Reagent spotting and assembly methods and systems

Abstract

The present disclosure generally relates to systems and methods for depositing droplets on a surface, for example, for use in digital micro fluidic devices, or for other applications. In some aspects, droplets may be deposited on a surface, e.g., of a digital microfluidic device, in a way that minimizes "splatter" or variability. For example, the droplets may be positioned on a surface to match the well locations of an ANSI microtiter plate of 96, 384, or 1536 wells. In some embodiments, the droplets may be deposited acoustically. The variability of the droplets may be minimized in certain cases by adding a charged molecule (e.g., an anionic polymer) and / or depositing the droplets under the influence of an electric field. Other aspects are generally directed to systems and methods involving such techniques, devices or cartridges prepared using techniques, systems or methods using such devices, or the like.

Description

[0001] REAGENT SPOTTING AND ASSEMBLY METHODS AND SYSTEMS

[0002] RELATED APPLICATIONS

[0003] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 549,080, filed February 2, 2024, entitled “Reagent Spotting and Assembly Methods and Systems,” by Hirukawa, et al. incorporated herein by reference in its entirety.

[0004] FIELD

[0005] The present disclosure generally relates to systems and methods for depositing droplets on a surface, for example, for use in digital microfluidic devices, or for other applications.

[0006] BACKGROUND

[0007] The use of microfluidic devices is attractive because they allow for miniaturization and parallelization of workflows. One type of microfluidic device is a digital microfluidic (DMF) device. Digital microfluidics provides methods of manipulating nanoliter to microliter volumes of liquids on an array of electrodes. By applying an electric potential to an electrode, these discrete droplets can be controlled in parallel, transported, mixed, reacted, and analyzed. However, using such microfluidic devices in a high-throughput context, where the device is compatible with standard lab automation tools both upstream and downstream of the workflow, has been a difficult task, and improvements are still needed.

[0008] SUMMARY

[0009] The present disclosure generally relates to systems and methods for depositing droplets on a surface, for example, for use in digital microfluidic devices, or for other applications. The subject matter of the present disclosure involves, in some cases, interrelated products, alternative solutions to a particular problem, and / or a plurality of different uses of one or more systems and / or articles.

[0010] In some cases, the surface may be the surface of a digital microfluidic chip. In some cases, the reagents may be reagents that allow CRISPR Cas9 genome editing, or other techniques. In certain embodiments, there may be a physical transfection site on a surface, which may have its own unique cargo or reagents, e.g., without requiring a unique onloading port. In some cases, assembly of cargo or reagents may be automated on the surface. This may be helpful, for example, for reducing dead volume, labor, automation time, or the like. Some embodiments involve methods for depositing nucleic acids on the surface (for example, the surface of a digital microfluidic device or chip), and then using the droplet fluidics to reconstitute that drop in a buffer, for example, by passing over that region and pulsating for a specified duration. This may allow for gene editing cargo to be assembled for downstream physical delivery into a cell. In some cases, the surface may be hydrophobic. In some cases, a nuclease can be directly added with cells in a buffer, e.g., prior to rehydrating the synthetic guide RNA(sgRNA). In some cases, the buffer may allow droplets to move with electrostatic force. In some cases, the buffer may be permissive of the ribonucleoprotein (RNP) formation. In certain embodiments, the buffer may allow electroporation to occur.

[0011] One aspect is generally directed to a device. In one set of embodiments, the device comprises a substantially planar- surface defining at least 50 regularly spaced regions on the surface, wherein at least 50% of the regions have a droplet positioned thereon, at least 95% of the droplets on the surface being positioned such that, for a region defining a center point, an area, and a maximum dimension, the droplet covers a surface area no more than 25% of the area of the region, and the droplet defines a center point that is positioned a displacement distance away from the center point of the region, wherein the displacement distance is less than 25% of the maximum dimension of the region, and wherein at least 95% of the droplets on the surface comprise an aqueous fluid and an anionic polymer.

[0012] In another set of embodiments, the device comprises a substantially planar surface defining at least 50 regularly spaced regions on the surface, wherein at least 50% of the regions have a droplet positioned thereon, at least 95% of the droplets on the surface being positioned such that, for a region defining a center point, an area, and a maximum dimension, the droplet has a volume of no more than 1 microliter, and the droplet defines a center point that is positioned a displacement distance away from the center point of the region, wherein the displacement distance is less than 25% of the maximum dimension of the region, and wherein at least 95% of the droplets on the surface comprise an aqueous fluid and an anionic polymer.

[0013] The device, in another set of embodiments, comprises a cartridge comprising a first electrode, a second electrode; and a substantially planar surface positioned between the first electrode and the second electrode, wherein the substantiality planar- surface has a plurality of concentrates positioned thereon, at least 95% of the concentrates on the surface comprising an anionic polymer and a nucleic acid. The device, in yet another set of embodiments, comprises a substantially planar surface defining at least 50 regularly spaced regions on the surface, wherein at least 5% of the regions have a droplet positioned thereon, the droplet covers a surface area no more than 25% of the area of the region, and at least 95% of the regions having a droplet positioned thereon having only a single droplet, and wherein at least 95% of the droplets on the surface comprise an aqueous fluid and an anionic polymer.

[0014] In still another set of embodiments, the device comprises a substantially planar surface defining at least 50 regularly spaced regions on the surface, wherein at least 5% of the regions have a droplet positioned thereon, the droplet having a volume of no more than 1 microliter, and at least 95% of the regions having a droplet positioned thereon having only a single droplet, and wherein at least 95% of the droplets on the surface comprise an aqueous fluid and an anionic polymer.

[0015] According to yet another set of embodiments, the device comprises a substantially planar surface defining at least 50 regularly spaced regions on the surface, wherein at least 20% of the regions have a concentrate positioned thereon, at least 95% of the concentrates on the surface being positioned such that, for a region defining a center point, an area, and a maximum dimension, the concentrate covers a surface area no more than 25% of the area of the region, and the concentrate defines a center point that is positioned a displacement distance away from the center point of the region, wherein the displacement distance is less than 25% of the maximum dimension of the region, and wherein at least 95% of the concentrate on the surface comprises an anionic polymer.

[0016] In another set of embodiments, the device comprises a cartridge comprising a first electrode, a second electrode; and a substantially planar surface positioned between the first electrode and the second electrode, wherein the substantiality planar surface has a plurality of droplets positioned thereon, at least 95% of the droplets on the surface comprising an anionic polymer and sgRNA.

[0017] Another aspect is generally drawn to a method. In one set of embodiments, the method comprises acoustically dispensing droplets onto a substantially planar hydrophobic surface in an electric field of at least 1 kV / cm, wherein at least 95% of the droplets on the surface comprise an aqueous fluid and an anionic polymer. In another set of embodiments, the method comprises providing a substantially planar surface defining at least 50 regularly spaced regions on the surface, wherein at least 20% of the regions have a concentrate positioned thereon, at least 95% of the concentrates on the surface being positioned such that, for a region defining a center point, an area, and a maximum dimension, the concentrate covers a surface area no more than 25% of the area of the region, and the concentrate defines a center point that is positioned a displacement distance away from the center point of the region, wherein the displacement distance is less than 25% of the maximum dimension of the region; and applying an electric field to move a droplet comprising a buffer and a cell to contact one of the concentrates positioned within the regions.

[0018] Yet another aspect is generally directed to systems and methods for on cartridge deposition of reagents on a digital microfluidic device. In some cases, this may be useful for certain gene editing applications. In addition, some aspects are generally directed to systems and methods for in-cartridge deposition of reagents on a digital microfluidic chip.

[0019] In another aspect, the present disclosure encompasses methods of making one or more of the embodiments described herein, for example, for a digital microfluidic device. In still another aspect, the present disclosure encompasses methods of using one or more of the embodiments described herein, for example, for a digital microfluidic device.

[0020] Other advantages and novel features of the present disclosure will become apparent from the following detailed description of various non-limiting embodiments of the disclosure when considered in conjunction with the accompanying figures.

[0021] BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Non-limiting embodiments of the present disclosure will be described by way of example with reference to the accompanying figures, which are schematic and are not intended to be drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is labeled in every figure, nor is every component of each embodiment of the disclosure shown where illustration is not necessary to allow those of ordinary skill in the art to understand the disclosure. In the figures:

[0023] Fig. 1 illustrates droplets on a surface, according to one embodiment;

[0024] Fig. 2 illustrates a droplet and a plurality of satellite droplets; Fig. 3 illustrates the evaluation of TCRa / b knock out and viability in primary human pan CD3+ T cells, in yet another embodiment;

[0025] Figs. 4A-4D illustrate validation of a reagent deposition process in still another embodiment;

[0026] Fig. 5 illustrates a DMF surface having deposited droplets, in yet another embodiment; and

[0027] Fig. 6 illustrates yet another DMF surface, in accordance with another embodiment.

[0028] DETAILED DESCRIPTION

[0029] The present disclosure generally relates to systems and methods for depositing droplets on a surface, for example, for use in digital microfluidic devices, or for other applications. In some aspects, droplets may be deposited on a surface, e.g., of a digital microfluidic device, in a way that minimizes “splatter” or variability. For example, the droplets may be positioned on a surface to match the well locations of an ANSI microtiter plate of 96, 384, or 1536 wells. In some embodiments, the droplets may be deposited acoustically. The variability of the droplets may be minimized in certain cases by adding a charged molecule (e.g., an anionic polymer) and / or depositing the droplets under the influence of an electric field. Other aspects are generally directed to systems and methods involving such techniques, devices or cartridges prepared using techniques, systems or methods using such devices, or the like.

[0030] Certain aspects are generally directed to systems and methods where aqueous fluid containing reagents (e.g., stabilized reagents) can be directly deposited on a surface, for example, using any of a variety of non-contact dispensing methods, such as acoustic dispensing. In some embodiments, such reagents may be deposited on the surface prior to the assembly of a device containing the surface, and later reconstituted during device operation. This may be useful, for example, in embodiments where the surface is relatively hydrophobic, or where unwanted reactions with the surface may later impede or reduce device performance. In some embodiments, the deposition of the aqueous fluid containing such reagents or other materials may be very precise, e.g., such that the deposition does not contaminate adjoining electrode locations, as this may result in sample contamination. Thus, certain embodiments are generally directed to reducing satellite droplet formation or “splatter,” especially when using acoustic or other non-contact dispensing methods, e.g., to allow for precise and accurate deposition of aqueous fluid or other materials. As an example, in one set of embodiments, a droplet (e.g., containing aqueous fluid, reagents, etc.), may be deposited on a surface in a specific region. In some cases, the droplet may be small, relative to the region. For instance, the droplet may cover a surface area of no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, or no more than 5% of the region. The surface may contain a variety of regions, which may be regularly or irregularly spaced around the surface. For instance, the regions may correspond to the well locations of an ANSI microtiter plate of 96, 384, or 1536 wells. A droplet may be deposited onto a specific region (e.g., corresponding to a single well location). As discussed herein, certain embodiments are generally directed to reducing or eliminating satellite droplet formation or “splatter,” e.g., as is shown in Fig. 2. This figure shows a main desired droplet 21 and satellite droplets 22 created when droplet 21 was deposited within region 20; in particular, in this example, it should be noted that the distribution of satellite droplets is such that some droplets may not actually be within region 20, and thus could present contamination issues under certain conditions.

[0031] A droplet may be positioned substantially in the center of a region; however, in some cases, the droplet may be positioned substantially off-center, although it is desired that the droplet is fully within the region. (See, e.g., Fig. 1, with droplet 15 within region 13.) In some cases, the center of the droplet may define a center point, and the region may define a center point. The center points may coincide, or be separated by a distance, e.g., a displacement distance. The displacement distance may be relatively small, compared to the region; for example, the displacement distance may be less than 50%, less than 25%, less than 10%, etc. of a maximum dimension of the region. In addition, it is desirable that there be very few or no satellite droplets. It should also be noted that not all regions need contain a droplet (for example, as is shown with region 16 in Fig. 1), although they can in some cases. In addition, although the regions are substantially rectangular- and regularly spaced in Fig. 1, this is by way of example only, and other shapes and / or other distributions (regular or irregular) are possible in other embodiments.

[0032] In some cases, after deposition, the droplets may be partially or fully dried, e.g., producing a concentrate. The concentrate may be liquid or solid, depending on the amount of drying, and may contain reagents, etc., such as are described herein. For instance, in one set of embodiments, after deposition of the droplets or a surface, of a plate, the plate may be used for digital microfluidics or other applications. In some cases, the concentrate may be reconstituted, for example by moving water or another fluid over the concentrate, e.g., as a droplet.

[0033] In certain cases, a fluid such as an aqueous fluid can be directly deposited on a surface, for example, using non-contact dispensing methods, e.g., where the only physical object coming into contact with the surface is the aqueous fluid. One non-limiting example is acoustic dispensing or acoustic liquid handling. However, as discussed, droplets may sometimes be accompanied by various “satellite” droplets that are also produced from the same fluid deposition mechanism, but which may impact the region in different locations, thereby creating satellite droplets around the main impact droplet. Surprisingly, it has been found that in accordance with some embodiments, by using a charged molecule (e.g., an anionic polymer) and depositing the droplets under the influence of an electric field, the amount of variability and production of satellite droplets may be reduced or eliminated. A variety of anionic polymers may be used, such as those described herein. In some cases, the anionic polymer may be selected to be substantially biocompatible, for example, with reagents that allow CRISPR or other techniques. Without wishing to be bound by any theory, it is believed that under the influence of the electric field, the charged molecules may alter the surface tension of the droplets, which may reduce the number of satellite droplets that are formed.

[0034] For example, in one set of embodiments, a nucleic acid, such as DNA or RNA, may be deposited on a surface along with certain reagents in a region, e.g., as discussed herein, e.g., to form droplets or concentrates. As a non-limiting example, the RNA may include sgRNA, e.g., for CRISPR or other applications. The surface may also include one or more electrodes, for example, which can be used to control the deposition of droplets as discussed herein. A fluid containing cells may be added to the surface, and moved to the region to reconstitute the reagents, for example, using digital microfluidics, electrowetting, gravity, or other suitable techniques. The fluid may also contain other species, for example, proteins or enzymes (e.g., Cas9), in various embodiments. In some cases, for example, for CRISPR applications, the fluid may be electroporated or “pulsed,” e.g., to allow the nucleic acid or reagents to enter the cells. In some embodiments, the same electrodes may be used for electroporation. Afterwards, the cells may be cultured, e.g., on the surface, transferred to other locations, or the like. In some embodiments, e.g., to allow reconstitution of nucleic acids such as sgRNA or DNA on a surface of a digital microfluidic device, a droplet containing buffer or buffer and cells may be directed to a deposited sgRNA region, and pulsed back and forth between 2 electrodes or between multiple electrodes, including but not limited to the electrode where the spot is localized, for example, to facilitate rehydration, ribonucleoprotein (RNP) assembly, or the like. In some cases, pulsing may be applied to solubilize the sgRNA, for example, while maintaining or regaining the hydrophobicity on the electrode. This may be useful, for example, to ensure droplet mobility, preventing cells or other reagents to be left behind, or the like. Following, the droplet may be moved off the electrode or region, e.g., without leaving a residual volume, preventing reagent loss, or surface contamination.

[0035] Various aspects are generally directed to non-contact methods of depositing a fluid on a surface. Non-limiting examples include piezoelectric droplet generation, electrophoretic deposition, acoustic dispensing or acoustic liquid handling, or the like. For example, a dispenser (e.g., a piezoelectric fluid dispenser, an electrophoretic dispenser, an acoustic fluid dispenser, etc.) may be positioned to transport fluid from a fluid reservoir to a surface. In acoustic dispensing or acoustic liquid handling, sound waves projected through a fluid may cause conical structures to be produced, from which droplets can emerge. This is commonly used, for example, in inkjet printers. By controlling the sound waves applied to the fluid, the volume of fluid that is transferred may be controlled. Other techniques control the creation of fluids using mechanical or electrical techniques.

[0036] In addition, in accordance with certain aspects, charged molecules may be present in the droplets. These may be useful, for example, for reducing the creation of satellite droplets. As discussed herein, without wishing to be bound by any theory, it is believed that under the influence of the electric field, the charged molecules may alter the surface tension of the droplets. A variety of charged molecules may be used. In some cases, the charged molecules may be selected to be substantially biocompatible, for example, with reagents that allow CRISPR or other techniques.

[0037] In some cases, the charged molecule may include a polymer. Charged polymers may be useful, in certain embodiments, as they can carry a relatively large number of charge groups per molecule. The polymers may include negatively charged or anionic polymers. One example of a charged polymer is poly-gamma-glutamic acid. Other examples include, but are not limited to, sodium carboxymethylcellulose, sodium alginate, dextran sulfate, heparin sulfate, hyaluronic acid, polyacrylic acid, sodium polyacrylatc, poly-l-aspartic acid, carrageenan, pectin, poly(acrylic acid-co -maleic acid), chondroitin sulfate, gamma-poly glutamic acid, poly(sodium 4-styrenesulfonate), poly (methacrylic acid), sodium polyitaconate, poly(acrylic acid-co-ethylene glycol), poly(acrylic acid-co-2-hydroxyethyl methacrylate), poly(methacrylic acid-co-ethyl acrylate), poly(maleic anhydride-alt- 1 -octadecene), etc. Other polymers may be useful in certain embodiments, e.g., in addition to and / or instead of these.

[0038] The charged molecules may be present at any concentration, and one or more than one type of charged molecule may be present, e.g., in a droplet. For example, the concentration may be at least 1 microgram / ml, at least 2 micrograms / ml, at least 3 micrograms / ml, at least 5 micrograms / ml, at least 10 micrograms / ml, at least 20 micrograms / ml, at least 30 micrograms / ml, at least 50 micrograms / ml, at least 100 micrograms / ml, at least 200 micrograms / ml, at least 300 micrograms / ml, at least 500 micrograms / ml, at least 1000 micrograms / ml, etc. In some cases, the concentration may be no more than 1000 micrograms / ml, no more than 500 micrograms / ml, no more than 300 micrograms / ml, no more than 200 micrograms / ml, no more than 100 micrograms / ml, no more than 50 micrograms / ml, no more than 30 micrograms / ml, no more than 20 micrograms / ml, no more than 10 micrograms / ml, no more than 5 micrograms / ml, no more than 3 micrograms / ml, no more than 2 micrograms / ml, no more than 1 microgram / ml, etc. Combinations of any of these are also possible in certain embodiments.

[0039] The charged molecules may be contained within water or another aqueous fluid, for example, saline, ethanol, or the like. In some embodiments, one or more salts may be dissolved in water to form an aqueous fluid. In some cases, the fluid may contain cell culture media, serum, or other species suitable for maintaining cells. Non-limiting examples of cell culture media include DMEM, MEM, RPMI, PBS, or the like.

[0040] In certain cases, some or all of the droplets on a surface may contain such charged molecules. For example, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the droplets on a surface may contain such charged molecules (for example, anionic polymers) In certain aspects, aqueous fluid containing such charged molecules may be deposited on a surface using non-contact methods of depositing a fluid onto a surface in the presence of an electric field. In some cases, the electric field may be created, at least in part, using one or more electrodes present on or near the surface. For example, in certain applications such as digital microfluidics, a surface may include one or more electrodes defining regions on the surface.

[0041] In some embodiments, the applied electric field may be at least 1 V / cm, at least 2 V / cm, at least 3 V / cm, at least 5 V / cm, at least 10 V / cm, at least 20 V / cm, at least 30 V / cm, at least 50 V / cm, at least 100 V / cm, at least 200 V / cm, at least 300 V / cm, at least 500 V / cm, at least 1 kV / cm, at least 2 kV / cm, at least 3 kV / cm, at least 5 kV / cm, at least 10 kV / cm, at least 20 kV / cm, at least 30 kV / cm, at least 50 kV / cm, at least 100 kV / cm, etc. In some cases, the electric field may be no more than 100 kV / cm, no more than 50 kV / cm, no more than 30 kV / cm, no more than 20 kV / cm, no more than 10 kV / cm, no more than 5 kV / cm, no more than 3 kV / cm, no more than 2 kV / cm, no more than 1 kV / cm, no more than 500 V / cm, no more than 300 V / cm, no more than 200 V / cm, no more than 100 V / cm, no more than 50 V / cm, no more than 30 V / cm, no more than 20 V / cm, no more than 10 V / cm, no more than 5 V / cm, no more than 3 V / cm, no more than 2 V / cm, no more than 1 V / cm, etc. In addition, in some cases, combinations of any of these may be used. For example, the applied electric field may be between 1 kV / cm and 10 kV / cm, between 3 kV / cm and 50 kV / cm, between 10 kV / cm and 100 kV / cm, etc.

[0042] In certain aspects, the droplets may be deposited on a surface. The surface may be substantially planar in some embodiments. In addition, the surface may be hydrophobic in some embodiments. For example, a hydrophobic surface may exhibit a water contact angle of at least 90°. In addition, in certain cases, the water contact angle may be at least 100°, at least 110°, at least 120°, etc.

[0043] In certain cases, the surface may contain a variety of regions, which may be regularly or irregularly spaced around the surface. The regions may have any shape and / or size, and different regions may independently have the same or different shapes and / or sizes. In some cases, there may be at least 5, at least 10, at least 20, at least 30, at least 50, at least 100, at least 200, at least 300, at least 500, at least 1000, at least 2000, at least 3000, at least 5000, at least 10,000, etc. regions on a surface. As non-limiting examples, the surface may have 6, 12, 24, 48, 96, 384, 1536, 3456, or 9600 regions. In some cases, the regions may each be defined by one or more electrodes. The regions may each independently have any suitable area. For example, the average area of the regions may be at least 0.5 mm2, at least 1 mm2, at least 5 mm2,at least 10 mm2, at least 20 mm2, at least 30 mm2, at least 50 mm2, at least 100 mm2, at least 200 mm2, at least 300 mm2, at least 500 mm2, at least 1,000 mm2, at least 2,000 mm2, etc.

[0044] The regions may also each independently have any shape, including circular, square, rectangular, triangular, hexagonal, irregular, etc. In some cases, a region may have a shape that is tessellatable, e.g., such that the shape may fill a plane with no gaps. Non-limiting examples include hexagons, squares, triangles, rectangles, parallelograms, and other regular- or irregular shapes. In addition, in some embodiments, a region may have a substantially square, rectangular, or hexagonal region that is formed by distorting opposing sides of a square, rectangle, or hexagon in exactly the same way. Non-limiting examples of such regions, including substantially square regions, can be seen in Fig. 6.

[0045] In some cases, at least some of the regions on a surface may correspond to the well locations of an ANSI microtiter plate of 96, 384, or 1536 wells. For instance, the centers of such regions may be substantially near the centers of the wells of a standard ANSI microtiter plate (also known as an SBS standard microplate), including ANSI / SBS 1-2004, ANSI / SBS 2-2004, ANSI / SBS 3-2004, and ANSI / SBS 4-2004. A typical microtiter plate has dimensions of 127.76 mm x 85.48 mm (+ / - 0.5 mm), and a variety of wells (e.g., 6, 12, 24, 48, 96, 384, 1536, or 3456 wells) arranged in a 2x3 ratio. For example, a 96- well plates would have a 9 mm well-to-well spacing, 384-well plates would have a 4.5 mm spacing, and 1536-well plates would have a 2.25 mm.

[0046] In addition, in some cases, a region may contain or be defined by an electrode, as discussed herein. In some cases, some or all of the regions may contain or defined by a single electrode, although in some cases, more than one electrode may be present. In addition, some regions may not necessarily contain electrodes in some embodiments.

[0047] In some aspects, the droplets may be small, relative to the region on a surface. For example, when deposited, the droplet may cover an area of no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, or no more than 10% of the area of the region. In some cases, the droplet may cover an area of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, etc. of the region. In addition, combinations of any of these are also possible. For example, a droplet may cover an area of between 35% and 55%, between 20% and 40%, between 45% and 60%, etc. of the region.

[0048] In some cases, the volume of a droplet on a surface may independently be at least 1 nl, at least 2 nl, at least 3 nl, at least 5 nl, at least 10 nl, at least 20 nl, at least 30 nl, at least 50 nl, at least 100 nl, at least 200 nl, at least 300 nl, at least 500 nl, or at least 1 microliter. In some embodiments, the volume of a droplet may independently be no more than 1 microliter, no more than 500 nl, no more than 300 nl, no more than 200 nl, no more than 100 nl, no more than 50 nl, no more than 30 nl, no more than 20 nl, no more than 10 nl, no more than 5 nl, no more than 3 nl, no more than 2 nl, or no more than 1 nl. Combinations of any of these are also possible. For example, a droplet may have a volume of between 20 nl and 50 nl, between 100 nl and 200 nl, between 5 nl and 10 nl, between 10 nl and 1 microliter, etc.

[0049] Not all of the regions may necessarily contain a droplet in certain cases, although in some embodiments, each region on a surface may contain a droplet. In certain embodiments, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the regions may contain a droplet. In some cases, no more than 95%, no more than 90%, no more than 85%, no more than 80%, no more than 75%, no more than 70%, no more than 65%, no more than 60%, no more than 55%, no more than 50%, no more than 45%, no more than 40%, no more than 35%, no more than 30%, no more than 25%, no more than 20%, no more than 15%, no more than 10%, or no more than 5% of the region may contain a droplet. Combinations of any of these ranges are also possible in other embodiments. For example, between 50% and 95%, between 20% and 60%, between 40% and 85%, etc. of the regions may have droplets,

[0050] As mentioned, in certain cases, the droplets may be positioned in a region such that the droplet is substantially centered in the region. In some cases, the center of the droplet may define a center point, and the region may define a center point. The center points may coincide, or be separated by a distance, e.g., a displacement distance. The displacement distance may be relatively small, compared to the region; for example, the displacement distance may be less than 50%, less than 40%, less than 30%, less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, etc. of a maximum dimension of the region (e.g., a diagonal or a diameter, etc.). In some cases, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% of the droplets on a surface may exhibit such displacement distances.

[0051] In some cases, the droplets on the surface may exhibit relatively low splatter. For example, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or substantially all of the regions that contain a droplet contain only a single droplet, or at least no more than 20 droplets (including satellite droplets), or no more than 15, no more than 10, no more than 5, no more than 4, no more than 3, or no more than 2 droplets, in some cases.

[0052] In some aspects, some or all of the droplets may be partially dried, e.g., to create a concentrate on the surface. The concentrate may be present, e.g., as a liquid (which may be relatively viscous in certain embodiments) or a solid, etc. In some cases, the concentrate may be reconstituted, e.g., upon exposure to an aqueous solution, such as a buffer, for example, as discussed herein.

[0053] The surface may form part of a device, such as a digital microfluidic device, according to certain embodiments. For instance, the surface may be the surface of a plate within a digital microfluidic device, which is prepared for later use, e.g., to allow CRISPR or other techniques. In some embodiments, a user may add cells and / or suitable proteins to the device, which already contains suitable reagents for performing such reactions.

[0054] As a non-limiting example, the surface may contain droplets or concentrate containing a nucleic acid, such as DNA or RNA (e.g., sgRNA). A user may add a fluid containing buffer, a protein, such as an enzyme (e.g., a nuclease such as Cas9), and / or a cell, which may be directed to the droplet or concentrate to reconstitute it to allow a reaction to occur, for example, using digital microfluidics. In addition, in some cases, one or more electrical pulses, may be applied, e.g., to allow transport into the cell to occur.

[0055] In some cases, the surface may include a plurality of electrodes, e.g., defining regions for the droplets. Various examples of electrodes are discussed herein. In some cases, such electrodes may be present in a first layer within the device. There may also be a second layer with one or more other electrodes, e.g., such that a voltage or voltage gradient can be applied from an electrode in the first layer to an electrode in the second layer. This may be used to impose such a voltage or voltage gradient across a region of the surface, for example, containing a droplet or a cell. In addition, in certain cases, the surface and the electrode layers may be present within a cartridge, e.g., that can be readily added or removed from a device.

[0056] Some aspects are generally directed to devices and methods for inserting biomolecules (such as CRISPR-associated ribonucleoproteins, genes such as sgRNA or Cas9, or other molecules described herein, etc.) into living cells, e.g., via electroporation, for example, for gene editing purposes. The target droplet may contain cells or other entities, for instance, to which a voltage or current is to be applied. This may be useful, for example, to cause electroporation to occur in cells within the droplet (e.g., to cause material such as DNA to be transported into cells), or for other applications such as those described herein.

[0057] For example, one aspect of the present disclosure is generally directed to systems and methods for applying relatively high voltages to one or more target droplets. The voltages experienced by a target droplet may be, for example, at least 1 V, at least 2 V, at least 3 V, at least 5 V, at least 10 V, at least 20 V, at least 30 V, at least 50 V, at least 100 V, at least 200 V, at least 300 V, at least 500 V, at least 1 kV, etc. In some cases, the voltage may be no more than 1 kV, no more than 500 V, no more than 300 V, no more than 200 V, no more than 100 V, no more than 50 V, no more than 30 V, no more than 20 V, no more than 10 V, no more than 5 V, no more than 3 V, no more than 2 V, no more than 1 V, etc. Combinations of any of these are also possible in certain embodiments, e.g., the voltage experienced by a target droplet may be between 50 V and 200 V, between 500 V and 1 kV, between 10 V and 20 V, etc. The voltages may be created using a using voltage generator or a voltage source. Such voltages may be continuous, and / or applied as electrical pulses, e.g., by using an electrical pulse generator. Those of ordinary skill in the art will be aware of a variety of voltage sources that may be used, some of which are available commercially. In addition, it should be understood that the present disclosure is not limited to only voltage sources, and that in certain embodiments, a current source may be used.

[0058] The voltage may be applied to a target droplet for a variety of applications. For example, in one embodiment, the voltage is applied to cause a reaction involving an entity such as a voltage-sensitive compound (e.g., voltage- sensitive dyes such as a quinone, for example, 2,5- dimethyl- 1 ,4-hydroquinone) within the droplet. As another example, in some embodiments, the voltage may be applied to cause electroporation to occur in cells within a target droplet. As is known by those of ordinary skill in the art, electroporation generally involves applying a voltage to cells to cause material to be transported into or out of cells. This can be used, for example, to introduce nucleic acids into cells, e.g., to transfect those cells with the nucleic acids. Nonlimiting examples of nucleic acids include DNA or RNA. A variety of genes (for example, contained in plasmids) may accordingly be transfected into cells for various applications such as gene editing, for instance, by using CRISPR genes for sgRNA and Cas9, etc. Other examples of genes that can be transfected include, but are not limited to, linear DNA, circular DNA, nucleic acids or fragments that are naturally occurring or are synthesized, etc. In addition, as another non-limiting example, a functional nucleic acid may be transported into a cell; examples include, but are not limited to, Aptamers, nucleic acid enzymes, aptazymes, ribozymes, deoxyribozymes, or the like. Other materials that may be transported into or out of cells include, but are not limited to, proteins, peptides, viruses, hormones, drugs, dyes such as fluorescent dyes, small molecules (e.g., with a molecular weight of less than 1 kDa or less than 2 kDa), charged compounds, chemotherapeutic agents, or the like.

[0059] The target droplets may contain various entities to which a voltage or current is to be applied. For example, according to certain applications, the voltage or current may be applied to cells. Thus, in some embodiments, the target droplet may contain any number of cells and / or cell types. For instance, the cells may be targeted for electroporation, or for other applications. The cells may be adherent, in some embodiments. The cell may be, for example, an isolated cell, a cell aggregate, or a cell found in a cell culture, in a tissue construct containing cells, or the like. Non-limiting examples of cells include immortal cells, primary cells, stem cells, germline cells, zygotes, embryos, or the like. Additional non-limiting examples of cells include, but are not limited to, a microbial cell, e.g., from bacterium or other single-cell organism, a plant cell, or an animal cell. If the cell is an animal cell, the cell may be, for example, an invertebrate cell (e.g., a cell from a fruit fly), a fish cell (e.g., a zebrafish cell), an amphibian cell (e.g., a frog cell), a reptile cell, a bird cell, or a mammalian cell, human or non-human mammal, such as a monkey, cow, sheep, goat, horse, rabbit, pig, mouse, rat, dog, or cat. If the cell is from a multicellular organism, the cell may be from any part of the organism. For instance, if the cell is from an animal, the cell may be a cardiac cell, a fibroblast, a keratinocyte, a heptaocyte, a chondracyte, a neural cell, an osteocyte, an osteoblast, a muscle cell, a blood cell, an endothelial cell, an immune cell (e.g., a T-cell, a B-cell, a macrophage, a neutrophil, a basophil, a mast cell, an eosinophil), etc. In one set of embodiments, the cells include mammalian cells. In another set of embodiments, the cells include non-mammalian cells.

[0060] The target droplet may contain a single cell type, or more than one cell type, e.g., from the same or different species, from the same or different organisms, etc. In some cases, the target droplet may contain only a single cell. However, in other cases, more than one cell may be present (e.g., which may be the same or different). For instance, a droplet may have at least 5, at least 10, at least 30, at least 50, at least 100, at least 300, at least 500, at least 1,000, at least 3,000, at least 5,000, at least 10,000, at least 30,000, at least 50,000, at least 100,000, at least 300,000, at least 500,000, or at least 1,000,000 cells. In some cases, a droplet may contain no more than 1,000,000, no more than 500,000, no more than 300,000, no more than 100,000, no more than 50,000, no more than 30,000, no more than 10,000, no more than 5,000, no more than 3,000, no more than 1,000, no more than 500, no more than 300, no more than 100, no more than 50, no more than 30, no more than 10, or no more than 5 cells. In addition, in certain embodiments, a droplet may contain a number of cells between any of these ranges. For instance, a target droplet may contain between 50,000 and 100,000 cells, between 5 and 50 cells, between 300 and 1,000 cells, etc.

[0061] The droplets (including target and other droplets) may be of any shape or size, and may each independently be the same or different sizes. For instance, in various embodiments, a droplet may have an average or characteristic diameter of less than 1 cm, less than 5 mm, less than 3 mm, less than 2 mm, less than 1 mm, less than 500 micrometers, less than 300 micrometers, less than 200 micrometers, less than 100 micrometers, less than 75 micrometers, less than 50 micrometers, less than 40 micrometers less than 30 micrometers, less than 25 micrometers, less than 20 micrometers, less than 15 micrometers, less than 10 micrometers, less than 5 micrometers, less than 3 micrometers, less than 2 micrometers, less than 1 micrometer, etc. The average or characteristic diameter of a droplet may also be at least 1 micrometer, at least 2 micrometers, at least 3 micrometers, at least 5 micrometers, at least 10 micrometers, at least 15 micrometers, at least 20 micrometers, at least 25 micrometers, at least 30 micrometers, at least 40 micrometers, at least 50 micrometers, at least 75 micrometers, at least 100 micrometers, at least 200 micrometers, at least 300 micrometers, at least 500 micrometers, at least 1 mm, at least 2 mm, at least 3 mm, at least 5 mm, at least 10 cm, etc. Combinations of any of the above are also possible. For example, the droplet may have an average or characteristic diameter of between 40 micrometers and 100 micrometers, between 50 micrometers and 1 mm, between 1 mm and 1 cm, etc. As mentioned, if a plurality of droplets are present (for example, a target droplet and one or more other droplets that are in contact or in ionic communication with the target droplet), then the droplets may independently be the same or different sizes, including any of the sizes discussed herein.

[0062] In some embodiments, a droplet may contain a medium suitable for the cells, e.g., the droplet may contain cell culture media, serum, or other species suitable for maintaining cells. Non-limiting examples of cell culture media include DMEM, MEM, RPMI, PBS, or the like. In one example, the medium may contain an aqueous fluid, e.g., containing one or more salts. As another non-limiting example, a droplet may contain a medium suitable for electroporation, e.g., electroporation buffer. Electroporation buffers may mimic the composition of the cytoplasm composition, and a variety of such electroporation buffers can be obtained commercially. In addition, other examples of fluids that may be contained within droplets include, but are not limited to, water, saline, ethanol, or the like. In some embodiments, one or more salts may be dissolved in water to form an aqueous solution that may be contained within a droplet.

[0063] The electrodes within the device may be made out of the same or different conductive materials, and may have the same or different shapes. Non-limiting examples of electrode materials include metals such as gold, silver, copper, platinum, steel, titanium, brass, palladium, or the like. Other materials may also be used in electrodes, for example, oxides (for example, titanium oxide, zirconium oxide, niobium oxide, tantalum oxide, ruthenium oxide, iridium oxide, platinum oxide, etc.), graphite, carbon, conductive polymers, or the like. It should be understood that since ion containment can be used in accordance with some embodiments to reduce or prevent contamination of a target droplet with ions or other chemical by-products created at the electrodes, a wide variety of materials can be used, including those that are known to create such by-products.

[0064] The electrodes may be positioned with any suitable spacing within the device. For example, the electrodes may be separated by at least 10 micrometers, at least 20 micrometers, at least 30 micrometers, at least 40 micrometers, at least 50 micrometers, at least 60 micrometers, at least 70 micrometers, at least 80 micrometers, at least 90 micrometers, at least 100 micrometers, at least 200 micrometers, at least 300 micrometers, at least 500 micrometers, at least 1 mm, at least 2 mm, at least 3 mm, at least 5 mm, at least 1 cm, at least 2 cm, at least 3 cm, at least 5 cm, etc. In some cases, the electrodes may be separated by no more than 5 cm, no more than 3 cm, no more than 2 cm, no more than 1 cm, no more than 5 mm, no more than 3 mm, no more than 2 mm, no more than 1 mm, no more than 500 micrometers, no more than 300 micrometers, no more than 200 micrometers, no more than 100 micrometers, no more than 50 micrometers, no more than 30 micrometers, no more than 20 micrometers, no more than 10 micrometers, etc. Combinations of any of these are also possible in some embodiments. For instance, the electrodes may be separated by a distance of between 20 micrometers and 30 micrometers, between 50 micrometers and 100 micrometers, between 1 mm and 1 cm, or the like. However, it should be understood that the physical distance between the electrodes need not necessarily define the length of the ionic communication pathway created between the electrodes when voltage is applied. For instance, a chain of droplets defining the ionic communication pathway between the electrodes may be non-linear, thereby defining a longer distance for current to flow.

[0065] In some cases, the voltage applied to the electrodes may be at least 10 V, at least 20 V, at least 30 V, at least 50 V, at least 100 V, at least 200 V, at least 300 V, at least 500 V, at least 1 kV, etc. In some embodiments, the voltage applied to the electrodes may be no more than 1 kV, no more than 500 V, no more than 300 V, no more than 200 V, no more than 100 V, no more than 50 V, no more than 30 V, no more than 20 V, no more than 10 V, etc. Combinations of any of these are also possible in certain embodiments, e.g., the voltage that is applied may be between 50 V and 200 V, between 500 V and 1 kV, between 10 V and 20 V, etc.

[0066] In addition, in certain cases, the voltage applied to the electrodes may be used to produce a voltage gradient within a target droplet of at least 1 V / cm, at least 2 V / cm, at least 3 V / cm, at least 5 V / cm, at least 10 V / cm, at least 20 V / cm, at least 30 V / cm, at least 50 V / cm, at least 100 V / cm, at least 200 V / cm, at least 300 V / cm, at least 500 V / cm, at least 1 kV / cm, at least 2 kV / cm, at least 3 kV / cm, at least 5 kV / cm, etc. In some cases, the target droplet may experience a voltage gradient of no more than 5 kV / cm, no more than 3 kV / cm, no more than 2 kV / cm, no more than 1 kV / cm, no more than 500 V / cm, no more than 300 V / cm, no more than 200 V / cm, no more than 100 V / cm, no more than 50 V / cm, no more than 30 V / cm, no more than 20 V / cm, no more than 10 V / cm, no more than 5 V / cm, no more than 3 V / cm, no more than 2 V / cm, no more than 1 V / cm, etc. In addition, in some cases, combinations of any of these may be used, e.g., the voltage applied to the electrodes that in used to produce a voltage gradient within a target droplet may be between 10 V / cm and 50 V / cm, between 50 V / cm and 100 V / cm, between 500 V / cm and 2 kV / cm, etc. Additionally, in some embodiments, the voltage or voltage gradient crated in a target droplet may be relatively uniform or homogenous. For instance, by using a fluid with a relatively high conductivity, a relatively uniform or homogenous electric field may be created within the target droplet.

[0067] In some cases, electrodes may be used to directly apply voltage or current to a target droplet, although in some cases, various droplets in contact with electrodes may be used to apply voltage or current to a target droplet, e.g., as discussed in Int. Pat. Apl. Pub. No. WO 2022 / 256604, incorporated herein by reference in its entirety.

[0068] A variety of devices and techniques may be used to produce droplet configurations such as those described herein. For example, in some cases, the droplets may be manually positioned between electrodes. In addition, in certain embodiments, a digital microfluidic device may be used to position droplets to connect between electrodes, e.g., able to apply voltages or currents such as are described herein to a target droplet. In a digital microfluidic device, droplets may be present on a substrate, e.g., a planar substrate, and moved around the substrate using techniques such as electrowetting, dielectrophoresis, immiscible-fluid flow, or the like.

[0069] A variety of electrodes may be present to manipulate or facilitate the movement of such droplets on the substrate, often defining a plurality of “pixels” or locations on the substrate where a droplet may be present. Those of ordinary skill in the art will be familiar with a variety of digital microfluidic devices and techniques for manipulating droplets therein, including moving, sorting, merging, mixing, splitting, etc. such droplets. In addition, it should be understood that the “droplets” within a digital microfluidic device are not necessarily spherical or circular, but may adopt a variety of other forms and shapes, for example, as defined by the pixels within the device.

[0070] Accordingly, in some embodiments, for instance, droplets, including a target droplet, may be moved into position between electrodes in the digital microfluidic device, for example, creating an ionic communication pathway between the electrodes, then a voltage or current applied using the electrodes to the target droplet. The digital microlluidic device may have 2, 3, 4, or more such electrodes able to supply voltages or currents to the target droplet, e.g., to cause electroporation within cells within a target droplet, and such electrodes may be the same as those used to manipulate droplets on the substrate, or different. In addition, in some cases, a digital microfluidic device may include parallel plates or substrates in which the droplets of fluid arc contained and manipulated between, and in some cases, the electrodes used for applying relatively high voltages (e.g., to cause electroporation, or other applications such as those described herein) may each be in the same plate or substrate, or in different plates or substrates. Non- limiting examples of such configurations can be seen in Figs. 4-6.

[0071] In some cases, droplets within a digital microfluidic device may be controlled to control a target droplet. For instance, a target droplet, and / or other droplets may be controlled within the digital microfluidic device to cause a certain voltage to be applied to the target droplet. Parameters such as the number of droplets, the compositions of the droplets, the positions of the droplets, the sizes of the droplets, the volume of the droplets, the electrical resistances of the droplets, etc. may be readily controlled using techniques for manipulating droplets within a digital microfluidic device, such as those previously discussed.

[0072] For instance, in certain embodiments, a target droplet may be moved within the digital microfluidic device to a first location, while other droplets may be moved to other locations (e.g., to be disposed of as waste). As a non-limiting example, if the target droplet contains cells, after applying a voltage (e.g., to cause electroporation within the cells), the target droplet may be moved within the digital microfluidic device to a location where the location cells are allowed to recover. In some cases, for example, the droplets containing the cells may be combined with droplets containing recovery buffer or cell media, etc.

[0073] Int. Pat. Apl. Pub. No. WO 2022 / 256604, published on December 8, 2022, is incorporated herein by reference in its entirety. In addition, U.S. Pat. Apl. Ser. No. 63 / 549,080, filed February 2, 2024, entitled “Reagent Spotting and Assembly Methods and Systems,” by Hirukawa, et al. is also incorporated herein by reference in its entirety.

[0074] The following examples are intended to illustrate certain embodiments of the present disclosure, but do not exemplify the full scope of the disclosure.

[0075] EXAMPLE 1

[0076] In this example, pan CD3+ T cells were isolated from the leukopack of a human donor using magnetic microbead negative selection. Cells were cultured in standard T cell media supplemented with 11-2 (200 U / ml) for 72 hours. T cells were resuspended in in specialized buffer containing the cells and Cas9 nuclease and loaded onto a digital microfluidics transfection device. The device included reagents deposited using an aqueous fluid containing an anionic polymer (vchiclc)and synthetic guide RNA (sgRNA), and were deposited using acoustic deposition in an electric field, as discussed herein. The Cas9 / cell mixes were incubated with synthetic guide RNA deposited directly onto the device for less than 10 minutes to allow for RNP (ribonucleoprotein) assembly and then passed to the transfection site on the device to allow for delivery of the RNP in the cells. The cells were then offloaded from the device into a sterile 96 well tissue culture plate and cultured for 48 hours in a tissue culture incubator. Cells were then recovered and assessed for viability and the TCRa / b expression at the protein level via flow cytometry.

[0077] Fig. 3 shows the evaluation of TCRa / b knock out and viability in primary human pan CD3+ T cells, transfected with RNP formed from sgRNA cargo deposited on chip. Editing efficiency of the TRAC sgRNA / Cas9 formed RNP was assessed at the protein level by staining for TCRa / b levels and viability (Live-Dye Stain) were assessed using flow cytometry. Cells were assessed 48 hours post cargo delivery. On-chip denotes cargo deposited on chip, off chip denotes cargo assembled off chip. These data demonstrate that on chip deposition and cargo assembly did not compromise knock out editing efficiency.

[0078] Figs. 4A-4D illustrate the validation of reagent deposition process on SBS-compatible DMF cartridge. Successful deposition of reagents on a DMF substrate was assessed by measuring the diameter and the distance from the center point of the electrode of a dehydrated droplet of 0.05% Trypan Blue which had been spotted by a non contact acoustic dispenser (Echo 655 Acoustic Dispenser, Beckman Coulter). A digital microscope was used to capture the images and imageJ to measure the diameter of the drop and the distance from the center point of the electrode.

[0079] Fig. 4A illustrates that the diameter of the spot (droplet) was consistent with different volumes. Fig. 4B illustrates that the site-to-site variation in spot location was less than 0.2 mm. Fig. 4C illustrates that the spot location was consistently in the center of the 2 mm x 2 mm electrode. Fig. 4D illustrates that 12.5 nL and 25 nL spots did not interfere with droplet movement.

[0080] Fig. 5 illustrates a DMF substrate with 0.05% Trypan Blue spots (12 of each volume 12.5 nL, 25 nL, 50 nL, 100 nL), illustrating consistent spotting within desired regions of the surface of the DMF substrate. EXAMPLE 2

[0081] One example embodiment of the present disclosure is now presented, for use in certain CRISPR applications.

[0082] Step 1: Nucleic acids (such as sgRNA) may be resuspended in a biocompatible negatively charged polymer (e.g., poly-gamma-glutamic acid) and deposited on the hydrophobic surface of a microfluidic device using a low volume liquid dispenser (e.g., a dispenser in the nanoliter volume range). Reagents may be spotted via non-contact dispensing directly on the hydrophobic surface of the DMF chip prior to cartridge assembly. Payload sgRNA libraries can also be directly deposited in an arrayed format onto a surface using standard low volume liquid dispensers such as the ECHO 655.

[0083] Step 2: The microfluidic chip is mated to another piece to create an enclose cartridge (assembly of the cartridge).

[0084] Step 3: A buffer containing cells and Cas9 nuclease is loading into one of the ports of the DMF cartridge. This buffer may be cell compatible without inhibiting cargo formation. Injection ports may be used to insert buffers, cells, and / or any reagents required for the experiment.

[0085] Step 4; A droplet of cells / Cas9 / buffer may be formed, and passed over the region of the chip that has the deposited nucleic acid. The droplet may be pulsed to remove the nucleic acid and incubated for a certain amount of time to allow the RNP (ribonucleoprotein) to form. Reagents may be moved to the electrode where the previously deposited reagent was located, and pulsed to resuspend the reagent of interest, e.g., followed by incubation for a programmable duration. RNP formation may occur on device, which may reduce upstream preparation time, or reduce dead volume loss experienced with conventional liquid handlers.

[0086] Step 5: The droplet containing the cells or newly formed RNP may be moved to a transfection site for delivery of the RNP cargo into the cell. Droplet structures may be exposed to an electric field in a specific droplet configuration with specific buffers to deliver payloads, for example, into low numbers of cells efficiently without disturbing viability.

[0087] Step 6: The cells may be offloaded from the chip into a tissue culture plate, e.g., to allow for the edit to take place.

[0088] It should be noted that no functionalization of the hydrophobic surface is required for nucleic acid deposition (reagent deposition), which may keep costs low, or simplify production. In addition, the number of unique cargo is not limited by the number of unique onloading ports in the device. In addition, having on-chip incubation and RNP assembly may reduce complexity, labor or time costs, dead volume, or the like.

[0089] While several embodiments of the present disclosure have been described and illustrated herein, those of ordinary skill in the ail will readily envision a variety of other means and / or structures for performing the functions and / or obtaining the results and / or one or more of the advantages described herein, and each of such variations and / or modifications is deemed to be within the scope of the present disclosure. More generally, those skilled in the ail will readily appreciate that all parameters, dimensions, materials, and configurations described herein are meant to be exemplary and that the actual parameters, dimensions, materials, and / or configurations will depend upon the specific application or applications for which the teachings of the present disclosure is / are used. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto, the disclosure may be practiced otherwise than as specifically described and claimed. The present disclosure is directed to each individual feature, system, article, material, kit, and / or method described herein. In addition, any combination of two or more such features, systems, articles, materials, kits, and / or methods, if such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent, is included within the scope of the present disclosure.

[0090] In cases where the present specification and a document incorporated by reference include conflicting and / or inconsistent disclosure, the present specification shall control. If two or more documents incorporated by reference include conflicting and / or inconsistent disclosure with respect to each other, then the document having the later effective date shall control.

[0091] All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0092] The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.” The phrase “and / or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.c., elements that arc conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and / or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and / or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0093] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, when separating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the claims, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e. “one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.”

[0094] As used herein in the specification and in the claims, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and / or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0095] When the word “about” is used herein in reference to a number, it should be understood that still another embodiment of the disclosure includes that number not modified by the presence of the word “about.”

[0096] It should also be understood that, unless clearly indicated to the contrary, in any methods claimed herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited. In the claims, as well as in the specification above, all transitional phrases such as

[0097] “comprising,” “including,” “carrying,” “having,” “containing,” “involving,” “holding,” “composed of,” and the like are to be understood to be open-ended, i.e., to mean including but not limited to. Only the transitional phrases “consisting of’ and “consisting essentially of’ shall be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

[0098] What is claimed is:

Claims

CLAIMS1. A device, comprising: a substantially planar surface defining at least 50 regularly spaced regions on the surface, wherein at least 50% of the regions have a droplet positioned thereon, at least 95% of the droplets on the surface being positioned such that, for a region defining a center point, an area, and a maximum dimension, the droplet covers a surface area no more than 25% of the area of the region, and the droplet defines a center point that is positioned a displacement distance away from the center point of the region, wherein the displacement distance is less than 25% of the maximum dimension of the region, and wherein at least 95% of the droplets on the surface comprise an aqueous fluid and an anionic polymer.

2. The device of claim 1, wherein the regions defined on the surface match well locations of an ANSI microtiter plate of 96, 384, or 1536 wells.

3. The device of any one of claims 1 or 2, wherein the planar surface is contained within a cartridge.

4. The device of any one of claims 1-3, wherein the planar surface is positioned between a first electrode and a second electrode.

5. The device of claim 4, wherein the planar surface is positioned between a plurality of first electrodes and a second electrode.

6. The device of claim 5, wherein the plurality of first electrodes are positioned to match well locations of an ANSI microtiter plate of 96, 384, or 1536 wells.

7. The device of any one of claims 1-6, wherein the device further comprising an acoustic fluid dispenser positioned to transport fluid from a fluid reservoir to the surface.

8. The device of any one of claims 1-7, wherein the substantially planar surface is the surface of a digital microfluidic device.

9. The device of any one of claims 1-8, wherein at least some of the regularly spaced regions are substantially circular.

10. The device of any one of claims 1-9, wherein at least some of the regularly spaced regions are substantially square.

11. A device, comprising: a substantially planar surface defining at least 50 regularly spaced regions on the surface, wherein at least 50% of the regions have a droplet positioned thereon, at least 95% of the droplets on the surface being positioned such that, for a region defining a center point, an area, and a maximum dimension, the droplet has a volume of no more than 1 microliter, and the droplet defines a center point that is positioned a displacement distance away from the center point of the region, wherein the displacement distance is less than 25% of the maximum dimension of the region, and wherein at least 95% of the droplets on the surface comprise an aqueous fluid and an anionic polymer.

12. A device, comprising: a cartridge comprising a first electrode, a second electrode; and a substantially planar surface positioned between the first electrode and the second electrode, wherein the substantiality planar surface has a plurality of concentrates positioned thereon, at least 95% of the concentrates on the surface comprising an anionic polymer and a nucleic acid.

13. The device of claim 12, wherein the nucleic acid is RNA.

14. The device of claim 13, wherein the nucleic acid is sgRNA.

15. The device of claim 13, wherein the nucleic acid is DNA.

16. A device, comprising: a substantially planar surface defining at least 50 regularly spaced regions on the surface, wherein at least 5% of the regions have a droplet positioned thereon, the droplet covers a surface area no more than 25% of the area of the region, and at least 95% of the regions having a droplet positioned thereon having only a single droplet, and wherein at least 95% of the droplets on the surface comprise an aqueous fluid and an anionic polymer.

17. The device of claim 16, wherein the regions defined on the surface match well locations of an ANSI microtiter plate of 96, 384, or 1536 wells.

18. A method, compri sin : acoustically dispensing droplets onto a substantially planar hydrophobic surface in an electric field of at least 1 kV / cm, wherein at least 95% of the droplets on the surface comprise an aqueous fluid and an anionic polymer.

19. The method of claim 18, further comprising applying the electric field to the surface using a first electrode and a second electrode, wherein the surface is positioned between the first electrode and the second electrode.

20. The method of any one of claims 18 or 19, wherein the electric filed is between 1 kV and 10 kV / cm.

21. The method of any one of claims 18-20, wherein the surface has a water contact angle of at least 90°.

22. The method of any one of claims 18-21 , wherein the surface is in contact with a conductive material layer.

23. A method, comprising: providing a substantially planar surface defining at least 50 regularly spaced regions on the surface, wherein at least 20% of the regions have a concentrate positioned thereon, at least 95% of the concentrates on the surface being positioned such that, for a region defining a center point, an area, and a maximum dimension, the concentrate covers a surface area no more than 25% of the area of the region, and the concentrate defines a center point that is positioned a displacement distance away from the center point of the region, wherein the displacement distance is less than 25% of the maximum dimension of the region; and applying an electric field to move a droplet comprising a buffer and a cell to contact one of the concentrates positioned within the regions.

24. The method of claim 23, wherein the droplet further comprises a protein.

25. The method of claim 24, wherein the protein comprises an enzyme.

26. The method of claim 25, wherein the enzyme comprises a nuclease.

27. The method of claim 26, wherein the nuclease comprises Cas9 nuclease.

28. The method of any one of claims 23-27, further comprising applying an electric pulse to the second droplet after contacting the droplet with the concentrate.

29. The method of claim 28, wherein the applied electric pulse is at least sufficient to electroporate the cell.

30. The method of any one of claims 28 or 29, wherein the applied electric pulse is at least 10 V31. A device, comprising: a substantially planar surface defining at least 50 regularly spaced regions on the surface, wherein at least 20% of the regions have a concentrate positioned thereon, at least 95% of the concentrates on the surface being positioned such that, for a region defining a center point, an area, and a maximum dimension, the concentrate covers a surface area no more than 25% of the area of the region, and the concentrate defines a center point that is positioned a displacement distance away from the center point of the region, wherein the displacement distance is less than 25% of the maximum dimension of the region, and wherein at least 95% of the concentrate on the surface comprises an anionic polymer.

32. A device, comprising: a cartridge comprising a first electrode, a second electrode; and a substantially planar surface positioned between the first electrode and the second electrode, wherein the substantiality planar surface has a plurality of droplets positioned thereon, at least 95% of the droplets on the surface comprising an anionic polymer and sgRNA.

33. A device, comprising: a substantially planar surface defining at least 50 regularly spaced regions on the surface, wherein at least 5% of the regions have a droplet positioned thereon, the droplet having a volume of no more than 1 microliter, and at least 95% of the regions having a droplet positioned thereon having only a single droplet, and wherein at least 95% of the droplets on the surface comprise an aqueous fluid and an anionic polymer.

34. A device, comprising: a substantially planar surface defining at least 50 regularly spaced regions on thesurface, wherein at least 5% of the regions have a droplet positioned thereon, at least 95% of the droplets on the surface being positioned such that, for a region defining a center point, an area, and a maximum dimension, the droplet covers a surface area no more than 75% of the area of the region, and the droplet defines a center point that is positioned a displacement distance away from the center point of the region, wherein the displacement distance is less than 25% of the maximum dimension of the region, and wherein at least 95% of the droplets on the surface comprise an aqueous fluid and an anionic polymer.

35. The device of claim 34, wherein the regions defined on the surface match well locations of an ANSI microtiter plate of 96, 384, or 1536 wells.

36. The device of any one of claims 34 or 35, wherein the planar surface is contained within a cartridge.

37. The device of any one of claims 34-36, wherein the planar surface is positioned between a first electrode and a second electrode.

38. The device of claim 37, wherein the planar surface is positioned between a plurality of first electrodes and a second electrode.

39. The device of claim 38, wherein the plurality of first electrodes are positioned to match well locations of an ANSI microtiter plate of 96, 384, or 1536 wells.

40. The device of any one of claims 34-39, wherein the device further comprising a nanovolume fluid dispenser positioned to transport fluid from a fluid reservoir to the surface.

41. The device of claim 40 wherein the nano volume fluid dispenser is configured to dispense 10 nl to 1000 nl of fluid.

42. The device of any one of claims 34- 1, wherein the device further comprising an acoustic fluid dispenser positioned to transport fluid from a fluid reservoir to the surface.

43. The device of any one of claims 34-42, wherein the substantially planar surface is the surface of a digital microfluidic device.

44. The device of any one of claims 34-43, wherein at least some of the regularly spaced regions are substantially circular.

45. The device of any one of claims 34-44, wherein at least some of the regularly spaced regions are substantially square.

46. A device, comprising: a substantially planar surface defining at least 50 regularly spaced regions on the surface, wherein at least 5% of the regions have a concentrate positioned thereon, at least 95% of the concentrates on the surface being positioned such that, for a region defining a center point, an area, and a maximum dimension, the concentrate covers a surface area no more than 75% of the area of the region, and the concentrate defines a center point that is positioned a displacement distance away from the center point of the region, wherein the displacement distance is less than 25% of the maximum dimension of the region, and wherein at least 95% of the concentrate on the surface comprises an anionic polymer.

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