Microfluidic Droplet Generator Research Articles

Fabrication of Crescent Shaped Microparticles for Particle Templated Droplet Formation.

Crescent-shaped hydrogel microparticles are shown to template uniform volume aqueous droplets upon simple mixing with aqueous and oil media for various bioassays. This emerging "lab on a particle" technique requires hydrogel particles with tunable material properties and dimensions. The crescent shape of the particles is attained by aqueous two-phase separation of polymers followed by photopolymerization of the curable precursor. In this work, the phase separation of poly(ethylene glycol) diacrylate (PEGDA, Mw 700) and dextran (Mw 40000) for tunable manufacturing of crescent-shaped particles is investigated. The particles' morphology is precisely tuned by following a phase diagram, varying the UV intensity, and adjusting the flow rates of various streams. The fabricated particles with variable dimensionsencapsulate uniform aqueous droplets upon mixing with an oil phase. The particles are fluorescently labeled with red and blue emitting dyes at variable concentrations to produce six color-coded particles. The blue fluorescent dye shows a moderate response to the pH change. The fluorescently labeled particles are able to tolerate an extremely acidic solution (pH 1) but disintegrate within an extremely basic solution (pH 14). The particle-templated droplets are able to effectively retain the disintegrating particle and the fluorescent signal at pH 14.

Abstract 533: Attenuating EZH2-mediated metastasis and tumor growth by pharmacological-based approaches using three-dimensional in vitro culture models

Abstract Triple Negative breast cancer (TNBC) accounts for 10-15% of all breast cancers where patients encounter poor clinical outcomes. Treatment modalities for TNBC are conventional cytotoxic chemotherapy and radiation leads to 35%-40% relapse within 5 years of diagnosis. There is a growing need for more effective targeted therapeutics to inhibit TNBC growth. The role of EZH2 in maintaining tumor growth and metastasis in TNBC is well documented. EZH2 promotes breast tumor-initiating cell expansion (BITC) and cancer progression by impairing the DNA damage repair process favoring activation of oncogenes. Despite the advancement in the discovery of inhibitors for EZH2 that attenuate its catalytic activity, resistance to these small molecules limits their use in solid tumors. The neurotransmitter dopamine via its D1 receptor activation in TNBC cell lines induces apoptosis and autophagy, as well as inhibits the invasion and regress in mammary tumors in vivo. Here we hypothesize that the presence of dopamine D1 receptor agonist (A77636) enhances the efficacy of EZH2 inhibitors (GSK126) to inhibit in vitro TNBC tumor growth and metastasis. To test the efficacy of the combination we employed a 3D culture system of MDA-MB-231 cells encapsulated in calcium-alginate microgels seeded from a microfluidic droplet generator. We also employed a 3D organ-on-chip-based microphysiological (MPS) platform (SynTumor) to study the effect of the drug combination on metastasis in vitro. The SynTumor MPS devices replicate the pathophysiological architecture of native vascularized breast tumors. The combination treatment enhanced a significant reduction of tumor spheroid diameter compared to the vehicle and indicated spheroid regression over treatment. Furthermore, the combination therapy induced necrosis and caused complete inhibition of EZH2 expression. The knockdown of EZH2 inhibited tumor spheroid formation and displayed cytoskeleton de-arrangement. In the microfluidic SynTumor model, circulating tumor cell numbers were reduced by half at 96 hrs after EZH2 combination treatment. Our data indicate that the combinatorial effect of DRD1 agonist and EZH2 inhibitor efficiently attenuates the EZH2-mediated in vitro tumor growth and metastasis (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). Citation Format: xilal Rima, Gautam Sarathy, Chunyu Hu, Divya S. Patel, Deborah Ramsey, Lara Rizotto, Dario Palmeri, Gwen Fewell, Bhuvaneswari Ramaswamy, Eduardo Reátegu, Eswar Shankar. Attenuating EZH2-mediated metastasis and tumor growth by pharmacological-based approaches using three-dimensional in vitro culture models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 533.

Modified capillary number to standardize droplet generation in suction-driven microfluidics

Modified capillary number to standardize droplet generation in suction-driven microfluidics

Dual-directional small-sampling deep-learning modelling on co-flowing microfluidic droplet generation

User-specified droplets generated by microfluidics are critical but requires intensive expertise and much time. Stimulated by data boosts from microfluidic experiments, deep learning has proven to be a powerful modeling approach in microfluidics with high accuracy. However, current deep learning approaches on microfluidics, emphasize droplet size prediction rather than actually obtaining droplets of the user-desired size. Such droplet size predictions invariably overlook the effect of the flow regime, a critical factor when determining the availability of the generated droplets because the flow regime influences the droplets’ dispersity. In addition, the big data prerequisite in deep learning models prohibits its extensive application in microfluidics. In order to solve these problems, this paper combines our current extensive experimental data (extracted by the Hough transformer) and the experimental data from previous literature as a co-flowing microfluidics-based droplet database. Based on this database, we established a dual-directional deep learning model where the droplet size variable resides not only in the output (for prediction), but also in the input (for acquisition) when considering the effect of flow regime. The new integrated active learning model fundamentally reduces the training dataset without sacrificing accuracy, thus pioneering small-sampling deep learning modelling in microfluidics. Compared to the single-directional modelling approach, the proposed dual-directional modelling demonstrates obvious improvements where the average relative error is only 9.90 % (obtained droplet size compared to the desired size), an increase of 45.6% compared with that of the single-directional model. The improved deep learning methodology here offers a universally accurate model for co-flowing microfluidic-based droplet acquisitions. It also has great prospects of becoming a prominent data processing structure that boosts, and possibly transforms existing microfluidic research and related fields.

Tailored Fluorosurfactants through Controlled/Living Radical Polymerization for Highly Stable Microfluidic Droplet Generation.

Droplet-based microfluidics represents a disruptive technology in the field of chemistry and biology through the generation and manipulation of sub-microlitre droplets. To avoid droplet coalescence, fluoropolymer-based surfactants are commonly used to reduce the interfacial tension between two immiscible phases to stabilize droplet interfaces. However, the conventional preparation of fluorosurfactants involves multiple steps of conjugation reactions between fluorinated and hydrophilic segments to form multiple-block copolymers. In addition, synthesis of customized surfactants with tailored properties is challenging due to the complex synthesis process. Here, we report a highly efficient synthetic method that utilizes living radical polymerization (LRP) to produce fluorosurfactants with tailored functionalities. Compared to the commercialized surfactant, our surfactants outperform in thermal cycling for polymerase chain reaction (PCR) testing, and exhibit exceptional biocompatibility for cell and yeast culturing in a double-emulsion system. This breakthrough synthetic approach has the potential to revolutionize the field of droplet-based microfluidics by enabling the development of novel designs that generate droplets with superior stability and functionality for a wide range of applications.

Tailored Fluorosurfactants through Controlled/Living Radical Polymerization for Highly Stable Microfluidic Droplet Generation

AbstractDroplet‐based microfluidics represents a disruptive technology in the field of chemistry and biology through the generation and manipulation of sub‐microlitre droplets. To avoid droplet coalescence, fluoropolymer‐based surfactants are commonly used to reduce the interfacial tension between two immiscible phases to stabilize droplet interfaces. However, the conventional preparation of fluorosurfactants involves multiple steps of conjugation reactions between fluorinated and hydrophilic segments to form multiple‐block copolymers. In addition, synthesis of customized surfactants with tailored properties is challenging due to the complex synthesis process. Here, we report a highly efficient synthetic method that utilizes living radical polymerization (LRP) to produce fluorosurfactants with tailored functionalities. Compared to the commercialized surfactant, our surfactants outperform in thermal cycling for polymerase chain reaction (PCR) testing, and exhibit exceptional biocompatibility for cell and yeast culturing in a double‐emulsion system. This breakthrough synthetic approach has the potential to revolutionize the field of droplet‐based microfluidics by enabling the development of novel designs that generate droplets with superior stability and functionality for a wide range of applications.

Emerging Advances in Microfluidic Hydrogel Droplets for Tissue Engineering and STEM Cell Mechanobiology.

Hydrogel droplets are biodegradable and biocompatible materials with promising applications in tissue engineering, cell encapsulation, and clinical treatments. They represent a well-controlled microstructure to bridge the spatial divide between two-dimensional cell cultures and three-dimensional tissues, toward the recreation of entire organs. The applications of hydrogel droplets in regenerative medicine require a thorough understanding of microfluidic techniques, the biocompatibility of hydrogel materials, and droplet production and manipulation mechanisms. Although hydrogel droplets were well studied, several emerging advances promise to extend current applications to tissue engineering and beyond. Hydrogel droplets can be designed with high surface-to-volume ratios and a variety of matrix microstructures. Microfluidics provides precise control of the flow patterns required for droplet generation, leading to tight distributions of particle size, shape, matrix, and mechanical properties in the resultant microparticles. This review focuses on recent advances in microfluidic hydrogel droplet generation. First, the theoretical principles of microfluidics, materials used in fabrication, and new 3D fabrication techniques were discussed. Then, the hydrogels used in droplet generation and their cell and tissue engineering applications were reviewed. Finally, droplet generation mechanisms were addressed, such as droplet production, droplet manipulation, and surfactants used to prevent coalescence. Lastly, we propose that microfluidic hydrogel droplets can enable novel shear-related tissue engineering and regeneration studies.

An approach for integrating droplet generation and detection in digital polymerase chain reaction applications based on a bifunctional microfluidic cross-structure

Digital polymerase chain reaction (dPCR) is an approach for absolute nucleic acid quantification with high sensitivity. Although several successful commercial dPCR devices have been developed to date, further miniaturizing device dimensions, decreasing cross-contamination, and improving automation level are still research highlights. In this study, we developed a fully contamination-free dPCR detection chip with fluorescence flow cytometry and micro droplet approach. A bifunctional cross-structure (BFCS) was designed to realize monodisperse sample droplet generation in forward flow and droplet detection in backward flow with simple pneumatic control and fixed chip position. In order to improve droplet detection efficiency and accuracy, droplets morphology and sequence pattern during microfluidic droplet generation and backward flow droplet detection at the same cross-structure were observed and analyzed under different pneumatic pressures. In addition, during backward flow droplet detection, an optimized declination angle of the chip was applied to increase droplet reflux rates. For the validation of PCR performance, temperature changing processes during PCR cycles were achieved by heating the monodispersed droplet array with a customized PCR amplification device. The fluorescence signal of each droplet right after passing the cross-structure was excitated and detected. The absolute quantification ability of our integrated dPCR microfluidic chip utilizing flow fluorescence cytometry was tested and verified with Influenza A virus gene (from 7.5 copies/μL to 30000 copies/μL). Thus, our platform provides a novel and integrated approach for ddPCR analysis.

Oil pollution remediation with mass-producible and recyclable amphiphilic magnetic Janus particles

The growing global energy demand and declining conventional oil supply have accelerated the use of unconventional heavy crude oils (UHCOs). However, high viscosities of UHCOs make conventional remediation methods ineffective for removing them from water. To address this challenge, we propose Pickering emulsification using amphiphilic magnetic Janus particles (AMJPs) as an efficient remediation method for UHCOs. AMJPs are produced using a multiplexed 3D-printed microfluidic droplet generator. The design of the generator is carefully optimized through a combination of experimental and numerical studies to achieve mass production of the monodisperse particles. These AMJPs have the remarkable ability to emulsify oil pollutants, with viscosities ranging from as low as 0.31 mPa∙s to as high as 97,100 mPa∙s. The Pickering emulsification using AMJPs results in rapid and highly efficient (>99.7%) recovery of UHCOs from water in a recyclable manner. We realize the first ever mass production of AMJPs via 3D-printed droplet generators. The use of these AMJPs in the Pickering emulsification approach has potential applications beyond UHCO remediation, including food, cosmetics, and pharmaceutical industries.

Numerical and artificial neural network analysis of an axisymmetric co-flow-focusing microfluidic droplet generator using active and passive control

Droplet generation in microscale has gained enormous attention in recent years especially in the pharmaceutical industry due to their application in targeted drug delivery into droplets. In most of these applications, monodispersity and uniformity of droplets are essential. Microfluidic devices can generate droplets at high throughput, enabling thousands of droplet compound encapsulation per second. The monodispersity of the droplets is ensured hydrodynamically through the dripping regime and their uniformity is controlled by active and passive microflow control methods. Here, we study numerically a microfluidic chip that uses a non-embedded co-flow-focusing geometry, so that the droplet generation throughput can take advantage of the flow-focusing devices while the non-embedded co-flow geometry forecloses the surfactant addition necessity. The continuous and dispersed phases were light mineral oil and water, respectively. We investigated the formation of droplets and studied how changing the external diameter of the chip affects the transition between the dripping regime (which corresponds to monodispersity) and the jetting regime. The number of parameters to be taken into account for the optimization of the device is enormous; therefore, in order to account for the effect of many geometrical and hydrodynamical parameters, we trained an artificial neural network based on our simulation data. Using this neural network, we evaluated droplet formation in 3240 different cases. This approach resulted in a remarkable reduction of computation time, from months to seconds. Examining numerous cases in such a short period lets us choose the optimum geometry and flow rate based on the application. The optimization was able to find the best geometry to extend the region of dripping regime in the flow rate map. Finally, to harness the droplet generation frequency, we also simulated a periodically switched laser and we were able to predict the generation of droplets with the same frequency as the switching frequency. Therefore, altering and controlling the frequency and dimensions of the droplets for a given flow rate ratio could be achieved with this technique, even without satellite droplets.

A universal model for continuous “one-to-two” high-efficient droplet generation in digital microfluidics

High-efficient droplet generation is crucial in microfluidics platforms in bio-applications. Among the microfluidic techniques, digital microfluidics, although with the capability of programmatically manipulating the droplets, is less effective in high-throughput droplets generation. In this Letter, a continuous “one-to-two” droplet generation strategy on digital microfluidics platform is achieved with an exponential generation efficiency, while overcoming the restriction on the droplet geometries (aspect ratio) in a wide range. An energy-based sub-droplet generation model is also proposed, as an expansion to the previous Laplace pressure-based geometrical model. The electrical parameters are taken into computing the sub-droplet generation on digital microfluidics from the aspect of energy. The proposed continuous “one-to-two” droplet generation strategy can generate sub-droplets with a minimized electrodes' number while with a higher energy input in each operation. The exponential efficiency will open the era for high-throughput droplets' application developments in digital microfluidics.

Abstract P2-16-05: Targeting EZH2 to overcome chemoresistance in Triple Negative Breast Cancers Employing Combinatorial Approach

Abstract Objective: Triple Negative Breast Cancers (TNBC) are highly invasive and 46% of patients develop distant metastases, leading to higher mortality. Chemotherapy remains the most efficacious option, however, there is still a largely unmet need to identify novel therapeutic targets in TNBC to increase treatment options and improve patient outcomes, survival. Enhancer of zeste homologue 2 (EZH2), a member of the catalytic subunit of the polycomb repressive complex 2, is a histone methyltransferase and its canonical function is to methylate lysine 27 of histone 3. EZH2 is a potential driver of TNBC metastasis, and its high expression strongly associates with the TNBC phenotype as compared with other molecular subtypes of breast cancer. Despite the advancement in the discovery of inhibitors for EZH2 that attenuate its catalytic activity. Currently, several EZH2 inhibitors are under development and undergoing clinical trials and these compounds have been proved to be effective in the treatment of hematological malignancies, sarcomas and malignant rhabdoid tumors. However these inhibitors do not affect the intrinsic protein stability of EZH2, but typically competes with the cofactor S-adenosylmethionine (SAM) and binds to the SET domain of EZH2. Hence, the EZH2 inhibitors are only effective for some malignant blood tumors, and have poor efficacy for solid tumors, such as TNBC. Several studies have shown the involvement of neurotransmitter dopamine in proliferation, apoptosis, tumor angiogenesis, and drug resistance among different cancers, including the breast1. Dopamine D1 receptor activation in TNBC cell line induces apoptosis, autophagy and phosphorylation of eukaryotic translation initiation factor 2-alpha (eIF2a)2. Also, D1R agonists inhibits the invasion of breast cancer cell lines MDA-MB-231 and BT-20 and regress mammary tumors3. The oncogenic nature of EZH2 in driving aggressiveness in TNBC cells led us to simultaneously target dopamine D1 receptor and EZH2 to completely ablate tumor growth and metastasis. Methods: Schrodinger protein modeling software was employed for docking studies. TNBC cells MDA-MB-231 cells were treated with the EZH2 inhibitor GSK126 and/or D1R agonists A77636 and SKF38393 for assessing cell viability, migration. Immuno precipitation to investigate if the combination could disrupt the PRC2 complex in TNBC cells. To model tumor burden and the effects of combination therapy on tumor growth in vitro, we tested the combinatory effect of GSK126 and D1R agonists in a 3D culture system of MDA-MB-231 cells encapsulated in calcium-alginate microgels seeded from a microfluidic droplet generator. Cell death was determined by Yo-pro-Propidium Iodide staining. Result: In silico analysis confirmed that D1R agonists exhibited strong stabilization of protein structures upon binding to the EZH2 catalytic active site, albeit with slightly weaker affinity than GSK126. Combination treatment with GSK126 and dopamine agonists A77636 or SKF 38393 led to significant and synergistic inhibition of cell viability (p< 0.01), migration, invasion, and EZH2 activity (p< 0.01) in TNBC cells. In addition, MDA-MB-231 cells formed tumor spheroids that were subjected to the EZH2 inhibitor alongside dopamine agonists, indicating tumor reduction relative to single agent treatment in 3D culture (p < 0.0001). The combination also led to increased necrotic cell death (p < 0.05). Immunoprecipitation of EZH2 in the presence of GSK126 and SKF38393 and A77636 dissociated the physical interaction between EZH2, EED, and SUZ12 in MDA-MB-231 cells. Conclusion: Our data suggest that the combination of GSK126 and Dopamine D1 agonists synergistically inhibits TNBC proliferation by disrupting EZH2 functions leading to necrotic cell death. (This work is supported by DOD: W81XWH2010065, for Eswar Shankar). 1) PMID: 21531818 2) PMID: 29773888 3) PMID: 26477316 Citation Format: Eswar Shankar, Sridhar SNC, Xilal Y. Rima, Prem Kushwaha, Shiv Verma, Gautam K. Sarathy, Anagh kulkarni, Dharmaraja Allimuthu, Eduardo Reátegui, Sanjay Gupta, Bhuvaneswari Ramaswamy. Targeting EZH2 to overcome chemoresistance in Triple Negative Breast Cancers Employing Combinatorial Approach. [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-16-05.

Computational simulation of the effects of interfacial tension in microfluidic flow focusing droplet generators

We present simulations of a square flow focusing droplet generator device exploring its performance characteristics over a range of interfacial surface tension values and varying neck width. Droplet generators have a wide range of applications from drug delivery to X-ray diffraction experiments. Matching the droplet frequency and volume to the experimental parameters is critical for maximising the data quality and minimising sample waste. Whilst varying the interfacial surface tension we observed that the lowest frequency of droplets is generated for surface tensions matching those typically reported for water-oil mixtures (around 40 mN/M). Decreasing or increasing the interfacial surface tension, for example by adding surfactant, results in an increase in droplet frequency. We also find that under the conditions simulated here, droplets are generated with much lower capillary numbers and higher Weber numbers than have typically been reported in the literature. The high ratio of flowrate-to-cross-section used here resulted in a velocity which was larger than has previously been reported for flow focusing devices and consequently we observe particularly large associated Reynolds numbers. However, in general, the simulated flow behaviour characteristics most closely match those typically observed for the jetting and tip-streaming regimes. The highest frequency of droplets achieved in our simulated devices was 36 kHz and 56 kHz corresponding to square neck channel widths of 12.5 and 25 µm respectively, an interfacial surface tension of 118.75 mN/m. We also examined the effect of varying neck width geometry for a fixed interfacial surface tension of 52 mN/m. We observed that the highest frequency droplet generation, 61 kHz, corresponded to a neck width of 37.5 µm with a corresponding droplet diameter of 22 µm. The high frequency, high monodispersity, and small droplet size predicted to occur through modification of the interfacial surface tension will have implications for the future design and optimisation of droplet-on-demand microfluidic devices.

Microfluidic Study of Oil Droplet Stability in Produced Water with Combinations of Production Chemicals

Discharged produced water contains traces of hydrocarbons after separation that might affect the eco-toxicity. These hydrocarbons can be oil droplets that are stabilized by added production chemicals and their combinations. The chemicals highly affect the treatment of the produced water before either reinjection or discharge to the sea. We apply microfluidic droplet generation for rapid evaluation of the synergistic effects of the combinations of production chemicals on the oil droplet stability. The coalescence frequency of dispersed droplets is calculated on the basis of high-speed camera video acquisition. The dispersed phase is either a model oil or a bottomhole crude oil sample, both in an aqueous medium. The individual and combined effects of a commercial demulsifier and a corrosion inhibitor are evaluated on the basis of the hydrophilic–lipophilic deviation theory. The presence of as low as 4 ppm of the water-in-oil demulsifier is demonstrated to stabilize oil-in-water droplets, while the corrosion inhibitor creates droplets in oil–water slug flow fashion. The presence of the corrosion inhibitor in conjunction with the demulsifier results in the droplet adherence to the channel surface. The importance of the right dosage and the right combination of production chemicals is demonstrated. The microfluidic droplet generation technique is a valuable tool for assessing the effects of production chemicals on oil droplet stability.

Scalable Production of Size-Controlled Cholangiocyte and Cholangiocarcinoma Organoids within Liver Extracellular Matrix-Containing Microcapsules.

Advances in biomaterials, particularly in combination with encapsulation strategies, have provided excellent opportunities to increase reproducibility and standardization for cell culture applications. Herein, hybrid microcapsules are produced in a flow-focusing microfluidic droplet generator combined with enzymatic outside-in crosslinking of dextran-tyramine, enriched with human liver extracellular matrix (ECM). The microcapsules provide a physiologically relevant microenvironment for the culture of intrahepatic cholangiocyte organoids (ICO) and patient-derived cholangiocarcinoma organoids (CCAO). Micro-encapsulation allowed for the scalable and size-standardized production of organoids with sustained proliferation for at least 21 days in vitro. Healthy ICO (n = 5) expressed cholangiocyte markers, including KRT7 and KRT19, similar to standard basement membrane extract cultures. The CCAO microcapsules (n = 3) showed retention of stem cell phenotype and expressed LGR5 and PROM1. Furthermore, ITGB1 was upregulated, indicative of increased cell adhesion to ECM in microcapsules. Encapsulated CCAO were amendable to drug screening assays, showing a dose-response response to the clinically relevant anti-cancer drugs gemcitabine and cisplatin. High-throughput drug testing identified both pan-effective drugs as well as patient-specific resistance patterns. The results described herein show the feasibility of this one-step encapsulation approach to create size-standardized organoids for scalable production. The liver extracellular matrix-containing microcapsules can provide a powerful platform to build mini healthy and tumor tissues for potential future transplantation or personalized medicine applications.

Phase-Optimized Peristaltic Pumping by Integrated Microfluidic Logic.

Microfluidic droplet generation typically entails an initial stabilization period on the order of minutes, exhibiting higher variation in droplet volume until the system reaches monodisperse production. The material lost during this period can be problematic when preparing droplets from limited samples such as patient biopsies. Active droplet generation strategies such as antiphase peristaltic pumping effectively reduce stabilization time but have required off-chip control hardware that reduces system accessibility. We present a fully integrated device that employs on-chip pneumatic logic to control phase-optimized peristaltic pumping. Droplet generation stabilizes in about a second, with only one or two non-uniform droplets produced initially.

Cell-like microparticles with tunable acoustic properties for calibrating devices

Mechanophenotype of biological cells has demonstrated correlation with biomolecular states and cell function. Hence, new methods to measure mechanophenotype at high throughput are of growing interest. Acoustophoretic microdevices can characterize cell mechanical features; however, calibration particles with physiologically relevant properties are needed to quantify and optimize device performance. Currently, conventional polymer microspheres are rigid and do not replicate cell deformation and compressibility. To address this, we developed monodisperse, tunable, cell-like microparticles (MPs) from polyacrylamide hydrogel, fabricated with a microfluidic droplet generator. Size and compressibility are adjusted by fabrication parameters, and density is adjusted by incorporation of nanoparticles (NPs). Here, we present for the first time microparticles of reduced density and acoustic contrast (lower than unloaded MPs) achieved by loading MPs with nanoparticles of low molecular weight alkanes. We produced the NPs by sonication and photopolymerization before addition to the MP precursor. NP-loaded MPs were less dense than unloaded MPs at 1005.9 and 1013.6kg/m3, respectively, and they exhibited negative acoustic contrast by acoustophoresis in aqueous medium while that of unloaded MPs was positive. These new particles extend the tunable range of acoustic contrast, mimicking and exceeding that of most biological cells and could also aid cell separation when conjugated to cells.

Direct measurement of vorticity using tracer particles with internal markers

Current experiment techniques for vorticity measurement suffer from limited spatial and temporal resolution to resolve the small-scale eddy dynamics in turbulence. In this study, we develop a new method for direct vorticity measurement in fluid flows based on digital inline holography (DIH). The DIH system utilizes a collimated laser beam to illuminate the tracers with internal markers and a digital sensor to record the generated holograms. The tracers made of the polydimethylsiloxane prepolymer mixed with internal markers are fabricated using a standard microfluidic droplet generator. A rotation measurement algorithm is developed based on the 3D location reconstruction and tracking of the internal markers and is assessed through synthetic holograms to identify the optimal parameter settings and measurement range (e.g., rotation rate from 0.3 to 0.7 rad/frame under numerical aperture of imaging of 0.25). Our proposed method based on DIH is evaluated by a calibration experiment of single tracer rotation, which yields the same optimal measurement range. Using von Kármán swirling flow setup, we further demonstrate the capability of the approach to simultaneously measure the Lagrangian rotation and translation of multiple tracers. Our method can measure vorticity in a small region on the order of 100 µm or less and can be potentially used to quantify the Kolmogorov-scale vorticity field in turbulent flows.

Low Cost, Ease-of-Access Fabrication of Microfluidic Devices Using Wet Paper Molds.

Rapid prototyping methods enable the widespread adoption of microfluidic technologies by empowering end-users from non-engineering disciplines to make devices using processes that are rapid, simple and inexpensive. In this work, we developed a liquid molding technique to create silicone/PDMS microfluidic devices by replica molding. To construct a liquid mold, we use inexpensive adhesive-backed paper, an acetate backing sheet, and an off-the-shelf digital cutter to create paper molds, which we then wet with predetermined amounts of water. Due to the immiscibility of water and PDMS, mold patterns can be effectively transferred onto PDMS similarly to solid molds. We demonstrate the feasibility of these wet paper molds for the fabrication of PDMS microfluidic devices and assess the influence of various process parameters on device yield and quality. This method possesses some distinct benefits compared to conventional techniques such as photolithography and 3D printing. First, we demonstrate that the shape of a channel’s cross-section may be altered from rectangular to semicircular by merely modifying the wetting parameters. Second, we illustrate how electrical impedance can be utilized as a marker for inspecting mold quality and identifying defects in a non-invasive manner without using visual tools such as microscopes or cameras. As a proof-of-concept device, we created a microfluidic T-junction droplet generator to produce water droplets in mineral oil ranging in size from 1.2 µL to 75 µL. We feel that this technology is an excellent addition to the microfluidic rapid prototyping toolbox and will find several applications in biological research.

Gigavalent Display of Proteins on Monodisperse Polyacrylamide Hydrogels as a Versatile Modular Platform for Functional Assays and Protein Engineering.

The assembly of robust, modular biological components into complex functional systems is central to synthetic biology. Here, we apply modular “plug and play” design principles to a solid-phase protein display system that facilitates protein purification and functional assays. Specifically, we capture proteins on polyacrylamide hydrogel display beads (PHD beads) made in microfluidic droplet generators. These monodisperse PHD beads are decorated with predefined amounts of anchors, methacrylate-PEG-benzylguanine (BG) and methacrylate-PEG-chloroalkane (CA), that react covalently with SNAP-/Halo-tag fusion proteins, respectively, in a specific, orthogonal, and stable fashion. Anchors, and thus proteins, are distributed throughout the entire bead volume, allowing attachment of ∼109 protein molecules per bead (⌀ 20 μm) —a higher density than achievable with commercial surface-modified beads. We showcase a diverse array of protein modules that enable the secondary capture of proteins, either noncovalently (IgG and SUMO-tag) or covalently (SpyCatcher, SpyTag, SnpCatcher, and SnpTag), in mono- and multivalent display formats. Solid-phase protein binding and enzymatic assays are carried out, and incorporating the photocleavable protein PhoCl enables the controlled release of modules via visible-light irradiation for functional assays in solution. We utilize photocleavage for valency engineering of an anti-TRAIL-R1 scFv, enhancing its apoptosis-inducing potency ∼50-fold through pentamerization.