BACKGROUND

[0001] Cell lysis on a digital microfluidic (DMF) system can be done mechanically or non- mechanically. For example, chemical (non-mechanical) lysis can be performed by combining a droplet containing the cell with a droplet containing a lysing agent. The lysate, however, is contaminated with the lysate agent and is diluted. The lysate contamination may degrade some of the analytes of interest and may also interfere with downstream analysis, thereby requiring additional removal steps. On the other hand, mechanical lysis can be performed in micro-devices, including nano-pillars and nano-scale barbs. While these adequately lyse cells, such micro-devices are typically not reusable and are difficult to fabricate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0002] FIG. 1 illustrates a sectional view of an example digital microfluidic (DMF) device having a closed- droplet configuration.

[0003] FIGs. 2A through 2C respectively illustrate a top view of an example digital microfluidic (DMF) device in operation.

[0004] FIGs. 3A through 3D respectively illustrate a sectional view of example digital microfluidic (DMF) devices having two or more thin film resistors.

[0005] FIG. 4 illustrates a sectional view of another example DMF device having a closed- droplet configuration.

[0006] FIGs. 5A through 5C respectively illustrate a sectional view of an example DMF device having an open droplet configuration.

[0007] FIG. 6 illustrates an example process for performing mechanical lysis to a cell using the DMF device of FIG. 1.

[0008] FIG. 7 illustrates an example DMF device having an array of lysis sites.

[0009] FIGs. 8A through 8H respectively illustrates an example lysis site of a DMF device.

DETAILED DESCRIPTION

[0010] There is an increasing demand for analysis of a large number of cells on a single cell level. Digital microfluidics allows a large number of droplets to be manipulated. Each droplet can contain a single cell and therefore a large number of cells can be manipulated on a single cell level. Many single cell analysis require the cells to be lysed. Traditionally on DMF platforms, this is done via chemical lysis (e.g. subjecting the cell to high pH) by combining a droplet containing the cell with a droplet containing a lysing agent. While this sometimes works, this is not desirable as cellular components of interests are then contaminated with the lysing agent and can be degraded (e.g. high pH solutions degrade RNA) or the lysing agent (e.g. surfactants) interferes with downstream analysis and needs to be washed away. Some surfactants may also be not compatible with DMF operation. Some cells are difficult to lyse purely with a chemical lysis but can be easily lysed with mechanical lysis. Furthermore, mechanical lysis is advantageous as it does not leave any contaminants in the cell lysis that may need to be cleaned up later. This disclosure provides a digital microfluidic (DMF) to perform non-chemical cell lysis therein, thereby creating a high throughput, single cell resolution analysis system.

[0011] FIG. 1 illustrates a sectional view of a digital microfluidic (DMF) device 100 having a thin film resistor 160 according to an example. As shown in FIG. 1, DMF device 100 includes a lower substrate 110 and a microfluidic layer 120 on lower substrate 110. In one example, microfluidic layer 120 can be covered by a transparent upper substrate 140 on which a transparent electrode 150 is deposited. Transparent electrode 150 is deposited on the surface of upper substrate 140 that faces microfluidic layer 120. In one example, transparent upper substrate 140 is made of an insulating material, e.g., glass or plastic, while transparent electrode 150 is made of a conductive material, e.g., ITO, and electrically coupled to the ground.

[0012] Microfluidic layer 120 can be filled with a carrier fluid 102 to facilitate multiphase movement of a cell containing droplet 104 (aqueous) within microfluidic layer 120. Carrier fluid 102 can be any suitable oil, such as, silicone oil (2 cSt, 5 cSt), FC40, FC75, Novec HFE7100, Novec HFE7300, Novec HFE 7500, and the like. In one example, a low contact angle hysteresis layer can be formed on a surface 122 of microfluidic layer 120, such that a cell containing droplet 104 is movable within microfluidic layer 120 with low friction. Thin film resistor 160 is embedded in lower substrate 110 and aligned with an area of microfluidic layer 120 to perform mechanical lysis to cell containing droplet 104 moved to the area. In one example, microfluidic layer 120 can be further confined to form a narrowed channel or conduit. [0013] In one example, microfluidic layer 120 has a thickness TM of about 200 microns or any suitable thickness in a range of about 10 to 3,000 microns. Cell containing droplet 104 can have a width WD of about 1 mm or any suitable width in a range of about 40 microns to 3 mm. Thin film resistor 160 can have a width WR of about 30 microns or any suitable width in a range of about 10- 100 microns. Thin film resistor 160 is made of a material that can generate high thermal flux in response to voltage pulses with a duration of about 1~10 micro seconds. In one example, thin film resistor 160 can generate a power density of about 0.1-2.0 GW/m ² .

[0014] In one example, DMF device 100 further includes one or more control electrodes 132 on lower substrate 110 for setting up the electric field to control movement of cell containing droplet 104 within microfluidic layer 120. A dielectric layer 130 can be formed between microfluidic layer 120 and lower substrate 110 to electrically insulate control electrodes 132 with cell containing droplet 104. Dielectric layer 530 can be made of, e.g., Kapton, ETFE, Paralyne, alumina, silica, aluminum nitride, aluminum oxide, and/or any other suitable materials.

[0015] In one example, all components above lower substrate 110 (e.g., control electrodes 132, dielectric layer 130, microfluidic layer 120, transparent electrode 150, and upper substrate 140) can be manufactured into a microfluidic cartridge 190 removable from lower substrate 110 and thus “consumable.” In another example, microfluidic cartridge 190 can be manufactured with another dielectric layer to define a contact interface between cartridge 170 and dielectric layer 130, such that when microfluidic cartridge 190 is removed, dielectric layer 130 and control electrodes 132 remain on lower substrate 110.

[0016] FIGs. 2A through 2C respectively illustrate a top view of digital microfluidic (DMF) device 100 in operation according to an example. As shown in FIGs. 2 A through 2C, in this example, microfluidic layer 120 of DMF device 100 in FIG. 1 is confined to form a microfluidic channel 220 with control electrode 132 and thin -film resistor 160 being aligned with a segment of microfluidic channel 220. Microfluidic channel 220 has an inlet 222 and an outlet 224 from which carrier fluid 102 and/or cell containing droplet 104 can be guided in and out of microfluidic channel 220. FIG. 2A shows that microfluidic channel 220 is empty. FIG. 2B shows that microfluidic channel 220 is primed with bovine whole blood. FIG. 2C shows lysed cells in microfluidic channel 220 after thin-film resistor 160 is activated to generate steam bubbles to lyse cells.

[0017] FIGs. 3A through 3D respectively illustrate a sectional view of digital microfluidic (DMF) device 100 having two or more thin film resistors according to various examples. Digital microfluidic (DMF) device 100 as shown in FIGs. 3A through 3D are substantially the same as that shown in FIG. 1, except that more thin film resistors are used in FIGs. 3A through 3D.

[0018] FIG. 3A shows an example of digital microfhiidic (DMF) device 100 that uses two thin film resistors 160 and 162, both being embedded in lower substrate 110 at different locations and electrically insulated from each other and from control electrode 132.

[0019] FIG. 3B shows that thin film resistors 160 is activated by an electric current to heat up cell containing droplet 104 thereby generating vapor bubbles 106 to perform mechanical lysis to a cell 108.

[0020] FIG. 3C shows an alternative example of digital microfhiidic (DMF) device 100 that uses two thin film resistors 160 and 162. As shown in FIG. 3C, first thin film resistor 160 is embedded in lower substrate 110 and electrically insulated from control electrode 132, while second thin film resistor 162 is embedded in upper substrate 140 and electrically insulated from transparent electrode 150.

[0021] FIG. 3D shows another example of digital microfhiidic (DMF) device 100 that uses five thin film resistors 160, 162, 164, 166, and 168. As shown in FIG. 3D, first and second thin film resistors 160 and 162 are embedded in lower substrate 110 at different locations and electrically insulated from each other and from control electrode 132. Third, fourth, and fifth thin film resistors 164, 166, 168 are embedded in upper substrate 140 and electrically insulated from each other and from transparent electrode 150.

[0022] FIG. 4 illustrates a sectional view of a digital microfhiidic (DMF) device 100 having a thin film resistor 160 according to another example. DMF device 100 as shown in FIG. 4 is substantially the same as that shown in FIG. 1, except that a first low contact angle hysteresis layer 170 is used in place of dielectric layer 130 and a second low contact angle hysteresis layer 180 is added between microfluidic layer 120 and transparent electrode 150. First and second contact angle hysteresis layers 170 and 180 can be made of a low surface energy coating material, e.g., Teflon, lluorosilane, Kapton FN, fhioroalkylsilane, 1H, lH,2H,2H-Perlluorodecyltriethoxysilane, Trichloro(lH, lH,2H,2H-perfhiorooctyl)silane, and/or any other suitable materials.

[0023] FIGs. 5A through 5C illustrate a sectional view of a digital microfluidic (DMF) device 500 having an open droplet configuration according to various examples. As shown in FIG. 5A, DMF device 500 includes a substrate 510 and a thin film resistor 560 embedded in substrate 510. One or more control electrodes 532 can be disposed on substrate 510 and electrically insulated from thin film resistor 560. A dielectric layer 530 can be formed on control electrodes 532 and over substrate 510 to electrically insulate control electrodes 132 with a cell containing droplet 504. Control electrodes 510 can set up the electric field to control movement of cell containing droplet 504 on dielectric layer 530. In addition, metal particles 580 may be embedded in dielectric layer 530 to enhance thermal conductivity and performance of DMF device 500.

[0024] DMF device 500 as shown in FIG. 5B is substantially the same as that shown in FIG. 5A, except that DMF device 500 in FIG. 5B additionally includes a low contact angle hysteresis layer 570 on dielectric layer 530 such that cell containing droplet 504 is movable on layer 570 with low friction. Further, DMF device 500 as shown in FIG. 5C is substantially the same as that shown in FIG. 5B, except that DMF device 500 in FIG. 5C additionally includes a planarization layer 580 between substrate 510 and dielectric layer 530. Planarization layer 580 can be made of, e.g., SU8, Paralyne, PDMS, acrylates, and/or any other suitable materials. As shown in FIGs. 5B and 5C, metal particles 580 maybe embedded in low contact angle hysteresis layer 570 to enhance thermal conductivity and performance of DMF device 500. It is appreciated that metal particles 580 can be embedded in one or more insulating layers (e.g., dielectric layer 530, low contact angle hysteresis layer 570 and planarization layer 580) between thin film resistor 560 and cell containing droplet 504. [0025] FIG. 6 illustrates an example process for performing mechanical lysis to a cell 108 contained in liquid 104 using digital microfluidic (DMF) 100 of FIG. 1. As shown in FIG. 6, in Step S610, control electrodes 132 moves cell containing droplet 104 to a location that is aligned with thin film resistor 160 so as to perform mechanical lysis there. For certain cells that are difficult to lyse mechanically, a droplet of chemical lysate (e.g., benzalkonium chloride, chlorhexidine digluconate, phenol, sodium dodecyl sulfate (SDS), and/or Triton X- 100 buffers) can be mixed with cell containing droplet 104 before mechanical lysis commences. In Step S620, an electric current is applied to thin film resistor 160 to generate vapor bubbles 106 in cell containing droplet 104. In Step S630, the membrane of cell 108 is mechanically pierced or cut due to vapor bubbles 106. The fast moving interface of vapor bubbles 106 produces high shear within droplet 104, which porates the cell membrane and thus lyses cell 108. In Step S640, the cell lysate diffuses in droplet 104. In Step S650, cell lysate is mixed uniformly throughout droplet 104 for further downstream analysis, which maybe, e.g., PCR, isothermal amplification, RT-PCR, CAS, ELISA, micro-array/sandwich assay, alpha-screen (modified sandwich assay using two-photon absorption), melting analysis, mass- spectrometry, etc. [0026] FIG. 7 illustrates an example DMF device 700 having an array of lysis sites 710. As shown in FIG. 7, each lysis site 710 includes a control electrode 712 and a thermal resistor 714. In this example, thermal resistor 714 is disposed in an area where control electrode 712 is cut off, such that control electrode 712 and thermal resistor 714 are formed on the same vertical level. It is appreciated that control electrode 712 and thermal resistor 714 can be formed on different vertical levels. Neighboring lysis sites 710 can be separated by a wall 702 such that each lysis site 710 constitutes a well structure to contain a cell containing droplet. Each lysis site 710 can have a square shape (or any other suitable shape, e.g., rectangle, circle, etc.) having a side length S of about 1 mm or any suitable width in a range of about 40 microns to 3 mm. In one example, DMF device 700 includes cartridge 190 of FIG. 1 removably disposed on lysis sites 710.

[0027] FIGs. 8A through 8H respectively illustrates an example lysis site 800 of a DMF device. In one example, lysis site 800 in FIGs. 8A through 8H includes one or more thermal resistors 820, each being capable of generating heat at the same power density.

[0028] As shown in FIG. 8A, in one example, lysis site 800 includes a control electrode 810 and a thermal resistor 820 located at a lower left corner of lysis site 800 for diffusion mixing.

[0029] As shown in FIG. 8B, in one example, lysis site 800 includes a control electrode 810 and two thermal resistors 820, respectively located at a upper and lower left corners of lysis site 800 for diffusion mixing. In one example, voltage pulses are transmitted to thermal resistors 820 in FIG. 8C asynchronously, such that the vapor bubbles so generated can create a chaos environment within a cell containing droplet for more effective lysing.

[0030] As shown in FIG. 8C, in one example, lysis site 800 includes a control electrode 810 and three thermal resistors 820, a first one being located at a lower left corners of lysis site 800, a second one being located at a upper left corners of lysis site 800, and a third one being located at central right side of lysis site 800 for diffusion mixing. In one example, voltage pulses are transmitted to thermal resistors 820 in FIG. 8C asynchronously, such that the vapor bubbles so generated can create a chaos environment within a cell containing droplet for more effective lysing.

[0031] As shown in FIG. 8D, in one example, lysis site 800 includes a control electrode 810 and an array of six thermal resistors 820 for mixing. Three of the six thermal resistors 820 are located at the left side of lysis site 800 while the other three of the six thermal resistors 820 are located at the right side of lysis site 800. In one example, voltage pulses are transmitted to thermal resistors 820 in FIG. 8D asynchronously, such that the vapor bubbles so generated can create a chaos environment within a cell containing droplet for more effective lysing.

[0032] As shown in FIG. 8E, in one example, lysis site 800 includes a control electrode 810 and a thermal resistor 820 located at a central portion of lysis site 800. It is appreciated that although a centrally positioned thermal resistor 820 may not be ideal for mixing, it can be successfully used for cell lysing.

[0033] As shown in FIG. 8F, in one example, lysis site 800 includes a control electrode 810 and a thermal resistor 820 located at a central portion of lysis site 800. Thermal resistor 820 in FIG. 8F is larger in area than thermal resistor 820 in FIG. 8E to deliver more vapor bubbles for more efficient lysing.

[0034] As shown in FIG. 8G, in one example, lysis site 800 includes a control electrode 810, a smaller thermal resistor 821 at an upper side of lysis site 800 (for diffusion mixing), and a larger thermal resistor 822 at a central upper side of lysis site 800 (for cell lysing). In one example, smaller thermal resistor 821 is in a rectangular shape and configured to quickly heat up and agitate/stir a cell containing droplet, while larger thermal resistor 822 is in a rectangular shape configured to slowly heat up the cell containing droplet. In one example, two terminals are formed at the left and right sides of smaller thermal resistor 821 to receive voltage pulses, while two terminals are formed at the top and bottom sides of larger thermal resistor 822 to receive voltage pulses. A length L of smaller thermal resistor 821 can be the same as a width W of larger thermal resistor 822.

[0035] As shown in FIG. 8H, in one example, lysis site 800 includes a control electrode 810 and a thermal resistor 820 having a spiral shape. First and second terminals 831 and 832 are respectively connected to two ends of thermal resistor 820. Third terminal 833 is connected to a position of thermal resistor 820 electrically closer to first terminal 831 such that the resistance between first and third terminals 831 and 833 is less than the resistance between second and third terminals 832 and 833. The section of thermal resistor 820 between first and third terminals 831 and 833 is configured to quickly heat up and agitate/stir a cell containing droplet, while section of thermal resistor 820 between second and third terminals 832 and 833 is configured to slowly heat up the cell containing droplet.

[0036] Advantages of the disclosed DMF device include the ability to perform complex operations with lysate from a large number of single cells and the ability to obtain genetic, proteomic, and other information from large number of single cells in an automated manner. Analytes from cells are self-contained in the droplet and inside the device, thereby reducing the probability of lysing reagent or other contamination. Because the disclosed DMF device provides electrically actuated lysis, no additional subsystems are needed.

[0037] For the purposes of describing and defining the present disclosure, it is noted that terms of degree (e.g., “substantially,” “slightly,” “about,” “comparable,” etc.) may be utilized herein to represent the inherent degree of uncertainty that may be attributed to any quantitative comparison, value, measurement, or other representation. Such terms of degree may also be utilized herein to represent the degree by which a quantitative representation may vary from a stated reference (e.g., about 10% or less) without resulting in a change in the basic function of the subject matter at issue. Unless otherwise stated herein, any numerical values appeared in this specification are deemed modified by a term of degree thereby reflecting their intrinsic uncertainty.

[0038] Although various embodiments of the present disclosure have been described in detail herein, one of ordinary skill in the art would readily appreciate modifications and other embodiments without departing from the spirit and scope of the present disclosure as stated in the appended claims.

WHAT IS CLAIMED IS: