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Publication Surface dissipation and foliar penetration of acetamiprid on tea leaves in the presence or absence of adjuvants(2024) Du, Xinyi; Gao, Wanjun; Gao, Zili; Dai, Haochen; Liu, Dongna; Doherty, Jeffery; Clark, John M; Hou, Ruyan; Luo, Fan; He, LiliThis study is to investigate the surface dissipation and foliar penetration of acetamiprid following application to live tea plants in the presence and absence of two adjuvants (Alligare 90 (R) (R) and Peptoil (R)). (R)). SERS mapping protocol with a self-assembled AuNP mirror as substrates was utilized for in situ and real time pesticide analysis. The surface dissipation analysis showed that acetamiprid with Alligare 90 (R) (R) had the lowest dissipation rate (34%) compared to acetamiprid alone (60%) or with Peptoil (R) (R) (44%), indicating the addition of Alligare 90 (R) (R) was more effective in reducing the surface dissipation of acetamiprid than Peptoil (R). (R). The study also investigated the foliar penetration of acetamiprid into tea leaves with and without adjuvants using SERS and LC-MS/MS. The study concludes that the two adjuvants tested can enhance spreadability, surface stability, and foliar penetration of acetamiprid applied on tea leaves. The information will facilitate the development and application of efficient and safe pesticide formulations.Publication Flipping out: role of arginine in hydrophobic interactions and biological formulation design(2025) Zajac, Jonathan William Patrick; Muralikrishnan, Praveen; Tohidian, Idris; Zeng, Xianci; Heldt, Caryn L; Perry, Sarah; Sarupria, SapnaArginine has been a mainstay in biological formulation development for decades. To date, the way arginine modulates protein stability has been widely studied and debated. Here, we employed a hydrophobic polymer to decouple hydrophobic effects from other interactions relevant to protein folding. While existing hypotheses for the effects of arginine can generally be categorized as either direct or indirect, our results indicate that direct and indirect mechanisms of arginine co-exist and oppose each other. At low concentrations, arginine was observed to stabilize hydrophobic polymer folding via a sidechain-dominated direct mechanism, while at high concentrations, arginine stabilized polymer folding via a backbone-dominated indirect mechanism. Upon introducing partially charged polymer sites, arginine destabilized polymer folding. Further, we found arginine-induced destabilization of a model virus similar to direct-mechanism destabilization of the charged polymer and concentration-dependent stabilization of a model protein similar to the indirect mechanism of hydrophobic polymer stabilization. These findings highlight the modular nature of the widely used additive arginine, with relevance in the information-driven design of stable biological formulations.Publication Brittle-to-Ductile Transitions of Polyelectrolyte Complexes: Humidity, Temperature, and Salt(2025) Ramírez Marrero, Isaac André; Kaiser, Nadine; von Vacano, Bernhard; Konradi, Rupert; Crosby, Alfred; Perry, SarahPolyelectrolyte complexation is an entropically driven, associative phase separation that results in a polymer-rich polyelectrolyte complex (PEC) and a polymer-poor supernatant. PECs show promise as a new class of sustainable materials since they can be processed using aqueous solutions rather than organic solvents. Previous reports have looked at the mechanical properties and glass transitions of PECs as a function of temperature, relative humidity (rH), and salt concentration (CS), but establishing a universal understanding of how these parameters affect PEC mechanics has yet to be achieved. We examined the effects of temperature, rH, and CS on the mechanical properties of PECs formed from poly(methacrylic acid) and poly(trimethyl aminoethyl methacrylate) with a goal of establishing design rules for their mechanical response. Relative humidity was shown to have the most dramatic effect on the mechanical properties, with temperature and salt concentration having far less of an impact. Furthermore, we observed that the glass transition of PECs is tied to both temperature and relative humidity, creating a glass transition rHg/Tg line that can be modulated by added salt. Finally, we looked at the thermodynamics behind the glass transition of PECs, which yielded similar energies as the condensation of water. We propose the use of water and/or salt as a low energy and efficient method of processing PECs for various applications.Publication Towards Stable Biologics: Understanding Co-Excipient Effects on Hydrophobic Interactions and Solvent Network Integrity(2025) Zajac, Jonathan W. P.; Muralikrishnan, Praveen; Heldt, Caryn L.; Perry, Sarah; Sarupria, SapnaThe formulation of biologics for increased shelf life stability is a complex task that depends on the chemical composition of both the active ingredient and any excipients in solution. A large number of unique excipients are typically required to stabilize biologics. However, it is not well-known how these excipient combinations influence biologics stability. To examine these formulations at the molecular level, we performed molecular dynamics simulations of arginine -- a widely used excipient with unique properties -- in solution both alone and with equimolar concentrations of lysine or glutamate. We studied the effects of these mixtures on a hydrophobic polymer model to isolate excipient mechanisms on hydrophobic interactions relevant in both protein folding and aggregation, crucial phenomena in biologics stability. We observed that arginine is the most effective single excipient in stabilizing hydrophobic polymer folding, and its effectiveness is augmented by lysine or glutamate addition. We decomposed the free energy of polymer folding/unfolding to identify that the key source of arginine-lysine and arginine-glutamate synergy is a reduction in destabilizing polymer-excipient interactions. We additionally applied principles from network theory to characterize the local solvent network embedding the hydrophobic polymer. Through this approach, we found arginine supports a more highly connected and stable local solvent network than in water, lysine, or glutamate solutions. These network properties are preserved when lysine or glutamate are added to arginine solutions. Taken together, our results highlight important molecular features in excipient solutions that establish the foundation for rational formulation design.Publication Determining the Bending Rigidity of Free-Standing Planar Phospholipid Bilayers(2023) Zabala-Ferrera, Oscar; Liu, Page; Beltramo, PeterWe describe a method to determine membrane bending rigidity from capacitance measurements on large area, free-standing, planar, biomembranes. The bending rigidity of lipid membranes is an important biological mechanical property that is commonly optically measured in vesicles, but difficult to quantify in a planar, unsupported system. To accomplish this, we simultaneously image and apply an electric potential to free-standing, millimeter area, planar lipid bilayers composed of DOPC and DOPG phospholipids to measure the membrane Young’s (elasticity) modulus. The bilayer is then modeled as two adjacent thin elastic films to calculate bending rigidity from the electromechanical response of the membrane to the applied field. Using DOPC, we show that bending rigidities determined by this approach are in good agreement with the existing work using neutron spin echo on vesicles, atomic force spectroscopy on supported lipid bilayers, and micropipette aspiration of giant unilamellar vesicles. We study the effect of asymmetric calcium concentration on symmetric DOPC and DOPG membranes and quantify the resulting changes in bending rigidity. This platform offers the ability to create planar bilayers of controlled lipid composition and aqueous ionic environment, with the ability to asymmetrically alter both. We aim to leverage this high degree of compositional and environmental control, along with the capacity to measure physical properties, in the study of various biological processes in the future.Publication Convective forces contribute to post‐traumatic degeneration after spinal cord injury(Wiley, 2025-01-14) Kwon, Hoi; Streilein, Christopher; Cornelison, ChaseSpinal cord injury (SCI) initiates a complex cascade of chemical and biophysical phenomena that result in tissue swelling, progressive neural degeneration, and formation of a fluid-filled cavity. Previous studies show fluid pressure above the spinal cord (supraspinal) is elevated for at least 3 days after injury and contributes to a phase of damage called secondary injury. Currently, it is unknown how fluid forces within the spinal cord itself (interstitial) are affected by SCI and if they contribute to secondary injury. We find spinal interstitial pressure increases from −3 mmHg in the naive cord to a peak of 13 mmHg at 3 days post-injury (DPI) but relatively normalizes to 2 mmHg by 7 DPI. A computational fluid dynamics model predicts interstitial flow velocities up to 0.9 μm/s at 3 DPI, returning to near baseline by 7 DPI. By quantifying vascular leakage of Evans Blue dye after a cervical hemi-contusion in rats, we confirm an increase in dye infiltration at 3 DPI compared to 7 DPI, suggestive of higher fluid velocities at the time of peak fluid pressure. In vivo expression of the apoptosis marker caspase-3 is strongly correlated with regions of interstitial flow at 3 DPI, and exogenously enhancing interstitial flow exacerbates tissue damage. In vitro, we show overnight exposure of neuronal cells to low pathological shear stress (0.1 dynes/cm2) significantly reduces cell count and neurite length. Collectively, these results indicate that interstitial fluid flow and shear stress may play a detrimental role in post-traumatic neural degeneration.Publication Scalable fabrication of an array-type fixed-target device for automated room temperature X-ray protein crystallography.(2025-01-02) Saha, Sarthak; Chen, Yaozu; Russi, Silvia; Marchany-Rivera, Darya; Cohen, Aina; Perry, SarahX-ray crystallography is one of the leading tools to analyze the 3-D structure, and therefore, function of proteins and other biological macromolecules. Traditional methods of mounting individual crystals for X-ray diffraction analysis can be tedious and result in damage to fragile protein crystals. Furthermore, the advent of multi-crystal and serial crystallography methods explicitly require the mounting of larger numbers of crystals. To address this need, we have developed a device that facilitates the straightforward mounting of protein crystals for diffraction analysis, and that can be easily manufactured at scale. Inspired by grid-style devices that have been reported in the literature, we have developed an X-ray compatible microfluidic device that can be used to trap protein crystals in an array configuration, while also providing excellent optical transparency, a low X-ray background, and compatibility with the robotic sample handling and environmental controls used at synchrotron macromolecular crystallography beamlines. At the Stanford Synchrotron Radiation Lightsource (SSRL), these capabilities allow for fully remote-access data collection at controlled humidity conditions. Furthermore, we have demonstrated continuous manufacturing of these devices via roll-to-roll fabrication to enable cost-effective and efficient large-scale production.Publication Polyelectrolyte–Carbon Dot Complex Coacervation(2025) Pandey, Pankaj Kumar; Sathyavageeswaran, Arvind; Holmlund, Nickolas Eric; Perry, SarahThis Letter presents complex coacervation between the biopolymer diethylaminoethyl dextran hydrochloride (DEAE-Dex) and carbon dots. The formation of these coacervates was dependent on both DEAE-Dex concentration and solution ionic strength. Fluorescence spectroscopy revealed that the blue fluorescence of the carbon dots was unaffected by coacervation. Additionally, microrheological studies were conducted to determine the viscosity of these coacervates. These complex coacervates, formed through the interaction of nanoparticles and polyelectrolytes, hold a promising role for future applications where the combination of optical properties from the carbon dots and encapsulation via coacervation can be leveraged.Publication Decoupling the Effects of Charge Density and Hydrophobicity on the Phase Behavior and Viscoelasticity of Complex Coacervates(American Chemical Society (ACS), 2024-05) Ramírez Marrero, Isaac André; Boudreau, Luke; Hu, Weiguo; Gutzler, Rainer; Kaiser, Nadine; von Vacano, Bernhard; Konradi, Rupert; Perry, SarahHere, we explore the effect of copolymer chemistry on the phase behavior and viscoelasticity of complex coacervates. To this end, we utilized a library of methacrylate copolymers with varying charge densities and hydrophobicity. Our results show that changing the charge density and hydrophobicity drastically affects the phase behavior─with charge density dictating the salt stability and hydrophobicity controlling the polymer concentration of the coacervates. Small-amplitude oscillatory shear measurements were used to study the viscoelastic response of the coacervates, leveraging knowledge of the coacervate phase behavior in tandem with time-salt superposition to construct a series of time-salt-copolymer master curves that highlight the effects of polymer charge density and hydrophobicity. These combined data show evidence of charge-dominated and hydrophobicity-dominated regimes, allowing for an understanding of how copolymer chemistry can be used to tune the mechanical properties of complex coacervates.Publication In situ small angle x-ray scattering, wide angle x-ray scattering, and Raman spectroscopy of microwave synthesis(2006) Tompsett, GA; Panzarella, B; Conner, WC; Yngvesson, KS; Lu, F; Suib, SL; Jones, KW; Bennett, SPublication In situ SAXS and WAXS of zeolite microwave synthesis(2007) Tompsett, GA; Panzarella, BA; Conner, WC; Bennett, S; Jones, KWPublication In situ SAXS/WAXS of zeolite microwave synthesis: NaY, NaA, and beta zeolites(2007) Panzarella, B; Tompsett, G; Conner, WC; Jones, KPublication Physical adsorption analysis of intact supported MFI zeolite membranes(2007) Hammond, KD; Tompsett, GA; Auerbach, SM; Conner, WCPublication Ultrasonic nozzle spray in situ mixing and microwave-assisted preparation of nanocrystalline spinel metal oxides: Nickel ferrite and zinc aluminate(2008) Nyutu, EK; Conner, WC; Auerbach, SM; Chen, CH; Suib, SLPublication Encapsulation of Inorganic Nanoparticles by Anionic Emulsion Polymerization of Diethyl Methylene Malonate for Developing Hybrid Microparticles with Tailorable Composition(2024) Beltramo, Peter J.Colloidal particle self-assembly into higher-ordered structures has been of great interest due to the promise of creating metamaterials with novel macroscopic properties. The physicochemical properties of these metamaterials can be tailored to achieve composites with tunable functionalities, either by controlling the assembly morphology and/or chemistry of the colloidal building blocks. This work describes a strategy of developing microparticles with a hybrid configuration that have an inorganic and an organic part. The inorganic part comprises functional nanoparticles, which are embedded within an organic polymer particle composed of diethyl methylene malonate polymer [p(DEMM)] prepared using anionic emulsion polymerization. DEMM polymerization is initiated entirely by the presence of hydroxyl anions and the resulting particle diameter can be tuned between 300 nm and 1 micrometer by reaction pH. Inorganic nanoparticles with varying chemistry (TiO2, CdTe, ZnO) can be loaded into the p(DEMM) particle with a controlled weight fraction, as confirmed by thermogravimetric analysis. The colloidal stability of the composite microparticles is seen to be dependent on the ligand coating attached to the inorganic constituent. These results provide a synthetic groundwork for creating hybrid, stimuli-responsive microparticles.Publication Apparatus for measuring physical adsorption on intact supported porous membranes(2007) Hammond, KD; Tompsett, GA; Auerbach, SM; Conner, WCPublication Microwave synthesis of zeolites. 2. Effect of vessel size, precursor volume, and irradiation method(2007) Panzarella, B; Tompsett, GA; Yngvesson, KS; Conner, WCPublication Genetic Circuits for Feedback Control of Gamma-Aminobutyric Acid Biosynthesis in Probiotic Escherichia coli Nissle 1917(2024) Lebovich, Matthew; Lora, Marcos A; Gracia-David, Jared; Andrews, LaurenEngineered microorganisms such as the probiotic strain Escherichia coli Nissle 1917 (EcN) offer a strategy to sense and modulate the concentration of metabolites or therapeutics in the gastrointestinal tract. Here, we present an approach to regulate the production of the depression-associated metabolite gamma-aminobutyric acid (GABA) in EcN using genetic circuits that implement negative feedback. We engineered EcN to produce GABA by overexpressing glutamate decarboxylase and applied an intracellular GABA biosensor to identify growth conditions that improve GABA biosynthesis. We next employed characterized genetically encoded NOT gates to construct genetic circuits with layered feedback to control the rate of GABA biosynthesis and the concentration of GABA produced. Looking ahead, this approach may be utilized to design feedback control of microbial metabolite biosynthesis to achieve designable smart microbes that act as living therapeutics.Publication Self-assembling polypeptides in complex coacervation(2024) Sathyavageeswaran, Arvind; Sabadini, Júlia Bonesso; Perry, Sarah L.Publication Design Rules for Sequestration of Viruses into Polypeptide Complex Coacervates(2023) Joshi, Pratik U.; Decker, Claire; Zeng, Xianci; Sathyavageeswaran, Arvind; Perry, Sarah L.; Heidt, Caryn L.Encapsulation is a strategy that has been used to facilitate the delivery and increase the stability of proteins and viruses. Here, we investigate the encapsulation of viruses via complex coacervation, which is a liquid–liquid phase separation resulting from the complexation of oppositely charged polymers. In particular, we utilized polypeptide-based coacervates and explored the effects of peptide chemistry, chain length, charge patterning, and hydrophobicity to better understand the effects of the coacervating polypeptides on virus incorporation. Our study utilized two nonenveloped viruses, porcine parvovirus (PPV) and human rhinovirus (HRV). PPV has a higher charge density than HRV, and they both appear to be relatively hydrophobic. These viruses were compared to characterize how the charge, hydrophobicity, and patterning of chemistry on the surface of the virus capsid affects encapsulation. Consistent with the electrostatic nature of complex coacervation, our results suggest that electrostatic effects associated with the net charge of both the virus and polypeptide dominated the potential for incorporating the virus into a coacervate, with clustering of charges also playing a significant role. Additionally, the hydrophobicity of a virus appears to determine the degree to which increasing the hydrophobicity of the coacervating peptides can enhance virus uptake. Nonintuitive trends in uptake were observed with regard to both charge patterning and polypeptide chain length, with these parameters having a significant effect on the range of coacervate compositions over which virus incorporation was observed. These results provide insights into biophysical mechanisms, where sequence effects can control the uptake of proteins or viruses into biological condensates and provide insights for use in formulation strategies.