Systems and methods for the assembly of charged agents on a surface

Abstract

Patterning a charged agent on a surface, the method comprising: providing a substrate assembly disposed on a working electrode, wherein the substrate assembly comprises a charge support layer disposed on a semiconductor; contacting the charge support layer of the substrate assembly with a suspension or solution comprising the charged agent dispersed or dissolved in a fluid carrier; positioning a counter electrode in electrical contact with the suspension or solution; applying an AC electric field within the substrate assembly using the working electrode and the counter electrode while projecting an image of a target pattern onto the semiconductor; ceasing application of the electric field; and allowing the charged agent to assemble on the charge support layer in the target pattern.

Description

[0001]Attorney Docket No.10046-557WO1 Systems and Methods for the Assembly of Charged Agents on a Surface CROSS-REFERENCE TO RELATED APPLICATIONS This application claims benefit of priority of U.S. Provisional Application No. 63 / 622,311, filed January 18, 2024, which is incorporated by reference in its entirety. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT This invention was made with government support under Grant numbers ECCS1930649 and 2219221 awarded by the National Science Foundation. The government has certain rights in the invention. BACKGROUND The patterning of components on a surface is important in a range of applications, including microelectronics, biodiagnostics, and optics. While many techniques, such as photolithography, have been investigated and in some cases widely employed, most existing strategies for patterning surfaces are complex, expensive, time-consuming, and limited terms of the surface area that can be patterned. Accordingly, there remains a need for improved methods of patterning agents on surfaces. SUMMARY Described herein are systems and methods for patterning charged agents (e.g., charged nanoparticles, ionic compounds, and molecules) with high precision and in a large scale. The methods described herein are impressively effective, efficient, and facile. In some embodiments, methods for patterning a charged agent on a surface can comprise providing a system comprising a substrate assembly disposed on a working electrode, wherein the substrate assembly comprises a charge support layer disposed on a semiconductor; contacting the charge support layer of the substrate assembly with a suspension or solution comprising the charged agent dispersed or dissolved in a fluid Attorney Docket No.10046-557WO1 carrier; positioning a counter electrode in electrical contact with the suspension or solution; applying an AC electric field within the substrate assembly using the working electrode and the counter electrode while projecting an image of a target pattern onto the semiconductor; ceasing application of the electric field; and allowing the charged agent to assemble on the charge support layer in the target pattern. Also provided herein are systems for performing these patterning methods. These systems can comprise a substrate assembly disposed on a working electrode, wherein the substrate assembly comprises a charge support layer disposed on a semiconductor; a sample well configured to position a fluid sample in contact with the charge support layer; a counter electrode configured to be in electrical contact with a fluid sample present in the sample well; and an image projector, such as a digital light projector, configured to project an image of a target pattern onto the semiconductor. DESCRIPTION OF DRAWINGS Figure 1A-1C. Figure 1A) Schematic of the DLP device used for large-area bubble patterning. Figure 1B) Close-up of the setup during bubble generation and subsequent particle patterning. Figure 1C) Laser pattern of a horse (top) and corresponding bubble pattern (bottom). Scale bar: 100 μm. Figures 2A-2F. Bubble generation: Figure 2A) Bubble radius linearly increases with laser intensity (inset: bubble volume vs laser intensity). Minimum required laser intensity as a function of Figure 2B) AC frequency (inset: profile in the low frequency regime), and Figure 2C) DC bias, and Figure 2D) Na2SO4 electrolyte concentration. Figure 2E) Schematic of the overall setup and corresponding equivalent circuit. Figure 2F) Effects of additives of H2O2and CTAC. Figures 3A-3D. Figure 3A) Laser profile and Figure 3B) corresponding bubble pattern of a Bevo superimposed on the map of Texas (Bevo is UT-Austin’s copyrighted logo). Figure 3C) Laser profile and corresponding Figure 3D) bubble pattern of the Max Planck Institute logo. Scale bars: 100 μm. Figures 4A-4F. Role of nanoparticles in the generation of bubble patterns: Figure 4A) Simultaneous generation of microbubbles over a larger area, respectively, with nanoparticles in solution (inset without nanoparticles in solution). Figure 4B) A microbubble is generated rapidly, within submilliseconds (a few µm) to less than a second Attorney Docket No.10046-557WO1 (~ 10 µm), and (inset) shrinks over tens of seconds. Figure 4C) The size of the nanoparticle assembly can be controlled by the bubble size from a few µm to >100 µm. Formation of nanoparticle-assemblies in an array by Figure 4D) the generation of an array of microbubbles (shown as an optical micrograph) in a solution containing polystyrene nanospheres, followed by the Figure 4E) shrinkage of the microbubble arrays that result in ordered nanoparticle assemblies. Figure 4F) The location of the nanoparticle assembly is within 1.0 ± 0.6 μm relative to the center of the bubble position. Scale bars: 100 μm. Figures 5A-5E. Demonstration of the microbubble-driven formation of nanoparticle clusters upon illumination of a light-pattern: Figure 5A) The interparticle distance of the deposits is controlled by the bubble size. Dependence of Figure 5B) bubble radius and separation of bubbles as a function of the E-field duration, and Figure 5C) bubble volume and bubble lifetime. Figures 5D-5E) Particle clusters formed by Figure 5D) circular and Figure 5E) rectangular light patterns (and the corresponding microbubbles). Simultaneously deposited particles (in the same color) and the concentric patterns made sequentially as denoted in red, green, and yellow. Scale bars: 100 μm. The contrasts of the images are modified so that the particles are more apparent. Figures 6A-6D. Assembly and SERS of Ag-nanoclusters with a precise co- localization of E-coli: SEM images of Figure 6A) bubble-deposited assemblies of silver nanoparticle and Figure 6B) E-coli cells. Scale bars: 1 μm, 3 μm, respectively. Figure 6C) Schematic of E-coli attraction to bubble-assembled silver nanoparticle arrays via light- directed dielectrophoresis. Figure 6D) Baseline-corrected SERS spectra with E-field on and off of one Ag-nanocluster from an assembly of 3 x 4 Ag-nanoclusters. Peaks labeled in orange font are from E-coli cells, those in black are from Ag nanoparticles alone. Analysis of peaks of E-coli is included in Table 2. Figure 7. Voltage sweep from +4 V to -4 V of the α-Si:H sample used in Example 1. Figure 8. Simulation results of heat generated by a focused 532 nm laser with an intensity of 100 mW / cm2. Figure 9. Photocurrent measurement results of an α-Si:H sample. Figure 10. Coffee ring patterns form when the surface is hydrophobic. Image has been enhanced by adjusting the contrast / brightness / sharpness to show more clearly the deposited particles. Scale bar: 50 μm. Attorney Docket No.10046-557WO1 Figure 11. The center of the bubble position is within 2.45 ± 1.08 μm relative to the center of the laser spot. Figures 12A-12F. Laser patterns used for line thickness vs bubble pattern determination: Figure 12A) 8 μm, Figure 12B) 16 μm and Figure 12C) 32 μm, and their corresponding bubble patterns. Figures 12D-12F) Scale bars: 50 μm. Figure 13. Bubble-assisted large-scale assembling of ordered arrays of nanospheres (total length: 2.56mm). Scale bar: 100 μm Figures 14A-14E. Figure 14A) Fluorescent and Figures 14B-14C) SEM images of 2 x 2 PS sphere deposits on a negatively charged surface. Scale bars: 25 μm. Figures 14D- 14E) SEM of a single Ag nanoparticle assembly on positively charged surface. Scale bars: 10 μm, 1 μm. Figure 15. Optical image of simultaneously deposited Ag nanoparticles in a 3x4 array. Scale bar: 100 μm. Figures 16A-16B. Figure 16A) Original and Figure 16B) normalized laser power vs relative position used for laser beam width determination with the 10 / 90 knife edge method. Figures 17A-17B. Analysis of bubble radius over time during bubble generation and collapse. Figure 17A) While most of the bubbles shrank similarly over time, Figure 17B) some bubbles showed a secondary expansion phase before shrinking again Figure 18. Surface Enhanced Raman Spectroscopy results taken at the background (Background_contro), on the silver aggregate without DEP attraction of E-coli (E-field off_control), and with DEP attraction of E-coli (E-field on). Figure 19. Schematic illustration of the system used to pattern a charged agent on a surface. Figure 20A. Illustration of the patterning of charged particles (fluorescent PS nanospheres) on a surface to reproduce the painting “Girl with a Pearl Earring.” Patterned particles (photographed through 10X objective) is shown on the left, the image projected to pattern the charged particles is shown on the right. Figure 20B. Enlargements of the patterned particles shown in Figure 20A (photographed through 20X objective). Figure 21A. Illustration of the patterning of charged particles (fluorescent PS nanospheres) on a surface to reproduce a photograph of an astronaut on the moon. Patterned particles (photographed through 10X objective) is shown on the left, the image projected to pattern the charged particles is shown on the right. Attorney Docket No.10046-557WO1 Figure 21B. Enlargements of the patterned particles shown in Figure 21A (photographed through 20X objective). Figure 22. Demonstration of the resolution observed using the patterning methods described herein. Figure 23. Demonstration of the impact of varying the number of 3 minute deposition steps on the density of particles deposited on the surface. Figure 24. Illustration of the patterning of 100 nm Au nanoparticles using the patterning methods described herein. Figure 25. Illustration of the patterning of 10 nm Au nanoparticles using the patterning methods described herein. Figure 26. Illustration of the patterning of positively charged polystyrene nanoparticles on a negatively charged charge support layer using the patterning methods described herein. Figure 27. Illustration of a method for negative image creation using the patterning methods described herein. Figure 28. Illustration of a method for negative image creation using the patterning methods described herein. Figure 29. SEM micrographs illustrating the sequential use of the methods described herein to perform multi-layer patterns on a surface. A first patterning was performed using the methods described herein (UVO + 3x PDDAPSS (3m) treatment – 30x AC deposition (1.5 kHz, 200ms, 12 Vpp)). A circle was used as the projected image. Subsequently, a second layer (1x PDDAPSS (3m) + 30x AC deposition), a third layer (1x PDDAPSS (3m) + 30x AC deposition) and a fourth layer (1x PDDAPSS (3m) + 30x AC deposition) were deposited, using circles of decreasing circumference. Figure 29 shows SEM micrographs following the second deposition (left), the third deposition (middle), and the fourth deposition (right). All three micrographs were captured using the same exposure time. The middle spot brightness seems to be similar for all conditions, and the pre- deposited spots seem to become darker (potentially due to additional PDDA / PSS layers on top of the deposited particles). Figures 30A-30B. SEM micrographs illustrating the deposition of particles in the first layer. Particle density difference can be seen within the two areas. Attorney Docket No.10046-557WO1 Figures 31A-31B. SEM micrographs illustrating the deposition of particles in the second layer. Particle density change is observed, but still on the single layer level (although some parts have more depositions). Figures 32A-32B. SEM micrographs illustrating the deposition of particles in the third layer. Particle density change is observed, but still on the single layer level (although some parts have more depositions). Figures 33A-33B. SEM micrographs illustrating the deposition of particles in the fourth layer. Particle density change is observed, but still on the single layer level (although some parts have more depositions). Figures 34A-34D. SEM micrographs illustrating the deposition of particles in the middle of the image. Particles were found to be densely packed, but not so much on the third or fourth layers. DETAILED DESCRIPTION Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. Provided herein are methods for patterning a charged agent on a surface. These methods can comprise providing a system comprising a substrate assembly disposed on a working electrode, wherein the substrate assembly comprises a charge support layer disposed on a semiconductor; contacting the charge support layer of the substrate assembly with a suspension or solution comprising the charged agent dispersed or dissolved in a fluid carrier; positioning a counter electrode in electrical contact with the suspension or solution; applying an AC electric field within the substrate assembly using the working electrode and the counter electrode while projecting an image of a target pattern onto the semiconductor; ceasing application of the electric field; and allowing the charged agent to assemble on the Attorney Docket No.10046-557WO1 charge support layer in the target pattern. In some embodiments, ceasing application of the electric field can further comprise ceasing projection of the image of the target pattern onto the semiconductor. Without wishing to be bound by theory, experiments indicate that charged agents can begin assembly while the AC electric field is applied within the substrate assembly and the image of the target pattern is projected onto the semiconductor. Assembly of the charged agent on the charge support layer can then continue after application of the AC electric field (and image projection) ceases. Thus, assembly of the charged agent on the charge support layer in the target can occur before and / or after application of the AC electric field (and image projection) ceases. Varying strategies can be used to control the density of the charged agent deposited on the charge support layer. The density of the charged agent on the surface can be controlled, if desired, by varying the target pattern projected on the surface. The density of the charged agent on the surface can also be controlled, if desired, by varying the varying the characteristics of the applied electric field (e.g., frequency, duration of applied field, etc.). The density of the charged agent on the surface can also be controlled, if desired, by removing (or altering the composition of) the suspension or solution comprising the charged agent dispersed or dissolved in a fluid carrier that is in contact with the charge support layer. For example, the suspension or solution comprising the charged agent can be removed to stop assembly. Alternatively, the concentration of the charged agent in the solution or suspension can be increased or decrease to increase or decrease, respectively, the density of the charged agent patterned on the surface. The ratio and / or identity of charged agent(s) in the solution or suspension can also be varied over time so as to deposit different combinations of charged agents on the charge support layer. In some embodiments, the charged agent can assemble on the charge support layer with a resolution of from 100 nm to 5 microns. In certain embodiments, the charged agent can assemble on the charge support layer with sub-micron resolution. In some embodiments, the charge support layer and the charged agent can exhibit the same surface charge. For example, in some embodiments, the charge support layer can be negatively charged and / or exhibits a negative charge when in contact with the suspension or solution comprising the charged agent. In other embodiments, the charge support layer can be positively charged and / or exhibits a positive charge when in contact with the suspension or solution comprising the charged agent. Attorney Docket No.10046-557WO1 In other embodiments, the charge support layer and the charged agent can exhibit an opposite surface charge. In the case of microbubble-enabled assembly (Example 1), the charges on the surface and the charged agent can have opposite charges. In these embodiments, nanobubbles can be created which assemble into isolated aggregates. The electrostatic interaction can assist the attachment of these charged aggregates to the surface. In the case of the patterning methods exemplified in Example 2, the charge of the charge support layer and the charged agent can have the same charge. Microbubbles can also form under controlled conditions (e.g. higher AC voltage), which can further assemble isolated nanoparticle aggregates on the printed charged agent pattern. The thickness of the charge support layer can be varied. In some embodiments, the charge support layer can have a thickness of less than 50 nm, such as a thickness of less than 25 nm, less than 20 nm, less than 15 nm, less than 10 nm, or less than 5 nm. In certain embodiments, the charge support layer can have a thickness of from 1 nm to 50 nm, such as a thickness of from 1 nm to 25 nm, from 1 nm to 20 nm, from 1 nm to 15 nm, from 1 nm to 10 nm, or from 1 nm to 5 nm. The charge support layer can be fabricated from any suitable material that can be adsorbed or otherwise deposited on a semiconductor substrate and can host and trap charged species. Generally, the material(s) that form the charge support layer will be insoluble in the suspension or solution comprising the charged agent. In some embodiments, the charge support layer can comprise a charged polymer or a polymer that exhibits a charge when in contact with the suspension or solution comprising the charged agent. Examples of charged polymers include, but are not limited to, sulfonated polysulfone (PSF-S03), sulfonated polyether sulfone (PES-S03), sulphonated polystyrene (PSS, poly(styrene sulfonate), PEI (poly(ethylene imine)), PAA (poly(allyl amine)), PDDA (poly(diallyldimethylammonium chloride)), PNIPAM (poly(N-isopropyl acrylamide), CS (Chitosan), PMA (poly(methacrylic acid)), PVS (poly(vinyl sulfate)), PAA (poly(amic acid)), PAH (poly(allylamine)), brush copolymers containing cationic or anionic sidechains, anionic or cationic proteins, copolymers thereof, and blends thereof. Other suitable materials include, for example, porous materials and frameworks, such as zeolitic materials or metal-organic frameworks. In certain embodiments, the charge support layer can comprise PSS, a copolymer thereof, or a blend thereof. In certain embodiments, the charge support layer can comprise PDDA, a copolymer thereof, or a blend thereof. Attorney Docket No.10046-557WO1 In some embodiments, the applied AC electric field can have a frequency of less than 20kHz, such as a frequency of from 1kHz to 20kHz, a frequency of from 1kHz to 15kHz, a frequency of from 1kHz to 10kHz, a frequency of from 1kHz to 8kHz, a frequency of from 1kHz to 6kHz, a frequency of from 1.5kHz to 6kHz, a frequency of from 1.5kHz to 5kHz, or a frequency of from 1.5kHz to 4.5kHz. In some embodiments, the image projected on the semiconductor can comprise electromagnetic radiation having an intensity of less than 10,000 mW / cm2at λmax, such as an intensity of less than 5,000 mW / cm2at λmax, an intensity of less than 2,000 mW / cm2at λmax, an intensity of less than 1,000 mW / cm2at λmax, or an intensity of less than 250 mW / cm2at λmax. In certain embodiments, the image projected on the semiconductor comprises electromagnetic radiation having an intensity of from 50 mW / cm2to 10,000 mW / cm2at λmax, such as an intensity of from 50 mW / cm2to 5,000 mW / cm2at λmax, an intensity of from 50 mW / cm2to 2,000 mW / cm2at λmax, or an intensity of from 50 mW / cm2to 1,000 mW / cm2at λmax. The image projected on the semiconductor can comprise electromagnetic radiation having a λmax in a region of the electromagnetic spectrum which induces a change in electrical conductivity in the semiconductor. As long as electromagnetic wave can enhance semiconductor’s electric conductivity, patterning can occur. So, the frequency range can be from UV to infrared or even longer wavelengths. By way of example, in some embodiments, the image projected on the semiconductor comprises electromagnetic radiation having a λmax in a visible region of the electromagnetic spectrum. In some of these embodiments, the semiconductor can comprise amorphous silicon. In another example, in some embodiments, the image projected on the semiconductor comprises electromagnetic radiation having a λmaxin an ultraviolet region of the electromagnetic spectrum. In some of these embodiments, the semiconductor can comprise titanium oxide. The image can be projected onto the semiconductor using any method which is compatible with the wavelength of electromagnetic radiation used as well as the desired resolution needed for patterning. In some examples, the image of the target pattern can be projected onto the semiconductor using a digital light projector. In other cases, the image of the target pattern can be projected onto the semiconductor by shining a light through a photomask. The electrodes can be fabricated from any suitable material. In some embodiments, at least the working electrode is transparent to a wavelength of electromagnetic radiation Attorney Docket No.10046-557WO1 used to project an image on the semiconductor. In some embodiments, the working electrode and the counter electrode are transparent to a wavelength of electromagnetic radiation used to project an image on the semiconductor. In some examples, the working electrode, the counter electrode, or a combination thereof can comprise a metal mesh. In other examples, the working electrode, the counter electrode, or a combination thereof can comprise a transparent conductive oxide (TCO) (e.g., fluoride-doped tin oxide (FTO), indium tin oxide (ITO), or aluminum-doped zinc oxide (AZO)), graphene, graphite, or thin metal film (e.g., a gold thin film). In some embodiments, the counter electrode can comprise a non-transparent electrode, such as a metal wire. The semiconductor can comprise any suitable semiconductor material. A variety of suitable semiconductors are known in the art, including silicon (e.g., amorphous silicon), germanium, diamond, silicon carbide, boron nitride, boron phosphide, boron arsenide, aluminum nitride, aluminum phosphide, aluminum arsenide, gallium nitride, gallium phosphide, gallium arsenide, indium nitride, indium phosphide, indium arsenide, cadmium selenide, cadmium sulfide, cadmium telluride, zinc oxide, zinc sulfide, zinc selenide, zinc telluride, cuprous chloride, copper sulfide, lead selenide, lead sulfide, lead telluride, tin sulfide, tin telluride, lead tin telluride, bismuth telluride, cadmium phosphide, cadmium arsenide, zinc phosphide, zinc diphosphide, zinc arsenide, titanium dioxide, copper oxide, and tin dioxide. In some embodiments, the semiconductor can comprise amorphous silicon. In some embodiments, the semiconductor can comprise a group IV semiconductor, such as germanium or silicon germanium, or a group III-V semiconductor, such as GaAs or InGaAs. In some embodiments, the semiconductor can comprise a metal oxide semiconductor (e.g., titanium dioxide, zinc oxide) or a 2-dimensional semiconductor material (e.g., tungsten sulfide). If desired, multiple charged agents can be sequentially patterned on a surface. For example, in some embodiments, one or more additional charged agents can be deposited on the a surface following patterning of a first charged agent using the methods described above. For example, in some embodiments, the methods described herein can further comprise contacting a charge support layer having a first charged agent assembled on the charge support layer in a first target pattern with a second suspension or solution comprising a second charged agent dispersed or dissolved in a fluid carrier; applying an AC electric Attorney Docket No.10046-557WO1 field within the semiconductor assembly using the working electrode and the counter electrode while projecting an image of a second target pattern onto the semiconductor; ceasing application of the electric field and image projection; and allowing the second charged agent to assemble on the charge support layer in the second target pattern, thereby generating a charge support layer having the first charged agent patterned in the first target pattern and the second target agent patterned in the second target pattern. The methods described herein can be used to deposit and / or pattern any suitable charged agent on a surface. Examples of suitable charged agents include, but are not limited to, charged ions, charged molecules, charged polymers, charged particles (including nanoparticles, microparticles, and irregular particles such as flakes), charged biomolecules (e.g., DNA, RNA, proteins, polysaccharides, etc.), cell, viruses, constructs such as nanowires, 3-dimensional materials, and combinations thereof. These methods can offer certain advantages over existing patterning technologies. For example, the low laser power requirement can result in minimal generation of heat, making these methods compatible with heat-sensitive materials such as biomolecules and cells. It also minimizes random solution flows and makes large-scale printing possible. Further, the optical pattern used to deposit particles can be independently controlled through a digital light projector, without the need to change the mask or to remove the sample from the system during the whole deposition process. This can allow for the sequential deposition of multiple patterns with minimal steps involved. In addition, the method can be performed without additional processes or special materials, such as photoresist and photomasks, greatly reducing the cost of deposition. These methods can be used to generate a variety of functional constructs by patterning appropriate materials, including for example high-precision lithography masks, functional chips based on synthesized semiconductor nanoparticles, reconfigurable optoelectronics, displays, and large-scale nanosensor arrays. These strategies can also be used to fabricate 3D structures on a surface via multistep deposition. Because deposition of the charged agent depends on the light pattern itself, the resolution of the technique depends on how accurately the light is focused onto the sample. Nevertheless, with well-developed deep-UV techniques that can create light patterns with features of tens of nanometers. Attorney Docket No.10046-557WO1 EXAMPLES The invention will be described in greater detail by way of specific examples. The following examples are offered for illustrative purposes and are not intended to limit the invention in any manner. Those of skill in the art will readily recognize a variety of non- critical parameters which can be changed or modified to yield essentially the same results. Example 1. Massively Parallel Microbubble Nano-Assembly Summary Microbubbles are an important tool due to their unique mechanical, acoustic, and dynamical properties. While there has been remarkable progress, it remains challenging to generate addressable microbubbles quickly in a parallel and controlled manner. In this example, an opto-electrochemical method is described that combines the precision of light- based imaging with the relatively low energy bubble formation by hydrolysis. In addition, the inclusion of nanoparticles enables individually addressable microbubbles to be formed in designed patterns. The size of the bubbles can be controlled from a few micrometers to over hundred micrometers with a spatial accuracy of ~ 2 - µm. The light intensity required is only ~0.1 W / cm2, which is on par with sunlight and several orders of magnitude lower than that required by other state-of-the-art techniques. The technique is general and permits a wide spectrum of particles to be assembled from suspension, ranging from 40-nm silver nanocrystals, 200 nm polymer nanospheres, to 2-μm-E-coli bacterial cells. Particle assembly in well-defined patterns is demonstrated. Further, Ag nanoparticle-bacterial-cell arrays can be formed that permit the spectroscopic detection of cell metabolites. Overall, the reported microbubble tool overcomes multiple key challenges in microbubble-based technologies and promises new opportunities in nanomanufacturing, nanophotonic patterning, nanorobotics, biosensing, and single-cell. Introduction Microbubbles exhibit special mechanical, acoustic, and dynamical properties and have become important tools in fields ranging from medical imaging to microfabrication. Their ability to interact with soft matter has thus been explored for diverse applications, including microfluidic pumping and mixing, chemical switching, drug delivery, shaping ultrasound fields, and in the propulsion of micro / nanorobots. Microbubbles that are ultrasonically excited can experience and also exert forces. The microbubbles can transduce forces directly, or indirectly via streaming or nonlinear effects. Microbubbles can thereby also move particles that are suspended in solution. In addition, microbubbles that are not Attorney Docket No.10046-557WO1 resonantly excited, can be used to induce local fluid flows and thus manipulate colloidal and nano-particulate matter. The latter often involves the generation of a single microbubble with a focused laser beam to deposit particles onto a substrate via convective flows that form around the microbubble. The laser beam can also be raster-scanned to move the micro- bubble and thereby drive complex nanoparticle deposits. However, this method is relatively slow as it involves a single microbubble and operates at elevated temperatures as the microbubbles are thermo-optically generated, which complicate the assembly of biological matter. In this example, we show an alternative scheme that uses low intensity light to obtain defined microbubble patterns and we show that these can be used to assemble colloidal matter, including biological cells at room temperature, into complex patterns. We demonstrate the co-localized controlled assembly of plasmonic nanoparticles with bacterial cells for surface enhanced Raman spectroscopy. The effect we describe relies on the use of a digital light projector in conjunction with a photoconductive substrate, such that low intensity light patterns can trigger the electrochemical generation of well-defined regular patterns of single microbubbles, including single large microbubbles with a diameter of tens of micrometers. The latter is surprising, as typically many small gas bubbles, several microns in size, form into clusters during an electrochemical reaction. Our approach overcomes this limitation and permits intricate patterns and clusters of nanoparticles and biological material to be formed on demand. Both the generation of the microbubbles and the assembly they drive are different in our method compared with existing techniques. Conventionally, microbubbles are formed via prefabricated templates, including concave microstructures, which are then used to trap bubbles that in turn can mediate acoustic forces. However, the bubble patterns are defined by the template and are thus static. Alternately, the use of laser-induced heating can form opto-thermal microbubbles flexibly at the focus of the laser. While the use of plasmonic Au / Ag nanoparticles or surfaces reduces the required laser power, the direct formation of a microbubble with laser light always requires relatively high intensities and also involves higher temperatures. These are estimated to range between 60° C and 250° C, depending on bubble size, in order to generate water vapor. As a serial method, the optothermal approach is thus difficult to adapt for larger-scale applications, and importantly precludes working with temperature-sensitive biological samples. Therefore, it is advantageous to consider alternative schemes to form Attorney Docket No.10046-557WO1 bubbles, such as electrolysis. While electrolysis can proceed at room temperature, it is challenging to define the size, the location where the bubble forms, or the number of bubbles that are generated. Overall, several drawbacks of existing state-of-the-art techniques to form microbubbles restrict practical applications of microbubbles (summarized in Table 1). Table 1. Summary of state-of-the-art bubble-based technologies. In this work, we first report how light projection can rapidly generate defined microbubble patterns. As we show, the size, position, and lifetime of the microbubbles can readily be controlled. We generate microbubbles with diameters that can vary between 2.5 and 140 µm, and controllably position these with a spatial accuracy of 2.45 µm. The microbubbles exist, depending on the experimental details, from about 23 ms to over several tens of minutes. As we show here, entire images can be formed with microbubbles and these can be refreshed at 6 Hz. The presence of nanoparticles in the same solution is shown to be crucial to obtain single microbubbles at the desired target location, instead of bubble clusters that would otherwise form. Further, nanoparticles in suspension can be patterned by the microbubbles. We trigger the formation of microbubbles via the hydrogen evolution reaction on a photoconducting hydrogen-terminated silicon, α-Si:H, substrate. This substrate is on one side in contact with an optically transparent fluorine doped tin oxide (FTO) surface that forms the working electrode, and on the other side it faces the fluid in which the assembly Attorney Docket No.10046-557WO1 takes place. An electric potential is applied between the FTO in contact with the α-Si:H substrate and another FTO substrate that forms the counter-electrode. In the absence of light the α-Si:H substrate is not conducting and it prevents any current from flowing and hence prevents the gas formation via electrolysis. However, when the α-Si:H substrate is illuminated with low intensity light – as little as ~0.1 W / cm2is sufficient – it becomes conducting and electrolysis proceeds in the illuminated region. We would like to stress that the intensity is several orders of magnitude lower compared to that required by various other light-based bubble techniques. Such a low-level light intensity enables the high scalability of our technique, as light projection can be used to illuminate entire images ‘in one shot’, and thus form complex bubble patterns. Furthermore, electrolysis often results in the formation of microbubble clusters in an activated region, but we show that the addition of nanoparticles can prevent the formation of small satellite bubbles, such that one can, for the first time, form individually light-addressable bubbles as large as ~140 µm in diameter. Next, we demonstrate that these room-temperature bubbles also drive the assembly of particles that are in the suspension. In contrast to opto-thermally generated bubbles, the deposition of particles from solution does not rely on thermocapillary or thermal convection currents and thus is also different to that of existing methods. Rather, the attraction of particles to the bubble surface can be attributed to surface tension and concentration gradient near a shrinking bubble, which will be discussed later. Finally, we show that these simultaneously generated microbubble patterns can be used to assemble nanoparticles in well-defined patterns, including millimeter-scale lattices. This allows us to position plasmonic-Ag nanoparticles to form nanosensors to spectroscopically record cell metabolites. The technique is general and permits a wide spectrum of particles to be assembled, ranging from 40-nm silver nanocrystals to 2-μm-E- coli bacterial cells. We expect that the fast, massively parallel nano assembly method presented herein, will enable further applications in nanomanufacturing, nanophotonic patterning, nanorobotics, biosensing, single-cell biology, and hybrid device fabrication. Materials and Methods The experimental setup included a digital light projector (DLP) which is illuminated by a 532 nm continuous wave laser and projects images to a photoconductive α-Si:H substrate (Figure 1A). A PDMS well, which houses a sodium-sulfate (Na2SO4) / nanoparticle solution, was assembled on the α-Si:H layer and covered with a second FTO substrate that formed the counter electrode (Figure 1B). While the light pattern formed by Attorney Docket No.10046-557WO1 the DLP illuminates the photoconductor, an electric signal of prescribed voltage and frequency was applied to the FTO-layer underneath the α-Si:H substrate and the FTO glass covering the PDMS well to generate bubbles that match the DLP laser pattern (Figure 1C). 200 nm fluorescent PS spheres used for particle deposition were purchased from Thermofisher Scientific. The carboxylate-terminated spheres have a surface charge of 0.3699 meq / g (technical data sheet). Sodium sulfate (anhydrous) and hydrogen peroxide (30%) were purchased from Fisher Chemical. Non-Functionalized silver nanoparticles (citrate capped, 40 nm) were purchased from NNCrystal US Corporation and washed with DI-water via centrifugation 3x and concentrated 4x before use. E-coli (K12 strain) were purchased from Carolina Biological Supply Company. The measured cell density of after washing 3x with DI water turned out to be 4.77 x 107cells / mL. Polydimethylsiloxane (Sylgard 184 silicone elastomer kit) was purchased from Dow Corning Corporation. Cetyltrimethylammonium chloride (25 wt. % in H2O) and Poly(diallyldimethylammonium chloride) (average Mw 200,000-350,000, 20 wt. % in H2O) was purchased from Sigma Aldrich. All reagents were used without further purification. FTO (Fluorine-doped Tin Oxide) glass (0.7 mm thickness and 13 – 15 Ohm / Sq sheet resistance), purchased from MSE Supplies (Product TEC 15), was used as the counter electrode. Sapphire glass–FTO glass–Hydrogen-terminated silicon substrates were fabricated at the University of Stuttgart. Equipment. The experiments are carried out on an inverted microscope (Olympus IX 70) equipped with a 532 nm laser (DJ532-40, Thorlabs) and a digital light projecting device (DLP, Model DLi4130 .7”VIS XGA, Digital Light Innovations). Scanning electron microscopy (Model Quanta 650, FEI, Model Apreo 2 SEM, Thermo Fisher Scientific) was used for the characterization of deposited particles. A function generator (Model 33250A, Agilent), coupled with a custom-made amplification circuit was used to create AC / DC fields with the desired electric field intensity. A reactive ion etcher (Model RIE-1C, Samco) or UVO-cleaner (Model 30, Jelight) was used to clean the surface of the silicon sample and etch the deposited PS spheres. The etcher was also used to etch away part of the silicon so that the FTO glass underneath could be exposed for electric connections. An e-beam and sputtering system (Model PVD75, Kurt J. Lesker Company) was used to deposit a thin film of Pt on top of a FTO-glass counter electrode substrate to test the effects on bubble generation. A 532 nm wavelength laser (Model Sapphire, Coherent) was used for the characterization of minimum laser intensity, and another 532 nm wavelength laser (DJ532- Attorney Docket No.10046-557WO1 40, Thorlabs), coupled with a digital light projector (Model DLi4130 .7”VIS XGA, Digital Light Innovations) was used for light pattern projection. Experimental Setup. In a typical experiment, a 100 to 250 μm-thick PDMS well (1.5 mm in diameter) is placed on the α-Si:H surface that has been cleaned by oxygen plasma or UV-ozone treatment. A Na2SO4 solution (0.18 μL) is mixed with the nanoparticles that are to be deposited and then dispersed into the well. The well is sealed with a FTO glass which serves as the counter-electrode. The FTO on the α-Si:H layer serves as the working electrode. An electric voltage is applied between the working and counter electrodes. The light-illuminated areas become conducting and here the bubbles are formed by electrolysis. Characterization of the Minimally Required Laser Intensity. A single laser beam with a diameter of 7.22 μm is used for the characterization of the bubble formation, measured by the 10 / 90 knife-edge method (Figures 16A-16B). Prior to the bubble generation, the surface of the silicon was gently cleaned using DI water and IPA. Unless stated otherwise, a 0.1 M Na2SO4 solution, -5 V DC / 12 Vpp at 1.5 kHz AC E-field was applied for ~225 ms using a 500 μm PDMS well, as a standard condition. The electrolyte solutions were prepared before use from a 1.5 M Na2SO4stock solution. When testing the effects of H2O2and CTAC these molecules were added to achieve a final concentration of 5% and 1.25 mg / mL, respectively. Microbubble Patterning and Particle Deposition. For bubble-driven nanoparticle assembly, the substrate surface is first cleaned with DI water and IPA. This is followed by a treatment with oxygen plasma for 1 m at 50 sccm, 50 W power, or with a UVO cleaner for 30 m. A 4% PDDA solution is then applied to the surface for 3 m to positively charge the surface and increase the hydrophilicity, after which the surface is washed with DI water and gently dried using a flow of nitrogen gas. A 0.66 – 0.8 M Na2SO4 solution containing 200 nm polystyrene nanoparticles is added to the PDMS well applied on the surface. A light pattern is projected via a digital mirror array controlled by a custom-made python program using a continuous 532 nm laser (DJ532-40, Thorlabs). An E-field of -5 V DC and 12 Vpp AC voltage at 1.5 kHz is applied between the working electrode underneath the α-Si:H surface and the counter FTO electrode. Microbubble patterns were formed with 15 – 50 ms AC bursts, while the bubble-assisted nanoparticle assembly were formed with bursts lasting 225 – 750 ms. Attorney Docket No.10046-557WO1 Silver nanoparticle deposition, E-coli detection and E-coli assembly. First, the silver nanoparticles were washed with DI water via centrifugation three times to remove any excess ions that might interfere with the SERS measurement. The bubble deposition was then repeated 5 times on the same spots to ensure that the clusters contain an adequate amount of silver nanoparticles on the surface for the subsequent SERS measurements. A suspension of E-coli bacteria was washed with DI water 5 times. No additional ions were required for the DEP, which proceeded when 20 Vppwas applied to the cell with a 1 MHz AC field. This ensured the DEP transport of the E-coli bacteria to the silver nanoparticles. For bubble-based live E-coli deposition, the E-coli was washed with DI water for 3 times and concentrated to 20x of the original solution (4.77 * 107cells / mL). The concentrated solution was mixed with a 1 M Na2SO4stock solution 1:1 to facilitate bubble growth, resulting in a 10x, 0.5 M Na2SO4 E-coli solution. For SEM imaging the original E- coli was fixed via washing with a 7.4 PBS solution 2 times and submersing in a 1:3 acetic acid:EtOH solution for 10 m. Then, the solution was washed by PBS solution twice and finally washed by DI water and concentrated to obtain a 20x higher E-coli cell density. The concentrated solution was mixed with a 1 M Na2SO4 stock solution 1:1 to facilitate bubble growth, resulting in a 10x, 0.5 M Na2SO4E-coli solution. After depositing the E-coli with bubbles, the solution was gently exchanged with DI water 10 times to suppress random deposition of E-coli on the surface, then dried. Before SEM imaging, a thin layer of gold / palladium was deposited to improve the imaging of the samples. Heating and simulation and laser heating. While many works utilize heat-induced thermophoretic and accompanying Marangoni flows to attract and trap particles onto the surface, the heat generation in our method in contrast, is minimal. Due to the low intensity, heating due to laser absorption is very small at the surface of the Si and the ohmic heating in our experiments is similarly minimal. Simulations (Figure 8) and calculations result in a heat increase of less than <<1 K for each source, due to the high photoconductivity of our substrate and the low laser intensity requirement to generate a bubble. Here, we provide the parameters relevant for ohmic heating and laser heating simulations: Attorney Docket No.10046-557WO1 Description Expression Thermal conductivity of a-Si 1.3 x 10-11(T-900)3+ 1.3 x 10-9(T-900)2+ 10-6(T- 900) + 10-2[W / cm∙K] Specific heat of a-Si 0.171 T / 1865 K + 0.952 [J / g∙K] Density of a-Si 2260 [kg / m3] Reflectivity of solid a-Si 0.58 Absorption coefficient of a-Si 5.02 x 103exp(T / 430) [ / cm] The temperature increase due to the absorption of light in the a-Si layer was simulated using COMSOL Multiphysics and further details are provided below. Effect of H2O2 and CTAC. We explore how various additives and surfactants such as H2O2and cetyltrimethylammonium chloride (CTAC) and a thin Pt catalyst layer on the counter electrode may affect the minimum laser intensity. Under normal conditions (a 0.5 M Na2SO4 solution with a -5 V DC bias superimposed on a 12 Vpp AC E-field with a frequency of 1.5 kHz), the average minimum laser intensity is 390.151 ± 17.477 mW / cm2. With the addition of H2O2, this number falls to 288.384 ± 36.120 mW / cm2, which represents a 26% drop in the required laser intensity. Despite having a slightly higher surface tension than water with a value of 80.4 mN / m, which would prohibit bubble formation, H2O2decreases the required laser intensity due to two factors: 1) H2O2has an acid dissociation constant (pKa) of 11.6, meaning that it is slightly acidic in nature; releasing H+ions into the solution which is the basis of the hydrogen bubbles generated by the E-field. 2) H2O2has been reported as an effective hole scavenger and can prevent hole- electron recombination during illumination. Indeed, as can be seen in equation (1), the increase of recombination lifetime leads to an increase of the photocurrent of amorphous silicon, leading to smaller minimum intensity values. CTAC, on the other hand, acts as a surfactant that reduces the surface tension of water at the liquid-gas interface, which can facilitate bubble growth. It has been reported that the addition CTAC can reduce the surface tension down to 36.1 mN / m, compared to the surface tension of plain water of 71.78 mN / m. For our experiments, we use a concentration of 1250 mg / L to ensure the highest decrease of surface tension. Indeed, as can be seen in Figure 2F, the addition of CTAC has shown to greatly decrease the required laser intensity down to 219.741 ± 51.093 mW / cm2. Attorney Docket No.10046-557WO1 Considerations on the formation of single vs multiple bubbles. A single larger bubble possesses a smaller surface area compared to multiple smaller bubbles that contain the same overall volume of gas, and hence minimization of the overall surface energy favors the formation of a single bubble. Hydrophobic or partially hydrophobic nanoparticles are known to attach themselves to gas bubbles and can in turn stabilize the bubbles. Smaller bubbles possess a larger curvature and are most probably not as well covered by nanoparticles, such that these smaller bubbles fuse to form larger, more stable ‘single’ bubbles. The exact mechanism of formation and hence the resulting size) is likely to be a complex process that is an interplay between the stabilizing effect of the nanoparticles, surface tension effects, as well as pressure-volume work and entropy considerations. A full exploration of all of the relevant parameters is beyond the scope of this work. Shrinkage of the H2 bubbles. The shrinkage and collapse of the bubbles are the result of competition between the outward H2diffusion pressure and the inward pressure of the surrounding liquid at the gas-liquid interface. Most of the bubbles show a similar trend, with an initial slow shrinking speed likely due to dynamic interactions with supersaturated hydrogen at the bubble surface vicinity, which is followed by a rapid collapse (Figure 17A). Here, most of the bubbles eventually shrank and collapsed under ~80 s. However, some of the bubbles exhibited a secondary expansion phase even after the E-field and laser pattern is turned off (Figure 17B). This additional growth of the bubbles increases the bubble lifetime, taking the total time to shrink and collapse as from ~ 125 s to over 300 s. Similar bubble growth has been found in bubbles in supersaturated water at the vicinity of an electrode. Consideration of the surface state and particle density. The versatile bubble- patterning technique is applied for capturing and assembling colloidal nanoparticles. For this purpose, it is crucial to control the surface state of the substate, as well as the shape and size of microbubbles. 1) On a native α-Si:H surface, a hydrogen bubble maximizes its contact and adapts to a truncated sphere due to the hydrophobic nature of both the hydrogen gas and the α- Si:H. As a result, the particles deposit into a coffee ring structure (Figure 10). We, therefore, tune the α-Si:H substrate to be hydrophilic to obtain single-spot particle deposition via a spherical, single-contact microbubbles. 2) When the particle density inside the solution is low, or no particles are inside the solution, multiple, instead of single, bubbles form under light spots (Figure 4A, inset), Attorney Docket No.10046-557WO1 which is not suitable for precisely positioning nanoparticles. The single-bubble formation in a nanoparticle colloid suspension, in contrast to multi-bubbles made without nanocolloids, could be attributed to a total energy reduction driven by surface-area minimization as discussed above. Gas-diffusion controlled process of microbubble growth. While a slope of 0.5 is often assumed for ideal diffusion-controlled growth, variations from the diffusion- controlled growth model may exist due to the following factors: 1) The research in references used solutions such as sulfuric acid in water, which exhibits a higher hydrogen ion concentration for hydrogen bubble growth compared to that in D.I. water used in our study. 3) The model assumes that the bubble is far away from any solid wall since the surface roughness may alter the bubble growth. While, we grew bubbles next to a substrate. 4) Last, we usually grew bubbles in a closely spaced array. It has been reported that the presence of close-by bubbles decreases a bubble’s growth. The reported power factor of a second bubble generated next to an already existing bubble is 0.44, which exactly agrees with that we observed in experiments. Deposition of hybrid Ag – E-coli assemblies. Ag– E-coli hybrid assemblies were obtained by depositing Ag nanoparticle assemblies using microbubbles as described in the text, followed by the deposition of E. coli cells. In between, the solution is exchanged. Specifically, we first deposited Ag clusters using a Ag particle solution. After the Ag nanoparticle deposition, the original Ag nanoparticle solution was removed by gently applying and removing fresh DI water to the surface 10 times. Then, the sample was dried, and an E. coli solution was dispersed in the contained, followed by the bubble assembly. Measurement of E-coli metabolites using SERS. Three SERS measurements are made with two measurements for control: 1) away from the silver nanoparticle deposits, where the silver nanoparticle density is low and not concentrated enough to generate ‘hot spots’ (Figure 18, Background_control). 2) At a silver nanoparticle deposit, but without any AC field to attract E-coli, leading to the generation of ‘hot spots’ but without any detection of E-coli (Figure 18, E-field off_control). 3) At a silver nanoparticle deposit with the AC field on, leading to the generation of ‘hot spots’ with E-coli at the vicinity of the deposit (Figure 18, E-field on). First, as expected, the SERS signals are low from the silicon surface alone, compared to when the measurement is taken at the silver nanoparticle assembly. The SERS measurement from the silver nanoparticle assembly in the absence of the E-field, shows stronger signals, but characteristic peaks associated with E-coli were not Attorney Docket No.10046-557WO1 observed. Last, when the E-field is turned on, multiple peaks are seen, indicating the attraction of E-coli by the DEP force. The plots are displayed in Figure 6D after a baseline correction. The peak positions are assigned after comparing the results with multiple references, which are summarized in Table 2. Table 2. Peak assignment of E-coli SERS measurement and its comparison with values reported in the literature. Results and discussion Working Mechanism, Characterization, and Optimization. In an electric field, we can rapidly create either individual or large arrays of microbubbles in a sodium-sulfate solution (Na2SO4) at the projected light points on the α-Si:H substrate. The application of both light and electric voltages is essential for the process, therefore, it is important to elucidate the working mechanism that generates microbubbles. We first determine the minimum required light intensity (MRLI) for the formation of bubbles. Upon light illumination with an applied electric field, a microbubble quickly forms. The diameter (2r) increases from ~12 μm to ~27 μm as the light intensity increases in an E- field (Vbias: -5 V, Vpp: 12 V at 1.5 kHz, 0.1 M Na2SO4concentration); the power coefficient Attorney Docket No.10046-557WO1 of 0.29 indicates the close-to-linear dependence of the bubble volume (V) on light intensity, where V ~ r3(Figure 2A). The MRLI depends on the frequency of the AC voltage and decreases from 12 W / cm2to 7.4 W / cm2when the AC frequency is lowered from 1 MHz to 1.5 kHz, respectively (Vbias: -5 V, Vpp: 12 V, 0.1 M Na2SO4concentration) (Figure 2B). At a given AC E-field (Vpp: 12 V at 1.5 kHz, 0.1 M Na2SO4 concentration), the MRLI is further reduced ~ 2.72-fold with an increasing superimposed DC bias from -3.5 to -5.5 V (Figure 2C). It should be noted that a constant positive DC voltage up to 9 V (or a positive bias superimposed on a symmetric AC voltage) does not generate bubbles, which suggest that the α-Si:H film conducts electrons under a negative electric bias, similar to p-type Si. This is further confirmed by a voltage sweep test from +4 V to -4 V (Figure 7). Increasing the electric conductivity of the electrolyte solution also promotes bubble generation, where the MRLI is lowered by over 4.5 fold from 7.4 to 0.16 W / cm2(Vbias: -5 V, Vpp: 12 V at 1.5 kHz) with the increase of Na2SO4concentration from 0.1 to. 1.5 M (Figure 2D). The requirement of light illumination and an applied electric voltage suggests three possible bubble-creation mechanisms: 1) electric ohmic heating of the light-illuminated α- Si:H, 2) thermal heating due to light absorption, and 3) light-controlled electrochemical hydrolysis. While thermal heating has been widely explored for making microbubbles,19this working mechanism requires a light intensity that is at least 5 orders of magnitude higher than in our system.34Further calculation and numerical simulation indicate that neither ohmic-resistive heating nor thermal heating can account for the observed bubble creation, the effects of which only increases the temperature by <<1 K (Figure 8). The electrochemical-reaction mechanism, in contrast, explains the observed bubble evolution and dependence on the laser intensity, the AC frequency, the DC bias, and the electrolyte concentration very well (see Figure 2A-2D). For instance, Figure 2A indicates the radius increases linearly with the light intensity (inset of Figure 2A), which is in agreement with the linear dependence of the photocurrent (ip) (of the α-Si:H substrate) participating in electrochemical reaction, with photon rate (N0) and thus light intensity, as shown in the following Equation (1): where R, α, d, η, τ, and ttare the surface reflection, surface absorption coefficient and depth, electron-hole pair generation efficiency, recombination lifetime, and carrier transit time, respectively. Attorney Docket No.10046-557WO1 We further model the system as an equivalent electrochemical circuit, which consists of an electric double layer (EDL) with a capacitance, CSi, next to the photoconductive Si electrode with a resistance of RSi, an electrolyte solution with a resistance of Rs, and a second EDL next to the FTO counter electrode (CR) (Figure 2E). Here, light turns on / off this electrochemical circuit path via controlling the Si-film’s local resistance. When light is switched on and RSi is significantly lowered (Figure 9), an applied voltage distributes across all the circuit elements, with the corresponding voltage drop proportional to each element’s resistance (impedance). For a capacitor, the impedance is given by Z =1 / (2πfC), and the voltage drop (VC_SI), increases with the decrease of AC frequency (f). Therefore, we observe that a low AC frequency and a negative DC bias both prompt the evolution of hydrogen-bubbles as both effects enhance the voltage drop on the EDL capacitor for electrochemical water reduction. Owing to the same principle, a reduced electrolyte resistance (Rs) improves hydrogen bubble evolution, by taking a smaller voltage drop and redistributing it to other circuit elements, including the EDL capacitor next to Si. The experimental observations, i.e., the improvement of the MRLI with the decrease of the AC frequency and the increase of negative DC bias and Na2SO4 electrolyte conductivity (Figure 2B-2D), all agree with the theoretical circuit-model analysis. Indeed, both theoretical analysis and experimental results indicate that the light-controlled electrochemical reaction is the dominating mechanism for bubble generation. It should also be noted that a DC field alone can generate microbubbles. However, we study the effects of an AC E-field due to its importance in understanding the working mechanism and practically employ this strategy for robust bubble creation. It is because we find that the Si surface is easily damaged with DC or low-frequency AC fields. This can be attributed to the FTO layer partially exposed to the solution due to the voids / grain boundaries in the 200 nm α-Si:H, in which case, the FTO competes with the reactions on the light-activated Si surface. If only a DC field is used, reactions also take place at the FTO layer, generating bubbles and damaging the Si layer in the process. The use of an AC field, on the other hand, lowers the conductivity of the Si’s capacitive component, and hence the total resistance of the Si. While this also reduces the effective voltage applied on the electric double layer, the reactions are shifted more to the Si with less reactions taking place on the FTO layer. Indeed, experimentally, at a 10 kHz AC field we seldom observe any bubbles from the FTO, which thus maintains its integrity. For the electrochemical formation of Attorney Docket No.10046-557WO1 bubbles, we therefore choose an optimized AC-frequency and further add a negative DC bias to obtain the best MRLI value while retaining surface integrity. The understanding of the bubble formation mechanism assists us in rationally improving the efficiency of the electrochemical system by adding hydrogen peroxide and surfactants (CTAC) for a lower redox voltage and surface tension, respectively (Figure 2F). With all conditions optimized, it only requires a light illumination of ~0.1 W / cm2, corresponding to the level of sunlight on Earth, to create a microbubble (532 nm laser; electrolyte: 1.5 M Na2SO4, 1.25 mg / mL CTAC, 5% H2O2; Vpp: 12 V at 1.5 kHz, square wave, Vbias: -6 V). Instant, Parallel, Versatile Microbubble Patterning. The above study and optimization enable the use of low-intensity light for making various microbubble patterns in a parallel, addressable, and versatile manner. The microbubble patterns can even be rapidly refreshed to form dynamic movies. Demonstrations include a bubble-made longhorn image, the symbol of UT Austin, which is overlaid on the map of the state of Texas (Figure 3A and 3B), the logo of Max Planck Society, Minerva (Figure 3C and 3D), as well as animations, including a running horse, a continuously expanding circle and fireworks. Applications: Microbubble-Actuators for Nanoparticle Capture, Assembly, and Printing High-Precision, Bubble-Actuated Nanoparticle Printing: The versatile bubble- patterning technique is applied for capturing and assembling colloidal nanoparticles. For this purpose, it is crucial to control the surface state of the substrate and the concentration of nanoparticle suspension. For instance, a native α-Si:H surface can only support truncated spherical hydrogen bubbles that results in coffee-ring-structured particle aggregation (Figure 10) due to the hydrophobic nature of both hydrogen gas and α-Si:H. Without nanoparticles in solution (or at a low concentration), multiple, instead of single, bubbles form under a light spot (Figure 4A), which is detrimental to precise particle printing. The formation of single or multiple bubbles can be understood when considering the thermodynamic factors driving the bubble formation. Nanoparticles are attracted to and cluster at the bubble interface likely due to concentration gradients and to minimize the surface tension of the microbubble. Finally, the particles are captured by a bubble and deposited at a single spot during its shrinkage (Figure 4B, inset). The above observations together with further considerations of surface charge and particle density indicate that a surface modification is needed to enable the formation of Attorney Docket No.10046-557WO1 single-spot particle aggregates. We first modify the α-Si:H surface with positive charges using oxygen plasma followed by poly-diallyldimethylammonium chloride (PDDA) coating (200-nm polystyrene nanospheres: 6 to 8 x 1010 / ml; Na2SO4 to 0.66 – 0.75 M). The efforts generate single, size-controlled, and position-defined bubble patterns at the point of the incident light with a positional accuracy of 2.45 ± 1.08 µm (Figure 11). The diameter of a bubble increases during the application of an E-field (Figure 4B) (Vbias: -5 V, Vpp: 12 V at 1.5 kHz) with the power-law coefficient determined as 0.44, close to 0.5 for gas-diffusion controlled bubble growth (Figure 4C). The PDDA functionalization of Si further enables single-point bubble formation due to the electrostatic attraction; it also effectively helps in fixing the nanoparticles to the oppositely charged substrate (Figure 4D-4E) In addition, the microbubbles shrink symmetrically during the process so that the nanoparticle clusters can be precisely localized near the center of the microbubble to within 1.03 ± 0.61 μm. This permits positioning to an order of magnitude smaller feature sizes compared to the size of the light pattern and bubbles (Figure 4F). As a result, with simple DLP light patterns, we successfully assemble arrays of nanocolloids via electrochemically- formed microbubbles (Figure 4E). Microbubbles for Rapid Programmable Nanoparticle Assembly: While small, individual light spots can be used to create corresponding individual microbubbles with precise position control, large-area simple light patterns can also be applied to simultaneously generate many microbubbles for rapid, large-scale, programmable particle assembly (Figure 5, Video S7). A single bar of light can thus be used to obtain an evenly spaced chain of bubbles (Figure 5a), where the spacing between bubbles can be controlled by the total E-field-application time (Figure 5b). Depending on the sizes of the light pattern and the bubbles, different arrangements of the patterns can be generated (inset, Figure 5b, blue and green rectangles in Figure 5b). When broadening the width of the light pattern, the bubbles first increase in size (Figures 12A-12F) and then a second row of bubbles forms. Further, the bubble size and hence the bubble lifetime can be varied over three orders-of-magnitude from 23 ms to 24 s (Figure 5C). The nanoparticle assembly arises at the microbubble and by choosing appropriate parameters based on the above observations, we achieve programmable well-ordered nanoparticle patterns consisting of evenly spaced spots along defined patterns (Figure 5D- 5E). Attorney Docket No.10046-557WO1 Millimeter-Scale Bubble Printing: Distinct from state-of-the-art bubble printing techniques, the reported opto-electrochemical bubble printing, allows for a massively parallel assembly. We demonstrate the printing of nanospheres into 2.56-mm-long nanoparticle lattices using opto-electrochemically patterned microbubble arrays, in only 7 depositions (Figure 13). The e aggregates are typically a few micrometers in size, and the deposition contrast can be further enhanced via surface charge modulation (Figure 14A- 14C). Patterning Nanosensors for Detecting Cell Metabolites. As is expected for a microbubble assembly process a wide range of materials can be used. Our electrochemical- microbubble printing technique has the distinct advantage that it is compatible with temperature-sensitive biological samples. We first use it to assemble metallic nanoparticles (Figure 6A) and then include the assembly of live bacterial cells (1-2µm) (Figure 6B). The method is versatile, also allowing the creation of Ag-E-coli hybrid assemblies. As a proof- of-concept, we assemble an array of plasmonic Ag nanoparticles (40 nm in diameter, 3x4) (Figure 14D-14E, Figure 15) and further trap live E-coli cells in a way that they co- localize with the Ag clusters (Figure 6C). The plasmonic assemblies made of aggregated Ag nanoparticles provide a large number of hotspots for sensitive surface-enhanced-Raman- spectroscopy (SERS) detection. Next, the E-coli cells are brought in contact with the Ag assemblies by light-controlled dielectrophoresis (DEP) at 1MHz, 20Vpp in deionized water (Figure 6D). The attraction of the E-coli cells to the Ag nanoparticle arrays can be seen from the change of light diffraction under laser when the AC field is off and on. The attraction to the Ag assemblies is further confirmed by the SERS detection of the metabolites released from the E-coli cells and cell membrane chemistry compared to that without E-coli cells or from randomly dispersed Ag nanoparticles (Figure 6D, Table 2). Summary We introduce an opto-electrochemical technique that permits the room-temperature formation and patterning of microbubbles with defined size in a parallel and scalable fashion. The required low light intensity (~0.1 W / cm2), a few orders of magnitude less than what has been reported previously, triggers the electrolysis-driven bubble formation. The size of the microbubbles determines their lifetime, and the preparation of the substrate enables the formation of well-defined microbubbles. The use of nanoparticles in the suspension facilitates the growth of single bubbles. The microbubbles then initiate and guide the assembly of nanoparticle clusters onto a substrate. The nanoparticle aggregates Attorney Docket No.10046-557WO1 can be positioned with an accuracy of ~ 1 µm, relative to the bubble position. We pattern Ag nanoparticle clusters and electrically guide E-coli to form nanosensor-cell pairs for the SERS detection of metabolites. The microbubble technique presented herein is general and can be used to quickly form assemblies from a range of structures and materials, including polymeric or metallic nanoparticles, colloidal microparticles, and live biological cells. This research overcomes longstanding challenges in obtaining microbubbles with high precision and high throughput. We expect a range of applications ranging from actuators, sensors, to particle assembly, cell-particle interfaced microchips, nanosensing, and micro / nanorobotics. References 1. Gao, Y., Wu, M., Lin, Y., Zhao, W. & Xu, J. Acoustic bubble-based bidirectional micropump. Microfluid. Nanofluidics 24, 1–10 (2020). 2. Tovar, A. R. & Lee, A. P. Lateral cavity acoustic transducer. Lab Chip 9, 41–43 (2009). 3. Orbay, S. et al. Mixing high-viscosity fluids via acoustically driven bubbles. J. Micromechanics Microengineering 27, 015008 (2017). 4. Ozcelik, A. et al. An acoustofluidic micromixer via bubble inception and cavitation from microchannel sidewalls. Anal. Chem. 86, 5083–5088 (2014). 5. Ahmed, D., Mao, X., Shi, J., Juluri, B. K. & Huang, T. J. A millisecond micromixer via single-bubble-based acoustic streaming. Lab Chip 9, 2738–2741 (2009). 6. Destgeer, G. et al. Adjustable, rapidly switching microfluidic gradient generation using focused travelling surface acoustic waves. Appl. Phys. Lett. 104, 10–15 (2014). 7. Ahmed, D. et al. Acoustofluidic chemical waveform generator and switch. Anal. Chem. 86, 11803–11810 (2014). 8. Jeong, J., Jang, D., Kim, D., Lee, D. & Chung, S. K. Acoustic bubble-based drug manipulation: Carrying, releasing and penetrating for targeted drug delivery using an electromagnetically actuated microrobot. Sensors Actuators, A Phys. 306, 111973 (2020). 9. Klibanov, A. L., Shevchenko, T. I., Raju, B. I., Seip, R. & Chin, C. T. Ultrasound- triggered release of materials entrapped in microbubble-liposome constructs: A tool for targeted drug delivery. J. Control. Release 148, 13–17 (2010). 10. Ma, Z. et al. Spatial ultrasound modulation by digitally controlling microbubble arrays. Nat. Commun. 11, 4537 (2020). 11. Chen, C. et al. Chemical / Light‐Powered Hybrid Micromotors with “On‐the‐Fly” Optical Brakes. Angew. Chemie 130, 8242–8246 (2018). Attorney Docket No.10046-557WO1 12. Yuan, K., De La Asunción-Nadal, V., Jurado-Sánchez, B. & Escarpa, A. 2D Nanomaterials Wrapped Janus Micromotors with Built-in Multiengines for Bubble, Magnetic, and Light Driven Propulsion. Chem. Mater. 32, 1983–1992 (2020). 13. Ren, L. et al. 3D steerable, acoustically powered microswimmers for single-particle manipulation. Sci. Adv. 5, eaax3084 (2019). 14. Li, J., Mayorga-Martinez, C. C., Ohl, C. D. & Pumera, M. Ultrasonically Propelled Micro- and Nanorobots. Adv. Funct. Mater. 32, 2102265 (2022). 15. Li, Y., Liu, X., Huang, Q., Ohta, A. T. & Arai, T. Bubbles in microfluidics: an all- purpose tool for micromanipulation. Lab Chip 21, 1016–1035 (2021). 16. Goyal, R., Athanassiadis, A. G., Ma, Z. & Fischer, P. Amplification of Acoustic Forces Using Microbubble Arrays Enables Manipulation of Centimeter-Scale Objects. Phys. Rev. Lett. 128, 254502 (2022). 17. Ghosh, S. et al. Directed Self-Assembly Driven Mesoscale Lithography Using Laser-Induced and Manipulated Microbubbles: Complex Architectures and Diverse Applications. Nano Lett. 21, 10–25 (2021). 18. Qiu, T. et al. Wireless Acoustic-Surface Actuators for Miniaturized Endoscopes. ACS Appl. Mater. Interfaces 9, 42536–42543 (2017). 19. Lin, L. et al. Bubble-Pen Lithography. Nano Lett. 16, 701–708 (2016). 20. Gargiulo, J., Cerrota, S., Cortés, E., Violi, I. L. & Stefani, F. D. Connecting Metallic Nanoparticles by Optical Printing. Nano Lett. 16, 1224–1229 (2016). 21. Armon, N. et al. Continuous Nanoparticle Assembly by a Modulated Photo-Induced Microbubble for Fabrication of Micrometric Conductive Patterns. ACS Appl. Mater. Interfaces 9, 44214–44221 (2017). 22. Chen, C., Karshalev, E., Guan, J. & Wang, J. Magnesium-Based Micromotors: Water-Powered Propulsion, Multifunctionality, and Biomedical and Environmental Applications. Small 14, 1704252 (2018). 23. Li, D., Liu, Y., Yang, Y. & Shen, Y. A fast and powerful swimming microrobot with a serrated tail enhanced propulsion interface. Nanoscale 10, 19673–19677 (2018). 24. Zhao, C. et al. Theory and experiment on particle trapping and manipulation via optothermally generated bubbles. Lab Chip 14, 384–391 (2014). 25. Choi, J. et al. Application of depletion attraction in mineral flotation: I. theory. Minerals 8, 1–15 (2018). Attorney Docket No.10046-557WO1 26. Flauraud, V. et al. Nanoscale topographical control of capillary assembly of nanoparticles. Nat. Nanotechnol. 12, 73–80 (2017). 27. Yeom, J. et al. Chiral templating of self-assembling nanostructures by circularly polarized light. Nat. Mater. 14, 66–72 (2015). 28. Venugopalan, P. L., Esteban-Fernández De Ávila, B., Pal, M., Ghosh, A. & Wang, J. Fantastic Voyage of Nanomotors into the Cell. ACS Nano 14, 9423–9439 (2020). 29. Fischer, P. & Ghosh, A. Magnetically actuated propulsion at low Reynolds numbers: Towards nanoscale control. Nanoscale 3, 557–563 (2011). 30. Law, J. et al. Micro / Nanorobotic Swarms: From Fundamentals to Functionalities. ACS Nano 17, 12971–12999 (2023). 31. Li, H. et al. Precise electrokinetic position and three-dimensional orientation control of a nanowire bioprobe in solution. Nat. Nanotechnol. 1–9 (2023). doi:10.1038 / s41565-023- 01439-7 32. Chen, P. et al. Multiplex serum cytokine immunoassay using nanoplasmonic biosensor microarrays. ACS Nano 9, 4173–4181 (2015). 33. Baysoy, A., Bai, Z., Satija, R. & Fan, R. The technological landscape and applications of single-cell multi-omics. Nat. Rev. Mol. Cell Biol. 1–19 (2023). doi:10.1038 / s41580-023-00615-w 34. Baffou, G., Polleux, J., Rigneault, H. & Monneret, S. Super-heating and micro- bubble generation around plasmonic nanoparticles under cw illumination. J. Phys. Chem. C 118, 4890–4898 (2014). 35. Loveland, R. J., Spear, W. E. & Al-Sharbaty, A. Photoconductivity and absorption in amorphous Si. J. Non. Cryst. Solids 13, 55–68 (1973). 36. Sakuma, G., Fukunaka, Y. & Matsushima, H. Nucleation and growth of electrolytic gas bubbles under microgravity. Int. J. Hydrogen Energy 39, 7638–7645 (2014). 37. Ma, Z., Joh, H., Fan, D. E. & Fischer, P. Dynamic Ultrasound Projector Controlled by Light. Adv. Sci. 9, 2104401 (2022). 38. Westerheide, D. E. & Westwater, J. W. Isothermal growth of hydrogen bubbles during electrolysis. AIChE J. 7, 357–362 (1961). 39. Higuera, F. J. A model of the growth of hydrogen bubbles in the electrolysis of water. J. Fluid Mech. 927, A33 (2021). 40. Huang, Y., Liang, Z., Alsoraya, M., Guo, J. & Fan, D. E. Light Gated Manipulation of Micro / Nanoparticles in Electric Fields. Adv. Intell. Syst. 2, 1900127 (2020). Attorney Docket No.10046-557WO1 41. Chiou, P. Y., Ohta, A. T. & Wu, M. C. Massively parallel manipulation of single cells and microparticles using optical images. Nature 436, 370–372 (2005). 42. Chao, Y. & Zhang, T. Optimization of fixation methods for observation of bacterial cell morphology and surface ultrastructures by atomic force microscopy. Appl. Microbiol. Biotechnol. 92, 381–392 (2011). Additional References 1. Bertin, N. et al. Bubble-based acoustic micropropulsors: active surfaces and mixers. Lab Chip 17, 1515–1528 (2017). 2. Conde, A. J., Keraite, I., Ongaro, A. E. & Kersaudy-Kerhoas, M. Versatile hybrid acoustic micromixer with demonstration of circulating cell-free DNA extraction from sub- ml plasma samples. Lab Chip 20, 741–748 (2020). 3. Ahmed, D. et al. Acoustofluidic chemical waveform generator and switch. Anal. Chem. 86, 11803–11810 (2014). 4. Jeong, J., Jang, D., Kim, D., Lee, D. & Chung, S. K. Acoustic bubble-based drug manipulation: Carrying, releasing and penetrating for targeted drug delivery using an electromagnetically actuated microrobot. Sensors Actuators, A Phys. 306, 111973 (2020). 5. Xie, Y. et al. Probing Cell Deformability via Acoustically Actuated Bubbles. Small 12, 902–910 (2016). 6. Hu, W., Fan, Q. & Ohta, A. T. An opto-thermocapillary cell micromanipulator. Lab Chip 13, 2285–2291 (2013). 7. Zhao, C. et al. Theory and experiment on particle trapping and manipulation via optothermally generated bubbles. Lab Chip 14, 384–391 (2014). 8. Lin, L. et al. Bubble-Pen Lithography. Nano Lett. 16, 701–708 (2016). 9. Meng, L. et al. Microbubble enhanced acoustic tweezers for size-independent cell sorting. Appl. Phys. Lett.116, (2020). 10. Ma, Z. et al. Spatial ultrasound modulation by digitally controlling microbubble arrays. Nat. Commun. 11, 4537 (2020). 11. Ma, Z., Joh, H., Fan, D. E. & Fischer, P. Dynamic Ultrasound Projector Controlled by Light. Adv. Sci. 9, 2104401 (2022). 12. Moo, J. G. S., Presolski, S. & Pumera, M. Photochromic Spatiotemporal Control of Bubble-Propelled Micromotors by a Spiropyran Molecular Switch. ACS Nano 10, 3543– 3552 (2016). Attorney Docket No.10046-557WO1 13. Li, Y. et al. Light-controlled bubble propulsion of amorphous TiO2 / Au Janus micromotors. RSC Adv. 6, 10697–10703 (2016). 14. Xie, C. et al. Identification of single bacterial cells in aqueous solution using confocal laser tweezers Raman spectroscopy. Anal. Chem. 77, 4390–4397 (2005). 15. Witkowska, E., Niciński, K., Korsak, D., Szymborski, T. & Kamińska, A. Sources of variability in SERS spectra of bacteria: comprehensive analysis of interactions between selected bacteria and plasmonic nanostructures. Anal. Bioanal. Chem. 411, 2001–2017 (2019). 16. Jarvis, R. M., Brooker, A. & Goodacre, R. Surface-enhanced Raman spectroscopy for bacterial discrimination utilizing a scanning electron microscope with a Raman spectroscopy interface. Anal. Chem. 76, 5198–5202 (2004). 17. Premasiri, W. R. et al. The biochemical origins of the surface-enhanced Raman spectra of bacteria: a metabolomics profiling by SERS. Anal. Bioanal. Chem. 408, 4631– 4647 (2016). 18. Efrima, S. & Zeiri, L. Understanding SERS of bacteria. J. Raman Spectrosc. 40, 277–288 (2009). 19. Gargiulo, J., Cerrota, S., Cortés, E., Violi, I. L. & Stefani, F. D. Connecting Metallic Nanoparticles by Optical Printing. Nano Lett. 16, 1224–1229 (2016). 20. Armon, N. et al. Continuous Nanoparticle Assembly by a Modulated Photo-Induced Microbubble for Fabrication of Micrometric Conductive Patterns. ACS Appl. Mater. Interfaces 9, 44214–44221 (2017). 21. Förster, J. & Vogt, H. Excimer Laser-Annealing of Amorphous Silicon Layers. 109, 47057 (2010). 22. Jellison, G. E. & Modine, F. A. Optical absorption of silicon between 1.6 and 4.7 eV at elevated temperatures. Appl. Phys. Lett. 41, 180–182 (1982). 23. Dotan, H., Sivula, K., Gr, M. & Warren, S. C. Probing the photoelectrochemical properties of hematite (α-Fe2O3) electrodes using hydrogen peroxide as a hole scavenger. Energy Environ. Sci. 4, 958–964 (2011). 24. Sreedhar, A. et al. Highly supportive hydrogen peroxide as a hole scavenger to improve the visible light water splitting activity of fl ake-like Co-doped ZnO thin fi lms. Sol. Energy 191, 151–160 (2019). 25. Chen, G. et al. Preparation and surface activity study of amino acid surfactants. Comptes rendus - Chim. 22, 277–282 (2019). Attorney Docket No.10046-557WO1 26. Chandran, P., Bakshi, S. & Chatterjee, D. Study on the characteristics of hydrogen bubble formation and its transport during electrolysis of water. Chem. Eng. Sci. 138, 99– 109 (2015). 27. Sakuma, G., Fukunaka, Y. & Matsushima, H. Nucleation and growth of electrolytic gas bubbles under microgravity. Int. J. Hydrogen Energy 39, 7638–7645 (2014). 28. Westerheide, D. E. & Westwater, J. W. Isothermal growth of hydrogen bubbles during electrolysis. AIChE J. 7, 357–362 (1961). 29. Higuera, F. J. A model of the growth of hydrogen bubbles in the electrolysis of water. J. Fluid Mech. 927, A33 (2021). 30. Aoki, K. J. Frequency-dependence of electric double layer capacitance without Faradaic reactions. JEAC 779, 117–125 (2016). 31. Choi, H., Li, C. & Peterson, G. P. Dynamic Processes of Nanobubbles: Growth, Collapse, and Coalescence. J. Heat Transfer 143, 1–23 (2021). Example 2. Light-Directed Large-Scale High-Precision Printing of Colloids, Compounds, and Molecules. In this Example, we describe a patterning technology that can deposit a charged agent (e.g., charged nanoparticles, ionic compounds, molecules, cells, etc.) with high precision and in a large scale. As a proof-of-principle, various sophisticated patterns of charged particles were made by assembling fluorescent nanocolloids to reproduce famous images, including the “Girl with a Pearl Earring” artwork and the picture of the first Astronaut on the Moon. The method is impressively effective, efficient, and facile. Patterning was performed using a substrate assembly (shown in Figure 19) that includes a semiconductor (a 200nm α-Si:H surface) on top of a fluoride-doped tin oxide (FTO) layer. A charge support layer (e.g., a layer of polystyrene sulfonate) was then deposited on the surface of the semiconductor. Deposition of the charge support layer was performed using the following four steps: 1. The semiconductor surface of the sample was cleaned gently with DI water / IPA solution, rubbed with a cotton swab, and dried with N gas. 2. The sample was then put into a UVO cleaner for 30m, removing any organic substances left on the surface and changing the surface with -OH. 3. A 4 wt% PDDA solution was applied on the semiconductor surface for 3m, washed with DI water, and dried with N gas. Attorney Docket No.10046-557WO1 4. A 4 wt% PSS solution was applied on the surface for 3m, washed with DI water, and dried with N gas. This method efficiently deposited a thin layer of polystyrene sulfonate on the semiconductor surface. Next, a 100um PDMS well was applied on top of the surface. The well was configured to contain a suspension or solution of a charged agent to be patterned in contact with the charge support layer. In this example, the PDMS well was filled with a 0.75 M solution of fluorescent charged 200 nm PS nanospheres. A piece of FTO glass, which acts as the counter electrode, was then used to cover the PDMS well. A pattern was projected to the surface of the sample from the back side using a 532 nm laser, which was controlled by a digital light projection (DLP) device. Then, the background light was turned off. An AC E-field (usually 1.5 kHz or 3 kHz, with 10 Vpp or 12 Vpp with controlled number of cycles or duration) was applied between the working electrode and the counter electrode. When the AC was off, the background light was then turned on again for observation. The counter FTO electrode was then removed, exposing the PDMS well and the solution inside. The charged solution inside the PDMS well was slowly exchanged to DI water, by applying and removing DI water on top of the PDMS well. After waiting for the DI water to evaporate, the surface of the charge support layer was observed under the microscope. We observed that the fluorescent charged PS nanospheres assembled on the charge support layer so as to duplicate the image projected onto the semiconductor. Figures 20A- 20B and Figures 21A-21B show examples of the assemblies formed when an image of “Girl with a Pearl Earring” and a photo of an astronaut on the moon were projected onto the semiconductor. Here, these complex photos were dissected into 6 sub-images that their overlapped image capture the fullness and shading effects, resulting in the accurate corresponding nanoparticle-made photos. We investigated the resolution obtainable using the patterning methods described herein. As shown in Figure 22, we readily achieved a resolution of less than 1 micron (approximately 200 nm in this example) when patterning lines on a surface. Further optimization of the charged agent, process parameters, and image projection can further refine resolution. Attorney Docket No.10046-557WO1 Studies also evaluated the deposition of charged agents over time. As shown in Figure 23, deposition of the charged agent (as measured by the density of particles present on the surface) occurred linearly over approximately thirty minutes before reaching a plateau. We also demonstrated the patterning of other types of charged agents (including gold nanoparticles of varying dimensions, Figures 24-25) as well as positively charged particles (Figure 26) which were patterned on positively charged PDDA polymer surfaces. As shown in Figures 27-28, the methods described herein can also be used to create negative images by etching nanoparticles randomly and uniformly distributed on a surface through a particle desorption strategy at optimized AC field conditions. Even more, the the deposition and etching strategies exemplified above can be readily combined and executed sequentially, resulting in the high-resolution patterning of ~200 nm-wide lines made of nanoparticles (Figure 22). We note that the solutions used in this example were ionic (aqueous Na2SO4solutions). This requires that charged agents possess sufficient surface charge to allow them to be suspended in this ionic solution. However, other charged species can be patterned by optimizing the pH value and ionic concentration of the solution, and / or functionalization of an agent to alter their charge. These methods were impressively effective, efficient, and facile. Patterning only utilized a projected light pattern and the application of a high-frequency AC field (< 10 kHz) using two electrodes (one of which is a working electrode connected to an amorphous Si surface. The same phenomena were also observed using ionic solutions, such as Na2SO4. In these cases, nanocolloids assemble according to the projected light pattern that defines the shape and local density of the colloidal printing, which forms during and after the turning off of electric voltage / light. The working mechanism is based on patterning of electric charges on the surface of amorphous Si that results in electrostatic attraction of nanocolloids and molecules; this working principle and the level of complexity of the obtained printing are distinct from those reported previously. For instance, the ligand-exchange-based patterning requires multi-step lithography and washing steps and only results in much simpler patterns compared to the reported technique. Furthermore, this technique permits stacked patterning for building up a printing with controlled local particle density. Thus, this patterning strategy can be used to print patterns including one or more charged agents in 2D or 3D. We Attorney Docket No.10046-557WO1 have also obtained arbitrary tunability in spatial density / fluorescent emission of nanoparticle printings via projecting a series of designed light patterns. Furthermore, we successfully demonstrated the direct deposition of ionic compounds from a solution, e.g., NaSO4, according to light patterns, onto the surface of Si, which has not been observed previously. This work proposes an original method to deposit various types of charged agents, including micro / nanoscale particles / colloids, charged molecules, and ionic compounds, onto a surface in a scalable designed pattern with high efficiency and precision. The deposition was achieved with a light intensity of 894.8 mW / cm2, which is expected to be even lower with further optimization. The technique can be easily scalable to large patterns with projected light. Furthermore, the technique permits multi-layer deposition of different patterns that stack vertically with the same setup, greatly increasing the versatility in creating hybrid patterned materials that can be made of particles / molecules of different types, shapes, and structures. The technique is efficient and low-cost. The methods described herein can also be performed sequentially on a surface to create multi-layer assemblies / patterns on a surface. In such embodiments, the same or different image can be projected from deposition step to deposition step. Likewise, the same or different charged agents can be used from deposition step to deposition step. Using such sequential, multi-layer assembly, complex architectures / multilayer assemblies / patterns can be generated, with particles of varying density and / or varying composition within different regions and / or layers of the multilayer assembly / pattern. Figure 29, Figures 30A- 30B, Figures 31A-31B, Figures 32A-32B, Figures 33A-33B, and Figures 34A-34D illustrate an example in which the sequential use of the methods described herein to perform multi-layer patterns on a surface. The systems and methods of the appended claims are not limited in scope by the specific systems and methods described herein, which are intended as illustrations of a few aspects of the claims. Any systems and methods that are functionally equivalent are intended to fall within the scope of the claims. Various modifications of the systems and methods in addition to those shown and described herein are intended to fall within the scope of the appended claims. Further, while only certain representative compounds, components, compositions, and method steps disclosed herein are specifically described, other combinations of the compounds, components, compositions, and method steps also Attorney Docket No.10046-557WO1 are intended to fall within the scope of the appended claims, even if not specifically recited. Thus, a combination of steps, elements, components, or constituents may be explicitly mentioned herein or less, however, other combinations of steps, elements, components, and constituents are included, even though not explicitly stated. The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of” and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments of the invention and are also disclosed. Other than where noted, all numbers expressing geometries, dimensions, and so forth used in the specification and claims are to be understood at the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, to be construed in light of the number of significant digits and ordinary rounding approaches. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed invention belongs. Publications cited herein and the materials for which they are cited are specifically incorporated by reference.

Claims

Attorney Docket No.10046-557WO1 WHAT IS CLAIMED IS:

1. A method of patterning a charged agent on a surface, the method comprising: providing a substrate assembly disposed on a working electrode, wherein the substrate assembly comprises a charge support layer disposed on a semiconductor; contacting the charge support layer of the substrate assembly with a suspension or solution comprising the charged agent dispersed or dissolved in a fluid carrier; positioning a counter electrode in electrical contact with the suspension or solution; applying an AC electric field within the substrate assembly using the working electrode and the counter electrode while projecting an image of a target pattern onto the semiconductor; ceasing application of the electric field; and allowing the charged agent to assemble on the charge support layer in the target pattern.

2. The method of claim 1, wherein the charge support layer and the charged agent exhibit the same surface charge.

3. The method of any of claims 1-2, wherein the charge support layer is negatively charged and / or exhibits a negative charge when in contact with the suspension or solution comprising the charged agent.

3. The method of any of claims 1-2, wherein the charge support layer is positively charged and / or exhibits a positive charge when in contact with the suspension or solution comprising the charged agent.

4. The method of any of claims 1-3, wherein the charge support layer has a thickness of less than 50 nm, such as a thickness of less than 25 nm, less than 20 nm, less than 15 nm, less than 10 nm, or less than 5 nm.

5. The method of any of claims 1-4, wherein the charge support layer has a thickness of from 1 nm to 50 nm, such as a thickness of from 1 nm to 25 nm, from 1 nm to 20 nm, from 1 nm to 15 nm, from 1 nm to 10 nm, or from 1 nm to 5 nm.Attorney Docket No.10046-557WO1 6. The method of any of claims 1-5, wherein the charge support layer comprises a charged polymer or a polymer that exhibits a charge when in contact with the suspension or solution comprising the charged agent.

7. The method of any of claims 1-6, wherein the charge support layer comprises polystyrene sulfonate, a copolymer thereof, or a blend thereof.

8. The method of any of claims 1-7, wherein the AC electric field has a frequency of less than 20kHz, such as a frequency of from 1kHz to 20kHz, a frequency of from 1kHz to 15kHz, a frequency of from 1kHz to 10kHz, a frequency of from 1kHz to 8kHz, a frequency of from 1kHz to 6kHz, a frequency of from 1.5kHz to 6kHz, a frequency of from 1.5kHz to 5kHz, or a frequency of from 1.5kHz to 4.5kHz.

9. The method of any of claims 1-8, wherein the image projected on the semiconductor comprises electromagnetic radiation having an intensity of less than 10,000 mW / cm2at λmax, such as an intensity of less than 5,000 mW / cm2at λmax, an intensity of less than 2,000 mW / cm2at λmax, an intensity of less than 1,000 mW / cm2at λmax, or an intensity of less than 250 mW / cm2at λmax.

10. The method of any of claims 1-9, wherein the image projected on the semiconductor comprises electromagnetic radiation having an intensity of from 50 mW / cm2to 10,000 mW / cm2at λmax, such as an intensity of from 50 mW / cm2to 5,000 mW / cm2at λmax, an intensity of from 50 mW / cm2to 2,000 mW / cm2at λmax, or an intensity of from 50 mW / cm2to 1,000 mW / cm2at λmax.

11. The method of any of claims 1-10, wherein the image projected on the semiconductor comprises electromagnetic radiation having a λmax in a region of the electromagnetic spectrum which induces a change in electrical conductivity in the semiconductor.Attorney Docket No.10046-557WO1 12. The method of any of claims 1-11, wherein the image projected on the semiconductor comprises electromagnetic radiation having a λmax in a visible region of the electromagnetic spectrum.

13. The method of any of claims 1-12, wherein the semiconductor comprises amorphous silicon.

14. The method of any of claims 1-11, wherein the image projected on the semiconductor comprises electromagnetic radiation having a λmax in an ultraviolet region of the electromagnetic spectrum.

15. The method of claim 14, wherein the semiconductor comprises titanium oxide.

16. The method of any of claims 1-15, wherein at least the working electrode is transparent to a wavelength of electromagnetic radiation used to project an image on the semiconductor.

17. The method of any of claims 1-16, wherein the working electrode and the counter electrode are transparent to a wavelength of electromagnetic radiation used to project an image on the semiconductor.

18. The method of any of claims 1-17, wherein the working electrode, the counter electrode, or a combination thereof comprise a metal mesh.

19. The method of any of claims 1-18, wherein the working electrode comprises a transparent conductive oxide (TCO) (e.g., fluoride-doped tin oxide (FTO), indium tin oxide (ITO), or aluminum-doped zinc oxide (AZO)), graphene, graphite, or thin metal film (e.g., a gold thin film).

20. The method of any of claims 1-19, wherein the counter electrode comprises a transparent conductive oxide (TCO) (e.g., fluoride-doped tin oxide (FTO), indium tin oxide (ITO), or aluminum-doped zinc oxide (AZO)), graphene, graphite, or thin metal film (e.g., a gold thin film).Attorney Docket No.10046-557WO1 21. The method of any of claims 1-20, wherein the semiconductor comprises amorphous silicon.

22. The method of any of claims 1-20, wherein the semiconductor comprises a group IV semiconductor, such as germanium or silicon germanium, or a group III-V semiconductor, such as GaAs or InGaAs.

23. The method of any of claims 1-20, wherein the semiconductor comprises a metal oxide semiconductor (e.g., titanium dioxide, zinc oxide) or a 2-dimensional semiconductor material (e.g., tungsten sulfide).

24. The method of any of claims 1-23, wherein the charged agent comprises a charged ion, a charged molecule, a charged polymer, a charged particle (flakes), a charged biomolecule (e.g., DNA, protein, etc.), a cell, a virus, a wire, a 3-dimensional material, or a combination thereof.

25. The method of any of claims 1-24, wherein the image of the target pattern is projected onto the semiconductor using a digital light projector.

26. The method of any of claims 1-24, wherein the image of the target pattern is projected onto the semiconductor by shining a light through a photomask.

27. The method of any of claims 1-26, wherein the charged agent is assemble on the charge support layer with a resolution of from 100 nm to 5 micron.

28. The method of any of claims 1-27, wherein the charged agent is assemble on the charge support layer with sub-micron resolution.

29. The method of any of claims 1-28, wherein the method further comprises contacting the charge support layer having the charged agent assembled on the charge support layer in the target pattern with a second suspension or solution comprising a second charged agent dispersed or dissolved in a fluid carrier;Attorney Docket No.10046-557WO1 applying an AC electric field within the semiconductor assembly using the working electrode and the counter electrode while projecting an image of a second target pattern onto the semiconductor; ceasing application of the electric field and image projection; and allowing the second charged agent to assemble on the charge support layer in the second target pattern, thereby generating a charge support layer having the first charged agent patterned in the first target pattern and the second target agent patterned in the second target pattern.

30. A system comprising: a substrate assembly disposed on a working electrode, wherein the substrate assembly comprises a charge support layer disposed on a semiconductor; a sample well configured to position a fluid sample in contact with the charge support layer; a counter electrode configured to be in electrical contact with a fluid sample present in the sample well; and an image projector, such as a digital light projector, configured to project an image of a target pattern onto the semiconductor. .

Images