Solvent Dependence of Ionic Liquid-Based Pt Nanoparticle Synthesis: Machine Learning-Aided In-Line Monitoring in a Flow Reactor.

Colloidal platinum nanoparticles (Pt NPs) possess a myriad of technologically relevant applications. A potentially sustainable route to synthesize Pt NPs is via polyol reduction in ionic liquid (IL) solvents; however, the development of this synthetic method is limited by the fact that reaction kinetics have not been investigated. In-line analysis in a flow reactor is an appealing approach to obtain such kinetic data; unfortunately, the optical featurelessness of Pt NPs in the visible spectrum complicates the direct analysis of flow chemistry products via ultraviolet-visible (UV-vis) spectrophotometry. Here, we report a machine learning (ML)-based approach to analyze in-line UV-vis spectrophotometric data to determine Pt NP product concentrations. Using a benchtop flow reactor with ML-interpreted in-line analysis, we were able to investigate NP yield as a function of residence time for two IL solvents: 1-butyl-1-methylpyrrolidinium triflate (BMPYRR-OTf) and 1-butyl-2-methylpyridinium triflate (BMPY-OTf). While these solvents are structurally similar, the polyol reduction shows radically different yields of Pt NPs depending on which solvent is used. The approach presented here will help develop an understanding of how the subtle differences in the molecular structures of these solvents lead to distinct reaction behavior. The accuracy of the ML prediction was validated by particle size analysis and the error was found to be as low as 4%. This approach is generalizable and has the potential to provide information on various reaction outcomes stemming from solvent effects, for example, differential yields, orders of reaction, rate coefficients, NP sizes, etc.

Multivariate Bayesian Optimization of CoO Nanoparticles for CO2 Hydrogenation Catalysis.

The hydrogenation of CO2 holds promise for transforming the production of renewable fuels and chemicals. However, the challenge lies in developing robust and selective catalysts for this process. Transition metal oxide catalysts, particularly cobalt oxide, have shown potential for CO2 hydrogenation, with performance heavily reliant on crystal phase and morphology. Achieving precise control over these catalyst attributes through colloidal nanoparticle synthesis could pave the way for catalyst and process advancement. Yet, navigating the complexities of colloidal nanoparticle syntheses, governed by numerous input variables, poses a significant challenge in systematically controlling resultant catalyst features. We present a multivariate Bayesian optimization, coupled with a data-driven classifier, to map the synthetic design space for colloidal CoO nanoparticles and simultaneously optimize them for multiple catalytically relevant features within a target crystalline phase. The optimized experimental conditions yielded small, phase-pure rock salt CoO nanoparticles of uniform size and shape. These optimized nanoparticles were then supported on SiO2 and assessed for thermocatalytic CO2 hydrogenation against larger, polydisperse CoO nanoparticles on SiO2 and a conventionally prepared catalyst. The optimized CoO/SiO2 catalyst consistently exhibited higher activity and CH4 selectivity (ca. 98%) across various pretreatment reduction temperatures as compared to the other catalysts. This remarkable performance was attributed to particle stability and consistent H* surface coverage, even after undergoing the highest temperature reduction, achieving a more stable catalytic species that resists sintering and carbon occlusion.

A Multistep, Multicomponent Extraction and Separation Microfluidic Route to Recycle Water-Miscible Ionic Liquid Solvents.

Recycling ionic liquid (IL) solvents can reduce the lifecycle cost of these expensive solvents. Liquid-liquid extraction is the most straightforward approach to purify IL solvents and is typically performed with an immiscible washing agent (e.g., water). Herein, we describe a recycling route for water-miscible ILs in which direct recycling is usually challenging. We use hydrophobic ILs as accommodating agents to draw the water-miscible IL from the aqueous washing stream. A biphasic slug flow of the mixed ILs and water is then separated by using a membrane. The water-miscible IL can then be drawn out from the mixed IL phase with acidified water and dried under vacuum. Both the water-miscible IL and the accommodating agent are then recycled. Here, we demonstrated a proof-of-concept of this process by recycling 1-butyl-3-methylimidazolium trifluoromethanesulfonate (BMIM-OTf) in the presence of the accommodating agent 1-butyl-3-methylimidazolium bis(trifluoromethylsulfonyl)imide (BMIM-NTf2) and acidified water. We then demonstrated the capacity to recycle 1-butyl-1-methylpyrrolidinium triflate (BMPYRR-OTf) from a realistic synthetic application: Pt nanoparticle synthesis in the water-miscible IL.

3D-printed microfluidic device for high-throughput production of lipid nanoparticles incorporating SARS-CoV-2 spike protein mRNA.

Lipid nanoparticles (LNPs) are drug carriers for protecting nucleic acids for cellular delivery. The first mRNA vaccines authorized by the United States Food and Drug Administration are the mRNA-1273 (Moderna) and BNT162b (BioNTech/Pfizer) vaccines against coronavirus disease 2019 (COVID-19). We designed a 3D printed Omnidirectional Sheath-flow Enabled Microfluidics (OSEM) device for producing mRNA-loaded LNPs that closely resemble the Moderna vaccine: we used the same lipid formulations to encapsulate mRNA encoding SARS-CoV-2 spike protein. The OSEM device is made of durable methacrylate-based materials that can support flow rates in the mL min-1 range and was fabricated by stereolithography (SLA), incorporating readily adaptable interfaces using commercial fluidic connectors. Two key features of the OSEM device are: 1) a 4-way hydrodynamic flow focusing region and 2) a staggered herringbone mixer (SHM). Superior to conventional planar fluid junctions, the 4-way sheath flow channel generates an evenly focused, circular center flow that facilitates the formation of LNPs with low polydispersity. Downstream, fluid mixing in the SHM is intensified by incorporating a zig-zag fluidic pathway to deliver high mRNA encapsulation efficiency. We characterized the mRNA-loaded LNPs produced in the OSEM device and showed that the enhanced 3D microfluidic structures enable a 5-fold higher throughput production rate (60 mL min-1) of LNPs compared to commercial multi-thousand-dollar micromixers. The device produced LNPs of diameter less than 90 nm, with low polydispersity (2-8%) and high mRNA encapsulation efficiency (>90%). The 3D-printed device provides a cost-effective and easily prepared solution for high-throughput LNP production.

Elucidating the Molecular Interactions between Lipids and Lysozyme: Evaporation Resistance and Bacterial Barriers for Dry Eye Disease.

Dry eye disease (DED) is a chronic condition characterized by ocular dryness and inflammation. The tear film lipid layer (TFLL) is the outermost layer composed of lipids and proteins that protect the ocular surface. However, environmental contaminants can disrupt its structure, potentially leading to DED. Although the importance of tear proteins in the TFLL functionality has been clinically recognized, the molecular mechanisms underlying TFLL-protein interactions remain unclear. In this study, we investigated tear protein-lipid interactions and analyzed their role in the TFLL functionality. The results show that lysozyme (LYZ) increases the stability of the TFLL by reducing its surface tension and increasing its surface pressure, resulting in increased TFLL evaporation and bacterial invasion resistance, with improved wettability and lubrication performance. These findings highlight the critical role of LYZ in maintaining ocular health and provide potential avenues for investigating novel approaches to DED treatment and patient well-being.

Curvature preference of cubic CsPbBr3 quantum dots embedded onto phospholipid bilayer membranes.

Curvature-mediated lipid-protein interactions are important determinants of numerous vital cellular reactions and mechanisms. Biomimetic lipid bilayer membranes, such as giant unilamellar vesicles (GUVs), coupled with quantum dot (QD) fluorescent probes, provide an avenue to elucidate the mechanisms and geometry of induced protein aggregation. However, essentially all QDs used in QD-lipid membrane studies encountered in the literature are of the cadmium selenide (CdSe) or CdSe core/ZnS shell type, which are quasispherically shaped. We report here the membrane curvature partitioning of cube-shaped CsPbBr3 QDs embedded within deformed GUV lipid bilayers versus that of a conventional small fluorophore (ATTO-488) and quasispherical CdSe core/ZnS shell QDs. In alignment with basic packing theory regarding cubes packed in curved confined spaces, the local relative concentration of CsPbBr3 is highest in areas of lowest relative curvature in the plane of observation; this partitioning behavior is significantly different from that of ATTO-488 (p = 0.0051) and CdSe (p = 1.10 × 10-11). In addition, when presented with only one principal radius of curvature in the observation plane, no significant difference (p = 0.172) was observed in the bilayer distribution of CsPbBr3versus that of ATTO-488, suggesting that both QD and lipid membrane geometry greatly impact the curvature preferences of the QDs. These results highlight a fully-synthetic analog to curvature-induced protein aggregation, and lay a framework for the structural and biophysical analysis of complexes between lipid membranes and the shape of intercalating particles.

Oxidation of Membrane Lipids Alters the Activity of the Human Serotonin 1A Receptor.

Lipid oxidation has significant effects on lipid bilayer properties; these effects can be expected to extend to interactions between the lipid bilayer and integral membrane proteins. Given that G protein-coupled receptor (GPCR) activity is known to depend on the properties of the surrounding lipid bilayer, these proteins represent an intriguing class of molecules in which the impact of lipid oxidation on protein behavior is studied. Here, we study the effects of lipid oxidation on the human serotonin 1A receptor (5-HT1AR). Giant unilamellar vesicles (GUVs) containing integral 5-HT1AR were fabricated by the hydrogel swelling method; these GUVs contained polyunsaturated 1-palmitoyl-2-linoleoyl-sn-glycero-3-phosphocholine (PLinPC) and its oxidation product 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) at various ratios. 5-HT1AR-integrated GUVs were also fabricated from lipid mixtures that had been oxidized by extended exposure to the atmosphere. Both types of vesicles were used to evaluate 5-HT1AR activity using an assay to quantify GDP-GTP exchange by the coupled G protein α subunit. Results indicated that 5-HT1AR activity increases significantly in bilayers containing oxidized lipids. This work is an important step in understanding how hyperbaric oxidation can change plasma membrane properties and lead to physiological dysfunction.

Construction of Model Lipid Membranes Incorporating G-protein Coupled Receptors (GPCRs).

Robust in vitro investigations of the structure and function of integral membrane proteins has been a challenge due to the complexities of the plasma membrane and the numerous factors that influence protein behavior in live cells. Giant unilamellar vesicles (GUVs) are a biomimetic and highly tunable in vitro model system for investigating protein-membrane interactions and probing protein behavior in a precise, stimulus-dependent manner. In this protocol, we present an inexpensive and effective method for fabricating GUVs with the human serotonin 1A receptor (5-HT1AR) stably integrated in the membrane. We fabricate GUVs using a modified hydrogel swelling method; by depositing a lipid film on top of a mixture of agarose and 5-HT1AR and then hydrating the entire system, vesicles can be formed with properly oriented and functional 5-HT1AR incorporated into the membrane. These GUVs can then be used to examine protein-membrane interactions and localization behavior via microscopy. Ultimately, this protocol can advance our understanding of the functionality of integral membrane proteins, providing profound physiological insight.

Compatibility of Popular Three-Dimensional Printed Microfluidics Materials with In Vitro Enzymatic Reactions.

3D printed microfluidics offer several advantages over conventional planar microfabrication techniques including fabrication of 3D microstructures, rapid prototyping, and inertness. While 3D printed materials have been studied for their biocompatibility in cell and tissue culture applications, their compatibility for in vitro biochemistry and molecular biology has not been systematically investigated. Here, we evaluate the compatibility of several common enzymatic reactions in the context of 3D-printed microfluidics: (1) polymerase chain reaction (PCR), (2) T7 in vitro transcription, (3) mammalian in vitro translation, and (4) reverse transcription. Surprisingly, all the materials tested significantly inhibit one or more of these in vitro enzymatic reactions. Inclusion of BSA mitigates only some of these inhibitory effects. Overall, inhibition appears to be due to a combination of the surface properties of the resins as well as soluble components (leachate) originating in the matrix.

In-situ transfer vat photopolymerization for transparent microfluidic device fabrication.

While vat photopolymerization has many advantages over soft lithography in fabricating microfluidic devices, including efficiency and shape complexity, it has difficulty achieving well-controlled micrometer-sized (smaller than 100 µm) channels in the layer building direction. The considerable light penetration depth of transparent resin leads to over-curing that inevitably cures the residual resin inside flow channels, causing clogs. In this paper, a 3D printing process - in-situ transfer vat photopolymerization is reported to solve this critical over-curing issue in fabricating microfluidic devices. We demonstrate microchannels with high Z-resolution (within 10 µm level) and high accuracy (within 2 µm level) using a general method with no requirements on liquid resins such as reduced transparency nor leads to a reduced fabrication speed. Compared with all other vat photopolymerization-based techniques specialized for microfluidic channel fabrication, our universal approach is compatible with commonly used 405 nm light sources and commercial photocurable resins. The process has been verified by multifunctional devices, including 3D serpentine microfluidic channels, microfluidic valves, and particle sorting devices. This work solves a critical barrier in 3D printing microfluidic channels using the high-speed vat photopolymerization process and broadens the material options. It also significantly advances vat photopolymerization's use in applications requiring small gaps with high accuracy in the Z-direction.

Light-Triggered Unique Shape Transformation of Giant Polymersomes with Tubular Protrusions.

Light-triggered unique shape transformation of calcein-loaded giant polymersomes with tubular protrusions, which serve as a reservoir membrane area during the shape transformation, is reported here. Under irradiation at the excitation wavelength of calcein, the tubular protrusions form strings of budded vesicles and then reintegrate into the mother vesicle. The initial giant polymersomes transform to two connected spherical vesicles via two pathways to alleviate the osmotic pressure imbalance across the vesicle membrane. The two connected spherical vesicles further transform to a mother vesicle with an inner daughter vesicle after switching off the light to relieve the bending energy. The finding provides a promising platform to mimic cell morphology changes.

An integrated microfluidic platform to fabricate single-micrometer asymmetric giant unilamellar vesicles (GUVs) using dielectrophoretic separation of microemulsions.

We present a microfluidic technique that generates asymmetric giant unilamellar vesicles (GUVs) in the size range of 2-14 µm. In our method, we (i) create water-in-oil emulsions as the precursors to build synthetic vesicles, (ii) deflect the emulsions across two oil streams containing different phospholipids at high throughput to establish an asymmetric architecture in the lipid bilayer membranes, and (iii) direct the water-in-oil emulsions across the oil-water interface of an oscillating oil jet in a co-flowing confined geometry to encapsulate the inner aqueous phase inside a lipid bilayer and complete the fabrication of GUVs. In the first step, we utilize a flow-focusing geometry with precisely controlled pneumatic pressures to form monodisperse water-in-oil emulsions. We observed different regimes in forming water-in-oil multiphase flows by changing the applied pressures and discovered a hysteretic behavior in jet breakup and droplet generation. In the second step of GUV fabrication, an oil stream containing phospholipids carries the emulsions into a separation region where we steer the emulsions across two parallel oil streams using active dielectrophoretic and pinched-flow fractionation separations. We explore the effect of applied DC voltage magnitude and carrier oil stream flow rate on the separation efficiency. We develop an image processing code that measures the degree of mixing between the two oil streams as the water-in-oil emulsions travel across them under dielectrophoretic steering to find the ideal operational conditions. Finally, we utilize an oscillating co-flowing jet to complete the formation of asymmetric giant unilamellar vesicles and transfer them to an aqueous phase. We investigate the effect of flow rates on properties of the co-flowing jet oscillating in the whipping mode (i.e., wavelength and amplitude) and define the phase diagram for the oil-in-water jet. Assays used to probe the lipid bilayer membrane of fabricated GUVs showed that membranes were unilamellar, minimal residual oil remained trapped between the two lipid leaflets, and 83% asymmetry was achieved across the lipid bilayers of GUVs.

Characterization of binding kinetics of A2AR to Gαs protein by surface plasmon resonance.

Because of their surface localization, G protein-coupled receptors (GPCRs) are often pharmaceutical targets as they respond to a variety of extracellular stimuli (e.g., light, hormones, small molecules) that may activate or inhibit a downstream signaling response. The adenosine A2A receptor (A2AR) is a well-characterized GPCR that is expressed widely throughout the human body, with over 10 crystal structures determined. Truncation of the A2AR C-terminus is necessary for crystallization as this portion of the receptor is long and unstructured; however, previous work suggests shortening of the A2AR C-terminus from 412 to 316 amino acids (A2AΔ316R) ablates downstream signaling, as measured by cAMP production, to below that of constitutive full-length A2AR levels. As cAMP production is downstream of the first activation event-coupling of G protein to its receptor-investigating that first step in activation is important in understanding how the truncation effects native GPCR function. Here, using purified receptor and Gαs proteins, we characterize the association of A2AR and A2AΔ316R to Gαs with and without GDP or GTPγs using surface plasmon resonance (SPR). Gαs affinity for A2AR was greatest for apo-Gαs, moderately affected in the presence of GDP and nearly completely ablated by the addition of GTPγs. Truncation of the A2AR C-terminus (A2AΔ316R) decreased the affinity of the unliganded receptor for Gαs by ∼20%, suggesting small changes to binding can greatly impact downstream signaling.

Carbon dioxide transport across membranes.

Carbon dioxide (CO2) movement across cellular membranes is passive and governed by Fick's law of diffusion. Until recently, we believed that gases cross biological membranes exclusively by dissolving in and then diffusing through membrane lipid. However, the observation that some membranes are CO2 impermeable led to the discovery of a gas molecule moving through a channel; namely, CO2 diffusion through aquaporin-1 (AQP1). Later work demonstrated CO2 diffusion through rhesus (Rh) proteins and NH3 diffusion through both AQPs and Rh proteins. The tetrameric AQPs exhibit differential selectivity for CO2 versus NH3 versus H2O, reflecting physico-chemical differences among the small molecules as well as among the hydrophilic monomeric pores and hydrophobic central pores of various AQPs. Preliminary work suggests that NH3 moves through the monomeric pores of AQP1, whereas CO2 moves through both monomeric and central pores. Initial work on AQP5 indicates that it is possible to create a metal-binding site on the central pore's extracellular face, thereby blocking CO2 movement. The trimeric Rh proteins have monomers with hydrophilic pores surrounding a hydrophobic central pore. Preliminary work on the bacterial Rh homologue AmtB suggests that gas can diffuse through the central pore and three sets of interfacial clefts between monomers. Finally, initial work indicates that CO2 diffuses through the electrogenic Na/HCO3 cotransporter NBCe1. At least in some cells, CO2-permeable proteins could provide important pathways for transmembrane CO2 movements. Such pathways could be amenable to cellular regulation and could become valuable drug targets.

Enabling Flow-Based Kinetic Off-Rate Selections Using a Microfluidic Enrichment Device.

Modern genomic sequencing efforts are identifying potential diagnostic and therapeutic targets more rapidly than existing methods can generate the peptide- and protein-based ligands required to study them. To address this problem, we have developed a microfluidic enrichment device (MFED) enabling kinetic off-rate selection without the use of exogenous competitor. We tuned the conditions of the device (bed volume, flow rate, immobilized target) such that modest, readily achievable changes in flow rates favor formation or dissociation of target-ligand complexes based on affinity. Simple kinetic equations can be used to describe the behavior of ligand binding in the MFED and the kinetic rate constants observed agree with independent measurements. We demonstrate the utility of the MFED by showing a 4-fold improvement in enrichment compared to standard selection. The MFED described here provides a route to simultaneously bias pools toward high-affinity ligands while reducing the demand for target-protein to less than a nanomole per selection.

Self-optimizing parallel millifluidic reactor for scaling nanoparticle synthesis.

We developed a 16-channel millifluidic reactor that uses a multiphase gas-liquid flow to continuously produce colloidal CsPbBr3 quantum dots with a throughtput of ∼1 L h-1. The optical properties of the product were monitored, and the reaction conditions were optimized in real time based on the in situ photoluminescence characteristics of the quantum dots.

An Exceptionally Mild and Scalable Solution-Phase Synthesis of Molybdenum Carbide Nanoparticles for Thermocatalytic CO2 Hydrogenation.

Transition metal carbides (TMCs) have demonstrated outstanding potential for utilization in a wide range of catalytic applications because of their inherent multifunctionality and tunable composition. However, the harsh conditions required to prepare these materials have limited the scope of synthetic control over their physical properties. The development of low-temperature, carburization-free routes to prepare TMCs would unlock the versatility of this class of materials, enhance our understanding of their physical properties, and enable their cost-effective production at industrial scales. Here, we report an exceptionally mild and scalable solution-phase synthesis route to phase-pure molybdenum carbide (α-MoC1-x) nanoparticles (NPs) in a continuous flow millifluidic reactor. We exploit the thermolytic decomposition of Mo(CO)6 in the presence of a surface-stabilizing ligand and a high boiling point solvent to yield MoC1-x NPs that are colloidally stable and resistant to bulk oxidation in air. To demonstrate the utility of this synthetic route to prepare catalytically active TMC NPs, we evaluated the thermochemical CO2 hydrogenation performance of α-MoC1-x NPs dispersed on an inert carbon support. The α-MoC1-x/C catalyst exhibited a 2-fold increase in both activity on a per-site basis and selectivity to C2+ products as compared to the bulk α-MoC1-x analogue.

Spectrophotometry in modular microfluidic architectures.

Assays for chemical biomarkers are a vital component in the ecosystem of noninvasive disease state assessment, many of which rely on quantification by colorimetric reactions or spectrophotometry. While modern advances in microfluidic technology have enabled such classes of devices to be employed in medical applications, the challenge has persisted in adapting the necessary tooling and equipment to integrate spectrophotometry into a microfluidic workflow. Spectrophotometric measurements are common in biomarker assays because of straightforward acquisition, ease of developing the assay's mechanism of action, and ease of tuning sensitivity. In this work, 3D-printed, discrete microfluidic elements are leveraged to develop a model system for assaying hyaluronidase, a urinary biomarker of bladder cancer, via absorbance spectrometry of gold nanoparticle aggregation. Compared to laboratory microtiter plate-based techniques, the system demonstrates equivalent performance while remaining competitive in terms of resource and operation requirements and cost.

Continuous Flow Methods of Fabricating Catalytically Active Metal Nanoparticles.

One of the obstacles preventing the commercialization of colloidal nanoparticle catalysts is the difficulty in fabricating these materials at scale while maintaining a high level of control over their resulting morphologies, and ultimately, their properties. Translation of batch-scale solution nanoparticle syntheses to continuous flow reactors has been identified as one method to address the scaling issue. The superior heat and mass transport afforded by the high surface-area-to-volume ratios of micro- and millifluidic channels allows for high control over reaction conditions and oftentimes results in decreased reaction times, higher yields, and/or more monodisperse size distributions compared to an analogous batch reaction. Furthermore, continuous flow reactors are automatable and have environmental health and safety benefits, making them practical for commercialization. Herein, a discussion of continuous flow methods, reactor design, and potential challenges is presented. A thorough account of the implementation of these technologies for the fabrication of catalytically active metal nanoparticles is reviewed for hydrogenation, electrocatalysis, and oxidation reactions.

Liposome production and concurrent loading of drug simulants by microfluidic hydrodynamic focusing.

Liposomes are spherical vesicles enclosed by phospholipid bilayers. Nanoscale liposomes are widely employed for drug delivery in the pharmaceutical industry. In this study, nanoscale liposomes are fabricated using the microfluidic hydrodynamic focusing (MHF) approach, and the effects of flow rate ratio (FRR) on liposome size and drug loading efficiency are studied. Fluorescein isothiocyanate modified dextran is used as a hydrophilic drug simulant and Nile red is used as a hydrophobic drug simulant. The experiment results show that hydrophilic drug simulant loading efficiency increases as FRR increases and eventually plateaues at around 90% loading efficiency. The hydrophobic drug simulant loading efficiency and FRR have a positive linear correlation when FRR varies from 10 to 50. Concurrent loading of both hydrophilic and hydrophobic drug simulants maintains the same loading efficiencies as those of loading each drug simulant alone. A negative correlation between liposome size and FRR is also confirmed. Unloaded liposomes and hydrophilic drug-loaded liposomes are of the same sizes, and are smaller than the ones loaded with the hydrophobic drug simulants alone or combined. The results suggest tunable liposome size and drug loading efficiency with the MHF technique. This provides evidence to encourage further studies of microfluidic liposome fabrication in the pharmaceutical industry.