Devices and methods for engulfing particles into droplets

Abstract

Provided are microfluidic devices. The devices comprise a chamber configured to contain a liquid medium. The devices further comprise a substrate comprising: i) a photoconductive layer forming a first wall of the chamber; and ii) first and second electrodes disposed in a planar arrangement within or adjacent to the photoconductive layer. The devices further comprise a microfluidic inlet channel connected to the chamber, and a microfluidic droplet generator connected to the chamber. Systems comprising the microfluidic devices of the present disclosure are also provided. The systems comprise a light pattern generator optically coupled to the photoconductive layer; and a voltage source electrically coupled to the first and second electrodes. Methods of engulfing a particle into a droplet implemented using a system of the present disclosure are also provided.

Description

[0001] Atty. Docket: STAN-2215WO (S24-293)

[0002] DEVICES AND METHODS FOR ENGULFING PARTICLES INTO DROPLETS

[0003] CROSS-REFERENCE TO RELATED APPLICATIONS

[0004] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 683,641 , filed August 15, 2024, which application is incorporated herein by reference in its entirety.

[0005] STATEMENT OF GOVERNMENT SUPPORT

[0006] This invention was made with Government support under contract GM 138716 awarded by the National Institutes of Health. The Government has certain rights in the invention.

[0007] INTRODUCTION

[0008] Picodroplet reactors - platforms for performing picoliter-volume reactions enabled by droplet microfluidics - are emerging as powerful tools for biochemical reactions with minimal reagent use and high precision1 5. With established droplet processing techniques including merging, splitting, mixing, incubation, and sorting, picodroplet reactors are versatile for biochemical workflows such as single-cell analysis and high-throughput screening for drug discovery and directed evolution6-8. However, many other important biochemical workflows, such as oligonucleotide synthesis, enzymatic assays, and immunoassays, require multi-step processes involving multiple reagent exchanges, which entail the sequential addition and subsequent removal of different reagents, as well as product extraction. Current picodroplet platforms cannot support such multi-step workflows, as they lack reliable mechanisms for controlled reagent exchange3.

[0009] Typically, multi-step biochemical workflows are performed on solid-phase platforms in column or multi-well plate formats9-11. In such platforms, microbeads (e.g., polystyrene or glass) are commonly used as solid supports to provide surfaces where reactant molecules can bind and undergo biochemical reactions. Because the reaction products remain tethered to the bead surface, product extraction is simplified, enabling high-throughput processing12 13. Despite these advantages, conventional solid-phase platforms face significant limitations. Processing millions of beads collectively in bulk leads to nonuniform reagent exposure and trapping of residual impurities within the bead matrix, resulting in incomplete reactions and error propagation across multiple reaction steps and cycles14-16.

[0010] While not originally developed to address limitations of conventional solid-phase systems, picodroplet microfluidics has been explored to encapsulate and isolate individual Atty. Docket: STAN-2215WO (S24-293) reaction sites (i.e., microbeads) within droplets. These efforts have focused primarily on enhancing detection sensitivity, enabling single-molecule assays, and performing digital quantification in biochemical and diagnostic applications through compartmentalization17’18. Although these systems can achieve significant sensitivity gains by reducing reaction volume and enhancing signal-to-noise ratios19 20, they again remain limited to single-step reactions due to the lack of mechanisms for reagent removal or bead decapsulation. Without the ability to exchange reagents between reaction steps, multi-step biochemical workflows remain inaccessible in these picodroplet systems.

[0011] SUMMARY

[0012] Provided are microfluidic devices. The devices comprise a chamber configured to contain a liquid medium. The devices further comprise a substrate comprising: i) a photoconductive layer forming a first wall of the chamber; and ii) first and second electrodes disposed in a planar arrangement within or adjacent to the photoconductive layer. The devices further comprise a microfluidic inlet channel connected to the chamber, and a microfluidic droplet generator connected to the chamber. Systems comprising the microfluidic devices of the present disclosure are also provided. The systems comprise a light pattern generator optically coupled to the photoconductive layer; and a voltage source electrically coupled to the first and second electrodes. Methods of engulfing a particle into a droplet implemented using a system of the present disclosure are also provided. In some embodiments, the methods comprise dispensing a particle suspended in the suspension medium from the microfluidic inlet channel into the chamber, and generating a droplet in the chamber using the microfluidic droplet generator. Such methods further comprise providing a voltage to the photoconductive layer using the first and second electrodes, and projecting a light pattern onto the photoconductive layer using the light pattern generator to engulf the particle into the droplet. The light pattern is selected to exert an electric field gradient on the droplet and / or the particle such that the particle is engulfed into the droplet.

[0013] BRIEF DESCRIPTION OF THE FIGURES

[0014] FIG. 1 : Schematic representation of a chamber in the devices disclosed herein and certain principles of OET.

[0015] FIG. 2A-2B: Design of the device for optoelectrically controlled encapsulation and decapsulation. A. Side view of the device. B. Top view of the device.

[0016] FIG. 3: Photo of an exemplary chamber from top view.

[0017] FIG. 4: An exemplary embodiment of a system of the disclosure.

[0018] FIG. 5: Schematic representation of optically induced dielectrophoresis controlled encapsulation and decapsulation of micro-beads using micro-droplets. The reaction chamber Atty. Docket: STAN-2215WO (S24-293) is filled with electrically insulating suspension medium (e.g., oil) and solid micro-beads are suspended therein. The droplets are generated in microfluidic channels and introduced into the chamber. Light patterns are used to manipulate droplets and beads.

[0019] FIG. 6: Droplet generation in the device of FIG. 4.

[0020] FIG. 7: Trapping and manipulation of microbeads and micro-droplets. Left panel shows droplet manipulation and middle and right panels show a bead (6 micron) being encapsulated by a micro-droplet. The bead and droplet were manipulated with light patterns.

[0021] FIG. 8: Parallel manipulation and encapsulation of two droplets and two beads. Encapsulation and decapsulation was simultaneously performed on two pairs of droplets and beads. In panels A-C, the voltage was ON. In panel D, the voltage was OFF.

[0022] FIG. 9: DNA “click” ligation cycle. DNA synthesis on a bead surface using encapsulation and decapsulation of the bead in reagent droplets.

[0023] FIG. 10A-10B: FIG. 10A: The DNA ‘click’ ligation cycle verified by standard polyacrylamide gel electrophoresis (PAGE) analysis of products from the beads. FIG. 10B: PAGE gel image in the Cy5 channel. It shows Atto 647-labeled products cleaved from beads after: (i) single click ligation step, (ii) full ligation cycle, and (iii) negative control.

[0024] FIG. 11 : On-chip DNA synthesis reaction. Demonstration of the proposed DNA ‘click’ ligation cycle containing both the enzymatic coupling and click ligation steps on a chip disclosed herein. Bar plot of the mean fluorescence intensity of bead samples from LAMPS after the three reactions. The error bars show the standard deviations.

[0025] FIG. 12A-12D: Encapsulation and decapsulation mechanism for reaction on LAMPS and its advantages. FIG. 12A, Limitations of nonuniform reagent exposure on conventional platform. Reaction errors can be introduced by insufficient reagent exposure area on the solid supports (i.e., microbeads) and impurities trapped within the bead matrix. FIG. 12B, Main feature of LAMPS: single bead is encapsulated and decapsulated by reagent picodroplet. The reaction is initiated when the bead (coated with reactant X) is encapsulated by the corresponding reagent droplet (containing reactant Y) and terminated when the bead is decapsulated by the droplet. The bead is 5 pm in diameter and the droplet is ~65 pL in volume (50 pm in diameter). FIG. 12C, 12D, Experimental images of encapsulation and decapsulation of a microbead by a reagent picodroplet. For decapsulation, the droplet is pulled towards the light patterns during the active period of the voltage, and retracts to its spherical shape during the inactive period. Scale bars: 25 pm.

[0026] FIG. 13A-13D: LAMPS platform design. FIG. 13A, Schematic showing top and side views of the LAMPS platform. Hexadecane is used as the oil medium in this study. FIG. 13B, Top-view optical image of the device. Four microfluidic droplet generation channels, each for a different reagent, are connected to a central chamber. Reagent droplets are generated at Atty. Docket: STAN-2215WO (S24-293) the intersections of the microfluidic channels and the central chamber (inset). Scale bars: 2 mm; 50 urn (inset). FIG. 13C, Numerical simulation of the electric field. FIG. 13D, Numerical simulation of the droplet motion during bead encapsulation. The gradient force exerted on the droplet from the light-activated virtual electrodes is sufficient to overcome its surface tension.

[0027] FIG. 14A-14C: Automated parallel 2D manipulation of picoreactor droplets achieved by LAMPS. FIG. 14A, Parallel encapsulation of two microbeads. FIG. 14B, Parallel manipulation and merging of two picodroplets. The two droplets move along preprogrammed paths that converge, resulting in their merging into a single droplet. FIG. 14C, Simultaneous parallel manipulation of four picodroplets in different directions along the preprogrammed paths. The preprogrammed paths are delineated by light blue arrows, and the droplets are outlined with orange dashed circles in (FIG. 14B) and (FIG. 14C). Scale bars: 100 pm.

[0028] FIG. 15A-15B: LAMPS demonstration with click ligation synthesis cycle. FIG. 15A, Eight-step templateindependent DNA click ligation synthesis cycle on the LAMPS platform. Each step is performed by encapsulation and decapsulation of the microbead with the corresponding reagent picodroplet. Step 1 : Azide addition. 3'-azide modified dNTP is coupled to DNA acceptor molecule using TdT. Steps 2 to 4: Washing steps with three PBS droplets. Step 5: Click ligation. 5'-alkyne modified donor molecule is ligated with 3'-azide acceptor molecule through CuAAC click chemistry. The ligated product has a triazole linkage. Steps 6 to 8: Washing steps with three PBS droplets. FIG. 15B, Fluorescence images of beads after three different reactions: (i) Click ligation step only with 3'-azide acceptor molecule; (ii) full ligation cycle, including both azide addition and click ligation steps, with 3'- hydroxyl acceptor molecule; and (iii) a negative control with 3'- hydroxyl acceptor molecule, where only the click ligation step was performed without the azide addition step and no ligation is expected. Low to no fluorescence in the control sample establishes a baseline for comparison. The fluorescence signal emitted by the ligated donor molecule labeled with Atto 647 after click ligation or full ligation cycle confirms successful reactions, (iv) A bright-field image of a representative bead is shown for reference. Scale bar: 5 pm.

[0029] DETAILED DESCRIPTION

[0030] Before the devices, systems and methods of the present disclosure are described in greater detail, it is to be understood that the devices, systems and methods are not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the devices, systems and methods will be limited only by the appended claims. Atty. Docket: STAN-2215WO (S24-293)

[0031] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the devices, systems and methods. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the devices, systems and methods, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the devices, systems and methods.

[0032] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the devices, systems and methods belong. Although any devices, systems and methods similar or equivalent to those described herein can also be used in the practice or testing of the devices, systems and methods, representative illustrative devices, systems and methods are now described.

[0034] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present devices, systems and methods are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0035] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation. Atty. Docket: STAN-2215WO (S24-293)

[0036] It is appreciated that certain features of the devices, systems and methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the devices, systems and methods, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present devices, systems and methods and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0037] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0038] MICROFLUIDIC DEVICES

[0039] Aspects of the present disclosure include microfluidic devices. The devices comprise a chamber configured to contain a liquid medium. The devices further comprise a substrate comprising: i) a photoconductive layer forming a first wall of the chamber; and ii) first and second electrodes disposed in a planar arrangement within or adjacent to the photoconductive layer. The devices further comprise a microfluidic inlet channel connected to the chamber, and a microfluidic droplet generator connected to the chamber. Systems comprising the microfluidic devices of the present disclosure are also provided.

[0040] Picoliter droplet reactors, enabled by droplet microfluidics, are revolutionary tools for biochemical reactions with high efficiency, precision, and minimal reagent use. They excel in single-step reactions and reagent addition through droplet merging. Integrating reagent exchange and washing capabilities directly into these platforms may enable complex multi- step processes, such as de novo oligonucleotide synthesis and multiplex immunoassays. However, current picodroplet microfluidics, lacking such capabilities, remain critically deficient in executing multi-step processes. The microfluidic devices and systems of the present disclosure, which employ light-orchestrated solid-phase picodroplet reactors, overcome these limitations. The microfluidic devices and systems employ optoelectronic tweezers to manipulate individual picodroplets and microbeads. This platform (sometimes referred to herein as “light-actuated multi-step picodroplet solid-phase reactors” or “LAMPS”) Atty. Docket: STAN-2215WO (S24-293) leverages optoelectronic tweezers (OET) to achieve programmable, sequential encapsulation and decapsulation of individual microbeads by corresponding reagent picodroplets. It is a microfluidic optoelectronic device that integrates on-demand droplet generation with nondestructive controlled manipulation of picodroplets and beads. Individual picoreactor droplets may be trapped and manipulated in parallel using programmable micro light patterns that are projected onto the device. It enables the reaction exchange capabilities that are missing in current droplet microfluidics. The LAMPS platform has several advantages over conventional solid-phase reactors as well. It ensures uniform reagent exposure, enhances reaction efficiency, and facilitates more effective washing steps between reaction steps. Additionally, replacing microtiter plates with picodroplet reactors allows reactions to be performed at significantly smaller volumes. The minimum reaction size is reduced from microliters to picoliters, minimizing reagent use and waste by up to a million-fold. By integrating reagent exchange capabilities into picoliter droplet microfluidics, the LAMPS platform enables solid-phase reactions in picodroplet reactors without inheriting the limitations of conventional solid-phase reactors. As demonstrated herein, this platform enables, e.g., an eight-step click chemistry-based DNA ligation synthesis cycle, with realtime in situ fluorescence detection of reaction products. This platform overcomes single-step limitations by achieving precise sequential encapsulation and decapsulation of beads with different reagent droplets, ensuring uniform reagent exposure and effective washing. Hence it mitigates reaction errors caused by nonuniform reagent exposure and trapped impurities in conventional bulk processing of microbeads. With minimal reagent consumption, real-time analysis, and programmable light- based control, the platform can be scaled and fully automated into a universal and versatile picoliter-scale reagent handling robot for miniaturizing and streamlining workflows in synthetic biology, drug discovery, and many other applications.

[0041] An optoelectronic tweezer (OET) device is illustrated in FIG. 1. A microfluidic device according to embodiments of the present disclosure is schematically illustrated in FIGs. 2A and 2B.

[0042] FIG. 2A provides a side view of the chamber. While conventional OET devices have a vertical electrode layout, the device disclosed herein has a planar electrode layout, as seen in the side view shown in FIG. 2A. A key challenge solved by the devices of the present disclosure is the manipulation of micro-objects (e.g., beads and / or droplets) in an electrically insulating medium. Most OETs employ a vertical electric field setup and cannot achieve this due to impedance mismatch issues. According to the devices of the present disclosure, a planar OET setup is employed where the electric field is in lateral direction. This planar OET Atty. Docket: STAN-2215WO (S24-293) setup does not suffer from impedance matching issues and can manipulate micro-objects as demonstrated herein.

[0043] The device of FIGs. 2A-2B has a planar electrode layout. Light patterns are projected from a spatial light modulator (SLM) onto the optoelectronic substrate, activating the photoconductive layer to form virtual electrodes. AC voltage is delivered to the physical electrodes present in the substrate. The physical electrodes along with the virtual electrodes activated by the light patterns form electric field gradients and exert a gradient force on the microparticles to manipulate them. The suspension medium is an electrically insulative and optically transparent.

[0044] In the example shown in FIG. 2A, the chamber has the height of about 110 microns, i.e., 0.11 mm. However, the chamber height can be from 0.05 to 0.15 mm, from 0.075 to 0.125 mm, or about 0.1 mm.

[0045] Also, in the example shown in FIGs. 2A-2B, the chamber has the length of about 5 mm and the width of less than 5 mm. However, the chamber can have a length and / or width of from 1 to 25 mm, from 2.5 to 7.5 mm, from 4 to 6 mm, or about 5 mm.

[0046] The top view of the chamber, as shown in FIG. 2B, shows droplet generators that introduce droplets into the chamber. The chamber can also have reagent inlets for providing reagents, such as enzymes and other reactants for the reactions carried out in the chamber. Further, the chamber can have an inlet for providing the suspension medium in the chamber. The schematic of FIG. 2B shows a droplet of about 50 microns diameter and a bead of about 5 microns diameter.

[0047] Particles, such as beads in a suspension medium are introduced through an inlet channel. Corresponding chemical reagents are introduced through the microfluidic channels and the reagent microdroplets are filled at the intersection of the microfluidic channels and the reaction chamber because of the step height between them.

[0048] The chamber shown in FIGs. 2A and 2B is fabricated with PDMS having various structures patterned therein. However, any suitable material or combination of materials can be used to fabricate the devices disclosed herein. In certain embodiments, the microfluidic device comprises one or more layers made of a material independently selected from silicon, glass, plastics, and ceramics, including glass ceramics. Suitable plastic materials include, but are not limited to, polycarbonates, polyacrylates such as polymethylmethacrylate (PMMA), and polyethyelenes such as polyethylene terephthalate (PET). Non-limiting examples of suitable glass materials include, but are not limited to, soda-lime silicate, aluminosilicate, alkali-aluminosilicate, borosilicate, alkaliborosilicate, aluminoborosilicate, alkali-aluminoborosilicate, and the like. In some embodiments, the microfluidic device is made of silicon, glass (e.g., borosilicate glass), or a combination thereof. For example, the Atty. Docket: STAN-2215WO (S24-293) microfluidic device may be made of a silicon portion bonded to a glass portion. In certain embodiments, the microfluidic device is made of a silicon portion etched to include a droplet generator channel, a reaction chamber and external electrical and fluidic connections, bonded to a glass portion etched to include electrodes and electrical contact points.

[0049] Another example of a device according to embodiments of the present disclosure is provided in FIG. 3, which shows an image of the device from the top. In this figure, the device 300 comprises microfluidic channels 301 , which can generate reagent droplets. The device has a substrate with interdigitated electrodes 302 that can supply electric current to the chamber 304. The device also has an oil inlet 303, which can provide oil to be introduced into the chamber as a suspension medium. In some embodiments, first and second electrodes of the interdigitated electrodes are separated from each other by a space of from 50 to 500 pm, from 75 to 125 pm, from 90 to 110 pm, or about 100 pm.

[0050] In some embodiments, the first and second electrodes of the interdigitated electrodes are two of a plurality of interdigitated electrodes in a planar arrangement within or adjacent to the photoconductive layer.

[0051] In certain embodiments, the microfluidic droplet generator is one of two or more microfluidic droplet generators connected to the chamber. Also, the microfluidic inlet channel can be one of two or more microfluidic inlet channels connected to the chamber.

[0052] In some embodiments, the microfluidic device further comprises one or more microfluidic outlet channels connected to the chamber.

[0053] The chamber can have a length extending along the photoconductive layer, wherein the length may be from 1 to 25 mm, from 2 to 10 mm, from 2.5 to 7.5 mm, from 4 to 6 mm, or about 5 mm. The chamber can have a height extending from the photoconductive layer, wherein the height may be from 0.05 to 0.15 mm, from 0.075 to 0.125 mm, or about 0.1 mm. In certain embodiments, the chamber has a width of from 1 to 25 mm, from 2.5 to 7.5 mm, from 4 to 6 mm, or about 5 mm.

[0054] The chamber can have any suitable shape and / or size, such as a rectangle, square, or any other suitable shape, including regular and irregular shapes and shapes with one or more curvilinear edges. In certain embodiments, the volume of the chamber is from 2 to 20 mm3, e.g., from 2 to 16 mm3, from 2 to 12 mm3, or from 4 to 8 mm3.

[0055] In some embodiments, one or more corners of the chamber are smooth, optionally wherein the one or more corners are rounded or sloped.

[0056] In certain embodiments, the photoconductive layer is made of a material comprising or consisting of hydrogenated amorphous silicon (a-Si:H), titanium oxide phthalocyanine (TiOPc), cadmium Sulfide (CdS), poly(3-hexylthiophene) (P3HT), and mixtures of P3HT. For Atty. Docket: STAN-2215WO (S24-293) example, in some instances, the mixture of P3HT is P3HT:phenyl C61 -butyric acid methyl ester (PCBM).

[0057] According to some embodiments, the electrode pattern comprises electrodes separated from each other by a space of from 50 to 500 pm, from 75 to 125 pm, from 90 to 110 pm, or about 100 pm.

[0058] SYSTEMS COMPRISING THE MICROFLUIDIC DEVICES

[0059] Aspects of the present disclosure further include systems comprising the microfluidic devices disclosed herein. As noted above, the devices disclosed herein can be used to optically manipulate micro-objects. To that end, the systems comprise the devices disclosed herein and can further comprise a light pattern generator optically coupled to the photoconductive layer, and a voltage source electrically coupled to the first and second electrodes disposed in a planar arrangement within or adjacent to the photoconductive layer.

[0060] In certain embodiments, the system further comprises (is configured to comprise) a pressure controller fluidically connected to the one or more of: the droplet generator, the inlet channel, and the outlet channel. The pressure controller can be a piezoelectric pressure controller.

[0061] In some embodiments, the light pattern generator of the system is SLM. The light pattern generator can be optically coupled to the photoconductive layer. In certain embodiments, the light pattern generator is optically coupled to the photoconductive layer via an objective lens.

[0062] In certain embodiments, the voltage source is adapted to deliver an AC voltage to the electrode pattern of the chamber. The AC voltage can have a frequency, e.g., within a range of from 100 Hz to 100 GHz.

[0063] In addition, the system can comprise an electrical apparatus for applying appropriate electric field to the chamber. As shown in FIG. 4, the electrical apparatus can comprise a signal generator and an RF amplifier, and, optionally, an oscilloscope.

[0064] In certain embodiments, the system can also comprise an optical apparatus for imaging the chamber. Such an optical apparatus can be used to observe the chamber, for example, the movements of particles and droplets in the chamber. Based on the observations, the light directed on the chamber substrate can be manipulated. Such optical apparatus can comprise, among other elements, an objective lens, a plate beamsplitter, and a camera. Certain examples of these features are illustrated in FIG. 4.

[0065] A system according to some embodiments of the present disclosure is illustrated in FIG. 4. As shown, this exemplary system comprises an optical system for pattern projection optically coupled to the microfluidic device (e.g., comprising an SLM, one or more lenses, Atty. Docket: STAN-2215WO (S24-293) and one or more mirrors), a fluidics system fluidically coupled to the microfluidic device (e.g., comprising a pressure controller and a reagent reservoir), an electrical system electrically coupled to the microfluidic device (e.g., comprising an oscilloscope, a waveform generator and an RF amplifier), and an optical system for imaging optically coupled to the microfluidic device (e.g., comprising a fluorescence excitation source (e.g., an LED) and suitably configured lenses, beam splitter, filters and a camera for observing the desired processes occurring within the microfluidics device).

[0066] The devices described herein can be fabricated using standard photo-lithography and soft-lithography. As such, it is comparable with industrial chip manufacturing processes. For mass-production, the cost per chip can be extremely low. This is in contrast to existing platforms.

[0067] The complete encapsulation of individual solid supports by the reagent droplets ensures uniform reagent exposure, which reduces the error rate of each reaction step and improve the final yield rate significantly. Furthermore, picoliter droplet reactors replacing microtiter plates reduce reagent consumption and waste generation by up to a million-fold, thereby enabling significantly lower fabrication costs. This also makes the process more sustainable.

[0068] METHODS OF ENGULFING PARTICLES

[0069] Aspects of the present disclosure further include methods of engulfing a particle into a droplet. In some instances, the methods are implemented using a system of the present disclosure. According to some embodiments, the methods comprise dispensing a particle suspended in the suspension medium from the microfluidic inlet channel into the chamber, generating a droplet in the chamber using the microfluidic droplet generator, providing a voltage to the photoconductive layer using the first and second electrodes, and projecting a light pattern onto the photoconductive layer using the light pattern generator to engulf the particle into the droplet. The light pattern is selected to exert an electric field gradient on the droplet and / or the particle such that the particle is engulfed into the droplet.

[0070] The suspension medium is a liquid medium. In certain embodiments, the liquid medium is inert, comprises a high dielectric strength, or both. By “inert” is meant a liquid that is not generally reactive. As used herein, a liquid having a “high dielectric strength” is a liquid having a dielectric strength of 1 x 105V / m or greater. In certain embodiments, the liquid has a dielectric strength of 1 x 106V / m or greater, such as 1 x 107V / m. According to some embodiments, the liquid medium which is inert and / or has a high dielectric point comprises, consists essentially of, or consists of an oil. Suitable oils include, but are not limited to, silicone oil. In certain embodiments, the liquid medium comprises an oil and a surfactant, e.g., a non-ionic surfactant. The surfactant may be present at a suitable percentage (e.g., to Atty. Docket: STAN-2215WO (S24-293) reduce the interfacial tension between water and oil), including from 0.5 to 10%, such as from 1 to 5%, e.g., about 2.5%.

[0071] The suspension medium can be an oil, such as a long chain alkane. A long chain alkane can be hexadecane. The oil can also be silicone oil, fluorinated oil (perfluorocarbons), vegetable oil, or mineral oil. Additional examples of suitable oils that could be used as a suspension medium in the devices and methods disclosed herein are well-known to a person of ordinary skill in the art and such embodiments are within the purview of the disclosure.

[0072] A schematic representation of the method of engulfing a particle in a droplet is provided in FIG. 5. In some embodiments, the particle is a bead or a cell.

[0073] A "particle" as used herein means a small, localized object to which can be ascribed a physical property such as volume, mass or average size. Particles may accordingly be of a symmetrical, globular, essentially globular or spherical shape, or be of an irregular, asymmetric shape or form. In certain embodiments, the particle is spherical, spheroid, rodshaped, disk-shaped, pyramid-shaped, cube-shaped, cylinder-shaped, nanohelical-shaped, nanospring-shaped, nanoring-shaped, arrow-shaped, teardrop-shaped, tetrapod-shaped, prism-shaped, or any other suitable geometric or non-geometric shape. The size of a particle may vary. The term “microparticle” refers to particles with a greatest dimension (e.g., diameter) in the nanometer and micrometer range. According to certain embodiments, the particle is a bead, e.g., a microbead. As used herein, the term “bead” refers to a small mass that is generally spherical or spheroid in shape. According to some embodiments, a microbead as used herein has an average diameter of from about 0.1 pm to about 500 pm, from about 0.50 pm to about 250 pm, e.g., from about 1 pm to about 100 pm.

[0074] Particles (e.g., microparticles, such as microbeads) as defined herein may comprise, consist essentially of, or consist of any suitable material, e.g., they may comprise, consist essentially of, or consist of inorganic or organic material. In certain embodiments, the particle comprises, consists essentially of, or consists of metal or an alloy of metals, or an organic material. According to certain embodiments, the particle comprises, consists essentially of, or consists of agarose, polystyrene, latex, polyvinyl alcohol, silica, or a ferromagnetic material (e.g., ferromagnetic metal) metal, alloys or composition materials.

[0075] In certain embodiments, the particle is a cell. The cell may be a prokaryotic or eukaryotic cell. According to some embodiments, the cell is an immune cell (e.g., a B cell, a T cell, or the like), an endothelial cell, an epithelial cell, a stem cell, or a neuron.

[0076] Any suitable approach may be employed for dispensing a particle into the liquid medium of the chamber of the microfluidic device. In certain embodiments, a particle (e.g., microbead) suspended in a solution (e.g., an oil solution) is introduced into the chamber Atty. Docket: STAN-2215WO (S24-293) through an inlet. The particle concentration may be tuned to ensure a single particle enters the vicinity of the electrodes and can be individually manipulated using light.

[0077] Similarly, any suitable approach may be employed for dispensing the droplet into the liquid medium of the chamber of the microfluidic device. In some embodiments, the microfluidic device comprises a droplet generator channel adapted to dispense a single droplet into the chamber at a time. According to certain embodiments, the droplet generator channel is from 10 to 40 pm wide, such as from 15 to 35 pm wide, or from 20 to 30 pm wide, e.g., about 25 pm wide. The droplet generator channel may be operably coupled to a fluidic port which is in turn operably coupled to a fluidic subsystem comprising a pressure controller (e.g., a piezo driven pressure controller) that allows for the dispensing of single droplets via the droplet generator channel into the chamber. In certain embodiments, a pressure pulse of from 1 to 20 mbar (e.g., from 5 to 15 mbar, e.g., about 10 mbar) is exerted using the pressure controller to dispense a single droplet into the chamber. In some embodiments, the volume of the droplet generated by the droplet generator (e.g., droplet generator channel) is from 10 to 100 picoliters, e.g., from 20 to 80 picoliters, such as from 40 to 60 picoliters.

[0078] In the example shown in FIG. 5, the particle and droplet are held in the chamber using the voltage applied to the electrodes and the light beams directed onto to the photoconductive layer, as shown in the left panel of FIG. 5.

[0079] To encapsulate the bead into the droplet, one or both of the light beams are moved so that the droplet is brought closer to the bead. The exerted dielectrophoretic force is sufficient to overcome the surface tension barrier and to force the droplet to engulf the bead, i.e., put the bead inside the droplet. This is shown in the middle panel of FIG. 5.

[0080] For decapsulation, i.e., taking the bead out of the droplet, a pulsed force is applied on the droplet which causes it to decapsulate the bead. This pulsed excitation causes the droplet to deform which facilitates the decapsulation process. This rapid deformation inducing pulse facilitates the bead to come out of the droplet when the droplet is moved using the light. The decapsulation is shown in the right panel of FIG. 5.

[0081] FIG. 6 provides an example of droplet generation in the device of the disclosure, for example, the device shown in FIG. 3 and discussed above. FIG. 6 shows an exemplary device in the left panel and a blown up portion of the chamber in the right panel.

[0082] As shown in the right panel, the device comprises the chamber 600 having the inlet 601 for generating droplets 602. Interdigitated electrodes 603 are also visible in this image.

[0083] FIG. 7 shows trapping and manipulation of a bead in a droplet. The left panel shows droplet manipulation using light. The middle panel shows a bead being brought closer to a Atty. Docket: STAN-2215WO (S24-293) droplet and the right panel shows the bead encapsulated by the droplet. The bead and the droplet were manipulated with light beams or patterns.

[0084] FIG. 8 shows parallel manipulation and encapsulation of two droplets in two beads. Panel A shows electrodes of interdigitated electrodes, two droplets (top and bottom) and two beads (top and bottom). Light beams were used to manipulate the droplets and to move them towards the beads, as shown in Panel B. Panel C shows that each droplet is brought sufficiently close to a corresponding bead so that the droplets encapsulate the beads. Panel D shows the two droplets encapsulating the two beads as the light beams are moved away from the droplets and beads.

[0085] The methods of manipulating beads and droplets find use in a variety of contexts. For example, in certain embodiments, the methods comprise modifying the particle engulfed in the droplet (e.g., conducting solid-phase synthesis on the particle, or the like) where it is demonstrated herein that the present methods provide for significantly higher fidelity modifications as compared to existing approaches and devices (e.g., benchtop platforms) for performing such modifications. Details regarding certain methods of the present disclosure are described later in the disclosure.

[0086] In some embodiments, the modifying of the beads comprises covalently or non- covalently attaching a molecule present in the droplet to the particle.

[0087] For example, the modifying can comprise covalently attaching a nucleotide or polynucleotide present in the droplet to the particle. The nucleotide or polynucleotide can be attached to the particle chemically and / or enzymatically. In certain embodiments, the nucleotide or polynucleotide is attached to the particle by azide addition followed by click ligation, which is described below in additional details.

[0088] In certain embodiments, the modifying comprises non-covalently attaching a nucleotide or polynucleotide present in the droplet to the particle. For example, the attaching can comprise base-pairing the nucleotide or polynucleotide to a nucleotide or polynucleotide present on the particle. In some embodiments, the nucleotide or polynucleotide is attached to the particle via a biotin-streptavidin interaction.

[0089] In further embodiments, the modifying comprises covalently attaching an amino acid or polypeptide present in the droplet to the particle. The amino acid or polypeptide can be attached to the particle chemically and / or enzymatically. For example, the amino acid or polypeptide can be attached to the particle by azide addition followed by click ligation.

[0090] In certain embodiments, the modifying comprises non-covalently attaching an amino acid or polypeptide present in the droplet to the particle. For example, an amino acid or polypeptide can be attached to the particle via a biotin-streptavidin interaction. Atty. Docket: STAN-2215WO (S24-293)

[0091] In even further embodiments, wherein the modifying comprises covalently or non- covalently attaching a sugar or carbohydrate present in the droplet to the particle.

[0092] In some embodiments, the surface of the particle may be functionalized (or “activated” / “derivatized”) with reactive groups to which the molecule present in the droplet is attached. For example, the surface may be functionalized with any useful / convenient reactive group, including but not limited to thiol groups (-SH), amine groups (-NH2), carboxyl groups (-COO), carbodiimide crosslinker chemistry, polyethylene glycol (PEG), and / or the like. Bioconjugation strategies that find use in attaching a molecule present in the droplet to a reactive group on the particle are described in Hermanson, “Bioconjugate Techniques,” Academic Press, 2nd edition, April 1 , 2008, Haugland, 1995, Methods Mol. Biol. 45:205-21 ; Brinkley, 1992, Bioconjugate Chemistry 3:2, and elsewhere.

[0093] According to embodiments in which the particle comprises silica (SiO2) on its surface, the surface silanol groups may be functionalized. The silica can be functionalized to bear free thiol / sulfhydryl groups either during the course of making the SiO2 surface (“direct modification”), or after the particle has been completely formed (“post-modification”). Reactions for the modification of silanol groups are known and include, but are not limited to, modification of the SiOs surface to present amines (e.g., by reaction with aminopropyl trimethoxysilane (APTMS)) or ethoxides (e.g., by reaction with 3-glycidyloxypropyl- trimethoxysilane (GPTMS)). Reagents also exist to incorporate sulfhydryls, carboxyl and other useful reactive groups for conjugation.

[0094] The molecule present in the droplet may already include a functional group useful for reacting with a reactive group present on the particle, or such a functional group may be provided to the molecule. Functional groups that may be used to bind a molecule present in the droplet to the particle include, but are not limited to, active esters, isocyanates, imidoesters, hydrazides, amino groups, aldehydes, ketones, photoreactive groups, maleimide groups, alpha-halo-acetyl groups, epoxides, azirdines, and the like. Reagents such as iodoacetamides, maleimides, benzylic halides and bromomethylketones react by S- alkylation of thiols to generate stable thioether products. For example, at pH 6.5-7.5, maleimide groups react with sulfhydryl groups to form stable thioether bonds. Arylating reagents such as NBD halides react with thiols or amines by a similar substitution of the aromatic halide by the nucleophile. Because the thiolate anion is a better nucleophile than the neutral thiol, cysteine is more reactive above its pKa(~8.3, depending on protein structural context). Thiols also react with certain amine-reactive reagents, including isothiocyanates and succinimidyl esters. The TS-Link series of reagents are available for reversible thiol modification. Atty. Docket: STAN-2215WO (S24-293)

[0095] With respect to amine reactive groups, primary amines exist at the N-terminus of polypeptide chains and in the side-chain of lysine (Lys, K) amino acid residues. Among the available functional groups in typical biological or protein samples, primary amines are especially nucleophilic, making them ready targets for conjugation with several reactive groups. For example, NHS esters are reactive groups formed by carbodiimide-activation of carboxylate molecules. NHS ester-activated crosslinkers and labeling compounds react with primary amines in physiologic to slightly alkaline conditions (pH 7.2 to 9) to yield stable amide bonds. The reaction releases N-hydroxysuccinimide (NHS). Also by way of example, imidoester crosslinkers react with primary amines to form amidine bonds. Imidoester crosslinkers react rapidly with amines at alkaline pH but have short half-lives. As the pH becomes more alkaline, the half-life and reactivity with amines increases. As such, crosslinking is more efficient when performed at pH 10 than at pH 8. Reaction conditions below pH 10 may result in side reactions, although amidine formation is favored between pH 8-10.

[0096] Numerous other synthetic chemical groups will form chemical bonds with primary amines, including but not limited to, isothiocyanates, isocyanates, acyl azides, sulfonyl chlorides, aldehydes, glyoxals, epoxides, oxiranes, carbonates, aryl halides, carbodiimides, anhydrides, and fluorophenyl esters. Such groups conjugate to amines by either acylation or alkylation.

[0097] According to certain embodiments, the modifying comprises covalently attaching a nucleotide or polynucleotide present in the droplet to the particle. The term “nucleotide” is intended to include those moieties that contain not only the naturally occurring purine and pyrimidine bases, but also other heterocyclic bases that have been modified. Such modifications include methylated purines or pyrimidines, acylated purines or pyrimidines, alkylated riboses or other heterocycles. In addition, the term “nucleotide” includes those moieties that contain hapten or fluorescent labels and may contain not only conventional ribose and deoxyribose sugars, but other sugars as well. Modified nucleosides or nucleotides also include modifications on the sugar moiety, e.g., wherein one or more of the hydroxyl groups are replaced with halogen atoms or aliphatic groups, or are functionalized as ethers, amines, or the like. As used herein, an “oligonucleotide” is a single-stranded multimer of nucleotides from 2 to 500 nucleotides, e.g., 2 to 200 nucleotides. Oligonucleotides may be synthetic or may be made enzymatically, and, in some embodiments, are 5 to 50 nucleotides in length (e.g., 9 to 50 nucleotides in length). Oligonucleotides may contain ribonucleotide monomers (i.e., may be oligoribonucleotides or “RNA oligonucleotides”) or deoxyribonucleotide monomers (i.e., may be oligodeoxyribonucleotides or “DNA oligonucleotides”). Oligonucleotides may be 5 to 9, 10 to 20, 21 to 30, 31 to 40, 41 to 50, 51 Atty. Docket: STAN-2215WO (S24-293) to 60, 61 to 70, 71 to 80, 80 to 100, 100 to 150 or 150 to 200, up to 500 or more nucleotides in length, for example. The term “nucleic acid” and “polynucleotide” are used interchangeably herein to describe a polymer of any length, e.g., greater than about 2 bases, greater than about 10 bases, greater than about 100 bases, greater than about 500 bases, greater than 1000 bases, greater than 10,000 bases, greater than 100,000 bases, greater than about 1 ,000,000, up to about 1010or more bases composed of nucleotides, e.g., deoxyribonucleotides or ribonucleotides, which can hybridize with naturally occurring nucleic acids in a sequence specific manner analogous to that of two naturally occurring nucleic acids, e.g., can participate in Watson-Crick base pairing interactions. Naturally-occurring nucleotides include guanine, cytosine, adenine, thymine, uracil (G, C, A, T and U respectively). DNA and RNA have a deoxyribose and ribose sugar backbone, respectively, whereas PNA’s backbone is composed of repeating N-(2-aminoethyl)-glycine units linked by peptide bonds. In PNA various purine and pyrimidine bases are linked to the backbone by methylenecarbonyl bonds. A locked nucleic acid (LNA), often referred to as inaccessible RNA, is a modified RNA nucleotide. The ribose moiety of an LNA nucleotide is modified with an extra bridge connecting the 2' oxygen and 4' carbon. The bridge “locks” the ribose in the 3'-endo (North) conformation, which is often found in the A-form duplexes. LNA nucleotides can be mixed with DNA or RNA residues in the oligonucleotide whenever desired. The term “unstructured nucleic acid,” or “UNA,” is a nucleic acid containing non-natural nucleotides that bind to each other with reduced stability. For example, an unstructured nucleic acid may contain a G' residue and a O' residue, where these residues correspond to non-naturally occurring forms, i.e., analogs, of G and C that base pair with each other with reduced stability, but retain an ability to base pair with naturally occurring C and G residues, respectively. Unstructured nucleic acid is described in US20050233340, which is incorporated by reference herein for disclosure of UNA.

[0098] When the modifying comprises covalently attaching a nucleotide or polynucleotide present in the droplet to the particle, the nucleotide or polynucleotide may be attached to the particle chemically. Approaches for chemical attachment of nucleotides or polynucleotides to the particle include, but are not limited to, click-chemistry based approaches. Click-chemistry reactions that may be employed include (i) nucleophilic substitutions; (ii) additions to C-C multiple bonds (e.g., Michael addition, epoxidation, dihydroxylation, aziridination); (iii) nonaldol like chemistry (e.g., N-hydroxysuccinimide active ester couplings); and (iv) cycloadditions (e.g., Diels-Adler reaction, Huisgen’s cycloaddition). Huisgen’s cycloaddition has been applied in various branches of chemistry. It consists of the condensation of organic azides with alkyne groups to form 1 ,2,3-triazole linkages. Azide and alkyne functionalities can be easily introduced in the scaffold of large organic constructs of biological relevance. Atty. Docket: STAN-2215WO (S24-293)

[0099] The reaction may be catalyzed by introducing copper(l). The Cu(l) core has a dual effect in that it activates the slow-reacting alkyne group thus accelerating the azide-alkyne condensation kinetics by ~107-108-fold, and it organizes the reacting groups by “templation” so that only a regiospecific 1 ,4-disubstituted adduct is formed. This reaction is known as the copper-catalyzed azide alkyne cycloaddition (CuAAC), and its compatibility with a wide range of biological substrates and synthetic conditions makes CuAAC the flagship among click conjugations. Since its discovery, Cu(l)-catalyzed azide alkyne cycloaddition has been widely used within the fields of biology, biochemistry, and biotechnology. Click-chemistry reactions that may be employed to attach the nucleotide or polynucleotide to the particle include, but are not limited to, Huisgen Azide-Alkyne 1 ,3-Dipolar Cycloaddition, Copper-Catalyzed Azide- Alkyne Cycloaddition (CuAAC), Ruthenium-Catalyzed Azide-Alkyne Cycloaddition (RuAAC), and the like. Details regarding click-chemistry with nucleic acids are found, e.g., in Fantoni et al. (2021 ) Chem. Rev. 121 (12)7122-7154.

[0100] When the modifying comprises covalently attaching a nucleotide or polynucleotide present in the droplet to the particle, the nucleotide or polynucleotide may be attached to the particle enzymatically. Enzymatic attachment may be performed by providing, e.g., a suitable nucleic acid ligase (e.g., a DNA ligase) and conditions suitable for enzymatic ligation reactions. Enzymatic attachment may also be performed by providing, e.g., a suitable nucleic acid polymerase and conditions suitable for polymerase-mediated extension reactions (e.g., extension of a primer disposed on the particle). Extension reactions where a nucleic acid present on the engulfed particle is extended within the droplet may be templated or non- templated. Enzymes that find use in templated extension reactions include, e.g., polymerases. Enzymes that find use in non-templated extension reactions include, e.g., terminal transferases. Terminal transferase (TdT) is a template independent polymerase that catalyzes the addition of deoxynucleotides to the 3' hydroxyl terminus of DNA molecules.

[0101] When the modifying comprises non-covalently attaching a nucleotide or polynucleotide present in the droplet to the particle, in certain embodiments, the attaching comprises base-pairing the nucleotide or polynucleotide to a nucleotide or polynucleotide present on the particle. Such base-pairing will typically be performed under hybridization conditions. As used herein, the term “hybridization conditions” means conditions in which a nucleic acid or oligonucleotide specifically hybridizes to a nucleotide or polynucleotide present on the particle. Whether such hybridization occurs is determined by such factors as the degree of complementarity and the temperature at which the hybridization occurs, which may be informed by the melting temperature (TM). The melting temperature refers to the temperature at which half of the nucleic acid duplexes remain hybridized and half of the duplexes dissociate into single strands. The Tm of a duplex may be experimentally Atty. Docket: STAN-2215WO (S24-293) determined or predicted using the following formula Tm = 81.5 + 16.6(log10[Na+]) + 0.41 (fraction G+C) - (60 / N), where N is the chain length and [Na+] is less than 1 M. See Sambrook and Russell (2001 ; Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor N.Y., Ch. 10). Other more advanced models that depend on various parameters may also be used to predict Tm of duplexes depending on various hybridization conditions. Approaches for achieving specific nucleic acid hybridization may be found in, e.g., Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology- Hybridization with Nucleic Acid Probes, part I, chapter 2, “Overview of principles of hybridization and the strategy of nucleic acid probe assays,” Elsevier (1993). When the modifying comprises non-covalently attaching a nucleotide or polynucleotide present in the droplet to the particle, in some embodiments, the nucleotide or polynucleotide is attached to the particle via a biotin-streptavidin interaction.

[0102] According to certain embodiments, the modifying comprises covalently attaching an amino acid or polypeptide present in the droplet to the particle. The term “amino acid” generally refers to any monomer unit that comprises a substituted or unsubstituted amino group, a substituted or unsubstituted carboxy group, and one or more side chains or groups, or analogs of any of these groups. Exemplary side chains include, e.g., thiol, seleno, sulfonyl, alkyl, aryl, acyl, keto, azido, hydroxyl, hydrazine, cyano, halo, hydrazide, alkenyl, alkynl, ether, borate, boronate, phospho, phosphono, phosphine, heterocyclic, enone, imine, aldehyde, ester, thioacid, hydroxylamine, or any combination of these groups. Other representative amino acids include, but are not limited to, amino acids comprising photoactivatable cross-linkers, metal binding amino acids, spin-labeled amino acids, fluorescent amino acids, metal-containing amino acids, amino acids with novel functional groups, amino acids that covalently or noncovalently interact with other molecules, photocaged and / or photoisomerizable amino acids, radioactive amino acids, amino acids comprising biotin or a biotin analog, glycosylated amino acids, other carbohydrate modified amino acids, amino acids comprising polyethylene glycol or polyether, heavy atom substituted amino acids, chemically cleavable and / or photocleavable amino acids, carbon- linked sugar-containing amino acids, redox- active amino acids, amino thioacid containing amino acids, and amino acids comprising one or more toxic moieties. As used herein, the term “amino acid” includes the following twenty natural or genetically encoded alpha-amino acids: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamine (Gin or Q), glutamic acid (Glu or E), glycine (Gly or G), histidine (His or H), isoleucine (lie or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V). The terms “polypeptide,” Atty. Docket: STAN-2215WO (S24-293)

[0103] “peptide,” and “protein,” used interchangeably herein, refer to a polymeric form of amino acids of any length, which can include genetically coded and non-genetically coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified peptide backbones. The term includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terminal methionine residues; immunologically tagged proteins; and the like.

[0104] In certain embodiments, the amino acid or polypeptide is attached to the particle chemically or enzymatically. When the amino acid or polypeptide is attached to the particle chemically, in certain embodiments, the attachment utilizes any of the click-chemistry approaches described above, e.g., Huisgen Azide-Alkyne 1 ,3-Dipolar Cycloaddition, Copper- Catalyzed Azide-Alkyne Cycloaddition (CuAAC), Ruthenium-Catalyzed Azide-Alkyne Cycloaddition (RuAAC), and the like. Details regarding click-chemistry with amino acids and polypeptides are found, e.g., in Parker & Pratt (2020) Cell 180(4) :605-632. When the amino acid or polypeptide is attached to the particle chemically, in certain embodiments, the attachment utilizes solid phase peptide chemistry, e.g., standard 9- fluorenylmethyloxycarbonyl (Fmoc)- or Boc-based solid phase peptide chemistry may be employed.

[0105] According to some embodiments, the modifying comprises non-covalently attaching an amino acid or polypeptide present in the droplet to the particle. In one non-limiting example, the amino acid or polypeptide is attached to the particle via a biotin-streptavidin interaction. Also by way of example, non-covalently attaching an amino acid or polypeptide present in the droplet to the particle comprised binding an antibody to the particle (e.g., a cell, a microbead, or the like).

[0106] According to certain embodiments, the modifying comprises covalently or non- covalently attaching a sugar or carbohydrate present in the droplet to the particle.

[0107] In certain embodiments, the particle comprises a protein on its surface, and the modifying comprises modifying the protein. Such protein modifications include, but are not limited to, post-translational modifications (PTMs) that occur in nature such as glycosylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, lipidation, and / or the like. By way of example, the modifying may comprise glycosylating the protein with a carbohydrate present in the droplet. Glycosylation is the enzymatic post-translational addition of carbohydrates (glycans) to proteins and lipids, resulting in "glycoproteins" and "glycolipids," respectively. Canonically, glycoprotein glycans can be N-linked (linkage to the amide group of Asn) or O-linked (linkage to the hydroxyl group of Ser, Thr). The particular glycan structures, the "glycoforms," of a glycoprotein impact the function, stability, folding, Atty. Docket: STAN-2215WO (S24-293) localization and ligand specificity of the glycoprotein, and play a role in cell adhesion and cell trafficking by modulating how cells interact with each other and with their extracellular matrix environment. Also by way of example, the modifying may comprise phosphorylating the protein with a phosphate group present in the droplet, e.g., the droplet may comprise a kinase enzyme, phosphate groups, an appropriate buffer, etc. to carry out phosphorylation of the protein within the droplet.

[0108] In some embodiments, when engulfed bead is assessed. For example, when the bead is a cell, the cell can be assessed for the expression of a polypeptide. The polypeptide can be a cytoplasmic polypeptide, a cell surface polypeptide, or a secreted polypeptide. The polypeptide can also be an antibody.

[0109] In certain embodiments, the droplet comprises a binding partner for the polypeptide, and the assessing comprises assessing for complexes comprising the binding partner and the polypeptide. The binding partner can be an antibody, a ligand, an aptamer, an antigen, or a small molecule.

[0110] According to some embodiments, once the particle is engulfed into the droplet, the methods further comprise assessing the engulfed particle. In certain embodiments, the methods comprise assessing the engulfed particle prior to and / or subsequent to modifying the engulfed particle. In some embodiments, the engulfed particle is assessed to determine whether the particle comprises the desired modification.

[0111] In certain embodiments, the particle is a cell, and the method comprises assessing the cell for expression of a polypeptide. The polypeptide may be a cytoplasmic polypeptide, a cell surface polypeptide, a secreted polypeptide, or the like. According to certain embodiments, the polypeptide is an antibody. In certain embodiments, the droplet comprises a binding partner for the polypeptide, and wherein the assessing comprises assessing for complexes comprising the binding partner and the polypeptide. Non-limiting examples of such binding partners include an antibody, a ligand, an aptamer, an antigen, or a small molecule.

[0112] By “antibody” is meant an antibody or immunoglobulin of any isotype (e.g., IgG (e.g., lgG1 , lgG2, lgG3, or lgG4), IgE, IgD, IgA, IgM, etc.), whole antibodies (e.g., antibodies composed of a tetramer which in turn is composed of two dimers of a heavy and light chain polypeptide); single chain antibodies (e.g., scFv); fragments of antibodies (e.g., fragments of whole or single chain antibodies) which retain specific binding to the cell surface molecule of the target cell, including, but not limited to single chain Fv (scFv), Fab, (Fab’)2, (scFv’)2, and diabodies; chimeric antibodies; monoclonal antibodies, human antibodies, humanized antibodies (e.g., humanized whole antibodies, humanized half antibodies, or humanized antibody fragments, e.g., humanized scFv); and fusion proteins comprising an antigen- Atty. Docket: STAN-2215WO (S24-293) binding portion of an antibody and a non-antibody protein. In certain embodiments, the antibody is selected from an IgG, single chain Fv (scFv), Fab, (Fab)2, (scFv’)2, or a single variable domain located on a heavy chain (VHH). According to some embodiments, the antibody is a VHH (sometimes referred to as a “nanobody”).

[0113] In certain embodiments, the particle is a B cell, and the method comprises assessing the B cell for expression of an antibody that specifically binds an antigen present in the droplet.

[0114] As used herein, a “ligand” is a substance that forms a complex with a biomolecule in nature to serve a biological purpose. The ligand may be a substance selected from a circulating factor, a secreted factor, a cytokine, a growth factor, a hormone, a peptide, a polypeptide, a small molecule, and a nucleic acid that forms a complex, e.g., with a cell surface molecule on the surface of the cell. In certain embodiments, the ligand is modified in such a way that complex formation with the cell surface molecule occurs, but the normal biological result of such complex formation does not occur. In certain embodiments, the ligand is the ligand of a cell surface receptor present on the cell. Cell surface receptors of interest include, but are not limited to, receptor tyrosine kinases (RTKs), non-receptor tyrosine kinases (non-RTKs), growth factor receptors, etc.

[0115] By “aptamer” is meant a nucleic acid (e.g., an oligonucleotide) that has a specific binding affinity for the cell surface molecule. Aptamers exhibit certain desirable properties, such as ease of selection and synthesis, high binding affinity and specificity, low immunogenicity, and versatile synthetic accessibility. Aptamers that bind to cell surface molecules are known and include, e.g., TTA1 (a tumor targeting aptamer to the extracellular matrix protein tenascin-C).

[0116] By “small molecule” is meant a compound having a molecular weight of 1000 atomic mass units (amu) or less. In certain embodiments, the small molecule is 750 amu or less, 500 amu or less, 400 amu or less, 300 amu or less, or 200 amu or less. In certain aspects, the small molecule is not made of repeating molecular units such as are present in a polymer. In certain aspects, the cell surface molecule is a receptor for which the ligand is a small molecule, and the small molecule is the small molecule ligand (or a derivative thereof) of the receptor.

[0117] In some embodiments, modifying comprises washing the engulfed particle to remove remnants of a previously contacted droplet by engulfing the droplet in a washing droplet. Such washing can be performed by encapsulating a bead in a droplet comprising an appropriate solution, such as phosphate buffer saline, water, or any other suitable washing solution. Atty. Docket: STAN-2215WO (S24-293)

[0118] In certain embodiments, the method further comprises ejecting the particle from the droplet. The ejecting the particle from the droplet can comprise applying a pulsed force on the droplet.

[0119] In even further embodiments, the ejected particle is subjected again to step (d) of the methods described above to re-engulf the particle in a droplet. When re-engulfed in a droplet, further steps can comprise repeating the modifying and / or washing steps as described above. Thus, in certain embodiments, the methods comprise performing solid-phase synthesis on the surface of the particle by iteratively engulfing, ejecting and re-engulfing the particle in droplets comprising monomers for solid-phase synthesis.

[0120] Accordingly, and with the benefit of the present disclosure, it will be appreciated that the present methods and devices find use in a variety of applications including but not limited to the following:

[0121] 1 . Enzymatic and / or chemical substrate ligation (e.g., DNA ligase)

[0122] • Initiator strand - primer for terminal transferase

[0123] • Cu(l)-catalyzed azide-alkyne cycloaddition (CuAAC)

[0124] 2. Enzyme-linked immunosorbent assay

[0125] • antigen on the particle; or antibody on the particle

[0126] 3. Biological / chemical combinatorial synthesis

[0127] 4. Combinatorial library generation (e.g., one particle one compound (OBOC) and split pool)

[0128] 5. High-throughput screening (e.g., drug discovery, substrate toxicity)

[0129] • different compounds in different droplets

[0130] 6. Hybridization assay (e.g., sandwich, competitive, nuclease, dual ligation)

[0131] • hybridize nucleic acid in droplet to nucleic acid on particle;

[0132] • error detection (quality control for sequence built onto the particle) o assess for hybridization under different conditions (temperature and / or salt concentration) o oligonucleotide error filtration (e.g., mismatches, deletions)

[0133] 7. Oligonucleotide read / write synthesis (e.g., DNA, RNA, peptide)

[0134] • fluorescent readout with information regarding which nucleotide was added

[0135] 8. Antibody capture (e.g., Protein-G, affinity) e.g., protein-G on particle to capture IgG bind a particular class of antibody onto the particle Atty. Docket: STAN-2215WO (S24-293)

[0136] 9. Particle surface functionalization (e.g., carbodiimide crosslinker chemistry, PEG)

[0137] • NHS, etc.

[0138] 10. Polymerase chain reaction (see also #16)

[0139] • primer on particle; template and reverse primer in reagent droplet; or bridgetype amplification

[0140] 11. Protein modification (e.g., phosphorylation, methylation, glycosylation)

[0141] • protein on particle, modification moiety in reagent droplet

[0142] 12. DNA Sequencing

[0143] 13. Biological cell sorting and manipulation

[0144] • isolate particular cell types (immune cells, red blood cells, etc.) o ligand or receptor on particle for binding partner on cell

[0145] 14. Substrate / oligonucleotide enzymatic functionalization (e.g., TdT + 3’ azide coupling)

[0146] 15. Oligonucleotide error filtration (e.g., mismatches, deletions)

[0147] 16. Polymerase chain assembly (e.g., oligonucleotide tiling and amplification)

[0148] 17. On-chip droplet library

[0149] • Each droplet of the library can contain a unique, sequence verified oligonucleotide. The oligonucleotide can contain, e.g., 3, 5, or 10 bases. Also, the oligonucleotide can contain 5' alkyne and / or 3' hydroxyl groups. Based on the desired target sequence, droplets with matching oligonucleotide composition can be selected and concatenated together in a specific order. Such concatenation can be performed using enzymatic and / or click ligation cycle(s). Post-ligation, the full-length product can be PCR-amplified on-bead. Alternatively, the full-length product can be released from the bead into solution and then PCR-amplified. An example library comprises 3-base codon-optimized and sequence-verified oligonucleotides each encoding a proteinogenic amino acid, e.g., one of the 22 commonly known proteinogenic amino acids. A nucleic acid encoding a desired polypeptide sequence can be produced by sequentially concatenating the relevant 3-base codon optimized oligonucleotides in the appropriate order.

[0150] An example of modifying the particle is provided in FIG. 9, which shows DNA ‘click’ ligation cycle implemented in the methods disclosed herein for preparation of ligated oligonucleotide. As shown in FIG. 9, the reaction cycle has two steps. The first step comprises azide addition, i.e., a nucleotide with 3’-azide is coupled to the DNA acceptor through enzymatic reaction using terminal transferase (TdT). Second step comprises a click Atty. Docket: STAN-2215WO (S24-293) ligation, i.e., 5’-alkyne DNA donor is ligated to the DNA acceptor through copper-catalyzed azide-alkyne cycloaddition (CuAAC) click chemistry.

[0151] Certain advantages of the methods disclosed herein are highlighted by this reaction cycle. The click ligation step uses -80% tert-butanol which is an organic solvent. The suspension medium is chosen to be chemically neutral and immiscible with organic reagents, such as tert-butanol, ethanol, dimethylformamide (DMF).

[0152] In some embodiments, the methods disclosed herein comprise concatenating single stranded DNA (ssDNA) fragments to make high-fidelity long DNA strands. This provides a template-free ligation that allows incorporating customized modifications.

[0153] FIG. 10 describes verification of the DNA click ligation using standard polyacrylamide gel electrophoresis (PAGE) analysis of the products from the bead. The PAGE analysis of FIG. 10 shows full length product with the highest molecular weight in the gel as compared to control OH acceptor DNA. FIG. 11 shows fluorescent imaging analysis showing successful on-chip DNA synthesis reaction.

[0154] The methods of the present disclosure are implemented using a microfluidic device. Non-limiting examples of microfluidic devices that find use in practicing the methods include those described in further detail below in the Devices and Experimental sections of the present disclosure. As used herein, a “microfluidic device” is a device comprising one or more channels comprising dimensions from tens to hundreds of microns. The one or more channels are configured to flow (and in some instances, manipulate and otherwise control) fluids in the range of microliters to picoliters. Any embodiments of the methods of the present disclosure may comprise imaging the particle during one or more, or each of, the dispensing, manipulating, and engulfing steps. Imaging may also be performed during any modification and / or assessment steps. For example, imaging may be performed to monitor and / or confirm that a desired modification of the particle has occurred. In certain embodiments, assessing the engulfed particle (e.g., performing any of the assessments described herein) comprises imaging the droplet, the particle therein, or both.

[0155] Current column-based solid-phase reactors suffer from reaction errors resulting from incomplete exposure to reagents which is an unavoidable consequence of tightly packing beads in the reaction column. As a result of accumulated errors, the yield and the maximum possible reaction cycles are limited. As our platform will use single-bead single-droplet reactors, we can bypass this issue, thereby enabling higher yield long strand oligonucleotide and peptide synthesis.

[0156] Current microarray based solid-phase synthesis platforms require precisely aligned droplets delivered to individual reaction spots. However, alignment errors in the droplet delivery can lead to reaction errors. The GET based technology described herein has a Atty. Docket: STAN-2215WO (S24-293) significantly higher spatial resolution with respect to droplet delivery. Thus, these methods are expected to outperform existing microarray platforms.

[0157] Typically, chemical reaction rates increase as the regent droplet volumes decrease. Due to the use of very small droplets (picoliter scale), the reaction rates in the platform described herein are much faster than the rates achieved in existing platforms.

[0158] The following examples are offered by way of illustration and not by way of limitation.

[0159] EXPERIMENTAL

[0160] Example 1 - OET-Orchestrated Microdroplet Reactor for Solid-Phase Reactions

[0161] Solid-phase synthesis is a driving force for manufacturing biological materials such as peptide and oligonucleotide. It is essential for biology and biochemistry researchers to synthesize and analyze the complex molecules that govern bioprocesses of living organisms, promoting drug development, gene engineering and personalized protein- and nucleic acidbased therapeutics. However, current solid-phase reactors suffer from limitations, such as synthesis errors introduced by unexposed solid support surface and impurities trapped within the bead matrix, and high reagent usage, leading to high cost. The excessive reagent usage also raises sustainability and disposal of toxic fluids concerns.

[0162] Introduced herein is a platform employing light-orchestrated microdroplet reactors for solid-phase reactions with high parallelizing capabilities. The devices disclosed herein integrates a microbead and microdroplet manipulation substrate and on-demand microdroplet generators. The microparticle manipulation substrate is a transparent substrate (e.g. glass, quartz, fused-silica, PDMS etc.) having first and second electrodes disposed in a planar arrangement (made of electrically conducting material such as gold, aluminum, copper, platinum, silver, indium tin oxide, carbon nanotubes, titanium etc.) and then deposited with a photoconductive layer (e.g., amorphous silicon, Titanyl phthalocyanine etc.). This optoelectronic substrate can manipulate microbeads and microdroplets when excited by light patterns (near-infrared to visible light) and AC voltage (50 Hz to1 GHz frequency). The manipulation scheme includes encapsulation and decapsulation of individual beads with droplets, making each droplet a microreactor. The manipulation scheme also includes on- chip merging / combining and mixing of different reagent droplets. Multiple integrated microfluidic channels are used to introduce single droplets of consistent size onto the optoelectronic substrate when driven by a high-precision piezoelectric pressure controller. The droplet generation structure is inexpensively fabricated using standard PDMS soft lithography mass manufacturing technology. Atty. Docket: STAN-2215WO (S24-293)

[0163] Thus, the device described in this Example integrates OET with one or more microfluidic droplet generators. The chip has a chamber (the reaction chamber) that is filled with electrically insulating oil (the suspension medium). The solid micro-beads are suspended in this oil. The droplets (composed of materials that are insoluble in the suspension medium) are generated with the integrated microfluidic channels and introduced in the chamber. Light patterns projected on the device (in micro scale) are used to manipulate the droplets and the beads. Along with the light, an electrical voltage is also necessary to achieve manipulation (alternating voltage of frequency in the range 100Hz to 100MHz).

[0164] The light pattern can be moved in real time to manipulate the droplet / bead arbitrarily. This is achieved by using an external spatial light modulator (SLM) focused through an objective lens and projected on to the chip. A custom python code is used to control the light patterns projected by the SLM in real time.

[0165] Integration of the droplet generators and OET allows dispensing reagent droplets and manipulating them in the same chip. This makes it significantly easier to perform on-chip chemistry without concerns about transporting fragile (and often unstable) micro-droplets from a separate source.

[0166] The droplet is moved with light pattern and brought closer to a bead. The exerted dielectrophoretic force by the OET is sufficient to overcome the surface tension barrier and force the droplet to encapsulate (i.e., put a bead inside a droplet) the bead. For decapsulation (i.e. , take the bead out of the droplet), a pulsed force is applied on the droplet which causes it to decapsulate the bead. This pulsed excitation causes the droplet to deform which facilitates the decapsulation process. Without this rapid deformation inducing pulse, the bead would not necessarily come out of the droplet when the droplet is moved.

[0167] An integrated droplet generator can produce on-demand monosized droplets. Step emulsification method was used to do this. The microfluidic channels are connected to a precision piezoelectric pressure controller. The pressure controller controls precise pressure pulses to generate exactly one droplet at a time of a fixed size. This control of size facilitates precise ratio and volumes / concentrations of the chemical reactions.

[0168] The devices and methods disclosed herein also allow real-time monitoring of the reaction, for example, by fluorescence imaging and other optical signals. Also, the devices provide for automated chemical reactions and for identifying optimal conditions for chemical reactions.

[0169] For example, fluorescence tagged bio-compounds can be used to test the reactions in the devices disclosed herein. Successful reactions are indicated by fluorescence emission from specific wavelengths which we can optically detect in real time. This allows monitoring reactions in real time. Mixing reagents at different ratios on-chip can be achieved by merging Atty. Docket: STAN-2215WO (S24-293) different number / volume of droplets corresponding to different reagents. In this manner, massive number of reaction conditions can be tested in parallel.

[0170] Not only single droplets and beads, but the devices and methods used herein could be used to project multiple light patterns simultaneously and control each of them independently in real time. Thus, multiple droplet / beads can be manipulated simultaneously and independently. Thus, the devices and methods disclosed herein allow parallel operation, i.e., simultaneous multiple bead-droplet reactors.

[0171] Integration of the droplet generators and OET allows dispensing reagent droplets and manipulating them in the same chip. This makes it significantly easier to perform on-chip chemistry without the need to transport fragile (and often unstable) micro-droplets from a separate source.

[0172] Example 2 - LAMPS platform design

[0173] The LAMPS platform, engineered as a solid-phase picodroplet reactor, enables reliable encapsulation and decapsulation of microbeads with reagent droplets. This ensures uniform reagent exposure over the surface of each bead followed by complete reagent removal postreaction. The LAMPS device consists of an optoelectronic trapping / manipulation surface bonded to a polydimethylsiloxane (PDMS) layer hosting the microfluidic components. A central reaction chamber (~ 5 mm x 5 mm) along with oil (i.e., suspension medium) inlet / outlet and four on-demand droplet generators are patterned in the PDMS layer. In our setup (FIG. 13A), 5 pm diameter magnetic beads, acting as solid supports, are suspended in the oil medium of hexadecane. This colloidal solution is then introduced into the central chamber of the device.

[0174] Hexadecane is chemically inert, optically transparent, and immiscible with the reagent chemicals involved in the CuAAC-based DNA ligation synthesis cycle, making it an ideal suspension medium. Its compatibility with aqueous solutions and prevalent solvents used in biochemical reactions, such as ethanol, dimethylformamide (DMF), and tert-butanol was experimentally confirmed. Droplets containing ~65 pL of reagent (50 pm in diameter) are generated on- demand into the chamber through microfluidic channels driven by a piezoelectric pressure controller. The step-emulsification droplet generation mechanism employed here depends primarily on the channel geometry and is not overly sensitive to pressure fluctuations. This ensures consistent on-demand droplet generation when using a standard benchtop pressure controller21-23.

[0175] The parallel trapping and manipulation of the picoreactor droplets is carried out using adapted optoelectronic tweezers (OET).24-26. An electric field gradient, created by light- activated virtual electrodes on a photoconductor, exerts trapping force on dielectric particles Atty. Docket: STAN-2215WO (S24-293) and droplets that have different material properties (i.e., permittivity and electrical conductivity) compared to the suspension medium. The low optical power requirement of OET makes them especially suitable for handing sensitive biomolecules that are prone to photothermal damage. Additionally, the low electrical conductivity of the suspension medium hexadecane mitigates the Joule heating effect that can also be detrimental to the biomolecules. On the other hand, the low conductivity of hexadecane makes the conventional vertical electrode configuration of OET ineffective for creating electric field gradients2427. Thus, an OET device with a planar electrode layout was adapted. The OET device features a 1 pm thick photoconductive layer of hydrogenated amorphous silicon (a:Si-H) deposited over gold interdigitated microelectrodes. This forms the optoelectronic trapping / manipulation surface. The interdigitated electrodes, with a width of 10 pm and spacings of 100 pm, are designed to work optimally for the 50 pm diameter picodroplets (FIG. 13B). Micro- sized light patterns are projected onto the photoconductive layer of the device by coupling the output of a spatial light modulator (SLM) to a custom inverted microscope, thereby creating localized high conductivity regions within the photoconductive layer. These regions, referred to as “virtual electrodes”, behave akin to the physical microelectrodes used in dielectrophoresis systems. With an AC voltage (9.3 V, 10 kHz) applied across the interdigitated electrodes, a nonuniform electric field is created near the virtual electrodes. The resulting field gradient is significant on the same spatial scale as the beads and the droplets and, therefore, can exert sufficient trapping forces on them. This is modeled through numerical simulations of the electric field (FIG. 13C) and the droplet motion (FIG. 13D) under the influence of the OET gradient forces (data not shown), as well as verified from experimental observations (FIG. 12C and 12D).

[0176] Employing a programmable SLM to steer the micro light patterns in real time, the LAMPS platform is capable of dynamic control, both spatial and temporal, of the trapping force, achieving precise and automated two-dimensional parallel manipulation of picodroplets (FIG. 14). FIG. 12C and FIG. 12D are images taken during experiment highlighting the moments of bead encapsulation and decapsulation, respectively. Once a droplet is generated near the microfluidic channel opening, two rectangular micro light patterns are used to seize the opposite ends of the droplet and subsequently guide its trajectory towards the trapped bead by translating the light patterns. Precise control over the light pattern translation is achieved through custom software to control the SLM. Desired light-pattern trajectories are preprogrammed to achieve semi- automated motion. As the droplet is pushed onto the bead surface, the applied gradient force is sufficient to overcome the surface tension of the droplet, leading to complete encapsulation of the bead (FIGs. 12C, 13D, and 14A). This ensures that the bead is fully exposed to the reagent over its entire Atty. Docket: STAN-2215WO (S24-293) surface during the reaction period. In addition, moving the droplet with the encapsulated bead induces fluid flow inside the droplet2829; this simulates a stirring mechanism that promotes reagent mixing for consistent bead surface exposure, which helps drive the reaction to completion.

[0177] To initiate decapsulation, the pair of rectangular light patterns are placed slightly away from the droplet and a pulse-modulated sinusoidal voltage (duty cycle 50%, period 2 s) is applied (FIG. 12D). This tailored voltage pattern triggers pronounced stretching and contraction of the droplet between the light patterns, a phenomenon attributable to both dielectrophoresis and electrowetting effects30-32. The stretching reduces the localized curvature of the droplet and enables bead decapsulation. During the period of reduced voltage, the droplet seeks to revert to its spherical shape due to surface tension, a characteristic that helps maintain droplet integrity and aids the decapsulation procedure.

[0178] Example 3 - Multi-step oligo assembly demonstration

[0179] By combining CuAAC click ligation and enzymatic azide addition, an eight step DNA ligation synthesis cycle on the LAMPS platform (including washing steps), depicted in FIG. 15A was developed. The cycle is designed to be iterated to concatenate sequence-verified oligonucleotides in a controlled manner for de novo DNA synthesis. In situ fluorescence analysis confirms the successful execution of the full reaction cycle (FIG. 15B and FIG. 11 further supporting data not shown).

[0180] Example 4 - CuAAC click chemistry-based DNA ligation

[0181] Traditional enzymatic DNA ligation methods face limitations such as the requirement for a template, difficulty joining fragments with repetitive sequences, and reduced compatibility with modified nucleotides33 34. Chemical ligation methods, however, may overcome these challenges and offer flexibility for diverse artificial modifications to DNA molecules35 36. The CuAAC click reaction facilitates the creation of covalent bonds between azide and alkyne groups. This enables the seamless integration of various molecular entities and the synthesis of complex molecules that are difficult or impossible to achieve using traditional methods37. Click DNA ligation with triazole formation has emerged as an important technique for synthesizing long DNA strands3338. Although the triazole linkage is different from the natural phosphate backbone of DNA, it is compatible with PCR and various downstream applications38-40. To illustrate the capability of LAMPS for template-independent DNA assembly, a solid-phase CuAAC click ligation in picodroplets was performed (FIG. 15A, Step 5). Atty. Docket: STAN-2215WO (S24-293)

[0182] Magnetic beads (diameter of 5 gm) as solid supports are initially coated with -500 attomoles of 3’-azide-modified DNA acceptor molecule. The donor molecule is a sequence-verified oligonucleotide modified with a 5'-alkyne and an internal Atto 647 fluorescent label. The 3’-end of the donor molecule is unmodified, retaining a hydroxyl group to prevent self-ligation. To avoid side reactions involving the 5’-alkyne of the donor molecule, copper(l) catalyst, and oxygen41 42, a droplet containing the donor molecule and another droplet containing the copper(l) catalyst are generated on-demand and introduced separately. The two droplets are then brought together with light patterns to merge and mix on-chip, forming a mixture droplet with minimized oxygen exposure. A single bead is encapsulated by the mixture droplet for the duration of the click reaction. The reaction is then terminated by decapsulation of the bead, followed by three sequential washing steps in which fresh phosphate-buffered saline (PBS) droplets are introduced in succession to encapsulate and decapsulate the bead; this removes any non-specific binding of unreacted donor molecule (FIG. 15A, Steps 6 to 8). The success of the click ligation reaction is verified in real time by in situ measurement of the fluorescence signal from the bead, which is emitted by the ligated donor molecule labeled with Atto 647 (FIG. 15B and FIG. 1 1 ).

[0183] Example 5 - TdT-based enzymatic azide addition

[0184] After the click ligation step, the DNA molecule bound to the beads have a 3’- hydroxyl group, which blocks further click reactions. To enable the addition of subsequent strands through click ligation, an azide addition step is incorporated into the reaction cycle (FIG. 15A, Step 1 ). This step is performed by the enzymatic action of TdT, a templateindependent DNA polymerase that adds nucleotides to the 3'-end of a DNA strand4344. With the 3'-azide modified deoxynucleotide triphosphate (dNTP), TdT couples precisely one nucleotide to the acceptor molecule, as the 3'- azide blocks additional nucleotide coupling and prevents uncontrolled polymerization. The completion of the enzymatic coupling reaction is then verified on the LAMPS platform, where the bead is encapsulated and decapsulated by an enzymatic addition droplet, followed by three sequential PBS washing steps to remove unreacted components (FIG. 15A, Steps 1 to 4). Moreover, the physical properties of the click ligation droplet and the azide addition droplet differ significantly. The ability to orchestrate both kinds of droplets demonstrates the robustness and versatility of the LAMPS platform. Atty. Docket: STAN-2215WO (S24-293)

[0185] Example 6 - Click ligation synthesis cycle

[0186] By combining enzymatic azide addition with subsequent CuAAC click ligation, the LAMPS platform enables a template-independent, stepwise DNA ligation synthesis cycle to concatenate sequence-verified oligonucleotides within picodroplets. To demonstrate a full ligation cycle on the LAMPS platform, the acceptor molecule initially bound to the beads is unmodified with a 3'- hydroxyl group, the dNTP used in the enzymatic azide addition step has a 3'-azide group without fluorescence labeling, and the donor molecule is modified with a 5'-alkyne group and internal Atto 647 label. Sufficient washing after each reaction step, performed by three sequential fresh PBS droplets, was found to be essential for completing the full reaction cycle. The success of the ligation cycle is verified by in situ detection of a fluorescence signal from the Atto 647-labeled donor molecule (FIG. 15B and FIG. 1 1 ). In this case, a fluorescence signal is only possible if azide was coupled to the acceptor, which makes it an accurate indicator of the reaction cycle efficacy.

[0187] FIG. 1 1 shows the mean bead fluorescence intensity for a completed ligation cycle compared to the negative control. Here, the negative control is an acceptor molecule with a 3'-hydroxyl where the click ligation is performed without azide addition. In the absence of the azide, the ligation step is expected to fail, and no fluorescence signal is expected for this negative control. The experimental results are consistent with this. In total, there are eight steps involving droplet encapsulation and decapsulation of the bead, which further demonstrates the robustness of the LAMPS for precise, multi-step biochemical processing.

[0188] Methods for Examples 2-6

[0189] Device design and fabrication

[0190] Step emulsification on-demand droplet generator

[0191] The droplet generators are fabricated using soft lithography techniques. To prepare the mask, a 10 pm-thick layer of SU-8 photoresist is spin-coated onto a bare silicon wafer, exposed to 365 nm UV light to define the four 50 pm-wide microfluidic channels, and post-exposure baked (PEB). The wafer is then coated with a 100 pm SU- 8 layer, aligned to the alignment marks, and exposed to UV light to define the chamber (~5 mm x 5 mm with filleted corners) and the oil inlet and outlet channels (600 pm wide). The sharp transitions between 10 pm-high channels and the 100 pm-high chamber facilitate on-demand droplet generation via Rayleigh-Plateau instability. After another Atty. Docket: STAN-2215WO (S24-293)

[0192] PEB, the wafer is developed in an SU-8 developer bath. Finally, the SU-8 mold is hard- baked. A 10:1 w / w mixture of PDMS prepolymer and curing agent is cast against the mold to fabricate the microfluidic component of the device.

[0193] Planar optoelectronic tweezers (OET)

[0194] The planar electrodes of OET are fabricated through bilayer lift-off process. A glass wafer is cleaned with piranha solution and primed with hexamethyldisilazane (HMDS). The wafer is then spin-coated with -250 nm of Lift-Off Layer 2000 (LOL 2000), baked at 170°C for 5 minutes, and coated with a 1 pm-thick layer of SPR3612 photoresist. The photoresist is then patterned (Hidelberg MLA 150, 120 mJ / cm2) to define the interdigitated electrode patterns (10 pm wide, 100 pm spacing). This configuration generates an electric field gradient sufficient for dielectrophoretic (DEP) trapping and manipulation of -50 pm-diameter reagent droplets in hexadecane. After developing, a 15 nm-thick chromium adhesion layer and a 200 nm gold layer are deposited by e-beam evaporation. The wafer is immersed in a Remover 1165 bath overnight and heated to 65°C for 30 minutes to lift-off excess material.

[0195] Before amorphous silicon deposition, the wafer is plasma cleaned for 1 min to remove organic contaminants (15 seem 02 flow, 15 Pa pressure, 250 W RF power). A -1 pm-thick layer of hydrogenated amorphous silicon (a-Si:H) is deposited on the wafer using plasma enhanced chemical vapor deposition (PECVD) with 700 seem SiH4 (5% in He), 1000 seem He flow, 1500 mTorr pressure, 25 W RF power, at 300°C.

[0196] Device assembly and surface treatment

[0197] The PDMS piece containing the droplet generators and chamber structure is bonded to the planar OET substrate using oxygen plasma bonding (2 seem 02 flow, 85 mTorr, 40 W RF power, 40 s). The bonded device is left under ambient conditions overnight to restore surface hydrophobicity, which is essential for reagent-in-oil droplet generation. Prior to experiments, the device is heated at 150-170°C to enhance the hydrophobicity of the a-Si surface. This treatment helps retain droplet integrity by preventing excessive spreading during trapping and manipulation.

[0198] Experimental Setup

[0199] The experimental setup, with the model numbers of the components labeled, is shown in FIG. 4. The setup consists of optical, electrical and fluidics systems.

[0200] The optical system consists of micro-pattern projection and imaging subsystems. The micro- pattern projection subsystem uses a custom inverted microscope with a spatial light modulator (SLM) to project programmable light patterns onto the device photoconductor surface. The imaging subsystem enables both bright-field and fluorescence imaging. Atty. Docket: STAN-2215WO (S24-293)

[0201] The imaging subsystem is a modified upright epifluorescence microscope. To monitor experiments in bright field mode, transmitted light from the SLM through the device collected by a top objective lens and directed to a scientific CMOS (sCMOS) camera. For fluorescence imaging, a shutter blocks the light from the SLM, and sample illumination is provided through the top objective lens by an LED coupled with an excitation filter. Emission light is collected by the same lens, filtered through an emission filter, and captured by the camera.

[0202] The electrical system is composed of a waveform generator (i.e., signal generator), a radio frequency (RF) amplifier, and an oscilloscope to monitor voltage signals. To ensure sufficient voltage levels for effective trapping and manipulation, the signal from the waveform generator is amplified by the RF amplifier before being directed to the interdigitated electrodes of the device.

[0203] The fluidics system provides pressure pulses for the droplet generators and regulates the oil inlet fluidic port. A piezoelectric pressure controller, connected to a pressurized nitrogen line and a vacuum pump, delivers precise pressure to fluidic reservoirs, enabling controlled reagent flow through microfluidic tubing attached to the device. A total of four fluidic reservoirs and corresponding tubing (each connected to separate channels on the pressure controller) allow for independent control of reagent flow and droplet generation. For simplicity, only one of these reservoirs is illustrated in FIG. 4.

[0204] Binding of DNA acceptor to magnetic beads

[0205] Streptavidin-coated magnetic beads (EPRUI, MagSA-5, 5 pm in diameter, -6300 beads / pL) are used as solid supports. A 100 pL aliquot of beads is washed twice with 1 mL PBS. An acceptor solution is prepared by diluting 5 pL of 100 pM 5’-biotinylated DNA acceptor with 95 pL of PBS (pH 7.4). The mixture of bead and acceptor is gently agitated using a tube rotator at 20 RPM for one hour to facilitate binding. Following incubation, the beads are washed multiple times with PBS and resuspended in 100 pL of PBS. The amount of DNA molecule bound per bead is estimated to be approximately 500 attomoles per bead.

[0206] Suspending beads in hexadecane

[0207] A 5 pL aliquot of beads bound with DNA acceptor is placed on a magnetic stand, and the supernatant is carefully removed to isolate the beads. The Eppendorf tube with isolated beads is left open for 20 minutes to air dry, removing any residual PBS. Hexadecane containing 4% v / v Span 80 is then added as the suspension medium. The bead in hexadecane solution is vortexed to create a uniform colloidal solution. Atty. Docket: STAN-2215WO (S24-293)

[0208] Droplet encapsulation and decapsulation of beads

[0209] Approximately 25 pL of each reagent is loaded into polyethylene microtubings (LD. x O.D.: 0.38 mm x 1 .09 mm) and connected directly to the corresponding reagent inlets on the device.

[0210] Individual droplets are generated at the sharp step transition between the shallow droplet generation channel and the deeper chamber by applying a pressure pulse of ~20 mbar to the droplet generator. Once a droplet is generated near the droplet generator opening, a pair of rectangular light patterns are projected onto the device to seize the left and right sides of the droplet, aligning with the interdigitated electrode orientation.

[0211] To trap and manipulate the droplet in two dimensions, a sinusoidal AC voltage (9.3 V, 10 kHz) is applied to the interdigitated electrodes. The droplet is trapped in the gap between the rectangular light patterns and moves in tandem with the rectangle pair. The trapping force generated by the virtual electrodes activated by the light patterns is sufficient to overcome the droplet’s surface tension, allowing the bead to be fully encapsulated within the droplet. For decapsulation, the droplet is positioned at the central of a pair of physical electrodes. The voltage is then turned off, and the rectangular light patterns are moved slightly away from the droplet along the physical electrode pair. A pulse-modulated sinusoidal voltage (50% duty cycle, 2-second period), similar to amplitude shift keying (burst mode), is applied. During the active phase of the duty cycle, the sinusoidal signal has a frequency of 20 kHz with a magnitude of 40 V. The left and right sides of the droplet are abruptly pulled toward the rectangular light patterns without breaking up the droplet. During the reduced voltage period, the droplet retracts to its spherical shape, leaving the bead behind and completing the decapsulation process.

[0212] Click ligation step and wash steps

[0213] The click ligation step starts with a bead bound with 3’-N3 DNA acceptors. The ligation process proceeds by generating two droplets: Click ligation (Part A) and Click ligation (Part B). Part A, which contains Atto 647- labeled donor molecules, is fluorescent, whereas Part B is non-fluorescent. Using two pairs of rectangular light patterns, these two droplets are maneuvered into proximity until they merge. The entire droplet becomes fluorescent immediately after merging, indicating thorough mixing of the components. After the bead is encapsulated by this merged droplet, it is incubated for 30 minutes before decapsulation. To enhance reagent exposure, the droplet is dragged around to stimulate internal fluid flow around the bead. This step is followed by three wash steps. In each wash step, the bead is encapsulated with a fresh PBS droplet (i.e., droplet from one wash step is not reused for the next wash step) for at least 30 seconds and then decapsulated. During each wash step, the Atty. Docket: STAN-2215WO (S24-293) droplet is moved back and forth by the light patterns to facilitate effective removal of unreacted materials. Fluorescence intensity of the bead decreases progressively over the first three wash steps, with no further reduction beyond the third wash. Therefore, the final fluorescence intensity of the bead is recorded after the third wash. For both the reaction and the negative control (using beads bound with 3’-OH DNA acceptors), the experiments are repeated five times.

[0214] Click ligation synthesis cycle

[0215] The click ligation synthesis cycle starts with a bead bound with 3’-OH DNA acceptors, which is encapsulated in an enzymatic azide addition droplet for 30 minutes, then decapsulated. This is followed by three PBS wash steps to remove unreacted components, resulting in 3’-N3 DNA product remain tethered to the bead. The wash steps are identical to the ones discussed in the previous section. The click ligation step then follows as previously described, with the final fluorescence intensity recorded after another three washes. While the demonstration on LAMPS focused on one full cycle, the iterative nature of the ligation synthesis cycle, which extends DNA primer molecules by defined sequences, indicates the method’s capability to produce extended oligonucleotides by simply repeating the steps already demonstrated.

[0216] Benchtop reactions and polyacrylamide gel electrophoresis (PAGE) analysis

[0217] Click ligation reactions, modified from established protocols, are initially tested in benchtop with bulk beads to validate benchtop performance before transitioning to on-chip reactions. Benchtop reactions are performed in 1 .5 mL Eppendorf tubes, with bulk reagent handling instead of the droplet encapsulation and decapsulation of individual beads. To perform benchtop reactions, 5 pL of DNA acceptor-bound beads are placed on a magnetic stand, the supernatant is removed, and 50 pL of reagent (enzymatic azide addition or Click ligation A & B mixture) is added. The mixture is vortexed briefly and incubated for 30 minutes with periodic agitation. Beads are washed three times with 50 pL PBS and resuspended in 50 pL water. A 0.5 pL aliquot is used for fluorescence microscopy.

[0218] For PAGE analysis, DNA is cleaved from beads by heating in 10 pL pure water at 80°C for 20 minutes. The supernatant is mixed with 10 pL 2x TBE-Urea buffer and heated to 80°C for 5 minutes. 8 pL of each sample is loaded onto a 15% TBE PAGE gel. Electrophoresis of the samples is performed at 100 V for 120 minutes. The gel is rinsed and imaged using the Cy5 channel on a gel imaging system, which can detect Atto 647 fluorescence. Bands representing full-length products are detected due to Atto 647 labeling of donor molecules. Atty. Docket: STAN-2215WO (S24-293)

[0219] Fluorescence image analysis

[0220] Fluorescence images are captured in monochrome at 1920 x 1080 resolution and saved as 16-bit TIFF files to preserve detail. Images are processed using a custom code that applies adaptive thresholding to convert gray-scale images to binary masks, compensating for background variations by subdividing the image into blocks. Individual beads are identified using image segmentation and a single orthogonal hop connectivity criterion. Each segmented region (sub- mask) corresponds to a detected bead. Multiplying each sub-mask with the original image extracts the pixels for the corresponding bead. Overlapping beads are grouped into a single sub-mask, but size normalization ensures no errors.

[0221] The raw fluorescence intensity value of the i-th object, / iraw,#, is calculated as: p sub s i where i = 1 , 2, •••, N$, N$ is the total number of detected objects, 7VPiXei,# is the number of non- zero pixels in the i-th object, Msea,# is the pixel value matrix of the i-th object, and each matrix element ranges from 0 to 65,535. This summing and averaging process automatically scales for bead size variations (e.g., a cluster of two beads).

[0222] The background intensity, / &■, is calculated by averaging from pixels outside detected objects. The corrected fluorescence intensity is then: lf,t = lf.rnv.t ~ hg •

[0223] Modeling and Simulations

[0224] The modeling and simulation efforts include electromagnetic simulations to determine the field distribution near the virtual electrodes and resulting OET gradient force, and fluid flow simulations to track droplet motion under this force.

[0225] Electromagnetic simulations

[0226] The field distribution near the virtual electrodes depends on the geometry, excitation AC voltage, and material properties. The key material properties, permittivity and electrical conductivity, are sourced from the literature. Simulations are performed using the Electric Current interface (AC / DC module) of COMSOL Multiphysics, with electric potentials applied as Dirichlet boundary conditions at the physical electrodes and two rectangular virtual electrodes modeling the light patterns. Atty. Docket: STAN-2215WO (S24-293)

[0227] The electromagnetic field distributions obtained from the simulation (FIG. 13C) allow for calculation of the force on the droplet using the Maxwell Stress Tensor (MST) method. Force components along x, y and z directions confirm that the droplet is attracted toward the virtual electrode gap, with sufficient gradient force generated near the electrodes to enable droplet manipulation.

[0228] Fluid flow simulations

[0229] To observe droplet motion under the gradient force, two-dimensional laminar two- phase flow simulations are performed using the phase-field method. The phase-field approach uses the variable “volume fraction” to represent the two fluids: values of 0 and 1 correspond to the droplet and the suspension medium, respectively, whereas intermediate values represent the boundary / transition region between the fluids. The fluid motions are governed by the Navier- Stokes equation in the laminar flow regime. The interfacial surface tension between the droplet (PBS) and the medium (hexadecane) is taken to be 53.1 mN / m. All the material parameter values are taken from the literature. Since PBS shares similar fluidic properties to water, water parameter values are used when PBS-specific data are unavailable.

[0230] In the two-phase flow, wetted-wall Navier-slip boundary conditions are set at the top and bottom surfaces of the fluid domain. The dielectrophoretic gradient force is coupled into the Multiphysics simulation as a volume force term defined in terms of the MST. The pressure field and the velocity field are initialized to zero. The droplet (radius ro = 25 pm) starts at x = -32 pm, z = 35 pm (10 pm above the bottom surface), while a bead is positioned at x = 0, z = 5 pm. A time-dependent fluid-flow simulation is performed to track the position of the droplet as it moves under the gradient force (FIG. 13D).

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[0283] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein.

Claims

Atty. Docket: STAN-2215WO (S24-293)WHAT is CLAIMED is:1 . A microfluidic device comprising: a chamber configured to contain a liquid medium; a substrate comprising: i) a photoconductive layer forming a first wall of the chamber; and ii) first and second electrodes disposed in a planar arrangement within or adjacent to the photoconductive layer; a microfluidic inlet channel connected to the chamber; and a microfluidic droplet generator connected to the chamber.

2. The microfluidic device of claim 1 , wherein the substrate further comprises a layer of transparent supporting material.

3. The microfluidic device of claim 1 or 2, wherein the first and second electrodes are disposed in a planar arrangement within the photoconductive layer.

4. The microfluidic device of any one of claims 1 to 3, further comprising a microfluidic outlet channel connected to the chamber.

5. The microfluidic device of claim 4, wherein the microfluidic outlet channel is one of two or more microfluidic outlet channels connected to the chamber.

6. The microfluidic device of any one of claims 1 to 5, wherein the microfluidic droplet generator is one of two or more microfluidic droplet generators connected to the chamber.

7. The microfluidic device of any one of claims 1 to 6, wherein the microfluidic inlet channel is one of two or more microfluidic inlet channels connected to the chamber.

8. The microfluidic device of any one of claims 1 to 7, wherein the chamber has a length extending along the photoconductive layer, wherein the length is from 1 to 25 mm, from 2 to 10 mm, from 2.5 to 7.5 mm, from 4 to 6 mm, or about 5 mm.

9. The microfluidic device any one of claims 1 to 8, wherein the chamber has a height extending from the photoconductive layer, wherein the height is from 0.05 to 0.15 mm, from 0.075 to 0.125 mm, or about 0.1 mm.Atty. Docket: STAN-2215WO (S24-293)10. The microfluidic device of any one of claims 1 to 9, wherein the chamber has a width of from 1 to 25 mm, from 2.5 to 7.5 mm, from 4 to 6 mm, or about 5 mm.11 . The microfluidic device of any one of claims 1 to 10, wherein one or more corners of the chamber are smooth, optionally wherein the one or more corners are rounded or sloped.

12. The microfluidic device of any one of claims 1 to 11 , wherein the photoconductive layer is made of a material comprising or consisting of hydrogenated amorphous silicon (a- Si:H), titanium oxide phthalocyanine (TiOPc), cadmium Sulfide (CdS), poly(3- hexylthiophene) (P3HT), and mixtures of P3HT.

13. The microfluidic device of claim 12, wherein the mixture of P3HT is P3HT:phenyl C61 -butyric acid methyl ester (PCBM).

14. The microfluidic device of any one of claims 1 to 13, wherein the electrode pattern comprises electrodes separated from each other by a space of from 50 to 500 pm, from 75 to 125 pm, from 90 to 110 pm, or about 100 pm.

15. The microfluidic device of any one of claims 1 to 14, wherein the first and second electrodes are two of a plurality of interdigitated electrodes in a planar arrangement within or adjacent to the photoconductive layer.

16. A system comprising: the microfluidic device of any one of claims 1 to 15; a light pattern generator optically coupled to the photoconductive layer; and a voltage source electrically coupled to the first and second electrodes.

17. The system of claim 16, further comprising a pressure controller fluidically connected to the one or more of: the droplet generator, the inlet channel, and the outlet channel.

18. The system of claim 17, wherein the pressure controller is a piezoelectric pressure controller.Atty. Docket: STAN-2215WO (S24-293)19. The system of claim 16 or 17, wherein the light pattern generator is spatial light modulator (SLM).

20. The system of any one of claims 16 to 19, wherein the light pattern generator is optically coupled to the photoconductive layer.21 . The system of claim 20, wherein the light pattern generator is optically coupled to the photoconductive layer via an objective lens.

22. The system of any one of claims 16 to 21 , wherein the voltage source is adapted to deliver an AC voltage within a frequency range of from 100 Hz to 100 GHz to the photoconductive layer.

23. A method of engulfing a particle into a droplet, the method being implemented using the system of any one of claims 16 to 22, wherein the chamber is filled with an electrically insulative suspension medium, wherein the method comprises:(a) dispensing a particle suspended in the suspension medium from the microfluidic inlet channel into the chamber;(b) generating a droplet in the chamber using the microfluidic droplet generator;(c) providing a voltage to the photoconductive layer using the first and second electrodes; and(d) projecting a light pattern onto the photoconductive layer using the light pattern generator to engulf the particle into the droplet, wherein the light pattern is selected to exert an electric field gradient on the droplet and / or the particle such that the particle is engulfed into the droplet.

24. The method of claim 23, wherein the suspension medium comprises or consists of an oil.

25. The method of claim 24, wherein the oil is a long chain alkane.

26. The method of claim 25, wherein the long chain alkane is hexadecane.

27. The method of claim 24, wherein the oil is silicone oil, fluorinated oil(perfluorocarbons), vegetable oil, or mineral oil.Atty. Docket: STAN-2215WO (S24-293)28. The method of any one of claims 23 to 27, wherein the particle is a microparticle.

29. The method of claim 28, wherein the microparticle is a bead.

30. The method of any one of claims 23 to 28, wherein the particle is a cell.31 . The method of any one of claims 23 to 30, wherein the method further comprises modifying the engulfed particle.

32. The method of claim 31 , wherein the modifying comprises covalently or non- covalently attaching a molecule present in the droplet to the particle.

33. The method of claim 32, wherein the modifying comprises covalently attaching a nucleotide or polynucleotide present in the droplet to the particle.

34. The method of claim 33, wherein the nucleotide or polynucleotide is attached to the particle chemically and / or enzymatically.

35. The method of claim 34, wherein the nucleotide or polynucleotide is attached to the particle by azide addition followed by click ligation.

36. The method of claim 32, wherein the modifying comprises non-covalently attaching a nucleotide or polynucleotide present in the droplet to the particle.

37. The method of claim 36, wherein the attaching comprises base-pairing the nucleotide or polynucleotide to a nucleotide or polynucleotide present on the particle.

38. The method of claim 37, wherein the nucleotide or polynucleotide is attached to the particle via a biotin-streptavidin interaction.

39. The method of claim 32, wherein the modifying comprises covalently attaching an amino acid or polypeptide present in the droplet to the particle.

40. The method of claim 39, wherein the amino acid or polypeptide is attached to the particle chemically and / or enzymatically.Atty. Docket: STAN-2215WO (S24-293)41 . The method of claim 34, wherein the amino acid or polypeptide is attached to the particle by azide addition followed by click ligation.

42. The method of claim 34, wherein the modifying comprises non-covalently attaching an amino acid or polypeptide present in the droplet to the particle.

43. The method of claim 42, wherein the amino acid or polypeptide is attached to the particle via a biotin-streptavidin interaction.

44. The method of claim 32, wherein the modifying comprises covalently or non- covalently attaching a sugar or carbohydrate present in the droplet to the particle.

45. The method of any one of claims 23 to 30, wherein the method further comprises washing the engulfed particle to remove remnants of a previously contacted droplet by engulfing the droplet in a washing droplet.

46. The method of claim 45, wherein the washing droplet comprises a phosphate buffer saline, water, or a binding buffer.

47. The method of any one of claims 23 to 46, wherein the method further comprises assessing the engulfed particle.

48. The method of claim 47, wherein the particle is a cell, and wherein the method comprises assessing the cell for expression of a polypeptide.

49. The method of claim 48, wherein the polypeptide is a cytoplasmic polypeptide, a cell surface polypeptide, or a secreted polypeptide.

50. The method of claim 48 or 49, wherein the polypeptide is an antibody.51 . The method of any one of claims 48 to 50, wherein the droplet comprises a binding partner for the polypeptide, and wherein the assessing comprises assessing for complexes comprising the binding partner and the polypeptide.

52. The method of claim 51 , wherein the binding partner is an antibody, a ligand, an aptamer, an antigen, or a small molecule.Atty. Docket: STAN-2215WO (S24-293)53. The method of any one of claims 23 to 51 , wherein the method further comprises ejecting the particle from the droplet.

54. The method of claim 53, wherein ejecting the particle from the droplet comprises applying a pulsed force on the droplet.

55. The method of claim 53 or 54, further comprising subjecting the ejected particle to step (d) to re-engulf the particle in a droplet.

56. The method of claim 55, further comprising modifying the re-engulfed particle.

57. The method of claim 56, configured to performing solid-phase synthesis on the surface of the particle by iteratively engulfing, ejecting and re-engulfing the particle in droplets comprising monomers for solid-phase synthesis.

58. The method of claim 57, wherein the monomers are nucleotides or amino acids.

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