Patent Description:
Single-molecule or single-cell assays (e.g., digital PCR, digital loop-mediated isothermal amplification (LAMP), digital ELISA, Drop-Seq) require fractionating or compartmentalizing a large volume to such a level that each smaller fractionated volume contains either none (<NUM>) or a single (<NUM>) entity of interest (i.e., a digital assay). Regardless of the compartment type, it is important that each fractionated compartment is relatively uniform in volume in order to allow reactions to proceed with similar properties in each fractionated volume. Currently, the main approaches to perform this compartmentalization in a uniform manner rely on (i) arrays of wells or (ii) the creation of monodisperse emulsions of drops or droplets using microfluidic approaches. However, there are significant disadvantages to microfluidic approaches given the cost for instruments, pumps, and microfluidic chips required to produce the droplets. Also, small sample volumes can be difficult to use because of the large dead-volumes contained within microfluidic pumping systems. In addition, solid surfaces for reaction or to release reagents and unique barcodes are desired for digital ELISA and single-cell RNAseq, but these are not easy to introduce in microwell arrays or droplets, and can be limited by Poisson statistics. For example, for digital ELISA assays there is often a bead that should be introduced into each volume that provides an affinity reagent to detect a protein of interest, while for single-cell nucleic acid amplification and sequencing assays, it is often desired to include a unique molecular barcode into each droplet such that the RNA amplified from each cell can be re-assigned to the cell of origin even after combining or pooling all of the nucleic acids for a sequencing run.

<NPL>, discloses free-floating amphiphilic picoliter microcarriers composed of an encoded hydrophobic hexagonal outer structure and a hydrophilic inner structure. While this allows multiplexed liquid loading, improvements remain desirable. <NPL>, discloses encapsulation of cells in porous particles but there is no disclosure of functionalization with a molecular capture region.

Therefore, there is a need to create simply operated methods of creating uniformly sized fluid compartments that also are associated with solid supports that allow reagent introduction into each compartment or volume.

The invention is directed to a particle system according to claim <NUM>.

The invention is directed to a method of performing an assay, according to claim <NUM>.

<FIG> illustrates one embodiment of a particle-drop system <NUM>. The particle-drop system <NUM> includes a plurality of three-dimensional drop-carrier particles <NUM>. The drop-carrier particles <NUM> are small, sub-millimeter scale solid particles that are formed having a particular geometric shape and have an interior region <NUM> and an exterior region <NUM>. The interior region <NUM> of the drop-carrier particle <NUM> defines a three-dimensional volume that holds a fluid droplet <NUM>. The fluid droplet <NUM> is the dispersed phase of an emulsion and, in one preferred embodiment, is an aqueous phase (e.g., formed from water). The interior region <NUM> of the drop-carrier particle <NUM> is, in one embodiment, hydrophilic. The hydrophilic nature of the interior region <NUM> may be achieved by the choice of material used during the manufacturing process used to make the drop-carrier particles <NUM> as explained herein. Alternatively, the interior region <NUM> may be rendered hydrophilic after formation of the drop-carrier particle <NUM>. The exterior region <NUM> of the drop-carrier particle <NUM> is, in one embodiment, hydrophobic. In another embodiment, the exterior region <NUM> of the drop-carrier particle <NUM> is fluorophilic. The hydrophobic or fluorophilic nature of the exterior region <NUM> may be achieved by the choice of material used during the manufacturing process used to make the drop-carrier particles <NUM> as explained herein. Alternatively, the exterior region <NUM> may be selectively rendered hydrophobic (or fluorophilic) after formation of the drop-carrier particle <NUM>.

With reference to <FIG>, when the drop-carrier particle <NUM> is loaded with the droplet <NUM>, the resulting construct is referred to herein as a particle-drop <NUM>. A plurality of these particle-drops <NUM> form an emulsion contained in a vial <NUM>. The particle-drop system <NUM> that is described herein has a plurality of particle-drops <NUM> that are disposed in oil <NUM> to form a particle-drop <NUM> emulsion. The oil <NUM> acts as the continuous phase while the aqueous-based droplet <NUM> acts as the dispersed phase. The oil <NUM> surrounds the particle-drops <NUM> to create a monodisperse particle-drop <NUM> emulsion. Monodisperse refers to the ability of the particle-drops <NUM> to retain substantially the same volume of fluid in each particle-drop <NUM>.

Importantly, the monodisperse particle-drop <NUM> emulsions are created without the need of any complex or expensive instruments. Notably, the assembly of drop-carrier particles <NUM> supports a unique volume of an aqueous droplet <NUM>, unlike droplets of multiple volumes supported by Pickering emulsions, such that a plurality of particle-drops <NUM> enables the formation of a monodisperse emulsion. As explained herein, drop-carrier particles <NUM> are formed from multiple material types into shaped particles with wetting surfaces that are strategically located, in some embodiments, on the interior of the drop-carrier particles <NUM>. For example, hydrophilic material is polymerized or crosslinked using light exposure on the interior cavity of the drop-carrier particle <NUM> while a separate hydrophobic material also polymerized or crosslinked using light surrounds the cavity or void as is illustrated in <FIG>. The drop-carrier particles <NUM> may be made from known polymer materials that can be polymerized or crosslinked using, for example, light-initiated polymerization as explained herein.

The drop-carrier particles <NUM> that are used to form the particle-drops <NUM> are sub-millimeter sized particles. Typically, the drop-carrier particles <NUM> have diameters or widths on the order of around <NUM>-<NUM> microns, although it should be appreciated that drop-carrier particles <NUM> of different sizes outside this specific range may also be used. While the embodiments described herein largely describe drop-carrier particles <NUM> having a hydrophilic interior region <NUM> and a hydrophobic exterior region <NUM>, it should be appreciated that these regions could be reversed with the interior region <NUM> being hydrophobic (or fluorophilic) and the exterior region <NUM> being hydrophilic. In such an embodiment, the fluid droplet <NUM> that is carried by the drop-carrier particle <NUM> would be a hydrophobic fluid such as oil while the continuous phase that surrounds the particle-drops <NUM> would be an aqueous solution.

In some embodiments, materials that comprise the hydrophobic exterior region <NUM> preferably will possess an interfacial tension with the continuous phase substantially close to zero. This enables mixing of the particle-drops <NUM> within the continuous phase without aggregation of the particle-drops <NUM> at their exterior surfaces. That is, the particle-drops <NUM> can remain well-suspended within the continuous phase. In order to form well-defined fluid drops <NUM>, the interfacial tension between the internal phase and interior surface or region <NUM> is less than interfacial tension between the internal phase and exterior surface or region <NUM>. In some embodiments a surfactant (e.g. Pluronic®, Pico-Surf™) is used to adjust the interfacial tensions between the phases to achieve these favorable conditions. Note that in this case the drop-carrier particle <NUM> still controls the shape and volume of the fluid drop <NUM>, which would vary over a much larger range with the use of a surfactant alone.

The drop-carrier particles <NUM> may be referred to as Janus particles because of their dual hydrophilic/hydrophobic surfaces. These Janus drop-carrier particles <NUM> can be designed with 3D shapes such that the drop-carrier particles <NUM> can encapsulate, support, and stabilize aqueous droplets <NUM> in the interior of the drop-carrier particles <NUM> while being suspended in an oil phase <NUM> to prevent coalescence of the droplets <NUM>. The interior hydrophilic region <NUM>, in some embodiments, can also be specifically functionalized to support nucleic acid barcodes or affinity capture reagents. For example, one or more biomolecules may be tethered (e.g., covalently attached to or through one or more linking moieties) to the surface of the interior region <NUM> of the drop-carrier particle <NUM>. As one illustrative example, antibodies may be bound to the interior hydrophilic region <NUM> of the drop-carrier particle <NUM> which is used to detect an antigen as explained herein.

Drop-carrier particles <NUM> can be easily mixed with small volumes of aqueous samples without complex protocols or instruments and moved between phases and solutions using gravitational, centripetal, or magnetic forces (for magnetic particle embedded drop-carrier particles <NUM> as explained herein). Similar to microfluidic droplets or microwells, particle-drops <NUM> can be incubated and reacted in oil-filled containers to perform a variety of chemical and biological reactions. Examples include, by way of illustration and not limitation, reverse transcription of RNA, nucleic acid amplification, enzymatic amplification, and other signal generation approaches. Reacted particle-drops <NUM> can be pooled in a new aqueous solution, or read out using standard microscopy, cost-effective wide-field lens-less imaging, or conventional flow cytometry devices; leading to low-cost complete solutions that can democratize digital molecular and single-cell assays in all research labs, and galvanize the development of point-of-care digital diagnostics that will ultimately improve health.

The hydrophilic interior region <NUM> of the drop-carrier particles <NUM> can vary in size and shape. The size of the drop-carrier particles <NUM> should be small enough that surface forces dominate (e.g., sub-millimeter) and control the assembly of fluid within the interior region <NUM> of the drop-carrier particle <NUM>, compared to gravity, fluid inertia, etc. The interior region size should be between about <NUM> micrometers and about <NUM> micrometers in an average linear dimension, defining a cavity with a holding volume between about <NUM> pL and about <NUM> nL. For example, a Bond Number (Bo), defined as the ratio of gravitational to surface tension forces preferably is smaller than unity (<NUM>). Here, <MAT>, where Δρ is the magnitude of the density difference between the interior and exterior liquid phases (e.g., water and oil), g is the acceleration due to gravity, L is a linear dimension of the interior hydrophilic region <NUM> of the drop-carrier particle <NUM>, and σ is the interfacial tension between the interior phase and the interior region <NUM>. The shape of the drop-carrier particle <NUM> should facilitate the entry of an interior liquid phase into the interior region <NUM> while preventing the assembly of a random number of multiple drop-carrier particles <NUM> around an interior liquid phase drop yielding uncontrolled and polydisperse volumes in a stabilized emulsion. The drop-carrier particle <NUM> shape preferably comprises an interior hydrophilic region <NUM> surrounded by an exterior hydrophobic region <NUM> over an angle of greater than <NUM>° around at least one axis. In other embodiments the shape of the drop-carrier particle <NUM> defines an interior hydrophilic region <NUM> surrounded by an exterior hydrophobic region <NUM> over an angle of greater than <NUM>° around at least one axis and an interior hydrophilic region <NUM> surrounded by an exterior hydrophobic region <NUM> over an angle of greater than <NUM>° around a second orthogonal axis. Exemplary designs with this characteristic are shown in <FIG> and <FIG>. In some embodiments the hydrophobic exterior region <NUM> surrounds the hydrophilic interior region <NUM> over an angle of at least <NUM>° around at least one axis. Exemplary drop-carrier particles <NUM> having hydrophobic exterior regions <NUM> surrounding interior regions <NUM> with an angle of <NUM>° around one axis are shown in <FIG>, <FIG>.

<FIG> illustrate another example of a drop-carrier particle <NUM> that includes a central or inner void <NUM> that is surrounded by a hydrophilic interior region <NUM> formed from hydrophilic material. The external or outer surface <NUM> of the drop-carrier particle <NUM> defines a hydrophobic region and is formed from a hydrophobic material. In this embodiment, the drop-carrier particle <NUM> is in the shape of a ring or annulus that has flat or planar top and bottom surfaces as best seen in <FIG>. This way, the drop-carrier particles <NUM> can be disposed between two optically transparent substrates (e.g., glass slides or glass slides with a hydrophobic surface coating) with the flat surfaces facing the two substrates. <FIG> illustrates a vial <NUM> containing a plurality of particle-drops <NUM> contained in an oil phase <NUM>.

<FIG> illustrate another example of a drop-carrier particle <NUM>. The drop-carrier particle <NUM> also includes a central or inner void <NUM> that defines a hydrophilic interior region <NUM>. The external or outer surface <NUM> of the drop-carrier particle <NUM> defines a hydrophobic region. In this embodiment, the external or outer surface includes a plurality of protrusions <NUM> that minimize the surface area of contact between drop-carrier particles <NUM> to prevent the drop-carrier particles <NUM> from aggregating together. <FIG> illustrates another embodiment of drop-carrier particles <NUM> that join together as illustrated in <FIG> to form a combined interior region <NUM> that is used to hold the fluid droplet <NUM> (not illustrated in <FIG>). In this embodiment, there are two crescent shaped drop-carrier particles 12a, 12b. The inner saddle-shaped surface <NUM> of the drop-carrier particles 12a, 12b is hydrophilic while the outer surface or region <NUM> is hydrophobic. Two of the crescent shaped drop-carrier particles 12a, 12b fit or nest together to form a single particle assembly <NUM> in which the combined hydrophilic surfaces <NUM> enclose a void or region <NUM> that holds the aqueous droplet <NUM>. In an alternative embodiment, the two different drop-carrier particles <NUM> may be oriented orthogonal to one another (i.e., rotated orthogonally to one another generally <NUM>°) to form the single particle assembly <NUM>. Various lock-and-key fitting arrangements between drop-carrier particles <NUM> can be utilized.

<FIG> illustrates another embodiment of a drop-carrier particle <NUM>. In this embodiment, the drop-carrier particle <NUM> contains a hydrogel in the interior portion <NUM> of the drop-carrier particle <NUM>. Examples of hydrogel materials that can be formed in the interior portion <NUM> of the drop-carrier particle <NUM> include polyethylene glycol (PEG)-based hydrogels such as poly(ethylene glycol) diacrylate (PEGDA). The hydrogel may be dried or partially wetted hydrogel material that is then exposed to an aqueous solution, which may also contain analytes, reagents, affinity probes or reagents, and the like, which can then enter the hydrogel void spaces. The porosity of the hydrogel may be tuned to control the ingress or egress of molecular species. For example, porosity may be tuned to trap molecules within the hydrogel or prevent larger molecules from entering the hydrogel. In some embodiments, the hydrogel may be biotinylated using a biotin modified PEG precursor. The biotinylated surface may be used to bind other biomolecules on or within the hydrogel. <FIG> illustrates one embodiment where protein (P) or nucleic acid (NA) molecules are loaded within the hydrogel filled interior portion <NUM> of the drop-carrier particle <NUM>. The hydrogel-containing particle-drops <NUM> can then be transferred to an oil solution to form the emulsion.

<FIG> illustrates another embodiment of a drop-carrier particle <NUM>. In this embodiment, a hydrogel layer <NUM> coats an interior portion <NUM> (e.g., inner surface) of the drop-carrier particle <NUM>. In this embodiment, the hydrogel layer <NUM> coats an inner surface of the drop carrier particle <NUM> but leaves a void <NUM> which may accommodate a fluid droplet <NUM>. The hydrogel layer <NUM> may contain various moieties or biomolecules therein. For example, as seen in <FIG>, nucleic acids or antibodies may be contained within the inside of the hydrogel layer <NUM>. Alternatively, or in conjunction with interior biomolecules or other moieties, surface bound nucleic acids, antibodies, or antigens may be located at the surface of the hydrogel layer <NUM>.

<FIG> illustrates another embodiment of the invention. In this embodiment, the drop-carrier particle <NUM> includes magnetic particles <NUM> contained in the exterior region <NUM> (e.g., the outer hydrophobic layer of the drop-carrier particle <NUM>). These magnetic particles <NUM> may be micro-sized (e.g., having a width or diameter of <NUM> and less than <NUM>) or nanometer-sized (e.g., having a size between <NUM> and <NUM>). The magnetic particles <NUM> may be made from iron oxide or other ferromagnetic materials. The magnetic particles <NUM> may be contained in one or more of the polymer or pre-polymer components that is flowed through the microfluidic device during the drop-carrier particle <NUM> formation process as explained herein. The magnetic particles <NUM> enable the drop-carrier particles <NUM> to be manipulated by an externally applied magnetic field which could be a permanent magnet or an electromagnet. For example, drop-carrier particles <NUM> may be pulled (or pushed) through various solutions (e.g., oil-based fluid, aqueous-based fluid, rinse fluids, wash fluids, reagent fluids, interfaces between two immiscible fluids) using an applied magnetic field. <FIG> illustrates a covalently linked fluorophore <NUM> to the outer surface of the drop-carrier particle <NUM>.

<FIG> illustrate another embodiment of a drop-carrier particle <NUM>. In this embodiment, the drop-carrier particles <NUM> are labelled with a unique indicia <NUM> that identify the particular drop-carrier particle <NUM>. The unique indicia <NUM> may also be referred to as a "barcode" because it provides a unique identifier for the drop-carrier particle <NUM>. The unique indicia <NUM> may be embodied in a number of different manifestations. For example, the unique indicia <NUM> may include holes or apertures formed in the drop-carrier particles <NUM>, shapes, patterns, or surface features formed on or in the drop-carrier particles <NUM>, fluorescent labels, markers, or the like. It should be appreciated that while the unique indicia <NUM> identifies a particular drop-carrier particle <NUM>, multiple different drop-carrier particles <NUM> may, in some embodiments, share the same unique indicia <NUM>. For example, a first plurality of drop-carrier particles <NUM> may contain a certain antibody bound or contained therein. All of these drop-carrier particles <NUM> may be labelled with the same unique indicia <NUM> so as to reflect that each of these drop-carrier particles <NUM> contains the same antibody. Of course, in other embodiments, each different drop-carrier particle <NUM> may contain different unique indicia <NUM>.

<FIG> illustrate another embodiment of a drop-carrier particle <NUM>. In this embodiment, the drop-carrier particle <NUM> is in the shape of a cylinder with the interior region <NUM> being hydrophilic and an exterior region <NUM> being hydrophobic. As seen in <FIG>, a central or inner void <NUM> is surrounded by a hydrophilic interior region <NUM>. An aqueous solution is isolated into a fluid droplet <NUM> (not illustrated in <FIG>) and is protected within the interior region <NUM> in contact with the interior hydrophilic material when contained in an oil solution. This embodiment is created through the flow of precursor materials through a series of nested cylindrical tubes to create a concentric layered flow of precursor materials (e.g. from inner to outer material in four concentric tubes, PEGDA, PEGDA+PI, PPGDA+PI, PPGDA) that can be photopolymerized downstream through a mask containing at least one rectangular opening as described herein.

<FIG> illustrate another embodiment of a drop-carrier particle <NUM>. In this embodiment, the drop-carrier particle <NUM> is created by consecutive deposition steps on a sacrificial spherical particle creating an object surrounding a spherical void, as described herein. The exterior region <NUM> is illustrated as the outer layer and forms the hydrophobic exterior region. The interior region <NUM> forms the interior hydrophilic region of the drop-carrier particle <NUM>. Aqueous solution is isolated and protected within the interior region <NUM> in contact with the interior hydrophilic material when mixed with aqueous solution and oil. <FIG> illustrates a sectional view in order see the internal structure of the interior region <NUM> of the drop-carrier particle <NUM>.

<FIG> illustrates another embodiment of a drop-carrier particle <NUM>. In this embodiment, the drop-carrier particle <NUM> includes tabs or flaps <NUM>. The tabs or flaps <NUM> are flexible and move in response to interfacial tension of the fluid droplet <NUM> when located in the drop-carrier particle <NUM> (fluid droplet not illustrated inside drop-carrier particle <NUM> in <FIG>). The exterior region <NUM> forms the hydrophobic exterior region. The interior region <NUM> forms the interior hydrophilic region of the drop-carrier particle <NUM> and is where the fluid droplet <NUM> is held when loaded therein. In this embodiment, upon encapsulating an aqueous fluid droplet <NUM> within the interior region <NUM> of the drop-carrier particle <NUM>, the flexible tabs or flaps <NUM> bends from the state of <FIG> to the state of <FIG> (in the direction of arrow A) due to the interfacial tension of the aqueous fluid droplet <NUM> with the exterior hydrophobic phase. The bent shape minimizes the total energy of the system including elastic and interfacial energy.

<FIG> illustrates another embodiment of a drop-carrier particle <NUM>. In this embodiment, the drop-carrier particle <NUM> has a plurality of tabs or flaps <NUM> that resemble flower petals that fold around the fluid droplet <NUM> (not illustrated) when encapsulating the aqueous phase. Arrow B illustrates how the drop-carrier <NUM> tabs or flaps <NUM> that contain the hydrophilic interior region <NUM> fold to envelope and encapsulate the aqueous phase. The hydrophobic exterior region <NUM> is maintained on the outside of the drop-carrier particle <NUM> after folding. This embodiment can be fabricated using standard surface micromachining and photolithography processes known in the art to create multilayered 2D structures using masking with a photoresist layer. For example a sacrificial layer is spin coated on a wafer (e.g., dextran solution) followed by evaporation of a gold layer (e.g., <NUM>) and chemical vapor deposition of a silicon dioxide layer (<NUM>) (optionally including an adhesion layer between silicon dioxide and gold, e.g., titanium <NUM>). The three-layer structure is photopatterned in the 2D shape shown unfolded in <FIG>, and the silicon dioxide and gold layers are etched. The dextran layer is then released in water to release the <NUM>-layer gold-silicon dioxide particle. The gold layer <NUM> is then coated with self-assembled monolayers to impart hydrophobic (using alkylthiol) or fluorophilic (perfluoralkylthiol) properties to the exterior layer <NUM>. The silicon dioxide hydrophilic interior layer <NUM> can then interact with aqueous solutions. In addition to evaporation and chemical vapor deposition processes, spin-coating or dip-coating of polymer materials with different surface properties or reactivity can be used to create the multi-layer structure.

The drop-carrier particles <NUM> can be designed in a manner such that bending around one or more axes requires reduced force by including thinned regions or regions with long lever arms (e.g., tabs or flaps <NUM>) that can bend with lower applied forces and torques. Drop-carrier particles <NUM> can also be designed to fold-up along more than one axis such as in origami folding to support interior aqueous droplets <NUM> that predominantly only interact with an interior region <NUM> hydrophilic phase. Drop-carrier particles <NUM> that bend to minimize interfacial energy have advantages in stabilizing particle-drops <NUM> once they are formed by undergoing this shape change which would require a higher activation energy due to thermal, mechanical or chemical means to overcome. Additionally there is less exposed surface area of the internal aqueous phase for interaction, further stabilizing the interior droplet <NUM>.

<FIG> illustrates an illustrative process for loading drop-carrier particles <NUM> with fluid droplets <NUM> to generate particle-drops <NUM>. In this embodiment, lyophilized powder of drop-carrier particles <NUM> are re-suspended in an oil phase <NUM>. This may include, by way of example, toluene, decanol, polypropylene glycol, lauryl alcohol, botanical oils, light mineral oil, heavy mineral oil, silicone oil, fluorinated oil (e.g., Fluorinert™ FC40, Novec™ <NUM>, Krytox™ oils). Next, the re-suspended drop-carrier particles <NUM> are transferred to a hydrophobic vessel (e.g., a Rain-X® coated glass vial, or low adhesion microwell plate). Next, the aqueous sample <NUM> is introduced. The aqueous sample <NUM> may contain analytes, reagents, biomolecules, stains, dyes, reporters that are to be loaded into the fluid droplet <NUM> volume that is loaded in the drop-carrier particle <NUM>. As seen in <FIG>, the particle and aqueous suspension is then mixed and/or vortexed. The mixture is then subject to centrifugation to load the aqueous phase (i.e., fluid droplets <NUM>) into the drop-carrier particles <NUM> to form particle-drops <NUM> suspended in the continuous oil phase. Optionally, if the particle-drop <NUM> is magnetic, an externally applied magnetic field can be used to mix the suspension and pull particle-drops into the continuous phase. While <FIG> describes the drop-carrier particles <NUM> first being re-suspended in an oil phase <NUM> prior to introduction of the aqueous sample <NUM>, the order may be reversed. For example, the drop-carrier particles <NUM> may be added first to the aqueous sample <NUM> followed by introduction of the oil phase <NUM>. As seen in <FIG>, the now formed particle-drops <NUM> may be used in one or more assays described herein. This may include incubation and reaction of the particle-drops <NUM> alone or the exchange or dilution with a new aqueous solution. Free drops of aqueous sample <NUM> not associated with drop-carrier particles <NUM> can be removed from the top of the vessel due to a difference in density or size (due to drop coalescence) compared to particle-drops <NUM>. After reaction, the particle-drops <NUM> may be imaged using one or more of the imaging modalities described herein. The particle-drops <NUM> may also be merged into a larger aqueous solution after the reaction (or after imaging) and subject to analysis as described in detail herein.

In one illustrative embodiment, particle-drops <NUM> may be generated in four (<NUM>) steps. First, drop-carrier particles <NUM> are taken out of particle stock solution and dried to remove ethanol. Second, drop-carrier particles <NUM> are resuspended in a proper oil phase solution, which has significant difference in interfacial energy between hydrophilic and hydrophobic layers. There are several options of organic solutions for the oil phase, including a toluene-ethanol-mix (ratio of <NUM>:<NUM>), decanol, and PPGDA. The particle-laden oil suspension is transferred to a <NUM> glass vial, which is treated by Rain-X® coating for two (<NUM>) days. Third, an aqueous phase (water) with a volume of the same order of magnitude as the multiplication of the drop-carrier particle <NUM> number and each individual cavity volume for a drop-carrier particle <NUM> is injected into the oil solution. Similarly, the integrated cavity volumes for a plurality of drop-carrier particles <NUM> can also be matched to a target aqueous sample volume. The combined solution is then pipetted up and down vigorously. Fourth, the vial is centrifuged for five (<NUM>) minutes at <NUM> rpm to bring the aqueous solution into the cavities of the drop-carrier particles <NUM>, generating particle-drops <NUM> that settle on the bottom of the glass vial.

The particle-drops <NUM>, in one embodiment, can then be incubated and reacted with one or more reactants. These reactants may be contained in separate aqueous solutions that the particle-drops <NUM> can be passed through or exposed to (e.g., to capture molecules or cells of interest with affinity reagents). Additional solutions may be exchanged that contain reagents or washes. The particle-drops <NUM> can then be subject to optical readout. For example, the particle-drops <NUM> may be on an optically transparent substrate such as glass or the like and imaged with an imaging device. The particle-drops <NUM> may also be loaded into wells in a microtiter plate or the like which can then be visualized. In some embodiments, the particle-drops <NUM> may be run through a conventional flow cytometer or fluorescence activated cell sorter (FACS) for the screening and sorting of particle-drops <NUM>. Alternatively, the emulsions can be broken and then molecules contained therein amplified and/or analyzed using various optical or nucleic acid sequence-specific detection schemes.

There are no commercially available particles with the desired characteristics or commercially available manufacturing methods for particles in the sub-millimeter length scale. The drop-carrier particles <NUM> described herein can be manufactured using a novel fabrication method, called high-throughput Optical Transient Liquid Molding (OTLM). In this method, microfluidic posts, pillars, or other protuberances are formed in a microfluidic channel and used to generate complex sub-millimeter scale particles with shapes that consist of the orthogonal intersection of horizontally and vertically-extruded 2D patterns in a highspeed manner. An example of OTLM particle fabrication techniques is found in International Patent Application Publication No. <CIT>,.

The horizontally and vertically-extruded 2D patterns are respectively determined by the cross-sectional shape of a flowstream of photo-crosslinkable polymer pre-cursor and the shape of an optical mask that is used to generate the other orthogonal cross-section. Inertial flow engineering is used to sculpt a single-phase flow stream into a complex and cross-sectional shape in a microchannel using the flow past a sequence of defined microstructures. The shape of the sculpted flow may be user-defined and programmed using software to define the microfluidic channel with the particular micropillar sequence necessary to create the final shape. For example, Wu et al. , which is incorporated by reference herein, describe a software µFlow ( available at http://biomicrofluidics. com/software. php) that allows for the design of 2D flow shapes with a simple graphical user interface (GUI) that can be used to predict and design particle shapes. See <NPL>).

Flowing through this microstructured channel creates a sculpted flow stream. The flow is then stopped using a pinch valve and the stream is illuminated using patterned UV light through an optical mask to achieve a complex 3D drop-carrier particle <NUM>. Automated control and microchannel design with an elongated illumination region downstream allows for a high production rate of ~<NUM>,<NUM> drop-carrier particles <NUM> per hour. Several embodiments of the drop-carrier particles <NUM> require concentric enclosed topologies, which can be achieved in a flow stream using recirculating secondary flows around offset pillars or posts <NUM>. Another flow channel design which achieves recirculating secondary flows which can be used for creating the concentric enclosed topology is a curving channel in which Dean flow creates circulation. These designs allow bending of the initial main co-flow from straight co-flowing regions to 2D full or partial encapsulation patterns consisting of concentric hydrophilic and hydrophobic layers. In one embodiment, the inner region <NUM> that holds a liquid compartment is formed in the flow stream by deforming a precursor co-flow with hydrophilic and hydrophobic polymer precursors that are flowing side by side into a curved or encapsulated shape with concentric regions consisting of an interior void, hydrophilic, and hydrophobic layers. The orthogonal UV exposure pattern with protruding shapes is designed to avoid the aggregation of drop-carrier particles <NUM> or introduce physical shape-based indicia <NUM>. This pattern is exposed through a mask <NUM> which contains the repeating pattern in a row along the flow direction to make many identical drop-carrier particles <NUM>.

Designs can include protruding shapes that avoid the aggregation of drop-carrier particles <NUM>, structured tabs, flaps, or overhangs <NUM> that optimize surface energy of particle-drops <NUM>, or indicia <NUM> for specific sets of drop-carrier particles <NUM> with unique chemical properties. Following synthesis, drop-carrier particles <NUM> can be stored as a dried or lyophilized powder or as a suspension in oil or aqueous solution. These complex 3D shapes are not possible with approaches like stop-flow lithography, and unlike stop-flow lithography which requires an oxygen quenching layer that prevents polymerized particles from sticking to the microchannel wall, the OTLM method enables fabrication of particles without an oxygen inhibition layer or specific channel wall materials that provide such a layer because the pre-polymer solution is sculpted to occupy regions away from the channel walls.

<FIG> illustrates a schematic or system level view of a microfluidic-based system <NUM> for the OTLM fabrication of drop-carrier particles <NUM>. The system <NUM> includes a microfluidic device <NUM> that includes a microfluidic channel <NUM> formed therein that includes a plurality of input channels <NUM> that, as explained herein, are used to deliver various polymer precursor components needed to make the final drop-carrier particles <NUM>. The precursor components include, for example, a hydrophilic precursor polymer, a hydrophobic precursor polymer, and a photoinitiator mixed with both solutions. As seen in <FIG>, one or more syringe pumps <NUM> are used to pump the pre-polymer components and photoinitiator/pre-polymer mixtures into the microfluidic channel <NUM>. The microfluidic channel <NUM> includes a sequence of posts or pillars <NUM> located in an upstream region A of the microfluidic channel <NUM> that, collectively, are used to generate the sculpted flow. The downstream region B of the microfluidic channel <NUM> is where ultraviolet light exposure takes place to crosslink the precursor polymers to form the drop-carrier particles <NUM>. The outlet of the microfluidic channel <NUM> is coupled to a pinch valve <NUM> operated by a microcontroller <NUM> that is actuated to stop flow within the microfluidic channel <NUM> during the light exposure step as described below. After the drop-carrier particles <NUM> are formed during the crosslinking process, the drop-carrier particles are collected in a collection vessel <NUM> (e.g., vial). An ultraviolet collimated light source <NUM> is provided and illuminates a mask <NUM> with a computer-controlled shutter <NUM>. The mask <NUM> includes one or more specifically shaped holes or apertures <NUM> (multiple drop-carrier particles <NUM> can be formed from a single exposure) formed therein that is used to define the shape of the drop-carrier particle <NUM> along one orthogonal axis. In some embodiments, the mask <NUM> may be secured to a z-adjust stage to control the size of the UV light projection on the shaped flow. The microfluidic channel <NUM> (or microfluidic device <NUM> that contains the microfluidic channel <NUM>) may include a xy-translation/rotation stage to align the microfluidic channel <NUM> with the UV crosslinking optical path defined by the plurality of apertures <NUM> in the mask <NUM>. A computer <NUM> is provided with software loaded thereon (e.g., LabVIEW™) that interfaces with and controls the syringe pump(s) <NUM>, pinch valve <NUM> (via microcontroller <NUM>), and collimated ultraviolet light source <NUM>, shutter <NUM>.

In one embodiment, to generate a concentric hydrophilic interior/hydrophobic exterior shape in the cross section of the polymer precursor stream, a co-flow with four (<NUM>) fluid streams, which include polypropylene glycol) diacrylate (PPGDA, MW-<NUM>) (the hydrophobic precursor in <FIG>), PPGDA added with photoinitiator (PI, <NUM>-hydroxy-<NUM>-methylpropiophenone) (the hydrophobic precursor + PI in <FIG>), poly(ethylene glycol) diacrylate (PEGDA, MW-<NUM>) added with PI (the hydrophilic precursor + PI in <FIG>), and PEGDA (the hydrophilic precursor in <FIG>). In this embodiment, these inputs are pumped into the microfluidic channel <NUM> with designed microstructures, e.g., posts or pillars <NUM>. The microfluidic channel <NUM> has, in one embodiment, a width of <NUM> micrometers and a height of 300micrometers, while the microstructures consist of six (<NUM>) pillars in series each having a diameter of <NUM> micrometers. The microfluidic channel <NUM> with the posts or pillars <NUM> is made up entirely with the same material, which is polydimethylsiloxane (PDMS) so the wetting properties of interface between PPGDA and PEGDA is the same on top and bottom walls and the deformation of the flow stream is symmetrical in terms of the middle plane of the microfluidic channel <NUM>.

In another embodiment, the hydrophilic precursor may include PEGDA while the hydrophobic precursor may include <NUM>,<NUM>-Hexanediol diacrylate (HDA), (<NPL>, available from Sigma-Aldrich, product number <NUM>,). PEGDA and HDA are used with an ultraviolet crosslinked transparent thiolene-based optical adhesive, NOA89 available from Norland Products, Inc. which is also used as the photoinitiator. Thus, with reference to <FIG>, the order of the four (<NUM>) input streams from top to bottom includes: HDA (top syringe), NOA89 (second from top syringe), PEGDA + <NUM>-hydroxy-<NUM>-methylpropiophenone (second from bottom syringe), and PEGDA (bottom syringe).

In another embodiment, the hydrophilic precursor may include PEGDA while the hydrophobic precursor may include a mixture of HDA and lauryl acrylate (<NPL>, available from Sigma-Aldrich, product number <NUM>) with lauryl acrylate ranging from between <NUM> to <NUM>% of the mixture on a volume basis. The photoinitiator (PI) used in this embodiment is <NUM>-hydroxy-<NUM>-methylpropiophenone (<NPL>, Darocur <NUM>, product number <NUM>, Sigma-Aldrich). Thus, with reference to <FIG>, the order of the four (<NUM>) input streams from top to bottom includes: HDA + lauryl acrylate (top syringe), HDA + lauryl acrylate + PI (second from top syringe), PEGDA + PI (second from bottom syringe), and PEGDA (bottom syringe).

A standard soft lithography process known to those skilled in the art is utilized to make the sealed microfluidic channel <NUM> with a modification of the bottom material, which is a glass slide having a <NUM> thickness with a thin (e.g., less than <NUM> microns) layer of PDMS. The flow rate of the hydrophilic portion (PEGDA+PI) is designed to be one fourth of the hydrophobic portion (PPGDA+PI) so particle-drops <NUM> formed inside the cavity can be preferentially surrounded and protected by an exterior hydrophobic layer. The PPGDA and PEGDA solutions used are diluted in ethanol to become <NUM> and <NUM> percentage of the polymer precursor respectively to match the liquid density and reduce the viscosity and the flow resistance required to drive the flow (i.e., volume fraction of PEGDA:ethanol=<NUM>:<NUM> and PPGDA: ethanol=<NUM>:<NUM>).

To simultaneously photo-crosslink the two polymer regions, the curing time for PPGDA and PEGDA is optimized to be within one (<NUM>) second by adding <NUM> and <NUM> percentage of PI respectively. The flow rates of PPGDA, PPGDA + photoinitiator, PEGDA + photoinitiator, and PEGDA are <NUM>, <NUM>, <NUM>, and <NUM>/min respectively. In addition to the design of the polymer precursor cross-section, there are an infinite degrees of freedom to design the second pattern exposed on top of the flow stream to determine the final 3D shape of the drop-carrier particles <NUM>. The shape of the optical mask <NUM> for one demonstration is designed to be a rectangular slit with dimensions of <NUM> micrometers parallel to the flow direction and <NUM> micrometers perpendicular to the flow direction respectively. There are more than a hundred transparent apertures <NUM> (e.g., slits) designed to be in an array on a chrome mask <NUM>. To accelerate the speed of photopolymerization of PPGDA and PEGDA, the power of UV light source <NUM>, which is collimated by an adaptor, is designed to be ~4W/cm<NUM> on the optical mask <NUM>.

In one embodiment, the microfluidic channel <NUM> is placed on the stage upside down. The inlet and outlet are connected to syringes installed on the syringe pumps <NUM> and pinch valve <NUM>, respectively. The optical mask <NUM> fixed with the holder is moved down using the z-translation axis to make contact with the glass of the microfluidic channel <NUM> with hard contact. The angle and xy location of the stage are tuned to ensure every slit is located along the same designed lateral position of the flow stream, i.e., the microfluidic channel <NUM>. Once all static alignments are finished, the polymer liquid precursor is pumped to start dynamic particle fabrication. In one embodiment, two syringe pumps <NUM> are utilized to introduce the four (<NUM>) streams into the microfluidic channel <NUM> at total flow rate of <NUM>/min to develop a precursor stream with concentric cross section. After five (<NUM>) minutes of flow to reach steady and fully developed channel flow, the pumps <NUM> are stopped and within -<NUM> micro seconds the pinch valve <NUM> downstream squeezes the tubing connecting to the outlet of the microfluidic channel <NUM> to fully stop the flow in ~<NUM> second. The shutter opens for one (<NUM>) second to apply a short period of UV exposure in the area of slits. Next, the pinch valve <NUM> is released and the pumps <NUM> are re-started to push the liquid again to re-develop the flow stream in the microfluidic channel <NUM>. The operation above is repeated multiple times by an automated LabVIEW™ program until the desired numbers of the drop-carrier particles <NUM> are reached.

After in-channel fabrication, all drop-carrier particles <NUM> are flushed out of the microfluidic channel <NUM> and collected together with waste of uncured precursor in a downstream vessel <NUM> such as a <NUM> conical tube. All drop-carrier particles <NUM> are purified by going through at least four (<NUM>) washes (including the time when the drop-carrier particles <NUM> are in the waste liquid) in ethanol. The rinse process includes centrifuging the tube to bring all drop-carrier particles <NUM> down to the bottom, removing the supernatant gently to avoid taking out liquid with drop-carrier particles <NUM>, and then flushing the tube with <NUM>~<NUM> ethanol. Later, the drop-carrier particles <NUM> are stored in ethanol at room temperature as a particle stock solution. There is no significant degradation and loss of functionality over more than two months of storage.

The hydrophilicity/hydrophobicity of the drop-carrier particles <NUM> can be optimized by changing the type or concentration of the precursor monomer. There are two main design variables for the photo-crosslinkable materials in the precursor co-flow which yield tradeoffs in the fabrication system: liquid viscosity and surface tension. Pure liquid precursors have generally high viscosity which increases the pressure to drive the flow sufficiently fast to achieve inertial flow shaping. Diluting the polymer precursors in solvents can reduce viscosity but hydrophilic and hydrophobic precursors are typically immiscible to each other once diluted in appropriate solvents. Alternative approaches to shape viscous polymer precursor streams at lower flow rates using herringbone or slanted grooves in the upper or bottom walls of the microfluidic channel <NUM> can also be used. These structured microfluidic channels <NUM> can create circulating flows at lower flow rates since they do not rely on fluid inertia to shape the flow.

Two solutions have been developed to design the precursor co-flow: a high-pressure flow method and a surface-energy-gradient flow method. In the high-pressure flow method, a co-flow with two miscible pure precursors (i.e. no dilution) is pumped with different hydrophilicity (e.g., poly(ethylene glycol) diacrylate/ fluorinated poly(ethylene glycol) acrylate, polypropylene glycol) diacrylate or Norland Optical Adhesive (NOA) UV adhesive) by applying higher pressure and designing a longer sequence of microstructures to provide sufficient flow deformation (a U-turn channel may be added to expand the downstream length microfluidic channel <NUM>). <FIG> illustrates the cross-sectional flow profiles PPGDA, PPGDA + PI, PEGDA + PI, and PEGDA undergoing high-pressure co-flow in the microfluidic channel <NUM>. The upper, non-shaped flow illustrates the cross-sectional profile prior to being shaped by the posts or pillars <NUM>. The lower, shaped flow illustrates the cross-sectional profile after being shaped by the posts or pillars <NUM>. In the surface-energy-gradient flow method, a co-flow is created with multiple flow streams where each stream is miscible or is immiscible but with a relatively low surface tension to the material streams neighboring it. The co-flow can be typically configured by merging three streams in order: diluted hydrophilic precursor, pure hydrophilic precursor (or hydrophobic precursor, miscible with each other), and diluted hydrophobic precursor.

In an alternative particle manufacturing process, if drop-carrier particles <NUM> cannot be manufactured using multiple materials with different hydrophobicity, or the hydrophobicity differences are not sufficient to create a stable emulsion one can use a previously demonstrated approach to create PEG particles with spatially varying chemistries and perform a second reaction after particle creation to introduce hydrophobic groups. For example, one can use PEG diacrylate co-flowed with PEG diacrylate / PEG acrylate succinimidyl carboxymethyl ester (JenKem Technology USA, Plano, TX) on the outer region. Then one can react the amine-reactive polymerized particles with long-chain amino alkanes (e.g., hexadecylamine) or amino fluoroalkanes to locally create a hydrophobic layer or shell over the outer part of the particle.

The biotinylation of the interior region <NUM> that forms the hydrophilic contact region is demonstrated using a modified chemical composition of the precursor in which biotinylated PEG precursors may be dosed into the hydrophilic layer to allow for the attachment of affinity reagents. A biotinylation solution is made by dissolving commercially available or synthesized biotin-PEG-acrylate in ethanol with <NUM>% (v/v) DMSO. The final precursor for in-channel fabrication is made of pure PEGDA, ethanol, and this biotinylation solution. The corresponding volume ratio depends on the degree of biotinylation for various applications but the sum of the volume of ethanol and biotinylation solution is kept to be <NUM>% of the total volume. Alternatively, biotinylation of the drop-carrier particle <NUM> may occur post-fabrication.

Molecules can be added to the hydrophilic interior region <NUM> of the drop-carrier particles <NUM> through covalent linkage or electrostatic association. For example, for a polyethylene glycol based hydrophilic region, standard chemistries like NHS-esters, acrylates, vinyl-sulfones, or maleimide groups introduced into the PEG backbone can be used to covalently link DNA, proteins, fluorophores, or other molecules. Alternatively, long DNA or other molecules can be introduced during the polymerization process that remain entangled or electrostatically adsorbed to the hydrophilic polymer matrix. Similarly, drop-carrier particles <NUM> could be soaked into reagents to enable entry within the hydrogel matrix, prior to lyophilizing. Drop-carrier particles <NUM> with the same indicia <NUM>, molecular barcodes or affinity reagents can be fabricated in a single batch and then combinations of these batches can be mixed together to obtain a mixture of drop-carrier particles <NUM> each with unique properties. Alternatively, molecular barcodes can be introduced into particles through split and recombine synthesis approaches to create nucleic acid barcodes.

There are several possible methods of using the particle-drops <NUM> to perform digital assays. Drop-carrier particles <NUM> starting as a lyophilized or dried powder can be added to an aqueous sample or vice versa. The quantity of drop-carrier particles <NUM> should be controlled such that the aqueous sample volume is approximately equal to the void volume that can be supported by the drop-carrier particles <NUM>. This allows for the majority of the sample to be associated with the drop-carrier particles <NUM> without loss of sample solution (i.e., high efficiency). In some cases a smaller amount of drop-carrier particles <NUM> may be used if only a fraction of the sample is desired to be analyzed, or drop-carrier particles <NUM> include affinity reagents to capture specific molecules from the solution (e.g., proteins or antigens with antibodies or aptamers, nucleic acids with complementary sequences or poly-T sequences to capture mRNA). An oil or other hydrophobic solvent can then be added to the particle-drop <NUM> solution at a volume to completely surround each particle-drop <NUM> with the hydrophobic continuous phase solution (volume fractions of > <NUM>:<NUM> continuous: disperse phase are best).

The sample of particle-drops <NUM> can then be suspended/dispersed in the continuous phase through a variety of methods. The solution could be vortexed, centrifuged, shaken or mixed. Magnetic force can be applied to pull magnetically-embedded drop-carrier particles <NUM> into the oil phase. If the oil phase is denser than the aqueous phase, and drop-carrier particles <NUM> are even more dense, they can be centrifuged to pass them into the oil phase. Suspended particle-drops <NUM> in the oil phase can then be reacted using reagents present in the aqueous sample solution or attached or eluting from the drop-carrier particles <NUM>. Reactions can be initiated through thermo-cycling e.g., for digital-PCR, other temperature increases (e.g., digital LAMP), or light activation.

In some embodiments, particle-drops <NUM> can be passed through separate aqueous solutions to first capture molecules or cells of interest with affinity reagents and then replace solutions with reagent solutions. Particle-drops <NUM> can be passed between solutions using magnetic force, centrifugation, or dilution or exchange with new solutions. The reactions in particle-drops <NUM> can be visualized or read-out using any number of types of optical systems. The suspension of particle-drops <NUM> could be spread on an optically transparent slide and compressed between cover-glass to readout an optical signal from each particle-drop <NUM>. Alternatively, the particle-drops <NUM> suspended in oil can be passed through a microfluidic channel and analyzed/and or sorted using flow cytometry optical setups and sorting approaches known in the art. Particle-drops <NUM> sized appropriately (e.g., < <NUM> micrometers in a long dimension) can be exchanged back into an aqueous solution (with or without the addition of surfactant) to pass through standard flow cytometer systems in a related embodiment. Particle-drops <NUM> may also be collected in wells of a microtiter plate. Fluorescent signal for nucleic acid amplification within particle-drops <NUM> can be accomplished using intercalator dyes or specific molecular affinity probes with quencher/fluorophore pairs. For protein recognition, an antibody-conjugated with an enzyme that turns over a fluorogenic or chromogenic substrate can be used. Other optical readout approaches known in the art for digital assays can be used in a similar manner for Particle-drops <NUM>. In the event that drop-carrier particles <NUM> coalesce, small amounts of surfactant (e.g., <NUM>-<NUM>% Pluronic) can be added following formation of the particle-drop emulsion <NUM> to further stabilize the separate aqueous compartments.

As one alternative manufacturing method to create drop-carrier particles <NUM>, spherical particle templates may be used to create particles of the type illustrated in <FIG>. In this method, spherical particle templates are arranged on a substrate (e.g., a silicon wafer) to pack in a regular array. For example, polystyrene particles of <NUM> micrometers in diameter create a close packed layer on a silicon wafer substrate. A first layer of silicon dioxide is deposited using low pressure chemical vapor deposition to a thickness of <NUM>. A layer of gold (<NUM>) is then evaporated on top of the silicon dioxide. The polystyrene particles are then dissolved away using a solvent (e.g., toluene) to leave a radially symmetric concentric shell structure with an internal silicon dioxide region and external gold region. The gold region or silicon dioxide region can then be selectively functionalized to impart hydrophilicity/hydrophobicity. For example, coating with a long-chain alkanethiol (<NUM> to <NUM> carbon length) forms a self-assembled monolayer on the gold layer which increases hydrophobicity, while the silicon dioxide remains hydrophilic creating a drop-carrier particle <NUM> with amphiphilic structure that can support a droplet of aqueous fluid <NUM> in the internal region <NUM>, and can be suspended in an oil phase.

<FIG> illustrates the use of particle-drops <NUM> for rapid digital assays using a sample. The sample may include cells, proteins, nucleic acids, and the like. Following rapid fractionation and exposure to the sample solution, solutions containing particle-drops <NUM> can be heated to perform amplification (e.g., nucleic acid amplification) and generate an optical signal (e.g., intercalator fluorescence). These signals can be read-out using low cost Smartphone-based fluorescence imagers <NUM> such as that disclosed in <CIT>, which is incorporated by reference, traditional flow cytometers <NUM>, or other microscope-based devices <NUM>. For example, in one exemplary workflow, a volume-calibrated number of drop-carrier particles <NUM> are introduced to a reaction mix with purified nucleic acids in a tube or other sample holder. Oil is added to the tube/sample holder and emulsified by gentle mixing. The reaction is then performed in the particle-drop <NUM> emulsion. The particle-drops <NUM> can then be spread on an optically transparent substrate (e.g., slide) and imaged using, for example, a mobile-phone based fluorescent imaging device.

In some cases, e.g., single-cell barcoded RNA-seq, following nucleic acid binding it is desirable to break the emulsion and exchange solutions to perform reverse transcription and amplification of the resultant cDNA. The pool all of the barcoded products can then be used to perform a single sequencing run at low cost. Breaking of a particle-drop <NUM> emulsion is possible through a variety of approaches. Using photodegradable crosslinkers during the fabrication of the drop-carrier particles <NUM> can allow photo-induced degradation of the drop-carrier particles <NUM> and merging of the aqueous solutions. Alternatively, magnetic or dense drop-carrier particles <NUM> can be transferred into a neighboring aqueous solution within a centrifuge or microfuge tube using magnetic force or centrifugation. Surfactants can also be introduced to the suspension that adsorb onto the hydrophobic exterior regions <NUM> of the drop-carrier particles <NUM> to make them more hydrophilic and break the particle-drop <NUM> suspension.

<FIG> illustrates a first protocol or method used to achieve single-cell RNA-sequencing using the particle-drop system described herein. The method uses the following operations: (<NUM>) prepare a single-cell suspension solution with a known cellular concentration; (<NUM>) mix the single-cell suspension solution and a solution of separate microspheres or microbeads with barcoded primers (or hydrogel microparticles with photocleavable barcoded primers) (i.e., a barcoding material); (<NUM>) add a dried drop-carrier particle <NUM> powder into the mixed solution and vortex it completely; (<NUM>) pour the mixed solution with particles into carrier oil (e.g., fluorinated fluid), or add oil into the mixed solution; (<NUM>) apply gravitational, centrifugal, or magnetic force to transport drop-carrier particles <NUM> from the mixed solution to the carrier oil to isolate a fraction of particle-drops <NUM> carrying a single cell and a barcoded microsphere/microbead (or hydrogel microparticle) inside each of the fluid droplets <NUM> of the drop-carrier particles <NUM>; (<NUM>) lyse the single cell in each droplet <NUM> physically, e.g., temperature increase, sonication and freeze-thaw, or chemically, e.g., adding lysis-buffer droplet suspensions (or another set of lysis-buffer-drop-carrier particles <NUM> with particle shape complimentary to the particles for cell suspension) in the carrier oil; (<NUM>) capture mRNAs of the single cell on its companion microsphere (or the primer photocleaved from the hydrogel microparticle after lysis); (<NUM>) transfer the drop-carrier particles <NUM> from the carrier oil back to an aqueous solution by adding large amount of the solution with reverse transcription (RT) reagents, which allows simultaneous production of cDNA with barcoded nucleic acid sequences; (<NUM>) conduct PCR (or other reduced biased amplification procedure to amplify all cDNA) and perform sequencing. The cDNA are sequenced and sourced back to the cell of origins by the barcode of the primer.

<FIG> illustrates a second protocol or method used to achieve single-cell RNA-sequencing using the particle-drop system described herein. The method uses the following operations: (<NUM>) prepare a single-cell suspension solution with a known cellular concentration; (<NUM>) add a quantity of dried powder of drop-carrier particles <NUM> that optionally have particle identifying indicia <NUM> or shapes with linked barcoded primers into the solution and vortex it completely (the volume supported by the drop-carrier particles should be matched to the volume of the solution); (<NUM>) pour the mixed solution into carrier oil or oil into the mixed solution;
(<NUM>) apply mixing, centrifugal or magnetic force to aid in transporting drop-carrier particles <NUM> from the aqueous solution into the carrier oil and isolate particle-drops <NUM> carrying zero or one cell in each; (<NUM>) lyse the single cells in each droplet <NUM> physically or chemically (e.g., as described above using monodisperse lysis buffer droplets or particle-drops <NUM> containing lysis buffer in the fluid droplet <NUM>); (<NUM>) hybridize single-cell released mRNAs to the primers on the interior surface of each drop-carrier particle; (<NUM>) transfer particle-drops <NUM> back to an aqueous solution with reverse transcriptase and perform reverse transcription to create barcoded cDNA; use template switching to link synthesized cDNA with a PCR handle; (<NUM>) utilize the cDNA product for PCR or other non-biased amplification and then sequencing. The mRNA transcripts are sequenced and sourced back to the cell of origins by the barcode of the primer.

There are a number of applications for particle-drops <NUM> with unique benefits because a set of solid-phases is associated with fluid drops <NUM> of substantially uniform volume. Many of these applications are enabled by isolating single entities (e.g., single-molecules or single-cells). Single-cell secretion analysis is an area that significantly benefits from an associated solid-phase to capture secreted molecules from cells within the aqueous phase internal to the particle-drop <NUM> but prevent cross-talk between neighboring cells in neighboring particle-drops <NUM>. For example, a secretion capture moiety may be present on the drop-carrier particle <NUM> with affinity which depends on the application. A secondary reporter molecule (e.g., antibody, aptamer, or enzyme) may also be present that binds to the secreted molecule at a different site. The secondary reporter molecule may have an attached fluorophore such that the accumulation of the secreted molecule and accumulation of the secondary reporter molecule leads to a local increase in fluorescence signal intensity which can be observed and quantified.

Particle-drops <NUM> can be used to enable the sorting and measurement based on secreted molecules. A drop-carrier particle <NUM> can be fabricated to include one or more molecular capture regions on the interior hydrophilic region <NUM> of the drop-carrier particle <NUM>. For example a biotinylated polyethylene glycol precursor can be used to create a biotinylated region on the drop-carrier particle <NUM>, bind streptavidin and then a biotinylated antibody. An exemplary application is in detecting cytokine secretions by leukocytes. In this case, the antibody would have affinity to the cytokine of interest. Multiple capture antibodies can also be used to cover the capture surface and have affinity for a variety of cytokines (e.g., IL-<NUM>, IL-<NUM>, IL-<NUM>, TNF-alpha, IFN-gamma). In this case, the binding of secreted molecules is distinguished by the fluorophore spectrum and intensity of the reporter molecules. The limitation of number of different secreted products that can be detected is based on spectral overlap between the reporter fluorophores (which limits to ~<NUM> separate fluorophores in the visible range). One can also create a plurality of molecular capture regions with spatial heterogeneity in location in the hydrophilic interior region <NUM> of the drop-carrier particle <NUM> to expand the number of secreted molecules that can be sensed. In this case biotin alone cannot be used and maintain selectivity and polymer precursor streams containing oligonucleotide capture regions with unique sequences can be used for each distinct capture location. The complementary oligonucleotide for each capture location can then be conjugated to antibodies or other capture agents to link them specifically to the desired spatial locations. In some embodiments the interior hydrophilic region <NUM> also contains a region with capture antibodies specific to cell-surface proteins (e.g., EpCAM, CD4, CD3, or CD8), enabling selective enrichment of cells with high level of expression of specific cell-surface proteins (e.g., epithelial cells, or CD4 T Cells) within the particle-drops <NUM>.

The process of measuring or sorting cells based on secretions using particle-drops <NUM> begins with creating an emulsion supported by the drop-carrier particles <NUM> with cells. Drop-carrier particles <NUM> are mixed with an aqueous solution of suspended and thoroughly washed cells, and added to a hydrophobic continuous phase and mixed as described to create the particle-drop <NUM> emulsion. Thorough washing is necessary to remove background secreted molecules. Reporter antibodies conjugated to fluorophores can be added to this washed cell solution, or can be added through a solution exchange operation as described herein. A suspension of particle-drops <NUM> containing single-cells in the fluid droplet <NUM> that is carried therein is then incubated at about <NUM> for <NUM> to <NUM> hours to accumulate the secretions of cells and allow binding to the functionalized particle-drop <NUM> surfaces. The reporter molecule then binds to the secretions localized to the particle-drop <NUM> surface creating a localized fluorescent signal. This localized fluorescent signal and the presence of the cell (through nuclear intercalating dyes, cell-surface stains or viability dyes) can be analyzed by imaging approaches in the suspended state. For example, automated microscopy can be used to identify particle-drops <NUM> containing single-cells with profiles of secretion that are desired based on the intensity and/or wavelength levels of fluorescent signals corresponding to secreted molecules. An analysis can be performed based on the distribution of single-cells with different classes of secretion profiles to create a diagnostic readout, for example in autoimmune disorders, sepsis, or transplant rejection. Besides use in diagnostics, cells with specific secretion profiles (e.g., antibody secretion with high titer and high affinity to antigen) can be selected using this approach for evolving high-secreting clones for antibody production for example.

Drop-particles <NUM> are also an excellent platform to capture the diversity of nucleic acids in a sample using an approach similar to beads, emulsions, amplification and magnetics (BEAMing). In this approach a pre-amplification step is performed on a sample to generate tag nucleic acid regions on a diverse set of amplified nucleic acids. These nucleic acids are captured onto the interior region <NUM> of the drop-carrier particles <NUM> based on hybridization to the tag region which is linked to the drop-carrier particle <NUM>. Linking of the tag region can be achieved using biotin-streptavidin linkages after covalently linking biotin into the interior hydrophilic region <NUM> of the drop-carrier particle <NUM>. Particle-drops <NUM> containing the nucleic acids are generated by adding an external oil phase. Unlike with BEAMing, using particle-drops <NUM> limits the number of solid phase particles per fluid compartment and amplification reaction. In addition each fluid droplet <NUM> has a more uniform volume and amount of reagents in the particle-drop approach. Combined, this yields better quantitative accuracy in the abundance of particular nucleic acids within a sample, instead of over counting based on multiple beads being encapsulated into a single droplet. Amplification is then performed within the drop-carrier particle <NUM> to amplify the signal from the enclosed nucleic acids within a particular particle-drop <NUM> that are immobilized on the drop-carrier particle <NUM>. Following amplification and immobilization, the emulsion is broken to bring the drop-carrier particles <NUM> back into aqueous solution. These drop-carrier particles <NUM> can be reacted to hybridize with various nucleic acid sequence-specific probes with attached fluorophores and read and sorted using flow cytometry or fluorescence-activated cell sorting.

A modification of the BEAMing protocol achieves a digital PCR-based solution using particle-drop technology. For digital PCR the pre-amplification step is not necessary and an overhanging tag region is introduced into primers that are specific to a particular target sequence. This tag sequence is also attached to the hydrophilic interior surface <NUM> of the drop-carrier particle <NUM>. For particle-drops <NUM> that contain the single nucleic acid sequence that is amplified, copies of the sequence are covalently linked to the drop-carrier particle <NUM> by incorporation of the immobilized tag sequence on the surface of the drop-carrier particle <NUM>. Following amplification and breaking the emulsion, specifically amplified and linked nucleic acids can be labeled with intercalating dyes or sequence specific probes and then analyzed by flow cytometry. A one-pot multiplexed digital nucleic acid amplification reaction and analysis can be performed using this approach. In this case no pre-amplification is performed and drop-carrier particles <NUM> are encoded to have primer tag sequences which are immobilized and have a universal region and specific region unique to target nucleic acid sequences. Amplification is performed in the particle-drop <NUM> which leads to amplification and attachment of nucleic acid sequences to the surface of the drop-carrier particle <NUM> in cases where one or more target nucleic acids was initially present. Once nucleic acids are attached, drop-carrier particles <NUM> can be transferred into an aqueous suspension where target specific probes are added or intercalator dyes are added to generate sequence-specific or double stranded DNA-specific fluorescent signal for amplified drops. Drop-carrier particles <NUM> can then be analyzed on a flow cytometer and the number of positive particles with intensity above a threshold can be counted in order to generate a measure of concentration of a specific nucleic acid. The system can be multiplexed using separate fluorophores conjugated to probes targeted different target nucleic acid sequences, or mixtures of two or more probes targeting a sequence with different ratios for each different target sequence to give even further increases in multiplexing capability. This assay can also be multiplexed at the level of different drop-carrier particles <NUM> mixed together that use separate fluorescent colors of the barcoded drop-carrier particles <NUM> or scatter signatures induced by different shape of drop-carrier particles <NUM> to distinguish different assays. For this application, drop-carrier particles <NUM> with embedded magnetic particles <NUM> can be used in order to enable easy separation and washing or drop-carrier particles <NUM> can be non-magnetic and transfer steps can be performed by centrifugation.

In a similar process to digital PCR and BEAMing, reduced bias whole genome amplification can be achieved by using particle-drops <NUM>. Whole genome amplification using approaches like multiple displacement amplification (MDA) can lead to bias since during exponential amplification small differences in kinetics can lead to several fold changes in amplification efficiency of different regions of DNA. This can lead to less reads in particular genes and reduced accuracy. Amplification of cDNA from a transcriptome can also benefit from reduced bias whole transcriptome amplification. First the fragmented DNA in an aqueous solution is mixed with drop-carrier particles <NUM>, following mixing, oil is added and mixing further disperses and forms uniform sized particle-drops <NUM>. Using the small particle-drop <NUM> compartments and amplification of only a single gene fragment in each compartment surrounded by an oil external phase allows for uniform amplification that can be run to completion in each compartment (i.e., fluid droplet <NUM>). Because the reaction amount depends on compartment volume, particle-drops <NUM> are advantageous because they create uniform sized compartments. Following amplification, the particle-drops <NUM> can be brought back into an aqueous solution and the amplified regions are retrieved into a bulk solution for further downstream sequencing or other analysis.

Particle-drops <NUM> can also provide a uniform emulsion for new techniques like DropSynth such as that described in <NPL>), which is incorporated therein by reference. In the DropSynth technique large genes, which would be difficult to synthesize using current techniques, are assembled in a massively parallel process by first having barcoded beads with tag nucleic acid sequences that will hybridize to the many oligonucleotide pieces that make up the larger gene. The beads are then prepared into an emulsion, the oligonucleotide pieces are digested to be cleaved from the beads and released into the droplet, and then assembled using enzymatic ligation. The interior solid phase of a drop-particle <NUM> can act to both template the initial oligonucleotide collection that will be incorporated in the reaction as well as create the emulsion with uniform droplet <NUM> size which allows for more uniform downstream reactions for gene assembly in the droplet <NUM>. The emulsion can then be broken to collect the synthesized genes or sorted based on barcoding of the drop-carrier particles <NUM>.

Besides digital nucleic acid amplification tests, digital immunoassays can be uniquely enabled by particle-drop technology. Digital versions of the enzyme-linked immunosorbent assay traditionally require a solid phase bead that has a capture antibody for a target biomolecule of interest (e.g., antigen), following capture of zero or one molecules per bead, remaining sample solution is washed away and a secondary antibody solution is introduced and secondary antibodies (polyclonal or monoclonal to a different site) bind to form a sandwich with the captured target. The secondary antibody or other recognition element is conjugated to an enzyme like HRP to generate amplified signal from fluorescent reporters. In the digital assay, the bead is confined in a fluid compartment (e.g., well or drop) so that fluorescent signal is contained within the small volume drop and is amplified to a high concentration from the initial single molecule. Challenges with the traditional digital assays include maintaining only a single bead per compartment and complex readout instruments that are limited to a certain field for microwell arrays.

Particle-drops <NUM> can provide significant advantages for digital ELISA assays. <FIG> illustrates a sequence of operations used to perform a digital ELISA assay. Implementing digital ELISA in particle-drops <NUM> first requires immobilizing capture antibodies on the interior hydrophilic region <NUM> of the drop-carrier particles <NUM>. This is accomplished as described herein using biotin-avidin binding interactions to biotinylated PEG hydrogel as the interior region <NUM> of the drop-carrier particles <NUM>, for example. Drop-carrier particles <NUM> with attached antibody are mixed with an aqueous solution of sample (containing an antigen) and allowed to bind with gentle mixing for <NUM>-<NUM> minutes. <FIG> illustrates in operation <NUM> an antigen-containing aqueous sample solution that is exposed to the drop-carrier particles <NUM> with attached antibody.

The sample is then centrifuged or magnetic drop-carrier particles <NUM> are collected to the bottom of a tube and washed a plurality of times (e.g., three times) to remove unbound sample. Then secondary antibody solution which contains the secondary antibody and a reporting enzyme is introduced as seen in operation <NUM> of <FIG> and washed <NUM>-<NUM> times to remove the unbound secondary antibody. Next, as seen in operation <NUM> of <FIG> the substrate of the enzyme linked to the secondary antibody is introduced. This may include, for example, a fluorogenic substrate or quencher/fluorophore pair. In operation <NUM> of <FIG>, the sample is then emulsified by mixing with an oil phase. Signal then accumulates in the particle-drops <NUM> which can be read for fluorescence intensity using microscopy of other fluorescent imaging technique to count the number of particle-drops <NUM> with intensity above a threshold that were considered positive which yields the target's concentration in the sample. <FIG> illustrates the resulting particle-drops <NUM> being labelled as negative ("<NUM>") or positive ("<NUM>"). In the case that the reaction is desired to be read out in a flow cytometer the particle-drops <NUM> need to be first transferred back to an aqueous solution. This transfer would lead to loss of accumulated fluorescent signal as the oil phase no longer maintains a barrier. In order to capture the amplified signal on the drop-carrier particle <NUM> tyramide signal amplification or catalyzed reporter deposition (CARD) techniques can be used. For example tyramide biotin (or tyramide AlexaFluor® <NUM>) can be covalently linked to neighboring tyrosine residues within peptides or proteins attached to the interior hydrophilic hydrogel matrix of the drop-carrier particle <NUM>. This covalent linkage is catalyzed by the presence of horse radish peroxidase attached to the secondary antibody. Following reaction and high efficiency linkage to the drop-carrier particle <NUM> while emulsified to localize signal, these drop-carrier particle <NUM> can be transferred to an aqueous phase for readout by flow cytometry for example, or reacted with streptavidin-conjugated to fluorophore and then run through a flow cytometer. The amount of particles with signal above threshold can be counted by gating on the flow cytometer to determine the concentration in the sample. Multiplexing can be conducted of multiple biomolecules by simultaneously mixing particles with different barcoding schemes as discussed herein that include separate capture antibodies targeting the set of biomolecules of interest. This multiplexed assay can use fluorescent colors of the barcoded drop-carrier particles <NUM> or scatter signatures to distinguish different assays. These other drop-carrier particles <NUM> with off-target antibodies can also serve as negative controls within a run.

The particle-drops <NUM> also provide advantages for single-cell RNA sequencing workflows. Current workflows encapsulate cells in droplets, merge these droplets with other droplets that contain spherical beads with mRNA capture moieties (e.g., an oligonucleotide containing a poly T region, a unique molecular identifier tag (UMI), and a uniform bead-specific tag) and cell lysis buffer. Following lysis, mRNA from a single cell is released and captured on the bead encapsulated in the same droplet. Once mRNA is captured, the emulsion is broken and the beads are washed and exchanged into solution with reverse transcriptase in order to generate cDNA that contains the captured sequence information, the UMI, and the bead-specific barcode sequence. This type of Drop-Seq barcoding technique is described, for example, in<NPL>), (including all Supplemental Information).

This cDNA can be further amplified and run through standard next-generation sequencing instruments (such as from Illumina, Inc. Using drop-carrier particles <NUM> a single-cell and "bead" (e.g., the drop-carrier particle <NUM> in this embodiment) are automatically brought together without the challenges of Poisson loading of droplets with beads. Cells can also be specifically captured on the drop-carrier particle <NUM> based on surface antigens targeting a particular cell type while in an aqueous phase prior to suspension in a surrounding oil phase. This also improves the cell loading in the particle-drops <NUM> beyond the current limitations of Poisson statistics which leads to large numbers of empty drops and waste of reagents in the current Drop-Seq single-cell protocols such as that described in <NPL> lysis can be performed by transferring a lysis solution into the particle-drop <NUM> following the initial formation of particle-drops <NUM>.

When no surfactant is used when mixing drop-carrier particles <NUM>, aqueous cell solution, and oil together particle-drops <NUM> form. For particle-drops <NUM> without surfactant coating of their free surfaces, the addition of new solution can lead to exchange of this solution into the particle-drop <NUM> without disrupting the particle-drop size. For example, one protocol for single-cell RNA-seq using drop-carrier particles in which the interior hydrophilic region contains barcoded capture oligonucleotides and cell capture antibodies includes the following steps. First, dehydrated drop-carrier particles <NUM> are mixed with cells in an aqueous solution to capture cells within drop-carrier particles <NUM>. Second, an oil phase is introduced and mixed to form suspended particle-drops <NUM> containing adhered cells within the fluid droplet <NUM>. Third, a lysis solution is introduced and rapidly mixing to enable entry into the particle-drops <NUM>. Fourth, the mixture is allowed to incubate in a quiescent state to allow cell lysis and capture of mRNA onto the interior surface of the drop-carrier particles <NUM>. Fifth, the drop-carrier particles <NUM> are brought back into an aqueous solution for downstream cDNA generation and sequencing. An example formulation for Cell lysis buffer that can be used is described by Macosko et al. (Drop-Seq Lysis Buffer - DLB, <NUM> Tris pH <NUM>, <NUM>% Ficoll PM-<NUM>, <NUM>% Sarkosyl, <NUM> EDTA). In a similar manner to single-cell RNA analysis, protein based barcoding for downstream single-cell analysis using sequencing can be achieved by including capture antibodies for proteins on the surface of the hydrophilic interior region <NUM> of the drop-carrier particles <NUM>. A secondary antibody sandwich can then include oligonucleotide barcodes that can be read and associated with a specific cell when pooled using nucleic acid sequencing as a final step. Such an approach for converting protein signal to nucleic acid signal is disclosed in <NPL>).

There are two main readout approaches for the products of particles-drops <NUM>. Optical readout of a fluorescent signal is a first approach that can be conducted using fluorescence microscopy, wide-field computational microscopy, flow cytometry, or imaging flow cytometry. Optical microscopy can be performed both while particle-drops <NUM> are surrounded in an oil phase or following transfer into an aqueous phase, especially if the generated signal is attached to the surface of the drop-carrier particle <NUM>. When transferring to an aqueous solution adhesion between particles along hydrophobic exterior surfaces can be mitigated to enable improved readout by addition of an optional surfactant that interacts with the hydrophobic exterior phase of the drop-carrier particle <NUM> (e.g. Pluronic®, Pico-Surf™, etc.), and allows for lower interfacial energy with the new surrounding aqueous phase. In other embodiments, the readout of single-cell RNA seq, single-cell DNA sequencing, or bias-free whole genome application includes further nucleic acid amplification and gene sequencing using standard commercial instruments such as those sold by Illumina, Inc.

In flow cytometry-based readout, particle-drops <NUM> are first transferred back to an aqueous phase. Fluorophores yielding a fluorescent signal are maintained attached to the drop-carrier particle <NUM> following transfer to an aqueous external phase due to affinity with a capture reagent on the interior region <NUM> of the drop-carrier particle <NUM>. In some embodiments, cells are also adhered or captured in the drop-carrier particle <NUM> where they can be stained in an aqueous phase with nuclear dyes or antibody-conjugated dyes to specific proteins and analyzed by flow cytometry. Once in an aqueous phase, suspensions of drop-carrier particles <NUM> (with molecular and/or cellular attached signals) are introduced into the flow cytometer for particles to be analyzed by forward and side scatter signals as well as attached fluorescence signals (from molecular binding events, presence of nucleic acids, or cells present). Drop-carrier particles <NUM> that are used in flow cytometry analysis should be relatively neutrally buoyant in the suspending medium to avoid drop-carrier particles <NUM> from settling, and sized to prevent clogging of commercial flow cytometer flow cells (e.g. < <NUM> micrometers in diameter for most flow cytometers from BD or Beckman Coulter, or < <NUM> micrometers in diameter for the Biosorter from Union Biometrica). Drop-carrier particles <NUM> can also be shaped or sized to elicit unique scattering signals from forward or side scatter. This is advantageous since multiple species of drop-carrier particles <NUM> that are specific to different target molecules or cells can be mixed together for analyzing a sample and can be distinguished by a scatter-based barcode or signature. For example drop-carrier particles <NUM> without sharp edges vs. particles containing more edges will yield different scatter signals, with the number of sharp edges correlating with the amount of scatter signal. Preferably an asymmetric shape of the drop-carrier particle <NUM> along at least two axes will yield alignment in the squeezing asymmetric sheath flow of a flow cytometer such that the scatter signal is more uniform between drop-carrier particles <NUM>.

Drop-carrier particles <NUM> with more or less holes, notches, or other surface features will yield unique side-scatter signatures. Drop-carrier particles <NUM> with <NUM>-<NUM> holes or notches in a 2x3 array yielding six (<NUM>) unique scatter signals can provide barcoding for six (<NUM>) separate reactions or molecules. The notches can be made in the surrounding hydrophobic external region <NUM> of the drop-carrier particle <NUM> or in the interior hydrophilic region <NUM> of the drop-carrier particle <NUM>. In addition to or instead of a scatter-based barcode, drop-carrier particle <NUM> may also possess a fluorescence "barcode" that consists of fluorophores or fluorescent particles embedded into or attached to the drop-carrier particle <NUM>. The barcode can include multiple fluorophores or a single fluorophore of multiple well-defined concentrations that when excited elicit well-defined intensities, or a combination of fluorophores and intensities in various permutations to yield a large number of individual barcode signatures (e.g., two (<NUM>) fluorophores and five (<NUM>) intensities yields twenty-five (<NUM>) unique barcodes). Fluorophores used for barcoding of drop-carrier particles <NUM> will preferably not overlap in emission spectra with fluorophores used in sensing on the drop-carrier particles <NUM>. In addition to analysis, sorting can be performed based on a combination of a barcode signal and molecular or cellular signal. Unique populations of barcoded drop-carrier particle <NUM> can be gated for sorting using standard gating tools in fluorescence activated cell sorters. In addition, unique populations of drop-carrier particle <NUM> with attached cell populations can be gated and sorted based on fluorescence signal intensity or combinations of fluorescence signals.

Drop-carrier particles <NUM> in an aqueous solution can also be read using commercial imaging flow cytometers (e.g., for these instruments, drop-carrier particle <NUM> size should be preferably between <NUM> micrometers and <NUM> micrometers in size). The imaging flow cytometer can characterize fluorescence intensity of the drop-carrier particle <NUM> associated with the signal from a target molecule or cell attached to the drop-carrier particle <NUM>, image an attached cell, or image a shape barcode of the drop-carrier particle <NUM> itself. The shape barcode can include changes in the overall morphology of the drop-carrier particle <NUM> envelope if extruded in 2D through the particle, or may include embedded notches, holes, or surface features that are included on a surface of the drop-carrier particle <NUM> in a 2D or linear array. The unique shape barcode can be associated with a unique molecular targeting agent on the surface of the drop-carrier particle <NUM> that would otherwise not be visible, and therefore allow distinguishing between different classes of drop-carrier particles <NUM> specific to different biomarkers. This would enable extreme multiplexing for detection of up to thousands of protein biomarkers for example.

In one embodiment, drop-particles <NUM> do not contain a cavity for an internal aqueous phase, but instead contain a hydrogel matrix that an aqueous solution can swell internal to an exterior region of the particle that remains hydrophobic. An example of this embodiment is illustrated in <FIG>. The mesh size and pore structure of this hydrogel matrix is important to control to enable molecular binding and enzymatic reactions within the material. For example, Xu et al. show that nucleic acid amplification reactions can be performed within PEG hydrogel gels. For example, the mesh size of PEG-based hydrogels formed using four-arm PEG crosslinkers is about <NUM> at <NUM>% (w/v), which allows diffusion of small molecules, oligonucleotides and enzymes but immobilizes cells and high-molecular-weight nucleic acids. Therefore, digital nucleic acid amplification reactions may be performed in the hydrogel matrix of the particle-drop <NUM> once encapsulated in an external oil phase. In addition this pore size is sufficiently large for antigen-antibody or antigen-aptamer binding reactions within the hydrogel matrix, as well as secondary antibody binding and signal generation from enzymes such as horse radish peroxidase or beta-galactosidase conjugated to secondary recognition elements (e.g., antibodies or aptamers).

In an alternative embodiment, the PEG gels can be functionalized to allow immobilization of low-molecular-weight species by attachment to the gel matrix. For example, acrydite modification on the <NUM>' end of one of the primers in a polymerase chain reaction can be used to covalently link the amplified DNA to the hydrogel matrix. See e.g., <NPL>), Alternatively, the primers can be linked to the hydrogel matrix using biotinylated primers that bind specifically to streptavidin immobilized within the hydrogel matrix. The amplified DNA linked to the matrix can then be assayed using intercalating dyes within the solution surrounded by an oil phase or once transferred to an aqueous phase. Other readout approaches that are sequence specific can also be incorporated, such as by hybridizing complementary fluorophore labeled nucleic acid probes to the immobilized and amplified nucleic acids. In other nucleic acid amplification reactions, e.g. loop-mediated isothermal amplification (LAMP) or rolling circle amplification, the nucleic acids produced are much longer and can be physically entrapped in the hydrogel matrix without the ability to leave, however, still allow exchange of other reagents and dyes (e.g., intercalator dyes such as EvaGreen® or SYBR® Green, or molecular beacons or other fluorophore labeled complementary sequences) upon transferring back to an aqueous external phase. Protein targets can be covalently linked to the gel matrix with the addition of a crosslinking agent and upon exposure to a crosslinking reaction. For example, as discussed in the work of Herr et el. , which is incorporated herein by reference, N-(<NUM>-((<NUM>-benzoylphenyl) formamido)propyl) methacrylamide can be used along with photo-activation to covalently link proteins to the hydrogel matrix. A UV light source capable of providing <NUM>-<NUM> ~<NUM> J/cm<NUM> of light can be used to link proteins on particle-drops <NUM>. This process can be performed while particle-drops <NUM> are in an oil suspension to covalently link protein targets to the interior region <NUM> of the drop-carrier particle <NUM>. The drop-carrier particles <NUM> can then be transferred back to an aqueous solution for further immune-labeling of protein biomarkers and analysis in aqueous solution.

An alternative embodiment to entrap molecular or cellular targets within a particle-drop <NUM> includes polymerizing a pre-polymer solution that acts as the internal aqueous phase <NUM> of the particle drop <NUM> following capture of cells, molecules, or other products of amplification reactions within the particle-drop <NUM>. The internal polymerization reaction also covalently links the target molecules or amplification products into the hydrogel or physically entraps cells or larger molecules within the hydrogel. Polymerization and covalent linkage or entrapment can be initiated with exposure to light (UV, white light) with the appropriate photoinitiator (e.g., Irgacure, Eosin Y), exposure to heat, or exposure to a pH change. For example, the LAMP reaction can proceed within <NUM>% <NUM>-arm PEG vinylsulfone, PEG dithiol precursor. Following this polymerization process, the drop-carrier particles <NUM> can be exchanged into an aqueous solution for downstream reactions, labeling, and flow cytometric or other readout processes.

As explained previously, the drop-carrier particles <NUM> enable the formation of monodisperse particle-drops <NUM> without the need of any complex or expensive instruments. <FIG> illustrates a graph of the nominal diameter of the fluid droplets <NUM> contained in particle-drops <NUM> compared to droplets formed with surfactant only. The latter was generated by following the same protocol except that no drop-carrier particles <NUM> were suspended in the oil phase and <NUM>% (w/v) surfactant (Pluronic®) was dissolved in the aqueous phase. The size of particle-drops <NUM> distributes narrowly in the range of <NUM> microns nominal diameter while the size of the droplets in the surfactant case spans widely. The particle-drops <NUM> thus can be used as small volume bioreactors having nearly identical volumes.

<FIG> also illustrates the ability of particle-drops <NUM> to create fluid droplets <NUM> with non-circular cross-sections. <FIG> illustrates the circularity of the fluid droplets <NUM> for particle-drops <NUM> and drops with surfactant only. As seen in <FIG>, a significant departure from circularity is seen for the particle-drops with the circularity concentrated around <NUM>. This is in contrast with drops formed with surfactant only which show a circularity of around unity. This shows the ability of the particle-drops <NUM> to generate non-circular fluid droplets <NUM>.

Claim 1:
A particle system (<NUM>) comprising:
a plurality of three-dimensional particles (<NUM>), each three-dimensional particle (<NUM>) having an interior region (<NUM>) defining a three-dimensional cavity or void and an exterior region (<NUM>), wherein the three-dimensional cavity or void is open to an external environment of the three-dimensional particle (<NUM>) and wherein the three-dimensional cavity or void has a volume between about <NUM> pL and about <NUM> nL and a molecular capture region disposed on the interior region (<NUM>) of the three-dimensional particle (<NUM>) configured to capture cells.