Alternating current droplet generation with hydrophobic nanoporous membrane

Abstract

Described herein is an alternating current electrospray technology comprising a hydrophobic nanoporous membrane that can generate monodisperse micron sized droplets at remarkably high throughput. The droplets can be used for ddPCR applications or for encapsulating extracellular vesicles or cells.

Description

[0001] ALTERNATING CURRENT DROPLET GENERATION WITH HYDROPHOBIC NANOPOROUS MEMBRANE

[0002] CROSS-REFERENCE TO RELATED APPLICATION(S)

[0003] This application claims priority to U.S. Provisional Patent Application No. 63 / 675,615, filed on July 25, 2024, which is incorporated by reference herein in its entirety.

[0004] BACKGROUND

[0005] Uniform droplets sizes are required for many biotechnical and diagnostic applications such as for droplet digital polymerase chain reaction (ddPCR) assays and coating cells.

[0006] What is needed is a rapid, high-throughput, and inexpensive approach to generate large numbers of monodisperse droplets suitable for ddPCR assays or encapsulation of extracellular vesicles or cells.

[0007] SUMMARY

[0008] One embodiment described herein is an apparatus for generating monodispersed liquid droplets, the apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying an alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium. In one aspect, the electrospray emitter comprises glass, the alternating current electrode is internal to the electrospray emitter, and the droplet chamber comprises a counter electrode. In another aspect, the droplet suspending medium comprises a carrier oil comprising a mineral oil, a fluorinated oil, a silicone oil, a surfactant, or combinations thereof. In another aspect, the droplet suspending medium comprises a mixture of polyethylene oxide and a nonionic surfactant. In another aspect, the alternating current electrospray device is configured to apply a frequency of alternating current ranging from about 1 kHz to about 1000 kHz. In another aspect, the alternating current electrospray device is configured to apply a potential of alternating current ranging from about 0.01 kV to about 100 kV. In another aspect, the hydrophobic nanoporous membrane comprises a porosity of about 50% to about 90%. In another aspect, the hydrophobic nanoporous membrane comprises a thickness of about 100 nm to about 50 pm. In another aspect, the hydrophobic nanoporous membrane comprises polyethylene terephthalate (PET), polycarbonate (PC), polystyrene (PS), poly-methyl-meta- acrylate (PMMA), cyclic olefin copolymer (COC), polyimide (PI), polydimethylsiloxane (PDMS), or combinations thereof. In another aspect, the hydrophobic nanoporous membrane further comprises an electrodeposited metal alloy layer on a membrane surface opposite the conduit and immersed in the droplet suspending medium. In another aspect, the metal alloy layer comprises nickel-iron, samarium-cobalt, aluminum-nickel-cobalt, nickel-iron-chromium, iron-chromium- cobalt, neodymium-iron-boron, or combinations thereof. In another aspect, the metal alloy layer has a thickness of about 50 nm to about 250 nm. In another aspect, the hydrophobic nanoporous membrane further comprises one or more gold layers in contact with the metal alloy layer. In another aspect, each gold layer has a thickness of about 5 nm to about 150 nm. In another aspect, the hydrophobic nanoporous membrane comprises asymmetrically shaped nanopores, each asymmetrically shaped nanopore comprising a first opening having a first diameter on a first membrane surface and being fluidly connected to the conduit of the electrospray emitter and a second opening having a second diameter on a second membrane surface and being configured to emit monodispersed liquid droplets into the droplet suspending medium, wherein the first diameter is greater than the second diameter. In another aspect, the hydrophobic nanoporous membrane comprises symmetrically shaped nanopores, each symmetrically shaped nanopore comprising a first opening having a first diameter on a first membrane surface and being fluidly connected to the conduit of the electrospray emitter and a second opening having a second diameter on a second membrane surface and being configured to emit monodispersed liquid droplets into the droplet suspending medium, wherein the first diameter is substantially equal to the second diameter. In another aspect, the second diameter of each asymmetrically shaped nanopore is about 50 nm to about 50 pm. In another aspect, the second diameter of each asymmetrically shaped nanopore is less than about 280 nm to 10 pm. In another aspect, the second diameter of each symmetrically shaped nanopore is about 50 nm to about 50 pm. In another aspect, the second diameter of each symmetrically shaped nanopore is less than about 280 nm to 10 pm. In another aspect, the hydrophobic nanoporous membrane comprises a combination of asymmetrically shaped nanopores and symmetrically shaped nanopores.

[0009] Another embodiment described herein is a method for generating monodispersed liquid droplets, the method comprising: applying an alternating current electric field to an apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying the alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium; introducing a pressurized fluid into the electrospray emitter; generating an electrospray by the electrospray emitter from the pressurized fluid in the droplet suspending medium; and collecting the monodispersed liquid droplets in the droplet suspending medium. In one aspect, the monodispersed liquid droplets have an average droplet diameter of about 5 pm to about 300 pm. In another aspect, the monodispersed liquid droplets have an average droplet diameter of about 10 pm to about 50 pm. In another aspect, the method generates monodispersed liquid droplets at a rate of about 1.0 x 104droplets per minute to about 1.0 x 106droplets per minute. In another aspect, the method generates at least 1.0 x 106monodispersed liquid droplets in about 5 minutes. In another aspect, the droplet suspending medium comprises a carrier oil comprising a mineral oil, a fluorinated oil, a silicone oil, a surfactant, or combinations thereof. In another aspect, the droplet suspending medium comprises a mixture of polyethylene oxide and a nonionic surfactant. In another aspect, the droplet suspending medium comprises a carrier oil concentration ranging from about 0.1 % v / v to about 5% v / v. In another aspect, the alternating current electrospray device comprises a pressure of about 1 kPa to about 10 kPa.

[0010] Another embodiment described herein is a method for performing a droplet digital polymerase chain reaction (PCR), the method comprising: applying an alternating current electric field to an apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying the alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium; introducing a pressurized fluid into the electrospray emitter, wherein the pressurized fluid comprises one or more template molecules and PCR reagents; generating an electrospray by the electrospray emitter from the pressurized fluid in the droplet suspending medium; amplifying an amplicon of one or more template molecules in a PCR thermal cycler; and detecting the amplicons.

[0011] Another embodiment described herein is a method for encapsulating extracellular vesicles or cells in an encapsulation medium, the method comprising: applying an alternating current electric field to an apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying the alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium; introducing a pressurized fluid into the electrospray emitter, wherein the pressurized fluid comprises one or more extracellular vesicles or cells and an encapsulation medium; generating an electrospray by the electrospray emitter from the pressurized fluid in the droplet suspending medium; and collecting encapsulated extracellular vesicles or cells. In one aspect, the encapsulation medium comprises alginate, collagen, norbornene-modified hyaluronic acid, or a combination thereof. In another aspect, the extracellular vesicles have a size of about 50 nm to about 150 nm.

[0012] DESCRIPTION OF THE DRAWINGS

[0013] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] FIG. 1 A shows a schematic overview of a multi-channel immersed AC electrospray (iACE) platform highlighting mechanism at the Magnetic Nanoporous Membranes (MNM) pores. FIG. 1 B shows a schematic overview of the function of the iACE platform. FIG. 1C shows an exemplary embodiment of an integrated semi-continuous spray platform.

[0015] FIG. 2A shows Magnetic Nanoporous Membranes (MNM) pores before (left) and after electroplating (right). FIG. 2B shows a graph illustrating representative droplet distributions produced using the platform shown in FIG. 1C. FIG. 2C shows a photograph of droplets produced using the platform showing in FIG. 1C.

[0016] FIG. 3 shows a micrograph of a membrane described herein and a cross-section of the asymmetric pore.

[0017] FIG. 4 shows a graphical representation of the pore symmetry and pressure, with depictions of pores at the top.

[0018] FIG. 5 shows a photograph of a miniature high voltage / frequency AC amplifier that can be used to generate AC current for the device described herein.

[0019] FIG. 6A show a schematic for generating encapsulated cells using a norbornene-modified hyaluronic acid (norHA). FIG. 6B shows a photomicrograph of encapsulated cells. FIG. 6C shows a graph illustrating the number of cells in microgel beads. FIG. 6D shows the diameters of cells laden microgels and cancer cells.

[0020] FIG. 7A show alginate, collagen, and norbornene-modified hyaluronic acid (norHA) microgels. FIG. 7B shows cell viability in alginate, collagen, and norbornene-modified hyaluronic acid (norHA) microgels.

[0021] DETAILED DESCRIPTION

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For example, any nomenclatures used in connection with, and techniques of biochemistry, molecular biology, immunology, microbiology, genetics, cell and tissue culture, and protein and nucleic acid chemistry described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0023] As used herein, the terms “amino acid,” “nucleotide,” “polynucleotide,” “vector,” “polypeptide,” and “protein” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0024] As used herein, terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The present disclosure also contemplates other embodiments “comprising,” “consisting essentially of,” and “consisting of’ the embodiments or elements presented herein, whether explicitly set forth or not. As used herein, “comprising,” is an “open- ended” term that does not exclude additional, unrecited elements or method steps. As used herein, “consisting essentially of” limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristics of the claimed invention. As used herein, “consisting of” excludes any element, step, or ingredient not specified in the claim.

[0025] As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.

[0026] As used herein, the term “or” can be conjunctive or disjunctive.

[0027] As used herein, the term “and / or” refers to both the conjunctive and disjunctive.

[0028] As used herein, the term “substantially” means to a great or significant extent, but not completely.

[0029] As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol means “about” or “approximately.”

[0030] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1 , 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points, or as described above in the definition of “about.”

[0031] As used herein, the terms “room temperature,” “RT,” or “ambient temperature” refer to the typical temperature in an indoor laboratory setting. In one aspect, the laboratory setting is climate controlled to maintain the temperature at a substantially uniform temperature or with a specific range of temperatures. In one aspect, “room temperature” refers a temperature of about 15-30 °C, including all integers and endpoints within the specified range. In another aspect, “room temperature” refers a temperature of about 15-30 °C; about 20-30 °C; about 22-30 °C; about 25-30 °C; about 27-30 °C; about 15-22 °C; about 15-25 °C; about 15-27 °C; about 20-22 °C; about 20-25 °C; about 20-27 °C; about 22-25 °C; about 22-27 °C; about 25-27 °C; about 15 °C ± 10%; about 20 °C ± 10%; about 22 °C ± 10%; about 25 °C ± 10%; about 27 °C ± 10%; ~20 °C, ~22 °C, ~25 °C, or ~27 °C, at standard atmospheric pressure.

[0032] As used herein, the terms “active ingredient” or “active pharmaceutical ingredient” refer to a pharmaceutical agent, active ingredient, compound, or substance, compositions, or mixtures thereof, that provide a pharmacological, often beneficial, effect.

[0033] As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.

[0034] As used herein, the term “dose” denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. “Formulation” and “composition” are used interchangeably herein.

[0035] As used herein, the term “prophylaxis” refers to preventing or reducing the progression of a disorder, either to a statistically significant degree or to a degree detectable by a person of ordinary skill in the art.

[0036] As used herein, the terms “effective amount” or “therapeutically effective amount,” refers to a substantially non-toxic, but sufficient amount of an action, agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. An effective amount may be based on factors individual to each subject, including, but not limited to, the subject’s age, size, type or extent of disease, stage of the disease, route of administration, the type or extent of supplemental therapy used, ongoing disease process, and type of treatment desired.

[0037] As used herein, the term “subject” refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), nonhuman primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human.

[0038] As used herein, a subject is “in need of treatment” if such subject would benefit biologically, medically, or in quality of life from such treatment. A subject in need of treatment does not necessarily present symptoms, particular in the case of preventative or prophylaxis treatments. As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given biological process, condition, symptom, disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.

[0039] As used herein, “treatment” or “treating” refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease. A treatment may be either performed in an acute or chronic way. The term “treatment” also refers to reducing the severity of a disease or symptoms associated with such disease prior to affliction with the disease. “Repressing” or “ameliorating” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject after clinical appearance of such disease, disorder, or its symptoms. “Prophylaxis of” or “preventing” a disease, disorder, or the symptoms thereof involves administering a cell, composition, or compound described herein to a subject prior to onset of the disease, disorder, or the symptoms thereof. “Suppressing” a disease or disorder involves administering a cell, composition, or compound described herein to a subject after induction of the disease or disorder thereof but before its clinical appearance or symptoms thereof have manifest.

[0040] As used herein, the term “monodispersed” refers to two or more liquid droplets having substantially the same size, substantially the same diameter, substantially the same radius, or substantially a uniform size in dispersed phase. Monodispersed liquid droplets can vary in size by, for example, ± 0.1 pm, ± 0.5 pm, ± 1 pm, ± 2 pm, ± 5 pm, ± 10 pm, ± 20 pm, ± 50 pm, ± 60 pm, ± 70 pm, ± 80 pm, ± 90 pm, ± 100 pm, ± 200 pm, ± 300 pm, ± 400 pm, ± 500 pm, ± 600 pm, ± 700 pm, ± 800 pm, ± 900 pm, or ± 1000 pm, or any integer within the range of 0.1-1000 pm. When a range of monodispersed liquid droplets is recited, the range refers to the size of all droplets, and not the different sizes of droplets. For example, “monodispersed liquid droplets having a diameter ranging from about 0.1 pm to about 1000 pm” means, for a particular diameter of liquid droplet within the range cited, all, a preponderance, or an average of the dispersed droplets have the same diameter, or a substantially similar diameter, for each of the droplet diameters in the range recited above.

[0041] As used herein, the terms “exosome,” “extracellular vesicle,” and “EV” are used interchangeably and refer to cell-derived vesicles having a diameter of between about 30-150 nm, such as between about 40-120 nm, or 50-100 nm, for example, a diameter of about 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 mm, 110 nm, 120 nm, or 130 nm. Exosomes may be isolated from any suitable biological sample from a mammal, including but not limited to, whole blood, serum, plasma, urine, saliva, breast milk, cerebrospinal fluid, amniotic fluid, ascitic fluid, bone marrow and cultured mammalian cells (e.g., immature dendritic cells (wild-type or immortalized), induced and non-induced pluripotent stem cells, fibroblasts, platelets, immune cells, reticulocytes, tumor cells, mesenchymal stem cells, satellite cells, hematopoietic stem cells, pancreatic stem cells, white and beige pre-adipocytes and the like). As one of skill in the art will appreciate, cultured cell samples will be in the cell-appropriate culture media (using exosome-free serum). Exosomes include specific surface markers not present in other vesicles, including surface markers such as tetraspanins, e.g., CD9, CD37, CD44, CD53, CD63, CD81 , CD82 and CD151 ; targeting or adhesion markers such as integrins, ICAM-1 , EpCAM and CD31 ; membrane fusion markers such as annexins, TSG101 , ALIX; and other exosome transmembrane proteins such as Rab5b, HLA-G, HSP70, LAMP2 (lysosome-associated membrane protein) and LIMP (lysosomal integral membrane protein). Exosomes may also be obtained from a non-mammal or from cultured non-mammalian cells. As the molecular machinery involved in exosome biogenesis is believed to be evolutionarily conserved, exosomes from non-mammalian sources include surface markers which are isoforms of mammalian surface markers, such as isoforms of CD9 and CD63, which distinguish them from other cellular vesicles. The term “non-mammal” is meant to encompass, for example, exosomes from microorganisms such as bacteria, flies, worms, plants, fruit / vegetables (e.g., corn, pomegranate), and yeast.

[0042] Methods for loading extracelluar vesicles are described in U.S. Pat. App. No. 19 / 146,809, filed on July 9, 2025, which is incorporated by reference herein for such teachings.

[0043] As used herein, the term “PCR reagent” or “PCR reaction mixture” is a term given ordinary meaning to a person skilled in the art and can include one or more PCR reagents. For example, a PCR reagent or PCR reaction mixture can comprise a polymerase enzyme (typically Taq Polymerase), template DNA, primers, deoxynucleotide (dNTP), MgCh, cofactors, and physiological buffers. For reverse-transcription PCR (rt-PCR), the PCR reagents or PCR reaction mixture can also comprise a reverse-transcription polymerase.

[0044] As used herein, the term “surfactant” refers to a surface-active agent capable of reducing the surface tension of a liquid in which it is dissolved, and / or the interfacial tension with another phase. A surfactant may incorporate both a hydrophilic portion and a hydrophobic portion, which may collectively confer a dual hydrophilic-hydrophobic character on the surfactant. In some embodiments, fluorosurfactants, or fluorinated surfactants, are used. Fluorosurfactants are organofluorine chemical compounds that have multiple fluorine atoms. They can be either polyfluorinated or perfluorinated. In some cases, the fluorosurfactant can be non-ionic. In some cases, the fluorosurfactant is a fluorosurfactant to the oil phase of the pristine oil HFE-7500 Engineered Fluid (3M Novec), having the formula C9H5F15O. In one aspect of the invention, the fluorosurfactant is RAN-008-FluoroSurfactant (RAN Biotechnologies), having the formula PFPE- PEG / PEO-PFPE. In another aspect, the fluorosurfactant is Krytox® 157 FSH (DuPont), having the formula PFPE-COOH. In other embodiments, a water-soluble surfactant is used to stabilize droplets in oil during the ddPCR assay. In one aspect, the water-soluble surfactant is the polyoxyalkylene block copolymer surfactant Pluronic® F127 (Sigma-Aldrich), having the formula PPO-PEO-PPO. In another aspect, the water-soluble surfactant is polyoxyalkylene block copolymer surfactant Poloxamer 188 (P188). Poloxamer 188 is a polyoxyalkylene block copolymer that has a slightly different molecular weight of the component blocks compared to Pluronic® F127. For droplet stabilization, Poloxamer 188 works just as good as Pluronic® F127. Other polyoxyalkylene block copolymers, including P123 and F108, can be used for droplet stabilization; however, Pluronic® F127 and Poloxamer 188 have been found to work the best.

[0045] As used herein, the term “electrospray” refers to an apparatus that employs electricity to disperse a fluid (i.e. , a liquid) into liquid droplets, or a fine aerosol. High voltage is applied to a liquid supplied through an “emitter” constructed of a glass capillary or metallic capillary. The tip of the emitter can be, for example, tapered to have a conical shape with an orifice or opening at the front of the tip, or can be connected to a membrane for droplet emission (e.g., a nanoporous membrane). For example, a Hamilton® micro-needle can serve as an emitter. High voltage which produces the electrospray can be from a direct current (DC) electric field, or an alternating current (AC) electric field. The electrospray emitter can be “configured,” for example, with an AC electrode in direct or indirect contact with the emitter. The electrode may also be inserted inside of, for example, a glass emitter. A liquid can be supplied, for example, through an inlet to the emitter by use of pressure. The pressure is applied, for example, through a manual syringe, a syringe pump, a diaphragm pump, a piston pump, a hydraulic pump, a compressed gas such as air or nitrogen, or any known apparatus or method of producing pressure, wherein the amount of pressure can be controlled, for example, by a pressure regulator.

[0046] As used herein, the term “fluid” refers to a liquid or a liquid mixture. The liquid can be, for example, water, an organic solvent, or an oil. The liquid can comprise a mixture of one or more miscible or immiscible liquids. The liquid can comprise a mixture of substances in the liquid, and the substances can be dissolved in the liquid, suspended in the liquid, or a combination thereof. The substances can include but are not limited to, for example, biological substances, proteins, DNA, RNA, enzymes, cells, various components of a cell, cancer cells, PCR reagents, surfactants, additives, salts, and oils. The fluid or the liquid can be in the form of a droplet, such as a liquid droplet or an aerosol produced by electrospray. As used herein, the term “droplet suspending medium” refers to, for example, an oil, water, an organic solvent, which is immiscible with the fluid (defined above). The droplet suspending medium is also immiscible with a liquid droplet, or an aerosol that is produced from a fluid, or immiscible with a liquid droplet, or an aerosol produced from an electrosprayed fluid.

[0047] As used herein, the term “chamber” refers to a vessel that is partially or fully enclosed. The chamber may comprise, among other things, a fluid, a liquid, an oil, water, a mixture, an emulsion, a suspension, or one or more droplets. The chamber, for example, may hold a volume of a liquid, or oil, or an emulsion for a certain length of time or allow such fluids to pass or flow through the chamber. The chamber may be constructed of any material compatible with the fluid or liquid it carries. The chamber may have internal dimensions suitable for the purposes of, for example, where an electrospray forms and emulsion, where PCR thermal cycling takes place, or where a sample is detected. The chamber may be substantially two-dimensional or may be three dimensional, depending on the intended use of the chamber within the context of this disclosure. For example, a droplet chamber can comprise a cylindrical or a cubical volume with inlets and outlets for the production and flow of emulsions. A PCR chip can comprise of a single serpentine channel of continuous “S”-shaped loops, or an array of multiple channels. A chamber can be configured for batch PCR. A two-dimensional chamber may be used, for example, to detect the signal from a sample that is substantially along a plane, or a three-dimensional chamber may be used, for example, to collect samples for three-dimensional imaging using a three-dimensional detector.

[0048] In some embodiments, the current disclosure combines AC electric field to generate droplets from a conic orifice or cappillary to achieve uniform droplets with a hydrophobic nanoporous membrane having asymmetric pores with conic through pores comprising surface modification of the ANM, to realize massively higher throughput for the generation of droplets (> 1 million droplet per minute) with small dimension (1-100 microns) for digital droplet Polymerase Reaction (ddPCR) and cell encapsulation applications. Alternating current (AC) droplet generators are described in U.S. Pat. No. 11 ,293,057, the teachings of which are hereby incorporated by reference in their entirety.

[0049] In some embodiments, the methods and apparatuses disclosed herein comprise coating ANM with a metal monolayer on the tip side so that it is hydrophobic on that side. The hydrophobic surface prevents adjacent droplets from coalescing. The AC field applied across the membrane can then produce droplets from each tip of the conic pore. The conic geometry reduces the hydrodynamic resistance so more droplets can be generated per pore. The large number of pores in each membrane allows massively parallel generation of droplets. The conic pore tip radius can be tuned from 10 nm to 1 micron, yielding monodispersed droplets from 1 to 100 microns. The pore density can also be tuned so that the droplet generation rate can span from 10,000 per minute to 1 million per minute.

[0050] In one embodiment described herein, an alternating current droplet generator comprises: (a) an electrospray emitter having a nanoporous membrane nozzle comprising a nanoporous membrane, and a conduit to the orifice for a pressurized fluid, wherein the emitter can generate liquid droplets from the pressurized fluid at the orifice by an alternating current; (b) an alternating current electrode and an optional counter electrode that are configured to provide the alternating current; and (c) a droplet chamber having one or more inlets for the electrospray emitter and a droplet suspending medium, wherein the conical end of the emitter is at least partially inserted into the chamber, and when the droplet suspending medium flows into the chamber the conical end is immersed in the medium; wherein more than 100 monodispersed liquid droplets per second can be produced from the pressurized fluid by the alternating current droplet generator in the droplet suspending medium to form an emulsion.

[0051] Embodiments of the droplet chamber can include one inlet for both the electrospray emitter and the droplet suspending medium, or the droplet chamber can include two or more inlets, wherein the electrospray emitter and the droplet suspending medium enter the droplet chamber from separate inlets, wherein the inlets can be configured in different positions, for example, a T-junction. In other embodiments, one or more inlets may be pressurized, or one or more inlets may be pressurized at different time intervals.

[0052] In yet other embodiments of the alternating current droplet generator, the AC droplet generator may be grounded, the AC droplet generator may comprise a counter electrode, or the droplet chamber may comprise a counter electrode.

[0053] Embodiments of the emitter include an inner diameter in the range of about 0.01 mm to about 0.5 mm, about 0.01 mm to about 0.25 mm, about 0.01 mm to about 0.15 mm, about 0.05 mm to about 0.1 mm, about 0.1 mm to about 0.5 mm, about 0.0001 mm to about 0.1 mm, about 0.0001 mm to about 0.01 mm, or about 0.001 mm to about 0.01 mm. Embodiments of the orifice include a diameter in the range of about 50 nm to about 10000 nm, about 100 nm to about 10000 nm, about 250 nm to about 10000 nm, about 500 nm to about 10000 nm, about 750 nm to about 10000 nm, or about 1000 nm to about 10000 nm. Embodiments of an orifice shape can include a variety of shapes that are not limited to, for example, a conical shape, or a round shape.

[0054] Embodiments of the capillary include an inner diameter in the range of about 0.01 mm to about 2 mm, 0.01 mm to about 1 mm, 0.01 mm to about 0.5 mm, about 0.01 mm to about 0.25 mm, about 0.01 mm to about 0.15 mm, about 0.05 mm to about 0.1 mm, about 0.1 mm to about 0.5 mm, about 0.0001 mm to about 0.1 mm, about 0.0001 mm to about 0.01 mm, about 0.001 mm to about 0.01 mm, about 50 nm to about 10000 nm, about 100 nm to about 10000 nm, about 250 nm to about 10000 nm, about 500 nm to about 10000 nm, about 750 nm to about 10000 nm, or about 1000 nm to about 10000 nm.

[0055] In another embodiment, the electrospray emitter comprises an alternating current electrode, and a droplet chamber comprises a counter electrode and a droplet suspending medium. In additional embodiments, the droplet chamber is without a counter electrode and comprises a droplet suspending medium. In various embodiments of this disclosure the droplet suspending medium is an oil, wherein the oil may be at atmospheric pressure, the oil may be at a pressure that is higher than atmospheric pressure, or the oil may be at a pressure that is lower than atmospheric pressure.

[0056] In other embodiments, the oil may be flowing, or the oil may not be flowing. Embodiments of the oil flow rate include a flow rate ranging from about 0.01 milliliters per minute to about 100 milliliters per minute, about 0.1 milliliters per minute to about 50 milliliters per minute, or about 0.5 milliliters per minute to about 10 milliliters per minute.

[0057] One embodiment for a method for producing liquid droplets comprises: (a) applying an alternating current electric field to an electrospray emitter; (b) introducing a fluid into the emitter; and (c) generating an electrospray by the emitter from the fluid at a rate of more than 100 liquid droplets per second in a droplet suspending medium to form a stable emulsion; wherein the electrospray produces monodispersed liquid droplets having a diameter ranging from about 0.1 micrometers to about 1000 micrometers in the droplet suspending medium.

[0058] Embodiments of monodispersed liquid droplets include droplet diameters ranging from about 0.1 micrometers to about 750 micrometers, about 0.1 micrometers to about 500 micrometers, about 0.1 micrometers to about 250 micrometers, about 0.1 micrometers to about 100 micrometers, about 0.1 micrometers to about 50 micrometers, about 0.1 micrometers to about 25 micrometers, about 0.1 micrometers to about 10 micrometers, about 0.1 micrometers to about 5 micrometers, about 0.1 micrometers to about 1 micrometer, or about 0.1 micrometers to about 0.5 micrometers.

[0059] Embodiments of monodispersed liquid droplets include droplet volumes of about 1 picoliter, about 2 picoliters, about 3 picoliters, about 4 picoliters, about 5 picoliters, about 10 picoliters, about 20 picoliters, about 50 picoliters, about 100 picoliters, about 200 picoliters, or about 500 picoliters.

[0060] In various embodiments, the diameter of a generated liquid droplet can be tuned to different diameters by varying the voltage of the electric filed. In other embodiments, the volume of a generated liquid droplet can be tuned to different volumes by varying the voltage of the electric filed. In yet other embodiments, the diameter of a generated liquid droplet can be tuned to different diameters by varying the frequency of the electric filed. In yet additional embodiments, the volume of a generated liquid droplet can be tuned to different volumes by varying the frequency of the electric filed.

[0061] In various embodiments of the disclosed methods, the frequency of the alternating current ranges from about 1 kilohertz to about 1000 kilohertz, about 10 kilohertz to about 500 kilohertz, about 25 kilohertz to about 250 kilohertz, or about 50 kilohertz to about 150 kilohertz. In various other embodiments, the potential of the alternating current ranges from about 0.01 kilovolts to about 1000 kilovolts, about 0.01 kilovolts to about 100 kilovolts, about 0.1 kilovolts to about 10 kilovolts, or about 0.1 kilovolts to about 1 kilovolts.

[0062] In yet other embodiments, the fluid comprises a template molecule, one or more PCR reagents, and water. In additional embodiments, the fluid further comprises a surfactant, the carrier oil further comprises a surfactant, or both the fluid and the carrier oil further comprise a surfactant. Other embodiments of surfactants comprise, but are not limited to, Tween, fluorinated surfactants, sodium dodecyl sulfate, any commercially available surfactant, or any surfactant that helps prevent droplets from coalescing.

[0063] In yet other various embodiments, the fluid is flowing at rate of about 0.1 microliters per minute to about 10000 microliters per minute, about 1 microliters per minute to about 1000 microliters per minute, about 1 microliters per minute to about 100 microliters per minute, about 1 microliters per minute to about 75 microliters per minute, about 1 microliters per minute to about 50 microliters per minute, about 1 microliters per minute to about 25 microliters per minute, or about 1 microliters per minute to about 10 microliters per minute. In various embodiments, the fluid is maintained at a constant flow rate by constant pressure, or the fluid flows to the electrospray continuously at a constant flow rate, or the fluid flows to the electrospray continuously at a constant flow rate by constant pressure. In other embodiments, the fluid comprising water under pressure flows to the electrospray continuously.

[0064] In additional embodiments, the droplet suspending medium comprises a flowing carrier oil. In other embodiments, the carrier oil is a mineral oil, a fluorinated oil, a silicone oil, or a combination thereof. Embodiments of the carrier oil include a flow rate of about 10 microliters per minute to about 100 microliters per minute, about 1 microliters per minute to about 75 microliters per minute, about 1 microliters per minute to about 50 microliters per minute, about 1 microliters per minute to about 25 microliters per minute, or about 1 microliters per minute to about 10 microliters per minute. In other various embodiments of the disclosed methods, the droplets to not substantially coalesce in the carrier oil. In additional; embodiments, the fluid comprises one or more biological substances. In yet other embodiments, the droplet suspending medium substantially comprises flowing water.

[0065] Various other embodiments of the disclosed methods include a method for performing droplet digital polymerase chain reaction (PCR) comprising: (a) applying an alternating current electric field to an electrospray emitter; (b) introducing a fluid into the emitter, wherein the fluid comprises one or more template molecules and PCR reagents; (c) generating an electrospray by the emitter from the fluid at a rate of more than 3000 liquid droplets per second in a droplet suspending medium to form a stable emulsion; (d) amplifying an amplicon of one or more template molecules in a PCR thermal cycler; and (e) detecting the amplicons; wherein the electrospray produces monodispersed liquid droplets having a diameter ranging from about 0.1 micrometers to about 1000 micrometers in the droplet suspending medium.

[0066] In other various embodiments, the amplicon detected is a polynucleotide of less than about 1000 nucleotides, less than about 750 nucleotides, less than about 500 nucleotides, less than about 250 nucleotides, less than about 100 nucleotides, less than about 50 nucleotides, or less than about 25 nucleotides. In yet further embodiments, the polynucleotide comprises a micro-ribonucleic acid (miRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), or a combination thereof. In other embodiments, the polynucleotide comprises a biomarker for a disease, such as cancer.

[0067] Embodiments of diseases include, but are not limited to, cancer, infectious diseases (for example, malaria and dengue), diseases relating to drug addiction, heart diseases that can cause cardiac arrest, mental health diseases (causing, for example, depression), Alzheimer's disease, and immune diseases such as HIV. Embodiments of this disclosure are applicable for said aforementioned diseases comprising pathogenic DNA, mRNA, or host miRNA biomarkers, diseases comprising nuclei acid biomarkers, or other diseases comprising polynucleotide biomarkers.

[0068] In additional embodiments, the biomarker comprises a biomarker found in a bodily fluid. In other additional embodiments, the fluid further comprises (a) a fluorescent reporter for PCR amplicons, (b) a fluorescent reporter for multiplex target quantification, (c) an optical barcode reporter for multiplex target quantification, or (d) a radioactive reporter.

[0069] The droplet generation system described herein using alternating current (AC) electrospray and a nanoporous membrane can generate greater than 3 x 103droplets per sec which is much greater speed than flow-focusing technology. It can offer the desirable droplet number, provide greater dynamic range, and reduce inhibitor cross-talking even for high mM concentration inhibitors like Ca2+in a reasonable assay time of less than one hour, without or with minimum pretreatment. In this disclosure, an imaging technology suitable to quantify the number of fluorescent droplets for the described high throughput platform is significantly less costly than the single-droplet optical detection technology used for current digital PCR technologies.

[0070] Alternating Current (AC) Nanoporous Membrane Platform

[0071] Due to their limitations, neither qPCR nor ddPCR technologies are ideal for accurate quantification of miRNA. Therefore, this disclosure provides the advantages of both systems while minimizing their shortcomings. Both DC and AC spray ionization methods are extensively used in mass spectrometry as a means of generating small, monodispersed droplets in air. AC electrospray is chosen for the purposes of this disclosure because it generates droplets with a desirable size (for example, 1-10 pm) that are weakly charged, making them more ideal for handling biological samples. In addition, it has been shown experimentally that a DC spray is very unstable when an aqueous phase is sprayed into oil, most likely due to an electric discharge into the oil phase. This instability is surmounted by employing an AC spray with a sufficiently high frequency so that the AC spray cone does not accumulate too much charge for a discharge to occur in the oil. By carefully controlling the parameters such as membrane pore size, frequency and voltage of the electric field, stable droplets ranging from 1-5 pm can be generated at a much higher throughput than the existing digital PCR technology, such that the ideal droplet number of is achieved in minutes. Furthermore, because the droplets generated by AC spray are smaller (1-5 pm droplet size), a larger number of droplets can be generated from the same volume (10 microliter) of sample. This advance effectively increases the dynamic range by 2 to 3 orders of magnitude and reduces or eliminates the need for dilution.

[0072] The asymmetric nanoporous membrane comprising a metal monolayer can be fabricated on polymer film membranes having asymmetrically shaped (i.e., conic) nanopores, with electrodeposition of the metal material only on a narrower tip side of the membrane. These asymmetric nanopore membranes (ANMs) are described in U.S. Pat. App. Pub. No. US 2022 / 0347686 A1 , the teachings of which are hereby incorporated by reference in their entirety. The ANM technology utilizes an asymmetric etching technique for commercial ion-track membranes to produce conic nanopores that can range from 10 nm to 200 nm on a narrower tip side and up to 2 microns on the wider base side. Track-etched membranes that have asymmetrically shaped pores (as opposed to the more conventional cylindrical or irregularly shaped pores in ultrafiltration membranes) offer important advantages. For example, one advantage of the asymmetrical pore shape is a dramatic 200-400% reduction in the applied pressure / force to drive a liquid sample through the filter membrane at the same throughput, compared to an analogous cylindrical pore membranes. This significant reduction in applied pressure prevents biomolecule deformation, lysing, and fusion. Moreover, the chance of clogging is significantly reduced due to a dramatic enhancement in the rate of transport through the membrane, relative to an analogous cylindrical pore membrane. This pore geometry design allows high yield and high throughput.

[0073] For liquid biopsy application of ddPCR for cancer screening, the key limitation is that it cannot detect cancer-causing mutation if the WT outnumbers the mutation by 100,000 to 1 . With the limited number of droplets (< 10,000) in the current technology, there will be too many WT per droplet to produce false positive droplets that are as bright as droplets with a single mutation. This severely limits the sensitivity of the liquid biopsy assay and prevents early cancer detection. By increasing the number of droplets 100-fold, the ddPCR liquid biopsy technology becomes more sensitive and hence increases its translatability significantly.

[0074] Also described herein is a method and compositions for single-cell encapsulation with gel for wound healing applications. This technology allows large-scale encapsulation of cells in gel droplets.

[0075] Also described are methods for encapsulating extracellular vesicles (EVs) having sizes from 50-100 nm, with an encapsulation medium. The asymmetric nanopore membranes have ideal dimensions for EVs. The extracellular vesicles contain biological markers such as DNA, RNA, proteins, or can be artifically produced to include drugs, CRISPR reagents, antibodies, or the like. The extracelluar vesicle delivery vehicle allows targeted delivery to certain cells because of the EV surface proteins. The encapsulating medium protects the EVs from the immune cells. The gel also adheres to the tissue so that the EVs can have longer residence time in vivo and are not depleted. The encapsulation medium also allows EV release when the gel encounters certain stimuli like pH, enzymes etc in a wound or near a tumor. Typical encapsulation media can be made from alginate, collagen, norbornene-modified hyaluronic acid, or a combination thereof.

[0076] One embodiment described herein is an apparatus for generating monodispersed liquid droplets, the apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying an alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium. In one aspect, the electrospray emitter comprises glass, the alternating current electrode is internal to the electrospray emitter, and the droplet chamber comprises a counter electrode. In another aspect, the droplet suspending medium comprises a carrier oil comprising a mineral oil, a fluorinated oil, a silicone oil, a surfactant, or combinations thereof. In another aspect, the droplet suspending medium comprises a mixture of polyethylene oxide and a nonionic surfactant. In another aspect, the alternating current electrospray device is configured to apply a frequency of alternating current ranging from about 1 kHz to about 1000 kHz. In another aspect, the alternating current electrospray device is configured to apply a potential of alternating current ranging from about 0.01 kV to about 100 kV. In another aspect, the hydrophobic nanoporous membrane comprises a porosity of about 50% to about 90%. In another aspect, the hydrophobic nanoporous membrane comprises a thickness of about 100 nm to about 50 pm. In another aspect, the hydrophobic nanoporous membrane comprises polyethylene terephthalate (PET), polycarbonate (PC), polystyrene (PS), poly-methyl-meta- acrylate (PMMA), cyclic olefin copolymer (COC), polyimide (PI), polydimethylsiloxane (PDMS), or combinations thereof. In another aspect, the hydrophobic nanoporous membrane further comprises an electrodeposited metal alloy layer on a membrane surface opposite the conduit and immersed in the droplet suspending medium. In another aspect, the metal alloy layer comprises nickel-iron, samarium-cobalt, aluminum-nickel-cobalt, nickel-iron-chromium, iron-chromium- cobalt, neodymium-iron-boron, or combinations thereof. In another aspect, the metal alloy layer has a thickness of about 50 nm to about 250 nm. In another aspect, the hydrophobic nanoporous membrane further comprises one or more gold layers in contact with the metal alloy layer. In another aspect, each gold layer has a thickness of about 5 nm to about 150 nm. In another aspect, the hydrophobic nanoporous membrane comprises asymmetrically shaped nanopores, each asymmetrically shaped nanopore comprising a first opening having a first diameter on a first membrane surface and being fluidly connected to the conduit of the electrospray emitter and a second opening having a second diameter on a second membrane surface and being configured to emit monodispersed liquid droplets into the droplet suspending medium, wherein the first diameter is greater than the second diameter. In another aspect, the hydrophobic nanoporous membrane comprises symmetrically shaped nanopores, each symmetrically shaped nanopore comprising a first opening having a first diameter on a first membrane surface and being fluidly connected to the conduit of the electrospray emitter and a second opening having a second diameter on a second membrane surface and being configured to emit monodispersed liquid droplets into the droplet suspending medium, wherein the first diameter is substantially equal to the second diameter. In another aspect, the second diameter of each asymmetrically shaped nanopore is about 50 nm to about 50 pm. In another aspect, the second diameter of each asymmetrically shaped nanopore is less than about 280 nm to 10 pm. In another aspect, the second diameter of each symmetrically shaped nanopore is about 50 nm to about 50 pm. In another aspect, the second diameter of each symmetrically shaped nanopore is less than about 280 nm to 10 pm. In another aspect, the hydrophobic nanoporous membrane comprises a combination of asymmetrically shaped nanopores and symmetrically shaped nanopores.

[0077] Another embodiment described herein is a method for generating monodispersed liquid droplets, the method comprising: applying an alternating current electric field to an apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying the alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium; introducing a pressurized fluid into the electrospray emitter; generating an electrospray by the electrospray emitter from the pressurized fluid in the droplet suspending medium; and collecting the monodispersed liquid droplets in the droplet suspending medium. In one aspect, the monodispersed liquid droplets have an average droplet diameter of about 5 pm to about 300 pm. In another aspect, the monodispersed liquid droplets have an average droplet diameter of about 10 pm to about 50 pm. In another aspect, the method generates monodispersed liquid droplets at a rate of about 1.0 x 104droplets per minute to about 1.0 x 106droplets per minute. In another aspect, the method generates at least 1.0 x 106monodispersed liquid droplets in about 5 minutes. In another aspect, the droplet suspending medium comprises a carrier oil comprising a mineral oil, a fluorinated oil, a silicone oil, a surfactant, or combinations thereof. In another aspect, the droplet suspending medium comprises a mixture of polyethylene oxide and a nonionic surfactant. In another aspect, the droplet suspending medium comprises a carrier oil concentration ranging from about 0.1 % v / v to about 5% v / v. In another aspect, the alternating current electrospray device comprises a pressure of about 1 kPa to about 10 kPa. Another embodiment described herein is a method for performing a droplet digital polymerase chain reaction (PCR), the method comprising: applying an alternating current electric field to an apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying the alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium; introducing a pressurized fluid into the electrospray emitter, wherein the pressurized fluid comprises one or more template molecules and PCR reagents; generating an electrospray by the electrospray emitter from the pressurized fluid in the droplet suspending medium; amplifying an amplicon of one or more template molecules in a PCR thermal cycler; and detecting the amplicons.

[0078] Another embodiment described herein is a method for encapsulating extracellular vesicles or cells in an encapsulation medium, the method comprising: applying an alternating current electric field to an apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying the alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium; introducing a pressurized fluid into the electrospray emitter, wherein the pressurized fluid comprises one or more extracellular vesicles or cells and an encapsulation medium; generating an electrospray by the electrospray emitter from the pressurized fluid in the droplet suspending medium; and collecting encapsulated extracellular vesicles or cells. In one aspect, the encapsulation medium comprises alginate, collagen, norbornene-modified hyaluronic acid, or a combination thereof. In another aspect, the extracellular vesicles have a size of about 50 nm to about 150 nm. It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.

[0079] Various embodiments and aspects of the inventions described herein are summarized by the following clauses:

[0080] Clause 1. An apparatus for generating monodispersed liquid droplets, the apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying an alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium.

[0081] Clause 2. The apparatus of clause 1 , wherein the electrospray emitter comprises glass, the alternating current electrode is internal to the electrospray emitter, and the droplet chamber comprises a counter electrode.

[0082] Clause 3. The apparatus of clause 1 or 2, wherein the droplet suspending medium comprises a carrier oil comprising a mineral oil, a fluorinated oil, a silicone oil, a surfactant, or combinations thereof.

[0083] Clause 4. The apparatus of any one of clauses 1-3, wherein the droplet suspending medium comprises a mixture of polyethylene oxide and a nonionic surfactant.

[0084] Clause s. The apparatus of any one of clauses 1-4, wherein the alternating current electrospray device is configured to apply a frequency of alternating current ranging from about 1 kHz to about 1000 kHz.

[0085] Clause 6. The apparatus of any one of clauses 1-5, wherein the alternating current electrospray device is configured to apply a potential of alternating current ranging from about 0.01 kV to about 100 kV.

[0086] Clause 7. The apparatus of any one of clauses 1-6, wherein the hydrophobic nanoporous membrane comprises a porosity of about 50% to about 90%.

[0087] Clause 8. The apparatus of any one of clauses 1-7, wherein the hydrophobic nanoporous membrane comprises a thickness of about 100 nm to about 50 pm.

[0088] Clause 9. The apparatus of any one of clauses 1-8, wherein the hydrophobic nanoporous membrane comprises polyethylene terephthalate (PET), polycarbonate (PC), polystyrene (PS), poly-methyl-meta-acrylate (PMMA), cyclic olefin copolymer (COC), polyimide (PI), polydimethylsiloxane (PDMS), or combinations thereof.

[0089] Clause 10. The apparatus of any one of clauses 1-9, wherein the hydrophobic nanoporous membrane further comprises an electrodeposited metal alloy layer on a membrane surface opposite the conduit and immersed in the droplet suspending medium.

[0090] Clause 11. The apparatus of any one of clauses 1-10, wherein the metal alloy layer comprises nickel-iron, samarium-cobalt, aluminum-nickel-cobalt, nickel-iron-chromium, iron- chromium-cobalt, neodymium-iron-boron, or combinations thereof.

[0091] Clause 12. The apparatus of any one of clauses 1-11 , wherein the metal alloy layer has a thickness of about 50 nm to about 250 nm.

[0092] Clause 13. The apparatus of any one of clauses 1-12, wherein the hydrophobic nanoporous membrane further comprises one or more gold layers in contact with the metal alloy layer. Clause 14. The apparatus of any one of clauses 1-13, wherein each gold layer has a thickness of about 5 nm to about 150 nm.

[0093] Clause 15. The apparatus of any one of clauses 1-14, wherein the hydrophobic nanoporous membrane comprises asymmetrically shaped nanopores, each asymmetrically shaped nanopore comprising a first opening having a first diameter on a first membrane surface and being fluidly connected to the conduit of the electrospray emitter and a second opening having a second diameter on a second membrane surface and being configured to emit monodispersed liquid droplets into the droplet suspending medium, wherein the first diameter is greater than the second diameter.

[0094] Clause 16. The apparatus of any one of clauses 1-15, wherein the hydrophobic nanoporous membrane comprises symmetrically shaped nanopores, each symmetrically shaped nanopore comprising a first opening having a first diameter on a first membrane surface and being fluidly connected to the conduit of the electrospray emitter and a second opening having a second diameter on a second membrane surface and being configured to emit monodispersed liquid droplets into the droplet suspending medium, wherein the first diameter is substantially equal to the second diameter.

[0095] Clause 17. The apparatus of any one of clauses 1-16, wherein the second diameter of each asymmetrically shaped nanopore is about 50 nm to about 50 pm.

[0096] Clause 18. The apparatus of any one of clauses 1-17, wherein the second diameter of each asymmetrically shaped nanopore is less than about 280 nm to 10 pm.

[0097] Clause 19. The apparatus of any one of clauses 1-18, wherein the second diameter of each symmetrically shaped nanopore is about 50 nm to about 50 pm.

[0098] Clause 20. The apparatus of any one of clauses 1-19, wherein the second diameter of each symmetrically shaped nanopore is less than about 280 nm to 10 pm.

[0099] Clause 21. The apparatus of any one of clauses 1-20, wherein the hydrophobic nanoporous membrane comprises a combination of asymmetrically shaped nanopores and symmetrically shaped nanopores.

[0100] Clause 22. A method for generating monodispersed liquid droplets, the method comprising: applying an alternating current electric field to an apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying the alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium; introducing a pressurized fluid into the electrospray emitter; generating an electrospray by the electrospray emitter from the pressurized fluid in the droplet suspending medium; and collecting the monodispersed liquid droplets in the droplet suspending medium.

[0101] Clause 23. The method of clause 22, wherein the monodispersed liquid droplets have an average droplet diameter of about 5 pm to about 300 pm.

[0102] Clause 24. The method of clause 22 or 23, wherein the monodispersed liquid droplets have an average droplet diameter of about 10 pm to about 50 pm.

[0103] Clause 25. The method of any one of clauses 22-24, wherein the method generates monodispersed liquid droplets at a rate of about 1.0 x 104droplets per minute to about 1.0 x 106droplets per minute.

[0104] Clause 26. The method of any one of clauses 22-25, wherein the method generates at least 1.0 x 106monodispersed liquid droplets in about 5 minutes.

[0105] Clause 27. The method of any one of clauses 22-26, wherein the droplet suspending medium comprises a carrier oil comprising a mineral oil, a fluorinated oil, a silicone oil, a surfactant, or combinations thereof.

[0106] Clause 28. The method of any one of clauses 22-27, wherein the droplet suspending medium comprises a mixture of polyethylene oxide and a nonionic surfactant.

[0107] Clause 29. The method of any one of clauses 22-28, wherein the droplet suspending medium comprises a carrier oil concentration ranging from about 0.1% v / v to about 5% v / v.

[0108] Clause 30. The method of any one of clauses 22-29, wherein the alternating current electrospray device comprises a pressure of about 1 kPa to about 10 kPa.

[0109] Clause 31. A method for performing a droplet digital polymerase chain reaction (PCR), the method comprising: applying an alternating current electric field to an apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying the alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium; introducing a pressurized fluid into the electrospray emitter, wherein the pressurized fluid comprises one or more template molecules and PCR reagents; generating an electrospray by the electrospray emitter from the pressurized fluid in the droplet suspending medium; amplifying an amplicon of one or more template molecules in a PCR thermal cycler; and detecting the amplicons.

[0110] Clause 32. A method for encapsulating cells in an encapsulation medium, the method comprising: applying an alternating current electric field to an apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying the alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium; introducing a pressurized fluid into the electrospray emitter, wherein the pressurized fluid comprises one or more extracellular vesicles or cells and an encapsulation medium; generating an electrospray by the electrospray emitter from the pressurized fluid in the droplet suspending medium; and collecting encapsulated extracellular vesicles or cells.

[0111] Clause 33. The method of clause 32, wherein the encapsulation medium comprises alginate, collagen, norbornene-modified hyaluronic acid, or a combination thereof.

[0112] Clause 34. The method of clause 32 or 33, wherein the extracellular vesicles have a size of about 50 nm to about 150 nm.

[0113] EXAMPLES

[0114] Example 1

[0115] Electrodeposited Magnetic Nanoporous Membrane (MNM)

[0116] Ni8oFe2o Deposition by Electroplating

[0117] Track-etched PET films (PET115745, Wuwei Kejin Xinfa) were used that were 11 -pm thick with a pore density of 5 x io7 / cm2. To fabricate the electroplated magnetic nanoporous membrane, 80 nm Au was deposited onto the track-etched PET films in an FC-1800 Evaporator. The gold layer provided good adhesiveness between the PET polymer film and NiFe and functioned as a seed layer for electroplating. The membrane was cut into 4 cm x 4 cm pieces. Copper tapes were used to fix membranes onto the support and electrically connected to the cathode. A nickel plate was used as the anode. The specific compounds used for the electroplating solution are shown in Table 1. The compounds were mixed with DI water and solved at 40 °C with agitation for 1 hour. Sodium hydroxide was used to adjust the pH of the solution to pH 3.0.

[0118] Table 1. Solutions for Electroplating Compound Concentration (g / 100 ml_)

[0119] NiSO4.6H2O 28.9

[0120] FeSO4.7H2O 6.4

[0121] H3BO3 4

[0122] 5-Sulfosalicylic acid dihydrate 0.890

[0123] 1,3,(6,7)-naphthalenetrisulfonic acid trisodium salt hydrate 0.304

[0124] Electroplating was performed at a constant current density at 2 mA / cm2using by a Keithley 2636A Dual-Channel System SourceMeter; voltage is monitored during the electroplating process. A custom electroplating stirring tank was designed for uniform deposition. The deposition rate was derived by SEM images on thicker samples grown under the same conditions. Another 10-nm Au was deposited on the top of the NiFe layer to reduce non-specific adsorption and chemical instability. Characterization of the membranes are described below.

[0125] NiSoFe2o Deposition by Sputtering

[0126] Similar to the electroplated samples, 80 nm Au was deposited onto the PET films initially. The sputtered samples were prepared at room temperature in a commercial UHV sputtering system Oerlikon DCSS using a NisoFe2o target. Ar gas flow was fixed to 20 cm3STP / min, and the plasma power was 50 W during deposition. The deposition rate was derived by means of a stylus profilometer and SEM images on thicker samples grown under the same conditions. After sputtering, 10 nm Au was deposited on top of the NiFe layer.

[0127] Characterization of Electroplated / Sputtered Magnetic Nanoporous Membranes

[0128] The structure of the NiFe layer was examined by both SEM and XRD. For XRD, a peak around 29 = 44.5° was investigated to determine the grain size of both electroplated and sputtered NiFe. No significant difference in the minimum grain size was found between the two (~30nm). However, large grain boundaries were observed on the sputtered NiFe but not the electroplated one under SEM. Such grain boundaries will result in lower magnetic permeability of the NiFe, which leads to lower magnetic force on the nanobeads.

[0129] Example 2

[0130] Alternating Current (AC) Electrospray Device with Magnetic Nanoporous Membrane

[0131] A multi-channel immersed AC electrospray (iACE) device was developed using Magnetic Nanoporous Membranes (MNM) (FIG. 1C, FIG. 2A). With a significantly reduced tip size compared to previous devices (e.g., 250 nm vs. 5 pm), this platform can produce monodispersed liquid droplets of smaller diameters more consistently (FIG. 2B-2C). By tuning the AC waveform and optimizing the number of exposed channels in the membrane, this device was demonstrated to rapidly generate 1 * 106droplets of 10-50 pm in diameter over the course of five minutes (FIG. 2B-C). A continuous droplet production platform was developed that uses the droplets’ innate buoyancy to selectively harvest the droplets without expending significant volumes of a fluorosurfactant (FIG. 2A).

Claims

CLAIMSWhat is claimed:

1. An apparatus for generating monodispersed liquid droplets, the apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying an alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium.

2. The apparatus of claim 1 , wherein the electrospray emitter comprises glass, the alternating current electrode is internal to the electrospray emitter, and the droplet chamber comprises a counter electrode.

3. The apparatus of claim 1 , wherein the droplet suspending medium comprises a carrier oil comprising a mineral oil, a fluorinated oil, a silicone oil, a surfactant, or combinations thereof.

4. The apparatus of claim 3, wherein the droplet suspending medium comprises a mixture of polyethylene oxide and a nonionic surfactant.

5. The apparatus of claim 1 , wherein the alternating current electrospray device is configured to apply a frequency of alternating current ranging from about 1 kHz to about 1000 kHz.

6. The apparatus of claim 1 , wherein the alternating current electrospray device is configured to apply a potential of alternating current ranging from about 0.01 kV to about 100 kV.

7. The apparatus of claim 1, wherein the hydrophobic nanoporous membrane comprises a porosity of about 50% to about 90%.

8. The apparatus of claim 1, wherein the hydrophobic nanoporous membrane comprises a thickness of about 100 nm to about 50 pm.

9. The apparatus of claim 1 , wherein the hydrophobic nanoporous membrane comprises polyethylene terephthalate (PET), polycarbonate (PC), polystyrene (PS), poly-methyl- meta-acrylate (PMMA), cyclic olefin copolymer (COC), polyimide (PI), polydimethylsiloxane (PDMS), or combinations thereof.

10. The apparatus of claim 1 , wherein the hydrophobic nanoporous membrane further comprises an electrodeposited metal alloy layer on a membrane surface opposite the conduit and immersed in the droplet suspending medium.

11. The apparatus of claim 10, wherein the metal alloy layer comprises nickel-iron, samariumcobalt, aluminum-nickel-cobalt, nickel-iron-chromium, iron-chromium-cobalt, neodymium- iron-boron, or combinations thereof.

12. The apparatus of claim 10, wherein the metal alloy layer has a thickness of about 50 nm to about 250 nm.

13. The apparatus of claim 10, wherein the hydrophobic nanoporous membrane further comprises one or more gold layers in contact with the metal alloy layer.

14. The apparatus of claim 13, wherein each gold layer has a thickness of about 5 nm to about 150 nm.

15. The apparatus of claim 1 , wherein the hydrophobic nanoporous membrane comprises asymmetrically shaped nanopores, each asymmetrically shaped nanopore comprising a first opening having a first diameter on a first membrane surface and being fluidlyconnected to the conduit of the electrospray emitter and a second opening having a second diameter on a second membrane surface and being configured to emit monodispersed liquid droplets into the droplet suspending medium, wherein the first diameter is greater than the second diameter.

16. The apparatus of claim 1 , wherein the hydrophobic nanoporous membrane comprises symmetrically shaped nanopores, each symmetrically shaped nanopore comprising a first opening having a first diameter on a first membrane surface and being fluidly connected to the conduit of the electrospray emitter and a second opening having a second diameter on a second membrane surface and being configured to emit monodispersed liquid droplets into the droplet suspending medium, wherein the first diameter is substantially equal to the second diameter.

17. The apparatus of claim 15, wherein the second diameter of each asymmetrically shaped nanopore is about 50 nm to about 50 pm.

18. The apparatus of claim 15, wherein the second diameter of each asymmetrically shaped nanopore is less than about 280 nm to 10 pm.

19. The apparatus of claim 16, wherein the second diameter of each symmetrically shaped nanopore is about 50 nm to about 50 pm.

20. The apparatus of claim 16, wherein the second diameter of each symmetrically shaped nanopore is less than about 280 nm to 10 pm.

21. The apparatus of claim 1, wherein the hydrophobic nanoporous membrane comprises a combination of asymmetrically shaped nanopores and symmetrically shaped nanopores.

22. A method for generating monodispersed liquid droplets, the method comprising: applying an alternating current electric field to an apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein theelectrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying the alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium; introducing a pressurized fluid into the electrospray emitter; generating an electrospray by the electrospray emitter from the pressurized fluid in the droplet suspending medium; and collecting the monodispersed liquid droplets in the droplet suspending medium.

23. The method of claim 22, wherein the monodispersed liquid droplets have an average droplet diameter of about 5 pm to about 300 pm.

24. The method of claim 22, wherein the monodispersed liquid droplets have an average droplet diameter of about 10 pm to about 50 pm.

25. The method of claim 22, wherein the method generates monodispersed liquid droplets at a rate of about 1.0 * 104droplets per minute to about 1.0 x 106droplets per minute.

26. The method of claim 22, wherein the method generates at least 1.0 x 106monodispersed liquid droplets in about 5 minutes.

27. The method of claim 22, wherein the droplet suspending medium comprises a carrier oil comprising a mineral oil, a fluorinated oil, a silicone oil, a surfactant, or combinations thereof.

28. The method of claim 27, wherein the droplet suspending medium comprises a mixture of polyethylene oxide and a nonionic surfactant.

29. The method of claim 27, wherein the droplet suspending medium comprises a carrier oil concentration ranging from about 0.1 % v / v to about 5% v / v.

30. The method of claim 22, wherein the alternating current electrospray device comprises a pressure of about 1 kPa to about 10 kPa.

31. A method for performing a droplet digital polymerase chain reaction (PCR), the method comprising: applying an alternating current electric field to an apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying the alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium; introducing a pressurized fluid into the electrospray emitter, wherein the pressurized fluid comprises one or more template molecules and PCR reagents; generating an electrospray by the electrospray emitter from the pressurized fluid in the droplet suspending medium; amplifying an amplicon of one or more template molecules in a PCR thermal cycler; and detecting the amplicons.

32. A method for encapsulating extracellular vesicles or cells in an encapsulation medium, the method comprising:applying an alternating current electric field to an apparatus comprising: a hydrophobic nanoporous membrane; and an alternating current electrospray device comprising: an electrospray emitter having a conduit connected to the hydrophobic nanoporous membrane for a pressurized fluid, wherein the electrospray emitter is configured to generate monodispersed liquid droplets from the pressurized fluid at the hydrophobic nanoporous membrane by applying the alternating current electric field; an alternating current electrode configured to provide the alternating current electric field; and a droplet chamber configured to hold a droplet suspending medium, the droplet chamber comprising one or more inlets for the electrospray emitter and the droplet suspending medium, wherein the hydrophobic nanoporous membrane is at least partially inserted into the droplet chamber and immersed in the droplet suspending medium; introducing a pressurized fluid into the electrospray emitter, wherein the pressurized fluid comprises one or more extracellular vesicles or cells and an encapsulation medium; generating an electrospray by the electrospray emitter from the pressurized fluid in the droplet suspending medium; and collecting encapsulated extracellular vesicles or cells.

33. The method of claim 32, wherein the encapsulation medium comprises alginate, collagen, norbornene-modified hyaluronic acid, or a combination thereof.

34. The method of claim 32, wherein the extracellular vesicles have a size of about 50 nm to about 150 nm.

Images