm to about 100 nm, about 100 nm to about 1 pm, about 1 pm to about 10 pm, about 100 nm to about 25 pm, about 1 pm to about 100 pm, or about 1 pm to about 50 pm, inclusive of any size ranges therebetween.

[0042] In various embodiments, the electrodes 120 in the plurality of electrodes 120 have an electrode-to-electrode separation distance between two adjacent electrodes from about 1 pm to about 5 mm, from about 1 pm to about 1 mm, from about 10 pm to about 500 pm, or from about 10 pm to about 1 mm, inclusive of any separation distance ranges therebetween.

[0043] In various embodiments, the electrode 120 and the one or more interconnects 130 include the same material. In various embodiments, the one or more interconnects 130 includes at least one of a transparent conducting material or a doped semiconducting material with sufficient electrochemical stability. In various embodiments, the transparent conducting material includes indium tin oxide, metal nanowire mesh, graphene, a doped graphene, a conducting polymer, a thin metal layer, an atomic-layer metal film, or any other suitable transparent conductor. [0044] Figure IB shows a zoomed-in schematic view of one of the electrodes 120 of the apparatus 100. In various embodiments, the apparatus 100 includes one electrode 120. As shown in Figures 1A and IB, the plurality of electrodes 120 are interconnected to each other via one or more interconnects 130 in a grid or in an array. In various embodiments, the plurality of electrodes 120 are interconnected to each other within a group that can include any number of electrodes 120, and the apparatus 100 can include any number of groups of electrodes 120.

[0045] Figure 1C shows a cross-sectional view (orthogonal to the view of Figure IB) of the apparatus 100, according to various embodiments. As shown in Figure 1C, the apparatus 100 includes the plurality of electrodes 120 and a counter-electrode 140. In accordance with various embodiments, each electrode 120 in the plurality of electrodes 120 can be a pair of electrodes, or any number of sets of electrodes disposed proximate the opening 125. In various embodiments, the counter-electrode 140 is a plane electrode that spans across a portion, a substantial portion, almost an entirety, or an entirety of the apparatus 100. For example, the counter-electrode 140 can be bigger than each of the plurality of electrodes 120. For example, the counter-electrode 140 can have a surface area that is bigger than a surface area of each of the individual electrodes 120. In various embodiments, the ratio of the surface area between the counter-electrode 140 and an electrode 120 can be about 1 : 1, 1.1 : 1, 2: 1, 5: 1, 10: 1, 50: 1, 100: 1, 1 million: 1, or any suitable ratios therebetween.

[0046] In various embodiments, the electrode 120 and the counter-electrode 140 have the same or substantially similar in size. In various embodiments, the electrode 120 and the counterelectrode 140 are disposed on the same plane.

[0047] As illustrated in Figure 1C, the plurality of electrodes 120 and the counter-electrode 140 are configured to receive a fluid (indicated as parallel arrows in Figure 1C) that flows in a channel 160 between the plurality of electrodes 120 and the counter-electrode 140. In various embodiments, the fluid that flows in the channel 160 can include, for example, but not limited to, an aqueous fluid, an aqueous buffer, an organic solvent, a hydrophobic fluid, or a gas.

[0048] In various embodiments, the fluid flows in the channel 160 at a flow rate between 0 to 10 mL/s. In various embodiments, the fluid is static and, therefore, has minimal to no flow rate. In various embodiments, the fluid flows from about 0.001 mL/s to about 0.1 mL/s, about 0.01 mL/s to about ImL/s, or about 0.1 mL/s to about 10 mL/s, inclusive of any flow rate ranges therebetween. [0049] Figure ID shows a zoomed-in cross-sectional view of one of the plurality of electrodes 120 of the apparatus 100. As shown in Figure ID, the apparatus 100 includes a membrane 110, the electrode 120, an interconnect 130, and a passivation layer 150. In various embodiments, the membrane 110 includes an electrically insulating material. In various embodiments, the membrane 110 includes an electrically insulating material, including, but not limited to silicon nitride, silicon oxide, a metal oxide, a carbide (such as, for example, SiCOH), a ceramic (such as, for example, alumina), and a polymer. In various embodiments, the membrane 110 includes an electrically conducting material, such as a metal or a doped semiconductor material. In various embodiments, the membrane 110 can be a single layer or a composite layer having a multilayer stack that includes any of the aforementioned materials.

[0050] In various embodiments, the wall forming the channel 160 comprises a channel material that can include, for example but not limited to, silicon, glass, plastic, or various elastomers such as, for example, poly(dimethyl siloxane) (PDMS), which can be used as structural materials for the fluidic layer. In various embodiments, the channel 160 has dimensions from about 1 nm to about 1 cm, from about 100 nm to about 100 mm, from about 200 nm to about 1 mm, or from about 200 nm to about 500 pm, inclusive of any dimensions therebetween. In various embodiments, the height of the channel 160 is set by the particle size being probed and in order to avoid clogging should be at least twice the diameter of the particle.

[0051] In various embodiments, the membrane 110 has a thickness between about 10 nm to about 1 cm. In various embodiments, the membrane has a thickness between about 10 nm to about 5 mm, between about 10 nm to about 1 mm, between about 10 nm to about 100 pm, about 50 nm to about 10 pm, about 50 nm to about 5 pm, about 100 nm to about 10 pm, about 100 nm to about 5 pm, or about 100 nm to about 2 pm, inclusive of any thickness ranges therebetween. In various embodiments, the membrane 110 or any layer of material comprising the membrane can be patterned.

[0052] Figure ID also shows a particle 165 that is suspended in the fluid that flows in the channel 160. In various embodiments, the particle 165 can include various types of particulate material or globular materials, including, but not limited to, any biological objects, cells, or non- biological objects. In various embodiments, the particle 165 can include a biological organism, a biological structure, a cell, a living cell, viruses, oil droplets, liposomes, micelles, reverse micelles, protein aggregates, polymers, surfactant assemblies, a vesicle, a micro-vesicle, a protein, a molecule, a microdroplet, or a non-biological particulate matter.

[0053] In various embodiments, the particle 165 can have a size between about 1 nm to about 1mm. In various embodiments, the particle 165 can have a size between about 10 nm to about 500 pm, about 50 nm to about 200 pm, about 200 nm to about 100 pm, about 300 nm to about 50 pm, about 100 nm to about 200 pm, about 100 nm to about 100 pm, or about 200 nm to about 50 pm, inclusive of any size ranges therebetween.

[0054] As shown in Figure ID, according to various embodiments, the membrane 110 is configured to separate the fluid from entering a compartment 180. Figure ID also shows the opening 125 of the apparatus 100. As shown in Figure ID, the opening 125 extends through the membrane 110 and the electrode 120. In various embodiments, the opening 125 extends through the membrane 110, the electrode 120, and the passivation layer 150. In various embodiments, the opening 125 may also serve as a capillary valve to isolate two fluid phases across the membrane 110 if the operation of the device requires more than one fluid phase (such as an ionic buffer and air or aqueous and organic solvents). In various embodiments, a wall of the opening 125 has a hydrophobic coating or a hydrophilic coating. In various embodiments, the opening 125 is made to be hydrophobic by a chemical treatment coating the interior walls of the opening 125. In various embodiments, the edge surface of the opening 125 on either side of the membrane and/or an inside of the wall (inner wall) of the opening 125 (also referred to herein as “pore interior”) are coated/chemically functionalized with a range of material classes including, for example, any small molecule, proteins, peptides, peptoids, polymers, or inorganic materials listed above in any suitable combination.

[0055] In accordance with various embodiments, the hydrophobic coating or hydrophilic coating are disposed (or deposited) on wall of the membrane 110 and/or the electrode 120 to prevent the fluid from entering into the compartment. In various embodiments, the coating is chemically and covalently attached to the relevant surfaces. In various embodiments, the hydrophobic coating can include a variety of classes such as azides, organosilanes, or fluorocarbons. In various embodiments, the hydrophilic coating can include a range of material classes including any small molecule, proteins, peptides, peptoids, polymers, or inorganic materials. In various embodiments, the wall of the opening 125 has a combination of patterned hydrophilic and hydrophobic coatings. [0056] In various embodiments, the hydrophobic coating has a contact angle between about 95° and about 165°. In various embodiments, the hydrophobic coating has a contact angle between about 100° and about 165°, about 105° and about 165°, about 110° and about 165°, about 120° and about 165°, about 95° and about 150°, about 95° and about 140°, or about 95° and about 130°, inclusive of any contact angle ranges therebetween.

[0057] In various embodiments, the hydrophilic coating has a contact angle between about 20° and about 80°. In various embodiments, the hydrophilic coating has a contact angle between about 25° and about 80°, about 30° and about 80°, about 35° and about 80°, about 40° and about 80°, about 20° and about 70°, about 20° and about 60°, or about 20° and about 50°, inclusive of any contact angle ranges therebetween.

[0058] According to various embodiments, a power source (not shown) can be electrically connected to the plurality of electrodes 120 and the counter-electrode 140 to provide an alternating current (AC) across the plurality of electrodes 120 and the counter-electrode 140 to generate an oscillating non-linear and/or non-uniform electric field for immobilizing (or trapping) the particle 165 suspended in the fluid that flows between the plurality of electrodes 120 and the counterelectrode 140. In various embodiments, an in-plane electric field with multiple electrodes can be applied to induce a local field minimum for alternate DEP field. In various embodiments, one or more AC or DC signals may be superposed on the DEP actuation signal for applications including impedance sensing, electrowetting, or electroporation.

[0059] In various embodiments, the AC across the plurality of electrodes 120 (electrode 120 if a single electrode or a pair of electrodes) and the counter-electrode 140 is supplied at a voltage between about 1 mV and about 300 V. In various embodiments, the AC across the plurality of electrodes 120 and the counter-electrode 140 is supplied at a voltage between about 5 mV and about 50 V between about 5 mV and about 20 V, about 250 mV and about 5 V, about 500 mV and about 50 V, about 750 mV and about 50 V, about 1 V and about 50 V, about 5 V and about 50 V, about 10 V and about 50 V, about 250 mV and about 40 V, about 250 mV and about 30 V, about 250 mV and about 20 V, about 250 mV and about 10 V, about 250 mV and about 8 V, about 250 mV and about 6 V, about 250 mV and about 5 V, about 500 mV and about 5 V, or about 1 V and about 5 V, inclusive of any voltage ranges therebetween. In various embodiments, the AC across the plurality of electrodes 120 (electrode 120 if a single electrode) and the counter-electrode 140 is supplied at a voltage between about 1 mV and about 20 V, between about 1 mV and about 10 V, between about 1 mV and about 8V, between about 1 mV and about 6 V, between about 1 mV and about 5 V, between about 1 mV and about 4 V, between about 1 mV and about 3 V, between about 1 mV and about 2 V, between about 1 mV and about 1 V, between about 1 mV and about 750 mV, between about 1 mV and about 500 mV, between about 1 mV and about 250 mV, between about 1 mV and about 200 mV, between about 1 mV and about 150 mV, between about 1 mV and about 100 mV, between about 1 mV and about 50 mV, inclusive of any ranges therebetween. [0060] In various embodiments, the AC across the plurality of electrodes 120 (electrode 120 if a single electrode or a pair of electrodes) and the counter-electrode 140 is supplied at an oscillating frequency between about 1 Hz and about 1 THz. In various embodiments, the AC across the plurality of electrodes 120 and the counter-electrode 140 is supplied at an oscillating frequency between about 10 Hz and about 100 GHz, about 10 Hz and about 10 GHz, about 100 Hz and about 10 GHz, about 1 kHz and about 1 GHz, about 10 kHz and about 1 GHz, about 100 kHz and about 1 GHz, about 500 kHz and about 1 GHz, about 1 MHz and about 1 GHz, about 10 MHz and about 1 GHz, about 100 MHz and about 1 GHz, about 10 kHz and about 500 MHz, about 10 kHz and about 100 MHz, about 10 kHz and about 50 MHz, about 10 kHz and about 30 MHz, about 10 kHz and about 20 MHz, about 10 kHz and about 10 MHz, about 100 kHz and about 10 MHz, or about 500 kHz and about 10 MHz, or about 1 MHz and about 10 MHz, inclusive of any frequency ranges therebetween.

[0061] In various embodiments, a direct current (DC) is applied across the plurality of electrodes 120 (electrode 120 if a single electrode or a pair of electrodes) and the counter-electrode 140. In various embodiments, the DC and AC can be superimposed when applied a current across the plurality of electrodes 120 (electrode 120 if a single electrode or a pair of electrodes) and the counter-electrode 140.

[0062] In various embodiments, the plurality of electrodes 120 and the counter-electrode 140 can be individually addressed, addressed in groups, or electrically short-circuited (e.g., shorted) together. In various embodiments, each in the pair of electrodes can be individually addressed, addressed in groups, or electrically short-circuited (e.g., shorted) together. For example, the AC can be supplied to each of the plurality of electrodes 120 and the counter-electrode 140 individually, or in groups. For example, the plurality of electrodes 120 and the counter-electrode 140 can be shorted for some of the plurality of electrodes 120 and the counter-electrode 140, and not the other electrodes 120 in the plurality of electrodes 120 and the counter-electrode 140. As such, any combination or configuration of arrangements between the plurality of electrodes 120 and the counter-electrode 140 can be implemented for the apparatus 100.

[0063] Also disclosed according to various embodiments is a pDEP cell capture chip using sets of electrodes in a membrane comprising one or more thin dielectric films (typically silicon oxide and silicon nitride), as described elsewhere. These can be designed to create a location of maximum field concentration (which may be referred to in herein as a “trapping site” or a “capture site”) between the tips of 2 or more electrodes when an AC voltage is applied. This phenomenon is illustrated in Figure 2B.

[0064] Also disclosed according to various embodiments is a set of fluidic pumps and sensors (either syringe pumps or displacement pumps and pressure sensors, or pressure pumps and flow rate sensors) for closed-loop control of flow rate.

[0065] This specification describe various exemplary embodiments of systems, processes and methods for single-cell trapping via dielectrophoresis. The disclosure, however, is not limited to these exemplary embodiments and applications or to the manner in which the exemplary embodiments and applications operate or are described herein. Moreover, any and all parameters, measurements, methods, and materials discussed herein are examples only and can vary based on need.

[0066] In describing the various embodiments, the specification may have presented a method and/or process as a particular sequence of steps. However, to the extent that the method or process does not rely on the particular order of steps set forth herein, the method or process should not be limited to the particular sequence of steps described. As one of ordinary skill in the art would appreciate, other sequences of steps may be possible. Therefore, the particular order of the steps set forth in the specification should not be construed as limitations on the claims. In addition, the claims directed to the method and/or process should not be limited to the performance of their steps in the order written, and one skilled in the art can readily appreciate that the sequences may be varied and still remain within the spirit and scope of the various embodiments. Similarly, any of the various system embodiments may have been presented as a group of particular components. However, these systems should not be limited to the particular set of components, now their specific configuration, communication and physical orientation with respect to each other. One skilled in the art should readily appreciate that these components can have various configurations and physical orientations (e.g., wholly separate components, units and subunits of groups of components, different communication regimes between components).

[0067] Although specific embodiments and applications of the disclosure have been described in this specification, these embodiments and applications are exemplary only, and many variations are possible.

III. Examples

[0068] The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Example 1: An Apparatus According to Various Embodiments

[0069] In accordance with various embodiments, a novel nanoinjection system is disclosed. The system can comprise but is not limited to a scalable microfluidic cell capture array separated by a porous membrane (Figure 2A). This system has the potential for delivery of genetic material with high precision and single-cell resolution if it is capable of on-demand capture and release of cells. Well-based or size-exclusion approaches to cell capture, while capable of high yields, present difficulties with individual addressability. The disclosed example of the scalable, addressable trapping array uses pairs of electrodes designed to create concentrated electric fields within defined regions using pDEP, similar to existing methods for single-molecule pDEP traps, as shown in Figures 2A and 2B.

[0070] In a fluid medium with permittivity E ¹, microparticles of radius a experience the DEP force given by,

F^DEP = 2 a³ef^lRe[K_CM\V\E„ns\², where Re [/_CM] is the real part of the so-called Clausius-Mossotti factor and E_rms is the root-mean- SP-sf^l square of an AC electric field. The CM factor K_CM = is dependent on the contrast

between the complex dielectric constant £ of the fluid medium and suspended particles at a particular electric field frequency. For HeLa cells, a core-shell model predicts negligible variation in CM factor in the low MHz frequency range.

[0071] Numerical simulation (Comsol Multiphysics®) was used to resolve fluid flow and electric fields in the device in order to enable design of experimental workflows and future rDEP iterations of electrode geometry. The non-dimensional trapping radius of the DEP site f

= -7^ ch. is determined by the balance between the DEP force F^DEP and the Stokes drag F^dras as presented in Fig. 2A depicting particle traces. The average inlet velocity U ^l and applied voltage (p^el control the drag and DEP forces, respectively. This enables design and development of selective single cell capture and release in a microfluidic device.

[0072] The time-averaged dielectrophoretic force on a homogeneous dielectric sphere in a dielectric medium is given by:

Where r is the radius of the particle, E_m* and 8p are the complex dielectric constants of the medium and particle respectively, given by 8_m = 8 H - .

Ct)

[0073] Three relevant parameters are seen in this equation:

1) The electric field gradient: DEP force is proportional to the square of the gradient of the electric field (which depends on the applied voltage and the shape of the trapping electrodes). called the Clausius-Mossotti factor, which varies between -1 and 1 depending

on the relative polarizability of the particle and the surrounding medium. For a given particlemedia system, this depends on frequency in a complex manner which can generally only be determined experimentally. In the disclosed system, positive dielectrophoresis is used, and therefore, media composition and frequency are chosen in order to keep the Clausius-Mossotti factor as close as possible to 1.

[0074] The radius of the particle r. With some caveats (see below), the DEP force will scale with r^A3 for a spherical particle.

[0075] Drag force on a spherical particle in low Reynolds number conditions is given by F_d = 6Ti[iUr where . is the dynamic viscosity of the fluid, U is the bulk fluid velocity, and r is the radius of the particle. Therefore, the force balance for the disclosed DEP capture system, ignoring forces from nonspecific adhesion and the correction to the drag force from the nearby wall and assuming that the electric field and velocity are relatively homogeneous over the size of the particle is:

While this inequality is true the particle will remain trapped. Because the electric field gradient is nonhomogeneous with a maximum at the trapping site, this is true in a finite volume of space around the trapping site at the local field maximum (e.g., “trapping volume”, which is due to the complex dependence on electrode volume that can be determined by simulation or experiment). [0076] From this force balance it can be seen that the trapping volume can be reduced certain parameters including but not limited to:

1) Increasing the fluid velocity;

2) Reducing the Clausius-Mossotti factor by changing the frequency; and/or

3) Reducing the magnitude of the electric field gradient by reducing the applied voltage.

[0077] The dependence of the trapping force on the cell size is complex. On one hand, the above equation suggests a quadratic scaling of trapping force with particle radius, suggesting that larger cells will be more easily trapped and more difficult to release. However, this ignores the geometry of the electric field and the trapping site - see caveat in italics above. Many pDEP electrode designs, including the disclosed system, necessarily produce a small region of high electric field. If a cell is too large, the DEP force will act over a fraction of the cell volume and drag force will continue to scale with size independently of DEP force, making very large cells more difficult to trap. The disclosed workflow has been tested with HeLa cells approximately 12 pm in diameter and is designed to function within a reasonable range of that size (for example between about 3 pm-50 pm diameter).

[0078] Parameter ranges for various embodiments can include, but are not limited to, the following:

1) Cell diameter: 3 pm-50 pm

2) Fluid (bulk) velocity: 10 pm/s - 50 mm/s

3) Applied voltage (amplitude of signal between electrodes): 100 mV-5 V 4) Frequency: 100 kHz-3 MHz (positive DEP)

[0079] In various embodiments, the apparatus can be fabricated using gold electrodes on a fused silica substrate. Fluidic channels (1.2 cm wide by 84 / m deep) are defined using laser-cut medical-grade acrylic/polyester spacer tape and capped with glass microscope slides cut to size. Cell capture is performed in a DMEM-based DEP buffer. Flow rates were varied from 10-100 /zL/min, corresponding to Uf^l = 0.17 - 1.7 mm/s. After loading HeLa cells (250,000 cells/mL), the flow rate was slowed to 10 qL/min and the DEP signal (f = 1MHz, (p^el = 2 to 4 V) was turned on.

[0080] Cell capture between the electrodes (see Figure 3D) subsequently occurred at the majority of sites. After the initial cell was captured, additional cells would form chains adjacent to the initial cell. Increasing the flow rate to l OOqL/min while keeping the DEP signal on removed these additional cells, leaving a single cell per site (see Figure 3H). The DEP signal was subsequently turned off and single cells were released.

[0081] The nonlinear relationship between capture volume, <p^el and Uf^l that arises from theory and simulation informs workflow design that enables single-cell capture using pDEP. As demonstrated above, effective single-cell capture can be achieved by increasing the flow rate to a value that reduces the trapping radius to less than the radius of a single cell, causing additional cells to be removed from the surface of the electrode by drag.

[0082] It has been successfully demonstrated single-cell pDEP capture using an array of microelectrode pairs compatible with the disclosed nanoinjection architecture. The disclosure may include optimization of electrode shape, denser arrays, and quantification of cell viability, as well as integration with the MEMS components of the nanoinjection system.

Example 2: A Workflow According to Various Embodiments

[0083] Example of workflows for single-cell pDEP capture includes one or more of the following:

1) Block surface with passivation buffer (e g., media containing Bovine Serum Albumin or similar blocking protein, Pluronic, etc.)

2) Load flow cell with DEP buffer. 3) Turn on DEP signal (1MHz, 1V-4V), flow cells onto chip (concentration about 250k cells/mL) at flow rate 1 (10 pL/min for the disclosed channel dimensions, which corresponds to an average fluid velocity of 170 pm/s). The fluid velocity is an important parameter which determines the drag force. Optimal fluid velocity for loading cells depends on the cell diameter, which determines the balance between Stokes drag and DEP force. At this flow rate, the DEP force is adjusted or changed to dominate over drag force, while ensuring that the flow rate is not so slow that cells simply settle and adhere to the surface. Continue to flow cells over chip under these conditions until a sufficient number of sites are filled. Most sites will have multiple cells trapped per site, with one cell at the center of the trap and additional cells trapped adjacent to this cell.

4) Remove all cells but the cell at the center of the trap - this cell will be most strongly held in place by the DEP force. This can be accomplished in one or a combination of several ways provided as follows: a) Increase the flow rate to flow rate 2 (>flow rate 1), washing the weakly adhered cells off of the chip. To do so, a flow rate of 100 pL/min can be used in various embodiments, corresponding to an average fluid velocity of 1.7 mm/s. The choice of this flow rate depends on the size of the cells and therefore on the ratio of the DEP force to the Stokes drag force (FDEP/FDrag ~ r^A3/r = r). This is an example method to implement this approach. b) Decrease the DEP force, either by decreasing the applied voltage or by decreasing the Clausius-Mossotti factor by changing the frequency. c) Physically the agitate the chip, for instance by applying a vibration or acoustic wave d) Wash the chip with a substance that decreases cell/cell or cell/surface adhesion, such as Trypsin.

IV. Recitation of Embodiments

[0084] Embodiment 1 : A method of capturing a cell, comprising: flowing a fluid at a first flow rate into a fluidic channel of an apparatus, wherein the fluid comprises a plurality of cells; generating a set of capture sites comprising one or more captured cells by applying a signal at a first signal strength to one or more electrodes disposed in the fluidic channel of the apparatus, thereby generating a non-uniform electric field at or in a vicinity of the one or more electrodes, wherein the fluidic channel comprises a capture site disposed approximate the one or more electrodes; and increasing the flow rate of the fluid to a second flow rate and/or changing the signal to a second signal strength to remove all but one captured cell at one or more of the capture sites in the set of capture sites.

[0085] Embodiment 2: The method of Embodiment 1, wherein prior to flowing the fluid at the first flow rate into the fluidic channel of the apparatus, the method further comprising: flowing a buffer solution for passivating a surface of the fluidic channel.

[0086] Embodiment 3: The method of Embodiment 2, wherein the buffer solution comprises a blocking agent.

[0087] Embodiment 4: The method of Embodiment 3, wherein the blocking agent comprises one or more proteins and/or one or more synthetic polymers.

[0088] Embodiment 5: The method of Embodiment 4, wherein the one or more proteins comprise an albumin protein.

[0089] Embodiment 6: The method of any one of Embodiments 1-5, wherein the first signal strength comprises an alternating current (AC) signal with a frequency within a range of about 0.5 MHz and about 50 MHz and/or a voltage within a range of about IV and about 5V.

[0090] Embodiment 7: The method of any one of Embodiments 1-6, wherein changing the signal to a second signal strength comprises: decreasing the strength of the signal via decreasing the voltage, and/or decreasing or increasing the frequency of the AC signal.

[0091] Embodiment 8: The method of any one of Embodiments 1-7, wherein the second signal strength comprises an alternating current (AC) signal with a frequency within a range of about 0.1 MHz and about 100 MHz, wherein the frequency is different from the frequency of the first signal strength, and/or a voltage within a range of about 0. IV and about 4V, wherein the voltage is lower than the voltage of the first signal strength.

[0092] Embodiment 9: The method of any one of Embodiments 1-8, wherein the first flow rate comprises a rate between about 1 pL/min and about 50 pL/min.

[0093] Embodiment 10: The method of any one of Embodiments 1-9, wherein the first flow rate has an average fluid velocity between about 10 pm/s and about 500 pm/s.

[0094] Embodiment 11 : The method of any one of Embodiments 1-10, wherein the second flow rate comprises a rate between about 80 pL/min and about 5,000 pL/min. [0095] Embodiment 12: The method of any one of Embodiments 1 -1 1, wherein the second flow rate has an average fluid velocity between about 1.4 mm/s and about 5.0 mm/s.

[0096] Embodiment 13: The method of any one of Embodiments 1-12, wherein the increasing the flow rate of the fluid to the second flow rate occurs from about 4 minutes to about 30 minutes after flowing the fluid at the first flow rate.

[0097] Embodiment 14: The method of any one of Embodiments 1-13, wherein the decreasing the signal to the second signal strength occurs from 4 minutes to about 30 minutes after applying the signal at the first signal strength.

[0098] Embodiment 15: The method of any one of Embodiments 1-14, wherein the fluid comprises between about 10,000 cells/mL and about 2,000,000 cells/mL.

[0099] Embodiment 16: The method of any one of Embodiments 1-15, wherein the plurality of cells comprise human cells.

[0100] Embodiment 17: The method of any one of Embodiments 1-16, wherein the apparatus comprises a membrane separating the fluidic channel from a compartment.

[0101] Embodiment 18: The method of Embodiment 17, wherein the membrane is disposed proximate to the one or more electrodes.

[0102] Embodiment 19: A method of capturing a cell, comprising: flowing a fluid at a first flow rate into a fluidic channel of an apparatus, the fluid comprising a plurality of cells; applying a signal at a first signal strength to one or more electrodes disposed in the fluidic channel of the apparatus, the fluidic channel comprising a capture site disposed approximate the one or more electrodes; generating a non-uniform electric field across the one or more electrodes based on the applied signal to immobilize one or more cells of the plurality of cells at one or more capture site; and adjusting the flowing of the fluid to a second flow rate and/or adjusting the signal to a second signal strength to remove all but one immobilized cell at the one or more capture sites.

[0103] Embodiment 20: The method of Embodiment 19, further comprising: prior to adjusting the flowing of the fluid, cleaning the fluidic channel by removing any cell that is not immobilized at the capture site.

[0104] Embodiment 21 : The method of any one of Embodiments 19-20, prior to flowing the fluid at the flow rate into the fluidic channel of the apparatus, the method further comprising: flowing a buffer solution for passivating a surface of the fluidic channel. [0105] Embodiment 22: The method of Embodiment 21 , wherein the buffer solution comprises a blocking agent.

[0106] Embodiment 23 : The method of Embodiment 22, wherein the blocking agent comprises one or more proteins and/or one or more synthetic polymers.

[0107] Embodiment 24: The method of Embodiment 23, wherein the one or more proteins comprise an albumin protein.

[0108] Embodiment 25: The method of any one of Embodiments 19-24, wherein the first signal strength comprises an alternating current (AC) signal with a frequency within a range of about 0.5 MHz and about 50 MHz and/or a voltage within a range of about IV and about 5 V.

[0109] Embodiment 26: The method of Embodiments 19-25, wherein adjusting the signal to a second signal strength comprises: decreasing the strength of the signal via decreasing the voltage, and/or decreasing or increasing the frequency of the AC signal.

[0110] Embodiment 27: The method of any one of Embodiments 19-26, wherein the second signal strength comprises an alternating current (AC) signal with a frequency within a range of about 0.1 MHz and about 100 MHz, wherein the frequency is different from the frequency of the first signal strength, and/or a voltage within a range of about 0.1V and about 4V, wherein the voltage is lower than the voltage of the first signal strength.

[oni] Embodiment 28: The method of any one of Embodiments 19-27, wherein the first flow rate comprises a rate between about 1 pL/min and about 50 pL/min.

[0112] Embodiment 29: The method of any one of Embodiments 19-28, wherein the first flow rate has an average fluid velocity between about 10 pm/s and about 500 pm/s.

[0113] Embodiment 30: The method of any one of Embodiments 19-29, wherein the second flow rate comprises a rate between about 80 pL/min and about 5,000 pL/min.

[0114] Embodiment 31 : The method of any one of Embodiments 19-30, wherein the second flow rate has an average fluid velocity between about 1.4 mm/s and about 5.0 mm/s.

[0115] Embodiment 32: The method of any one ofEmbodiments 19-31 , wherein the increasing the flow rate of the fluid to the second flow rate occurs from about 4 minutes to about 30 minutes after flowing the fluid at the first flow rate.

[0116] Embodiment 33: The method of any one of Embodiments 19-32, wherein the decreasing the signal to the second signal strength occurs from 4 minutes to about 30 minutes after applying the signal at the first signal strength. [0117] Embodiment 34: The method of any one of Embodiments 19-33 wherein the fluid comprises between about 10,000 cells/mL and about 2,000,000 cells/mL.

[0118] Embodiment 35: The method of any one of Embodiments 19-34, wherein the plurality of cells comprise human cells.

[0119] Embodiment 36: The method of any one of Embodiments 19-35, wherein the apparatus comprises a membrane separating the fluidic channel from a compartment.

[0120] Embodiment 37: The method of Embodiment 36, wherein the membrane is disposed proximate to the one or more electrodes.

[0121] Embodiment 38: A method for capturing a single cell at a capture point in a fluidic channel, comprising: flowing a fluid into the fluidic channel at a first flow rate, wherein the fluid comprises a plurality of cells and wherein the fluidic channel comprises a plurality of capture points; applying a dielectrophoretic force at a first strength on the plurality of cells, wherein the first flow rate and the first dielectrophoretic force are balanced to generate one or more captured cells from the plurality of cells at one or more capture points of the plurality of capture points; and changing the dielectrophoretic force to a second strength on the plurality of cells and/or flowing the fluid into the fluidic channel at a second flow rate to dislodge all but one cell of the captured cells.

[0122] Embodiment 39: The method of Embodiment 38, wherein prior to flowing the fluid at the first flow rate, the method further comprising: flowing a buffer solution for passivating a surface of the fluidic channel.

[0123] Embodiment 40: The method of Embodiment 39, wherein the buffer solution comprises a blocking agent.

[0124] Embodiment 41 : The method of Embodiment 40, wherein the blocking agent comprises one or more proteins and/or one or more synthetic polymers.

[0125] Embodiment 42: The method of Embodiment 41, wherein the one or more proteins comprise an albumin protein.

[0126] Embodiment 43: The method of any one of Embodiments 38-42, wherein the first strength comprises an alternating current (AC) signal with a frequency within a range of about 0.5 MHz and about 50 MHz and/or a voltage within a range of about IV and about 5V. [0127] Embodiment 44: The method of any one of Embodiments 38-43, wherein changing the di electrophoretic force comprises: decreasing the strength of the signal via decreasing the voltage, and/or decreasing or increasing the frequency of the AC signal.

[0128] Embodiment 45: The method of any one of Embodiments 38-44, wherein the second strength comprises an alternating current (AC) signal with a frequency within a range of about 0.1 MHz and about 100 MHz, wherein the frequency is different from the frequency of the first strength, and/or a voltage within a range of about 0. IV and about 4V, wherein the voltage is lower than the voltage of the first strength.

[0129] Embodiment 46: The method of any one of Embodiments 38-45, wherein the first flow rate comprises a rate between about 1 pL/min and about 50 pL/min.

[0130] Embodiment 47: The method of any one of Embodiments 38-46, wherein the first flow rate has an average fluid velocity between about 10 pm/s and about 500 pm/s.

[0131] Embodiment 48: The method of any one of Embodiments 38-47, wherein the second flow rate comprises a rate between about 80 pL/min and about 5,000 pL/min.

[0132] Embodiment 49: The method of any one of Embodiments 38-48, wherein the second flow rate has an average fluid velocity between about 1.4 mm/s and about 5.0 mm/s.

[0133] Embodiment 50: The method of any one of Embodiments 38-49, wherein the flowing of the fluid at the second flow rate occurs from about 4 minutes to about 30 minutes after flowing the fluid at the first flow rate.

[0134] Embodiment 51 : The method of any one of Embodiments 38-50, wherein the changing the di electrophoretic force occurs from 4 minutes to about 30 minutes after applying the signal at the first signal strength.

[0135] Embodiment 52: The method of any one of Embodiments 38-51, wherein the fluid comprises between about 10,000 cells/mL and about 2,000,000 cells/mL.

[0136] Embodiment 53: The method of any one of Embodiments 38-52, wherein the plurality of cells comprise human cells.

[0137] Embodiment 54: A method of capturing a cell, comprising: flowing a fluid at a flow rate into a fluidic channel of an apparatus, the fluid comprising a plurality of cells; applying a signal to one or more electrodes disposed in the fluidic channel of the apparatus, thereby generating a non-uniform electric field at or in a vicinity of the one or more electrodes, wherein the fluidic channel comprises a capture site disposed approximate the one or more electrodes; and adjusting the flow rate of the fluid and/or a strength of the signal to immobilize one or more cells of the plurality of cells at the capture site, and/or to remove any cell that is not immobilized at the capture site.

[0138] Embodiment 55: The method of Embodiment 54, further comprising: adjusting the strength of the signal to immobilize one or more cells of the plurality of cells at the capture site.

[0139] Embodiment 56: The method of Embodiments 54 or 55, wherein adjusting the flow rate includes decreasing the flow rate to ensure at least one cell is immobilized at the capture site.

[0140] Embodiment 57: The method of Embodiments 55 or 56, wherein adjusting the strength of the signal includes increasing the strength of the signal to ensure at least one cell is immobilized at the capture site.

[0141] Embodiment 58: The method of any one of Embodiments 54-57, further comprising: increasing the flow rate to remove any cell that is not immobilized at the capture site.

[0142] Embodiment 59: The method of any one of Embodiments 54-58, prior to flowing the fluid at the flow rate into the fluidic channel of the apparatus, the method further comprising: flowing a buffer solution for passivating a surface of the fluidic channel.

[0143] Embodiment 60: The method of Embodiment 59, wherein the buffer solution comprises a media containing Bovine Serum Albumin or similar blocking protein, Pluronic, etc.

[0144] Embodiment 61 : The method of any one of Embodiments 54-60, wherein the signal is an alternating current (AC) signal with a frequency of about 1 MHz and/or an operating voltage within a range of about IV and about 4V.

[0145] Embodiment 62: The method of Embodiment 61, wherein adjusting the strength of the signal to remove any cell that is not immobilized at the capture site comprises: decreasing the strength of the signal via decreasing the operating voltage, and/or decreasing the frequency of the AC signal.

[0146] Embodiment 63 : The method of any one of Embodiments 54-62, wherein the flow rate ranges between about 1 pL/min and about 200 pL/min.

[0147] Embodiment 64: The method of any one of Embodiments 54-63, wherein the fluid has an average fluid velocity between about 10 pm/s and about 3000 pm/s.

[0148] Embodiment 65: The method of any one of Embodiments 54-64, wherein the fluid comprises between about 10,000 cells/mL and about 2,000,000 cells/mL. [0149] Embodiment 66: A method of capturing a cell, comprising: flowing a fluid at a flow rate into a fluidic channel of an apparatus, the fluid comprising a plurality of cells; applying a signal to one or more electrodes disposed in the fluidic channel of the apparatus, the fluidic channel comprising a capture site disposed approximate the one or more electrodes; generating a non- uniform electric field across the one or more electrodes based on the applied signal; and immobilizing one or more cells of the plurality of cells at the capture site by adjusting a strength of the signal and/or adjusting the flow rate.

[0150] Embodiment 67: The method of Embodiment 66, further comprising: cleaning the fluidic channel by removing any cell that is not immobilized at the capture site by increasing the flow rate.

[0151] Embodiment 68: The method of Embodiments 66 or 67, wherein immobilizing the one or more cells comprises: decreasing the flow rate to immobilize the one or more cells of the plurality of cells at the capture site.

[0152] Embodiment 69: The method of Embodiments 67 or 68, wherein adjusting the strength of the signal includes increasing the strength of the signal to ensure at least one cell is immobilized at the capture site.

[0153] Embodiment 70: The method of any one of Embodiments 66-69, further comprising: increasing the flow rate to remove any cell that is not immobilized at the capture site.

[0154] Embodiment 71 : The method of any one of Embodiments 66-70, prior to flowing the fluid at the flow rate into the fluidic channel of the apparatus, the method further comprising: flowing a buffer solution for passivating a surface of the fluidic channel.

[0155] Embodiment 72: The method of Embodiment 71, wherein the buffer solution comprises a media containing Bovine Serum Albumin or similar blocking protein, Pluronic, etc.

[0156] Embodiment 73: The method of any one of Embodiments 66-72, wherein the signal is an alternating current (AC) signal with a frequency of about 1 MHz and/or an operating voltage within a range of about IV and about 4V.

[0157] Embodiment 74: The method of Embodiment 73, wherein removing any cell that is not immobilized at the capture site comprises: decreasing the strength of the signal via decreasing the operating voltage, and/or decreasing the frequency of the AC signal.

[0158] Embodiment 75: The method of any one of Embodiments 66-74, wherein the flow rate ranges between about 1 pL/min and about 200 pL/min. [0159] Embodiment 76: The method of any one of Embodiments 66-75, wherein the fluid has an average fluid velocity between about 10 pm/s and about 3000 pm/s.

[0160] Embodiment 77: The method of any one of Embodiments 66-76, wherein the fluid comprises between about 10,000 cells/mL and about 2,000,000 cells/mL.

5 [0161] Embodiment 78: A system configured to perform the method of any one of Embodiments 1-77.

[0162] Embodiment 79: The system of Embodiment 78, further comprising one or more of (1) a membrane for separating a fluid from a compartment; (2) one or more electrodes disposed proximate to the membrane; (3) a counter-electrode, wherein the one or more electrodes and the fO counter-electrode are configured to generate a non-uniform electric field across the one or more electrodes and the counter-electrode; and (4) a power source for providing an alternating current (AC) across the one or more electrodes and the counter-electrode, thereby generating an oscillating non-uniform electric field for immobilizing a particle suspended in the fluid that flows between the one or more electrodes and the counter-electrode.

[0163] Embodiment 80: The system of Embodiment 78, further comprising one or more electrodes and a counter-electrode configured for generating a non-uniform electric field for immobilizing a particle suspended in a fluid that flows between the one or more electrodes and the counter-electrode; and a membrane disposed proximate a surface of the one or more electrodes, the surface of the one or more electrodes distal the counter-electrode, wherein the membrane is

20 configured for separating the fluid from a compartment, and has an opening configured to allow for insertion of a sharp member disposed in the compartment.

[0164] While this specification contains many specific embodiment details, these should not be construed as limitations on the scope of any inventions or of what may be claimed, but rather as descriptions of features specific to particular embodiments of particular inventions. Certain features that are described in this specification in the context of separate embodiments can also be implemented in combination in a single embodiment. Conversely, various features that are described in the context of a single embodiment can also be implemented in multiple embodiments separately or in any suitable sub-combination. Moreover, although features may be described above as acting in certain combinations and even initially claimed as such, one or more features

30 from a claimed combination can in some cases be excised from the combination, and the claimed combination may be directed to a sub-combination or variation of a sub-combination. [0165] Similarly, while operations are depicted in the drawings in a particular order, this should not be understood as requiring that such operations be performed in the particular order shown or in sequential order, or that all illustrated operations be performed, to achieve desirable results. In certain circumstances, multitasking and parallel processing may be advantageous. Moreover, the separation of various system components in the embodiments described above should not be understood as requiring such separation in all embodiments, and it should be understood that the described program components and systems can generally be integrated together in a single software product or packaged into multiple software products.

[0166] References to “or” may be construed as inclusive so that any terms described using “or” may indicate any of a single, more than one, and all of the described terms. The labels “first,” “second,” “third,” and so forth are not necessarily meant to indicate an ordering and are generally used merely to distinguish between like or similar items or elements.

[0167] Various modifications to the embodiments described in this disclosure may be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of this disclosure. Thus, the claims are not intended to be limited to the embodiments shown herein, but are to be accorded the widest scope consistent with this disclosure, the principles and the novel features disclosed herein.

Claims

What is claimed is:

1. A method of capturing a cell, comprising: flowing a fluid at a first flow rate into a fluidic channel of an apparatus, wherein the fluid comprises a plurality of cells; generating a set of capture sites comprising one or more captured cells by applying a signal at a first signal strength to one or more electrodes disposed in the fluidic channel of the apparatus, thereby generating a non-uniform electric field at or in a vicinity of the one or more electrodes, wherein the fluidic channel comprises a capture site disposed approximate the one or more electrodes; and increasing the flow rate of the fluid to a second flow rate and/or changing the signal to a second signal strength to remove all but one captured cell at one or more of the capture sites in the set of capture sites.

2. The method of claim 1, wherein prior to flowing the fluid at the first flow rate into the fluidic channel of the apparatus, the method further comprising: flowing a buffer solution for passivating a surface of the fluidic channel.

3. The method of claim 2, wherein the buffer solution comprises a blocking agent.

4. The method of claim 3, wherein the blocking agent comprises one or more proteins and/or one or more synthetic polymers.

5. The method of claim 4, wherein the one or more proteins comprise an albumin protein.

6. The method of any one of claims 1-5, wherein the first signal strength comprises an alternating current (AC) signal with a frequency within a range of about 0.5 MHz and about 50 MHz and/or a voltage within a range of about IV and about 5 V.

7. The method of any one of claims 1-6, wherein changing the signal to a second signal strength comprises: decreasing the strength of the signal via decreasing the voltage, and/or decreasing or increasing the frequency of the AC signal.

8. The method of any one of claims 1-7, wherein the second signal strength comprises an alternating current (AC) signal with a frequency within a range of about 0.1 MHz and about 100 MHz, wherein the frequency is different from the frequency of the first signal strength, and/or a voltage within a range of about 0.1V and about 4V, wherein the voltage is lower than the voltage of the first signal strength.

9. The method of any one of claims 1-8, wherein the first flow rate comprises a rate between about 1 pL/min and about 50 pL/min.

10. The method of any one of claims 1-9, wherein the first flow rate has an average fluid velocity between about 10 pm/s and about 500 pm/s.

11. The method of any one of claims 1-10, wherein the second flow rate comprises a rate between about 80 pL/min and about 5,000 pL/min.

12. The method of any one of claims 1-11, wherein the second flow rate has an average fluid velocity between about 1.4 mm/s and about 5.0 mm/s.

13. The method of any one of claims 1-12, wherein the increasing the flow rate of the fluid to the second flow rate occurs from about 4 minutes to about 30 minutes after flowing the fluid at the first flow rate.

14. The method of any one of claims 1-13, wherein the decreasing the signal to the second signal strength occurs from 4 minutes to about 30 minutes after applying the signal at the first signal strength.

15. The method of any one of claims 1-14, wherein the fluid comprises between about 10,000 cells/mL and about 2,000,000 cells/mL.

16. The method of any one of claims 1-15, wherein the plurality of cells comprise human cells.

17. The method of any one of claims 1-16, wherein the apparatus comprises a membrane separating the fluidic channel from a compartment.

18. The method of claim 17, wherein the membrane is disposed proximate to the one or more electrodes.

19. A method of capturing a cell, comprising: flowing a fluid at a first flow rate into a fluidic channel of an apparatus, the fluid comprising a plurality of cells; applying a signal at a first signal strength to one or more electrodes disposed in the fluidic channel of the apparatus, the fluidic channel comprising a capture site disposed approximate the one or more electrodes; generating a non-uniform electric field across the one or more electrodes based on the applied signal to immobilize one or more cells of the plurality of cells at one or more capture site; and adjusting the flowing of the fluid to a second flow rate and/or adjusting the signal to a second signal strength to remove all but one immobilized cell at the one or more capture sites.

20. The method of claim 19, further comprising: prior to adjusting the flowing of the fluid, cleaning the fluidic channel by removing any cell that is not immobilized at the capture site.

21. The method of any one of claims 19-20, prior to flowing the fluid at the flow rate into the fluidic channel of the apparatus, the method further comprising: flowing a buffer solution for passivating a surface of the fluidic channel.

22. The method of claim 21, wherein the buffer solution comprises a blocking agent.

23. The method of claim 22, wherein the blocking agent comprises one or more proteins and/or one or more synthetic polymers.

24. The method of claim 23, wherein the one or more proteins comprise an albumin protein.

25. The method of any one of claims 19-24, wherein the first signal strength comprises an alternating current (AC) signal with a frequency within a range of about 0.5 MHz and about 50 MHz and/or a voltage within a range of about IV and about 5 V.

26. The method of any one of claims 19-25, wherein adjusting the signal to a second signal strength comprises: decreasing the strength of the signal via decreasing the voltage, and/or decreasing or increasing the frequency of the AC signal.

27. The method of any one of claims 19-26, wherein the second signal strength comprises an alternating current (AC) signal with a frequency within a range of about 0.1 MHz and about 100 MHz, wherein the frequency is different from the frequency of the first signal strength, and/or a voltage within a range of about 0.1V and about 4V, wherein the voltage is lower than the voltage of the first signal strength.

28. The method of any one of claims 19-27, wherein the first flow rate comprises a rate between about 1 pL/min and about 50 pL/min.

29. The method of any one of claims 19-28, wherein the first flow rate has an average fluid velocity between about 10 pm/s and about 500 pm/s.

30. The method of any one of claims 19-29, wherein the second flow rate comprises a rate between about 80 pL/min and about 5,000 pL/min.

31. The method of any one of claims 19-30, wherein the second flow rate has an average fluid velocity between about 1.4 mm/s and about 5.0 mm/s.

32. The method of any one of claims 19-31, wherein the increasing the flow rate of the fluid to the second flow rate occurs from about 4 minutes to about 30 minutes after flowing the fluid at the first flow rate.

33. The method of any one of claims 19-32, wherein the decreasing the signal to the second signal strength occurs from 4 minutes to about 30 minutes after applying the signal at the first signal strength.

34. The method of any one of claims 19-33 wherein the fluid comprises between about 10,000 cells/mL and about 2,000,000 cells/mL.

35. The method of any one of claims 19-34, wherein the plurality of cells comprise human cells.

36. The method of any one of claims 19-35, wherein the apparatus comprises a membrane separating the fluidic channel from a compartment.

37. The method of claim 36, wherein the membrane is disposed proximate to the one or more electrodes.

38. A method for capturing a single cell at a capture point in a fluidic channel, comprising: flowing a fluid into the fluidic channel at a first flow rate, wherein the fluid comprises a plurality of cells and wherein the fluidic channel comprises a plurality of capture points; applying a di electrophoretic force at a first strength on the plurality of cells, wherein the first flow rate and the first dielectrophoretic force are balanced to generate one or more captured cells from the plurality of cells at one or more capture points of the plurality of capture points; and changing the dielectrophoretic force to a second strength on the plurality of cells and/or flowing the fluid into the fluidic channel at a second flow rate to dislodge all but one cell of the captured cells.

39. The method of claim 38, wherein prior to flowing the fluid at the first flow rate, the method further comprising: flowing a buffer solution for passivating a surface of the fluidic channel.

40. The method of claim 39, wherein the buffer solution comprises a blocking agent.

41. The method of claim 40, wherein the blocking agent comprises one or more proteins and/or one or more synthetic polymers.

42. The method of claim 41, wherein the one or more proteins comprise an albumin protein.

43. The method of any one of claims 38-42, wherein the first strength comprises an alternating current (AC) signal with a frequency within a range of about 0.5 MHz and about 50 MHz and/or a voltage within a range of about IV and about 5 V.

44. The method of any one of claims 38-43, wherein changing the dielectrophoretic force comprises: decreasing the strength of the signal via decreasing the voltage, and/or decreasing or increasing the frequency of the AC signal.

45. The method of any one of claims 38-44, wherein the second strength comprises an alternating current (AC) signal with a frequency within a range of about 0.1 MHz and about 100 MHz, wherein the frequency is different from the frequency of the first strength, and/or a voltage within a range of about 0.1V and about 4V, wherein the voltage is lower than the voltage of the first strength.

46. The method of any one of claims 38-45, wherein the first flow rate comprises a rate between about 1 pL/min and about 50 pL/min.

47. The method of any one of claims 38-46, wherein the first flow rate has an average fluid velocity between about 10 pm/s and about 500 pm/s.

48. The method of any one of claims 38-47, wherein the second flow rate comprises a rate between about 80 pL/min and about 5,000 pL/min.

49. The method of any one of claims 38-48, wherein the second flow rate has an average fluid velocity between about 1.4 mm/s and about 5.0 mm/s.

50. The method of any one of claims 38-49, wherein the flowing of the fluid at the second flow rate occurs from about 4 minutes to about 30 minutes after flowing the fluid at the first flow rate.

51. The method of any one of claims 38-50, wherein the changing the di electrophoretic force occurs from 4 minutes to about 30 minutes after applying the signal at the first signal strength.

52. The method of any one of claims 38-51, wherein the fluid comprises between about 10,000 cells/mL and about 2,000,000 cells/mL.

53. The method of any one of claims 38-52, wherein the plurality of cells comprise human cells.

54. A system configured to perform the method of any one of claims 1-53.

55. The system of claim 54, further comprising one or more of

(1) a membrane for separating a fluid from a compartment;

(2) one or more electrodes disposed proximate to the membrane;

(3) a counter-electrode, wherein the one or more electrodes and the counter-electrode are configured to generate a non-uniform electric field across the one or more electrodes and the counter-electrode; and

(4) a power source for providing an alternating current (AC) across the one or more electrodes and the counter-electrode, thereby generating an oscillating non-uniform electric field for immobilizing a particle suspended in the fluid that flows between the one or more electrodes and the counter-electrode.

56. The system of claim 55, further comprising one or more electrodes and a counter-electrode configured for generating a non-uniform electric field for immobilizing a particle suspended in a fluid that flows between the one or more electrodes and the counter-electrode; and a membrane disposed proximate a surface of the one or more electrodes, the surface of the one or more electrodes distal the counter-electrode, wherein the membrane is configured for separating the fluid from a compartment, and has an opening configured to allow for insertion of a sharp member disposed in the compartment.