Browsing by Author "Zauscher, Stefan"

Biosensing is a critical area of research that involves detecting and measuring biological molecules. Among the various types of biosensors, acoustic biosensors are attractive for their simplicity, robustness, and low cost, particularly in point-of-care (POC) applications. However, the quality factor (Q-factor) of acoustic biosensors is often low, limiting their sensitivity and accuracy in terms of in-liquid gravimetric detection for biosensing applications. In this dissertation, we present a novel approach that eliminates nearly all dissipation and damping from sample liquids, rendering a significant improvement in Q-factor for in-liquid gravimetric detection. We constructed rigid microfluidic channels to confine liquids and the associated acoustic energy, thereby eliminating acoustic radiation damping. We also used the channels' side walls to create pressure waves, confining the liquids within and suppressing acoustic damping due to the viscous layer. The quartz crystal microbalance (QCM) was selected as the model system for implementing the new paradigm due to its widespread usage in various applications, simplicity, cost-effectiveness, and relevance of its principles to other types of acoustic biosensors. We hypothesized that the ratio of the wavelength of the pressure wave to the width of the channels is a crucial determining factor for optimal performance. We then tested the hypothesis by building the microfluidic QCM (the µ-QCM) to improve the Q-factor of conventional QCM. The combination of experiments, simulations, and theoretical studies demonstrated a 10-fold improvement in the Q-factor. The new system offers many other advantages, including direct data interpretation, minimized sample volume requirement, and easier temperature control for in-liquid gravimetric detection. Additionally, the same principles can be applied to other acoustic biosensors, benefiting the entire field.

Virtually every consumer product available on the market today contains some form of fossil fuel-based polymer. However, these materials pose environmental, human health, and economic concerns due to their enduring presence in the global ecosystem and their degradation products. Addressing this crisis necessitates scalable production of biodegradable alternatives, such as polyhydroxyalkanoates (PHAs). PHAs are presented as promising substitutes due to their biodegradability, biocompatibility, and the potential for complete renewable utilization post-degradation, but a current challenge to widespread use of these materials lies in understanding the quantitative relationship between the structural characteristics of PHAs, their environmental interactions, and their degradation rates to enhance their industrial production and distribution. To bridge this knowledge gap, the dissertation outlines a comprehensive approach involving the development of a specialized dataset, the application of machine learning (ML) models to predict degradation rates based on structural and environmental factors, and the experimental validation of these predictions. The first part of this research focuses on assembling a manually curated dataset from the extensive, available open-access literature, aimed at understanding the effects of structural and environmental features on PHA degradation. The second part leverages this dataset through ML modeling, employing techniques like random forest regression to predict degradation profiles with over 80% accuracy. This methodology enables a deeper understanding of the complex interplay between chemical structures and degradation properties, surpassing traditional trial-and-error approaches. The final part of this research aims to complete an iterative workflow for dataset development by validating ML model predictions through physical experiments, enriching the original dataset with comprehensive experimental data on PHA degradation in hydrolytic environments with contact angle, molecular weight, and thermal property characterizations. The incorporation of experimental findings into the ML dataset, particularly through expanded ML techniques that emphasize pairwise feature importance such as explainable boosting machines (EBM), helps in pinpointing critical factors influencing PHA degradation, such as environmental temperature and material properties. The model performances indicate a strong performance of manually assembled literature-based datasets when predicting degradation rate for PHAs. In conclusion, a data science-based framework has been developed for exploring PHA biopolyester degradation and explores the combination of features of the material and its environment that integrates the structure, properties, and experimentally verified degradation profiles of the material. This workflow will be a useful and generalizable pipeline for PHAs and other polymers to expand the biopolymer design space with degradation in mind.

Human joints are able to withstand millions of loading cycles with loads regularly more than 3 times an individual's body weight in large part due to the unique bearing properties of articular cartilage, a strong, slippery tissue that covers the ends of long bones. PRG4 is a boundary lubricating glycoprotein present on the cartilage surface and in the synovial fluid surrounding it. While evidence that PRG4 lubricates and preserves normal joint function is strong, little is known of its effect on cartilage surface properties, the mechanism by which it lubricates, or its postulated role of preventing wear on joints. The effect of PRG4 on cartilage friction, wear, structure, morphology, and the mechanisms by which it mediates these factors are studied here. Methods to study these parameters at the microscale using atomic force microscopy are also developed.

Cartilage of mice with the Prg4 gene (which expresses PRG4) deleted is shown to be different in a number of ways from wild type cartilage. The uppermost layer is thicker and less uniform and the surface is rougher and softer. There is also a loss of proteoglycans, structural components of cartilage, from the underlying superficial tissue, and apparent tissue damage in some cases. Wear in the presence of PRG4 in shown to be significantly lower than in its absence, a finding which may have direct implications for prevention and treatment of osteoarthritis. It appears that PRG4 needs to be present in solution, not merely on the cartilage surface to have this effect, indicating that adsorption properties are important for wear prevention.

Cadmium sulfide (CdS) is one of the most commonly used II/VI semiconductor materials because of its electron energy band edge positions. CdS nanoparticles (NPs) are widely used in applications such as photodegradation of organic molecules, photocatalysis of water splitting, and as building blocks of photovoltaic devices. Bacterial precipitation of CdS NPs provides an innovative, environmentally friendly route for the synthesis of NPs with controllable electronic properties. Our previous research shows that CdS NPs can be extracellularly precipitated with tunable CdS crystallite sizes ranging from 5 nm to over 15 nm in diameter. In this thesis, I investigated the potential application of these bacterially precipitated CdS NPs for photodegradation of organic molecules, photocurrent generation, and for photoelectrochemical (PEC) hydrogen evolution. The results show that the bacterially precipitated CdS NPs and their devices performed competitively when compared with their counterparts that were synthesized via chemical bath deposition (CBD). In photodegradation experiments, the bacterially precipitated CdS NPs showed a slower rate of degradation than CBD CdS. In transient photocurrent response experiments, the devices incorporating bacterially precipitated CdS NPs showed a higher current response to visible light. Furthermore, in electrochemical hydrogen generation experiments, the bacterially precipitated CdS NP device showed a lower onset potential to trigger the reaction when irradiated with light. Collectively, the preliminary results show that biosynthesized CdS NPs have potentially promising applications for the photodegradation of organic molecules and for the photoelectrochemical hydrogen generation.

Engineering thin, organic materials with tailored properties requires both the understanding of the conformation of thin organic films and their conformational response to changes in the environment, and the accurate characterization the mechanical properties of the materials as a thin layer on surfaces. These issues have not yet been sufficiently addressed due to the paucity of appropriate tools and data interpretation approaches to reveal the nanometer scale conformation and mechanics of surface-grafted, thin, organic films. In this dissertation, I report on the characterization of conformational and mechanical properties of thin organic films, and the development of techniques that allow more detailed and reliable measurement of these material properties. First, I co-developed a novel approach to evaluate neutron reflectivity data and to simulate the conformational structure for thin stimulus-responsive polymer brushes. In this approach, we used a molecular-based lattice mean-field theory, augmented with experimentally obtained parameters to describe the polymer chains. The approach and fitting results required fewer fitting parameters, and captured the thermal response of the sample self-consistently.

Second, I demonstrated the capability of force-modulation microscopy in imaging surface-grafted, organic thin films in aqueous environments, with high spatial resolution and sensitivity to conformational details that affect the contact mechanics. To this end, I developed a new parameter-selection approach. This approach allowed both highly sensitive mapping of subtle differences in the molecular packing of thiol molecules on the substrate surface, and generation of high-contrast contact-stiffness images of end-grafted protein patterns on a surface. Finally, I discussed model selection and error estimation in calculating the reduced Young's modulus of soft materials on surfaces. I found that the detailed characterization of probe apex profiles, using probe-reconstruction techniques, provide only marginal improvements in calculating the reduced Young's modulus of thin films, compared with analytical models of equivalent probe radii; however, I found that a hybrid worn-cone model is appropriate for large indentations on soft materials, and benefits from the characterization of the probe apex profile. Additionally, we rendered error maps of several common scenarios, referenced to indentation and probe radius values, in the determination of the reduced Young's modulus.

Articular cartilage covers the ends of bones in synovial joints, and undergoes 5000 loading cycles during a normal daily activity. This is over 108 loading cycles over an 80-year life span, which leads to mechanical wear and degeneration of the cartilage tissue resulting in pathologies such as Osteoarthritis (OA). OA is one of the most common forms of arthritis and is associated with functional limitations and pain for individuals with the disease. Various therapeutic interventions such as drugs and viscosupplements (artificial lubricants) are being developed to treat OA, but suffer from low residence time in the knee joint and/or inadequate distribution across and within the cartilage tissue. In this dissertation, I design a cationic branched poly lysine nanocarrier with many functional groups that can adhere and penetrate through full thickness cartilage tissue. This proof-of-concept study shows how the incorporation of cartilage interacting moieties such as cationic molecules can be used to enhance cartilage binding and can be leveraged for drug conjugation.Cartilage is naturally lubricated by synovial fluid which consists of diverse but unique molecules that work synergistically to reduce friction and wear from loading, and facilitates smooth joint loading. For example, highly hydrated hyaluronic acid (HA) interacts with lubricin to be localized at the surface of the cartilage. However, following joint injury and/or the onset of inflammatory joint diseases, composition of synovial fluid changes and its lubrication properties are compromised. The compromised synovial fluid is inadequate to provide lubrication and further contributes to cartilage degeneration. Intra-articular injections of high molecular weight viscosupplements, formulated with HA, are currently used to treat OA. Despite the importance of surface-adhered molecules in multiple modes of lubrication, current viscosupplements lack the ability to adhere to cartilage. Herein, I leverage these branched poly-lysine molecules to improve localization of HA molecules at the cartilage surface by conjugating BPL molecules into HA polymer chains. The BPL molecules present on the HA polymer chains interact with the cartilage tissue via electrostatic interactions. This study provides new insights into leveraging electrostatic interactions to improve lubricants, lubricant-cartilage interactions, and their role in different modes of lubrications. Finally, I have developed an in vitro “joint”-on-a-chip platform to recapitulate cartilage mechanical loading associated with various gait motions. Employing this platform, I studied how physiologic or hyperphysiologic mechanical loads affect cartilage health. While in this thesis research, I only examined the effect of mechanical loading on cartilage health this device can be applied to optimize the design of OA therapeutics, such as drugs or lubricants, before testing them in vivo. Overall, this dissertation offers new findings and design principles regarding scalable and easy to manufacture cationic branched poly-L-lysine molecules which can adhere and penetrate across cartilage tissue and its application to design cartilage adhering HA-based lubricants. I also developed microphyisiological model (Joint-on-Chip) that can accommodate explants and can be used to mimic various attributes of the knee joint. This device can be used as a screening platform, and reduce the dependence on animal models to study OA and discover therapeutics.

Characterization of thin films, prominently including self-assembled monolayers is important to the understanding of interfacial events in both biological and manufactured systems. To facilitate such work, a surface plasmon resonance device, or SPR, was constructed, and tests were conducted to evaluate the performance of the system relative to current systems and mathematical models. First, relevant analytical equations are introduced to describe the behavior of the system. In subsequent chapters, the design of the device, its calibration, and operating procedure are explained. Finally, the system is tested against samples with known behaviors, and the experimental and analytical results are compared.

Kidney stones disease (KSD) is infamous for the morbidity it renders to an afflicted individual by causing intense pain through abrasions the urinary tract during stone passage and/or by causing increased fluid pressure caused by outflow blockage. Symptomatic kidney stones are typically treated through lithotripsy, in which stones are broken into fragments which are small enough for active retrieval or spontaneous passage. However, many individuals experience incomplete passage and some fragments remain in the kidney indefinitely. These residual stone fragments (RSF) serve as nuclei for further stone growth and cause KSD recurrence. RSFs can grow through two mechanisms: 1) calcium and other ionic stone precursors can directly crystalize on the surface of a fragment, and 2) small urinary crystallites become coated in an adhesive layer of urinary protein and adhere to the RSF. While dietary changes and a variety of medications have been shown to be effective at inhibiting this growth, and ultimately disease recurrence, a lack of patient compliance severely limits the efficacy of these approaches. In this work, we designed an analogue of mucins, biological surface coatings employed by the body as surface protectants and lubricants, to adsorb to the surface of RSFs and inhibit both mechanisms of stone growth. To do so, we performed phage display to identify peptides which bind to kidney stones. We designed genes for these peptides and expressed them as fusion peptides with elastin-like polypeptides to facilitate expression. We also used a calcium depletion assay to probe their ability to inhibit growth of kidney stone. The peptides discovered by phage display were unable to inhibit growth of RSFs through calcium adsorption. Instead, we used oligoanionic binders to synthesize analogue mucins composed of elastin-like polypeptides and synthetic polymers. We characterized these mucin analogues at each step using a combination of NMR, IR, and GPC when appropriate. Stone-targeted mucin analogues successfully inhibited the growth of calcium oxalate monohydrate stone models. Finally, to monitor adsorption of these mucin analogues to model kidney stones, we functionalized sensors with calcium oxalate monohydrate using polyacrylate as an adhesive layer. In sum, this work explores the ability to synthesize mucin analogues to inhibit recurrent kidney stone disease and have potential to shift the paradigm of kidney stone treatment.

The use of DNA as a polymeric building material transcends its function in biology and is exciting in bionanotechnology for applications ranging from biosensing, to diagnostics, and to targeted drug delivery. These applications are enabled by DNA’s unique structural and chemical properties, embodied as a directional polyanion that exhibits molecular recognition capabilities. Hence, the efficient and precise synthesis of high molecular weight DNA materials has become key to advance DNA bionanotechnology. Current synthesis methods largely rely on either solid phase chemical synthesis or template-dependent polymerase amplification. The inherent step-by-step fashion of solid phase synthesis limits the length of the resulting DNA to typically less than 150 nucleotides. In contrast, polymerase based enzymatic synthesis methods (e.g., polymerase chain reaction) are not limited by product length, but require a DNA template to guide the synthesis. Furthermore, advanced DNA bionanotechnology requires tailorable structural and self-assembly properties. Current synthesis methods, however, often involve multiple conjugating reactions and extensive purification steps.

The research described in this dissertation aims to develop a facile method to synthesize high molecular weight, single stranded DNA (or polynucleotide) with versatile functionalities. We exploit the ability of a template-independent DNA polymerase−terminal deoxynucleotidyl transferase (TdT) to catalyze the polymerization of 2’-deoxyribonucleoside 5’-triphosphates (dNTP, monomer) from the 3’-hydroxyl group of an oligodeoxyribonucleotide (initiator). We termed this enzymatic synthesis method: TdT catalyzed enzymatic polymerization, or TcEP.

Specifically, this dissertation is structured to address three specific research aims. With the objective to generate high molecular weight polynucleotides, Specific Aim 1 studies the reaction kinetics of TcEP by investigating the polymerization of 2’-deoxythymidine 5’-triphosphates (monomer) from the 3’-hydroxyl group of oligodeoxyribothymidine (initiator) using in situ 1H NMR and fluorescent gel electrophoresis. We found that TcEP kinetics follows the “living” chain-growth polycondensation mechanism, and like in “living” polymerizations, the molecular weight of the final product is determined by the starting molar ratio of monomer to initiator. The distribution of the molecular weight is crucially influenced by the molar ratio of initiator to TdT. We developed a reaction kinetics model that allows us to quantitatively describe the reaction and predict the molecular weight of the reaction products.

Specific Aim 2 further explores TcEP’s ability to transcend homo-polynucleotide synthesis by varying the choices of initiators and monomers. We investigated the effects of initiator length and sequence on TcEP, and found that the minimum length of an effective initiator should be 10 nucleotides and that the formation of secondary structures close to the 3’-hydroxyl group can impede the polymerization reaction. We also demonstrated TcEP’s capacity to incorporate a wide range of unnatural dNTPs into the growing chain, such as, hydrophobic fluorescent dNTP and fluoro modified dNTP. By harnessing the encoded nucleotide sequence of an initiator and the chemical diversity of monomers, TcEP enables us to introduce molecular recognition capabilities and chemical functionalities on the 5’-terminus and 3’-terminus, respectively.

Building on TcEP’s synthesis capacities, in Specific Aim 3 we invented a two-step strategy to synthesize diblock amphiphilic polynucleotides, in which the first, hydrophilic block serves as a macro-initiator for the growth of the second block, comprised of natural and/or unnatural nucleotides. By tuning the hydrophilic length, we synthesized the amphiphilic diblock polynucleotides that can self-assemble into micellar structures ranging from star-like to crew-cut morphologies. The observed self-assembly behaviors agree with predictions from dissipative particle dynamics simulations as well as scaling law for polyelectrolyte block copolymers.

In summary, we developed an enzymatic synthesis method (i.e., TcEP) that enables the facile synthesis of high molecular weight polynucleotides with low polydispersity. Although we can control the nucleotide sequence only to a limited extent, TcEP offers a method to integrate an oligodeoxyribonucleotide with specific sequence at the 5’-terminus and to incorporate functional groups along the growing chains simultaneously. Additionally, we used TcEP to synthesize amphiphilic polynucleotides that display self-assemble ability. We anticipate that our facile synthesis method will not only advance molecular biology, but also invigorate materials science and bionanotechnology.

Ferroelectric thin films (FETFs) offer a promising new platform for advancing liquid-phase interfacial sensing devices. FETFs are capable of expressing surface charge densities that are an order of magnitude higher than those of traditional charged surfaces in liquid environments (e.g., common oxides, self-assembled monolayers, or electrets). Furthermore, the switchable polarization state of FETFs enables patterning of charge-heterogeneous surfaces whose charge patterns persist over a range of environmental conditions. Integration of FETFs into liquid-phase interfacial sensing devices, however, requires the fabrication of films with nanometer-scale surface roughness, high remnant polarization values, and interfacial stability during prolonged exposure. The objectives of my research were to i) fabricate ferroelectric ultra-smooth lead zirconium titanate (US-PZT) thin films with nanometer-scale surface roughness, ii) establish the interfacial stability of these films after prolonged exposure to aqueous environments, iii) measure the interfacial forces as a function of film polarization and ionic strength, iv) calculate the surface potential of the US-PZT surface using electric double layer (EDL) theory, and v) demonstrate the guided deposition of charged colloidal particles onto locally polarized US-PZT thin films from solution.

I demonstrate the use of ferroelectric US-PZT thin films to manipulate EDL interaction forces in aqueous environments. My work conclusively shows that the polarization state of US-PZT controls EDL formation and can be used to induce the guided deposition of charged colloidal particles in solution.

I present a robust fabrication scheme for making ferroelectric US-PZT thin films from a sol-gel precursor. By optimizing critical thermal processing steps I am able to minimize the in-plane stress of the film and reliably produce US-PZT thin films on the wafer-scale with mean surface roughness values of only 2.4 nm over a 25 μm2 area. I then establish US-PZT film stability in water by measuring changes in film topography, crystallinity, surface chemistry, and electrical properties as a function of exposure duration. My results show that fabrication of crack-free US-PZT thin film is critical for long-term film fidelity in aqueous environments. Furthermore, I found no change in film topography or bulk composition with increasing exposure duration. Prolonged exposure to aqueous environments, however, gradually oxidizes the surface of the US-PZT wich results in a decrease in film resistivity and polarization saturation. Next, I used colloidal probe force microscopy (CPFM) to measure the EDL interaction force as a function of separation distance between polarized US-PZT thin films and a clean borosilicate probe. CPFM measurements were performed on oppositely polarized US-PZT thin films, which expressed either a positive or negative surface charge, and over a range of ionic strengths. The inner-Helmholtz plane (IHP) potential of the US-PZT was determined by fitting the CPFM force-separation data to number of EDL models, including; an analytical EDL model using a constant potential boundary condition with a Stern layer, a charge regulation EDL model, and a numerical EDL model using the non-linear Poisson-Boltzmann equation. Each model provides good agreement with the experimentally measured and predict high IHP surface potential for the polarized US-PZT thin films in solution. Finally, I demonstrate the use of polarized US-PZT to induce the guided deposition of positively or negatively charged colloidal particles from aqueous environments. I explore the effects of ionic strength, particle size, surface roughness, and pH on particle deposition.

Overall, this work demonstrates, for the first time, that FETFs can be used as a platform to manipulate colloidal particles in aqueous environments. The experimental results demonstrate that the surface charge of the FETF is reduced by charge shielding and perform similarly to traditional, charged surfaces in aqueous environments.

2D materials, e.g., graphene, and heterostructures have extraordinary properties compared to their 3D counterparts, and have great potential for a broad range of applications, including flexible electronic devices, nanocomposites, and transistors. However, in most of these applications the 2D materials need to interface with other materials such as substrates or other 2D heteroststructures for not only device functionality but also mechanical stability. The interfacial properties of 2D materials and heterostructures greatly affect the performance of these 2D materials-based devices and thus call for further investigation.

In this dissertation, advanced scanning probe microscopy (SPM) techniques, including contact resonance atomic force microscopy (CR-AFM) and piezoresponse force microscopy (PFM), are applied to study the interfacial mechanical and piezoelectrical properties of graphene and 2D materials heterostructures. For the first time, CR-AFM is demonstrated with the sensitivity to local stiffness changes that arise from a single atomic layer of a van-der-Waals-adhered material. To this end, a new approach, combining CR-AFM with first-principles calculations and continuum mechanics modeling, is introduced, which can yield a quantitative subsurface atomic structure fingerprint for 2D materials and heterostructures, as demonstrated on an ideal model system – epitaxial graphene on SiC (0001). This model system is further investigated with PFM, which revealed a new source of piezoelectricity in graphene layers that arises from the presence of interfacial dipole moments induced by the polarization in the substrate. The last part of the dissertation discusses the interfacial mechanical properties of graphene deposited onto self-assembled-monolayers (SAMs). CR-AFM experiments and molecular dynamics (MD) simulations show that the surface energy of the SAM strongly affects the amount of water molecules present at the graphene-SAM interface, which in turn influences the elastic modulus of these graphene-SAM heterostructures. The SPM methods used in this dissertation can provide rich structure-property information about interfaces and surfaces, and can be used to understand other interfacial problems of fundamental and practical interest in 2D materials and heterostructures, such as nanoconfined water and 2D layered hybrid organic-inorganic perovskites.

Friction is the resistive force that arises when two contacting surfaces move relative to each other. Frictional interactions are important from both engineering and biological perspectives. In this research I focus on the fundamental understanding of friction on polymeric and biological surfaces in aqueous environments. First, I examine the frictional properties of a stimulus-responsive hydrogel, poly-N-isopropylacrylamide (pNIPAAm), to understand how different phase states affect its tribological properties. My measurements indicate that gels in a collapsed conformation at low shear rates, exhibit significantly larger friction than swollen gels. These differences arise from changes in surface roughness, adhesive interactions, and chain entanglements of the gel surfaces associated with the phase transition. Importantly, I show that the changes in friction, triggered by an external stimulus, are reversible.

Second, I examine details of the boundary lubrication mechanism involved in mediating friction and wear in diarthrodial joints. Specifically, I looked at the constituents of the synovial fluid, lubricin and hyaluronic acid (HA) and examined their interactions on model substrates, (1) to determine the effect of surface chemistry on adsorption using surface plasmon resonance (SPR), and (2) to study normal force interactions between these surfaces using colloidal probe microscopy (CPM). I found that lubricin is highly surface-active, adsorbed strongly onto hydrophobic, hydrophilic and also collagen surfaces. Overall, lubricin develops strong repulsive interactions. This behavior is in contrast to that of HA, which does not adsorb appreciably, nor does it develop significant repulsive interactions. I speculate that in mediating interactions at the cartilage surface, an important role of lubricin is one of providing a protective coating on cartilage surfaces that maintains the contacting surfaces in a sterically repulsive state.

Primary OAG (POAG) is the second leading cause of irreversible blindness in the US and its prevalence is expected to worsen in the coming years. Major pathological changes have been attributed to the Juxtacanicular Tissue (JCT) and Inner Wall (IW) of Schlemm’s Canal (SC) within the human conventional outflow pathway. Biological tissues like the JCT/IW are viscoelastic in nature with both intra-cellular and extra-cellular mechanisms by which to store and dissipate applied forces. How these events contribute towards regulation of the local mechanobiology in the dynamic reciprocitybetween cells, their extracellular matrix (ECM), and in ultimately regulating outflow resistance, is poorly understood. Furthermore, the viscoelastic properties of the human trabecular meshwork (hTM) tissue are poorly understood. As a first step to develop more insight into the role of viscoelasticity, it was our goal to determine the localized dynamic mechanical properties of different regions of the hTM as a function of dexamethasone treatment. To explore the viscoelastic properties of the different tissue regions in the hTM comprising the hTM, we applied our co-located AFM-based rheometer/CLSM method to frontal sections of hTM under control and early ocular hypertensive conditions across a broad frequency range (1 Hz-1 kHz). We specifically considered the storage and loss moduli in the ocular pulse-relevant frequency range (1-10 Hz) and their changes across regions under dex treatment for two donors.

Among soft matter materials, block copolymers can form ordered structures with high regularity by microphase separation, triggered by the selective incompatibility of the blocks with each other. Many current studies of block-co-polypeptides are focused on their self-assembly in dilute solutions. This work expands previous research on the self-assembly of block-co-polypeptides into micellar structures in dilute solutions to the microphase separation in concentrated solutions and on surfaces, by using a model family of resilin-like/elastin-like block-co-polypeptides. The effects of four parameters, including relative block lengths, temperature, concentration, and deposition methods, on microphase separation are investigated. The results show that the presence of microphase separation and the morphologies of microphase separated structures are predictable from our study of thermodynamic theory. This work provides an understanding of how sequence design of stimulus-responsive block-co-polypeptides is related to their microphase separation.

One promising HIV-1 vaccine target is the membrane-proximal external region (MPER) of viral gp41. MPER is poorly immunogenic, however, the two rare neutralizing antibodies (NAbs), 2F5 and 4E10, bind to MPER with great neutralizing ability. Although their neutralizing mechanism represents a promising framework for the design of new HIV-1 liposomal vaccine candidates, this mechanism remains poorly understood. It is known that 2F5 and 4E10 are required to first associate with HIV-1 lipids before binding to the target MPER antigen, however, little is known about how lipid membranes contribute to NAb-antigen binding. To this end we have developed model membrane systems to study NAb and antigen lipid interactions.

We first created a surface plasmon resonance (SPR) spectroscopy based assay that monitors antibody binding to thiol monolayers, which mimic the surface chemical properties of lipid membranes. Next, we focused on mimicking the lipid phase organization (i.e., domain formation) of native membranes by using supported lipid bilayers (SLBs). We used simple SLB compositions to model the liquid-disordered (Ld) and gel phases. To model the HIV-1 envelope, we used a complex SLB composition that contains an Ld and liquid-ordered (Lo) phase. To reliably create model HIV-1 SLBs, we developed an SLB formation technique that uses amphipathic, α-helical peptides as a catalyst to generate complex SLBs that have a high cholesterol content and contain multiple lipid types. For all SLB surfaces we used atomic force microscopy (AFM) to visualize membrane domains, antigen presentation, and antibody-membrane interactions.

Results from experiments using thiol surfaces showed that NAb binding to hydrophobic thiol surfaces was significantly greater than that of control monoclonal antibodies. This supports the hypothesis that these NAbs embed into the hydrophobic membrane core. Our results demonstrate that 2F5/4E10 do not interact with the highly ordered gel and Lo domains in the SLB but exclusively bind to the Ld phase. This suggests that 2F5/4E10 require low membrane order and weak lateral lipid-lipid interactions to insert into the hydrophobic membrane interior. Thus, vaccine liposomes that primarily contain an Ld phase are more likely to elicit the production of lipid reactive, 2F5- and 4E10-like antibodies, compared to liposomes that contain an Lo or gel phase. In the context of liposomal antigen presentation, our results show that the presence of the MPER656 antigen can severely limit the Ld area available for antibody interactions. Subsequently, this reduces the amount of MPER656 that is accessible for 2F5/4E10 binding, since MPER656 preferentially localizes to the Ld area. If Ld forming lipid components are used in vaccine liposomes, it is important to ensure that the presence of antigen does not inhibit large-scale Ld formation.