<p>Pathogen detection via viscosity quantification in biological systems has long been an essential aspect of biomedical research. The importance of persistent testing of pathogens such as <em>V. cholerae</em> and HIV has consistently been recognized but limited in regions where systematic and financial resources are unavailable. Current methods require the samples be transported to research labs primarily in large cities or different countries. For consistent pathogen testing to be performed in remote areas, detection methods must be designed for portability with laboratory standards and simplicity for use without much technical background in place. </p> <p>Particle Diffusometry is a visualization method on the result of the amplification of pathogen by quantifying the Brownian motion of suspended particles in a solution. The amplification usually occurs in the specialized machine; then, the fluid sample gets inserted into the microfluidic chip for optical observation for Brownian motion. The technique has been used in particle sizing and measuring viscosity change in the biomolecular solution. In use with limitations, I present the improvements on the existing Particle Diffusometry technique to expand its use in broader biomedical applications.</p> <p>We address the portability of the technique. In the emerging and fast-growing mobile technology market, we have developed a smartphone-based portable platform capable of performing par quality tasks compared to traditional lab-based microscopy. We successfully measure the presence of <em>V. cholerae</em> as few as 6 cells/reaction, a waterborne pathogen, where its DNA is spiked into environmental water sources in just under 35 minutes. To further make the overall technology portable, we developed an on-chip amplification method accompanied by the portable heating unit. A mobile heating unit removes the need for the qPCR machine to amplify the biomolecular structure. Also, it opens the capability of on-chip amplification, further simplifying the steps needed to identify the pathogen in the source. We confirm the validity of the developed setup by measuring the presence of as low as 50 SARS-CoV-2 virus particles within 10% saliva. </p> <p>Addressing two main limitations of the existing Particle Diffusometry technique, improvements in the algorithm occur. First, we improve the algorithm to calculate diffusion coefficients even when the particles suspended in the sample are experiencing unified patterns, hence the flow, when recording is taking place. The improved algorithm correctly identified the diffusion coefficient within  margin of error using simulation and experimental verification for the sample under simple shear flow types, uniform, Couette, and Poiseuille. Second, we address the mismatch between the frame rate of the camera and the Brownian motion of particles at elevated temperatures. By configuring the correction equation for the frame mismatch behavior, we corrected the deviation of the diffusion coefficient in the range of 3E-13 to 3E-12 m²/s. Ultimately, we applied the improved flow algorithm to the elevated temperature simulation, showing the error propagation does not differ by the temperature; the percentage of error in computing the diffusion coefficient for the sample exhibiting flow only depends on the flow velocity. </p> <p>Applying these two improvements, we perform measurements on over-time viscosity change using the hydrogel formation. We characterize the hydrogel formation time using the diffusion gradient plane and variation of the initiator. By applying the addressed improvements on the real-time detection of HIV amplification on-chip, we further validate the applicative nature of the extended Particle Diffusometry technique. </p> <p>Real-time flow-adjusted Particle Diffusometry is, therefore, a feasible method for detecting viscosity changes in both chemical and biomolecular solutions in real-time. This approach opens up an alternative method for measuring biological amplification in real-time. The improvements further open the existing Particle Diffusometry technique to be widely used in the field involving rheology and pathogen detection not only in the traditional lab-based setting but also out in the field. </p>

This dissertation focuses on the fundamental understanding of liquid dynamics confined in polymer membranes. Such knowledge guides the development of better polymer membranes for practical applications and contributes to the general understanding of confined liquid dynamics in various nanoporous materials. First, we investigate the membrane transport by experimental measurements on a PFSA membrane and computer modeling of the confined liquid molecules. We probe the nano-scale environment in the ionomer membrane by determining the activation energy of diffusion. We notice two structural features of the PFSA membrane that dominate membrane transport. At relatively high hydrations, the nano-scale phase-separation creates bulk-like water in the ionomer membrane and prompts fast transport of mobile species. At relatively low hydrations, the nanoconfinement of the polymer matrix leads to the ordering of confined water and prompts a high energy barrier for transport. We then delve deeper into the confinement effect by molecular modeling of various nanoconfining geometries, including carbon nanotubes, parallel graphene sheets, and parallel rigid rods. We notice retarded water dynamics under hydrophobic confinement regardless of the geometry. We further investigate the confined water by determining the residence time of water around water, which evaluates the timescale of associations between water molecules. We learn that a decreasing confinement size prompts longer associations among water molecules. Further, we propose that the prolonged associations are responsible for the retarded water dynamics under hydrophobic confinement. Next, we turn our attention to the effect of interactions between mobile species (mostly water molecules) and a confining surface. In ionomer membranes, interactions between mobile species and the ionic groups dominate the water-surface interactions. We start by looking at water-ion interactions in bulk solutions. Using solutions at varying concentrations, we notice a temperature-concentration superposition behavior from diffusion coefficients of water molecules and ions in the solutions in both experimental and computational results. Observation of this superposition behavior in bulk solutions is unprecedented. The temperature-concentration superposition parallels the well-known time-temperature superposition. We are able to extract the offset of reciprocal temperature, which fits well to a Williams-Landel-Ferry type equation. The temperature-concentration superposition points to the new perspective that the effect of ions on water dynamics can be similar to the effect of lowering temperature. We further investigate the effect of ions by modeling ions/charges onto confining geometries. Remarkably, we reveal that the presence of ions can break the ordered water structure induced by confinement. The hydrophobic confinement prompts the ordering of water molecules, which leads to slower diffusion and higher activation energy. The presence of ions/charges on the confining surface has multiple effects on the dynamics of confined water. First, the ions associate strongly with neighboring water molecules while breaking the hydrogen-bonding network between water molecules. Second, the disruption of the hydrogen-bonding network leads to decreased activation energy of diffusion and enhanced water mobility. At relatively high ion density, the water-ion interactions overcome the structure-breaking effect and lead to retarded water diffusion. Overall, the studies presented in this dissertation augment our understanding of water transport in nanostructures by revealing the rich behavior of liquid-water dynamics under both hydrophobic and ionic confinement. / Doctor of Philosophy / Polymer separations membranes contribute to important applications such as fuel cells and water desalination. Optimizing the separation ability of polymer membranes improves their practical performance. The transport property of a polymer membrane depends on its nanoscale and microscale structures. This dissertation focuses on the nanoscale structure-transport relations in ionic polymer membranes. We utilize nuclear magnetic resonance techniques and molecular dynamics simulations to probe the transport properties. We investigate the effects of membrane nanostructure and water-ion interactions on the dynamics of confined water. Such knowledge not only guides the development of high-performance membranes but also contributes to the fundamental understanding of liquid dynamics in nanoporous materials.

Block copolymers have been extensively applied in diverse fields including packaging, electrolytes, delivery devices, and biosensors. Multiple investigations have been carried out on polymeric materials for cargo delivery purpose to understand how they behave over time. Block copolymer micelles (BCMs) have demonstrated superiority to deliver cargo, especially in drug delivery due to their encapsulation of hydrophobic agents. This dissertation will mainly study BCMs for potential applications in cargo delivery. Methods to study BCMs, including NMR spectroscopy, relaxometry and diffusometry, can provide valuable molecular information, such as chemical structure, translational motion, inter- or intramolecular interaction, dynamics, and exchange kinetics. Therefore, this dissertation describes applications of versatile NMR methods to reveal the fundamental behaviors of block copolymer self-assemblies, such as their dynamic stability, cargo partitioning, polymer chain exchange, and chain distribution in solution. We have investigated two BCM systems. Poly(ethylene oxide)-b-(ε-caprolactone) (PEO-PCL) is a model system to study BCM dynamic stability. PEO-PCL can self-assemble into spherical micelles at 1% w/v in D2O-THF-d8 mixed solvents. We used NMR diffusometry to quantify diffusion coefficients and populations of micelles and unimers (i.e. free polymer chains in solution) over a range of temperature (21 – 50 °C) and solvent composition (10 – 100 vol % THF-d8). By mapping the micelle-unimer coexistence phase diagrams, we are able to enhance our ability to understand and design micelle structure and dynamics. Moreover, we can also probe the chain exchange kinetics between micelles using a new technique we developed – time-resolved NMR spin-lattice relaxation (T1) or TR-NMR. This technique is an analog to time-resolved small-angle neutron scattering (TR-SANS), which can monitor specific signal intensity changes caused after mixing of isotope-labeled micelle solutions. A second system, Pluronic® F127 (PEO99PPO69PEO99) is a test system to study BCM structure and dynamic changes upon drug uptake. Pluronic® F127 is a commercial copolymer that can solubilize different hydrophobic drugs in micelles. We successfully encapsulated three model drugs into Pluronic® F127 BCMs and investigated the effects of polymer concentration and drug composition on drug partitioning fractions. Also, we proposed to design and synthesize a series of block copolymers with varied glass transition temperatures in core-forming blocks. Using NMR diffusometry, we have measured the existing unimer concentrations in micellar solutions and correlated these results with chain mobility and internal chemical composition. Lastly, we have extended our expertise in NMR and polymers into the study of ion-containing polymer systems (polyelectrolytes). A critical problem in polymer science is the inability to reliably measure the molecular weight of polyelectrolytes. We are developing methods to solve this problem by using NMR diffusometry, rheology, scattering, and scaling theories to accomplish general molecular weight measurements for polyelectrolytes. In short, this dissertation describes studies to provide more perspectives on structural and dynamic properties at various time and length scales for polymeric materials. NMR measurements, in combination with many other advanced techniques, have given us a better picture of soft matter behaviors and provided guidance for synthesis and processing efforts, especially in block copolymer micelles for delivery purposes. / Doctor of Philosophy / Block copolymers have been extensively applied in diverse fields in packaging, electrolytes and nano-scale drug delivery carriers. In the area of cancer treatment, only a limited number of drug nanocarriers have been approved for clinical applications. Therefore, it is very important to understand the principles behind drug delivery for targeted purposes. There have been many studies on polymeric delivery carriers but their behaviors have not been completely understood. Therefore, we have tremendous interest in unraveling the mysteries in those polymeric systems. Among a multitude of techniques to study block copolymer materials, the NMR method serves as a potent tool for its non-destructive, chemical-specific and isotope-selective merits. NMR can provide basic information about block copolymer self-assembly and other polymeric properties, such as chemical structure, molecular interactions and diffusion coefficients of species of interests. Chapters 3, 4, 5, 6, and 7 have investigated different classes of polymeric materials, mainly block copolymer micelles, for their structure and stability, exchange kinetics of polymer chains or cargo, and translational properties. Greater understanding about the fundamental properties of these polymeric systems, is essential for enabling new applications and new research areas.

This dissertation aims to investigate and correlate structure, dynamics and transport properties of several novel soft materials systems using multiple Nuclear Magnetic Resonance (NMR) methodologies, including solid-state NMR (SSNMR), diffusometry, and imaging, and with the help of X-ray scattering. First, we report the investigation of structure and dynamics of three polymeric membranes: hydroxyalkyl-containing imidazolium homopolymers, poly(arylene ether sulfone) segmented copolymers, and disulfonated poly(arylene ether sulfone) random copolymers using a wide array of SSNMR techniques, including: 1) ¹³C cross-polarization magic angle spinning (CPMAS) with varying cross-polarization (CP) contact time, 2) ¹³C single-pulse magic angle spinning (MAS) with varying delay time, 3) ²³Na single-pulse MAS, 4) two dimensional phaseadjusted spinning sideband (2D PASS), 5) proton spin−lattice relaxation (T₁), 6) rotating frame spin−lattice relaxation (T₁ρ), and 7) center-band-only detection of exchange (CODEX). These various types of SSNMR spectroscopic methods provide a wealth of structural and dynamic information over a wide range of time scales from a few nanoseconds to seconds. We further present a picture of rich structural and transport behaviors in supramolecular assemblies formed by amphiphilic wedge molecules using a combination of ²³Na solid-state NMR, ¹H/²H PFG NMR diffusion, relaxation and grazing-incidence small-angle X-ray scattering. Our results show that the liquid crystalline domains in these materials undergo a transition from columnar to bicontinuous cubic phases with a simple increase in humidity, while the amorphous domain boundaries consist of individual wedge molecules with a significant fraction (~ 10%) of total wedge molecules. Multiple-component diffusion of both wedges and water further confirms the structural and dynamic heterogeneity, with the bicontinous cubic phase being able to facilitate much faster water and ion transport than the columnar phase. We then develop a quantitative approach to probe the migration of two novel “theranostic” polymeric agents (combining “therapeutic” and “diagnostic” functions) into bulk hydrogels using two distinct time-resolved magnetic resonance imaging (MRI) methods. To the best of our knowledge, this is the first work that combines time-resolved MRI experiments to reliably quantify diffusivity of paramagnetic and superparamagnetic nanoparticles in bulk biological media. Our results agree closely with those obtained from fluorescence techniques, yet the capability of our approach allows the analysis of actual nanoparticles diffusion through biogels on mm to cm scales during a range of time periods. Finally, we employ a combination of NMR techniques to obtain a comprehensive understanding of ion clustering and transport behaviors of ionic liquids inside the benchmark ionic polymer Nafion. Spin relaxation shows that anion relaxation is more influenced by the fixed sulfonate groups than cation relaxation. 2D ¹H-¹⁹F heteronuclear Overhauser effect spectroscopy (HOESY) and 1D ¹⁹F¹⁹F selective nuclear Overhauser effect (NOE) spectroscopy confirm our assumption of the formation of ion clusters at low water content in the ionomer. While we observe non-restricted diffusion behavior for cations, anion diffusion is strongly restricted both between domain boundaries and within domains in the absence of water. The restricted anion diffusion can serve as a reliable probe for detailed multiscale structures of the ionomer. / Ph. D.

The purpose of this study was to investigate the fundamental characteristics of a chelating surfactant in terms of solution behaviour, chelation of divalent metal ions, and interaction in mixtures with different foaming agents and divalent metal ion, as well as examining its prospects in some practical applications. Chelating surfactants are functional molecules, with both surface active and chelating properties, which are water soluble and therefore suitable for chelation in many aqueous environments. The dual functionality offers the possibility to recover the chelating surfactant as well as the metals. The DTPA (diethylenetriaminepentaacetic acid)-based chelating surfactant 4-C12-DTPA (2-dodecyldiethylenetriaminepentaacetic acid) was synthesized at Mid Sweden University. In the absence of metal ions, all eight donor atoms in the headgroup of 4-C12-DTPA are titrating and the headgroup charge can be tuned from +3 to -5 by altering the pH. The solution properties, studied by surface tension measurements and NMR diffusometry, were consequently found strongly pH dependent. pH measurements of chelating surfactant solutions as a function of concentration was used to extract information regarding the interaction between surfactants in the aggregation process. Small differences in the conditional stability constants (log K) between coordination complexes of DTPA and 4-C12-DTPA, determined by competition measurements utilizing electrospray ionization mass spectrometry (ESI-MS), indicated that the hydrocarbon tail only affected the chelating ability of the headgroup to a limited extent. This was further confirmed in hydrogen peroxide bleaching of thermomechanical pulp (TMP) treated with 4-C12-DTPA. Interaction parameters for mixed systems of 4-C12-DTPA and different foaming agents were calculated following the approach of Rubingh’s regular solution theory. The mixtures were also examined with addition of divalent metal ions in equimolar ratio to the chelating surfactant. Strong correlation was found between the interaction parameter and the phase transfer efficiency of Ni2+ ions during flotations. Furthermore, a significant difference in log K between different metal complexes with 4-C12-DTPA enabled selective recovery of the metal ion with the highest log K. The findings in this study contribute to the understanding of the fundamental characteristics of chelating surfactants, which can be further utilized in practical applications.

<div>Pathogen identification at the point of use is critical in preventing disease transmission and enabling prompt treatment. Current rapid diagnostic tests suffer from high rates of false negatives because they are not capable of detecting the inherently low concentrations of pathogens found in early stages of infection or in environmental reservoirs. The gold standard method for timely pathogen identification is a nucleic acid amplification assay called polymerase chain reaction. Although polymerase chain reaction is extremely sensitive and specific, it requires expensive laboratory equipment and trained personnel to perform the sample preparation, cyclical heating, and amplicon analysis. Isothermal nucleic acid amplification assays are better suited for field use because they operate at a single temperature and are robust to common sample matrix inhibitors. Thus, there is a need to translate isothermal amplification assays to the point of use for rapid and sensitive detection of pathogens in complex samples.</div><div><br></div><div>Here, I outline an approach to bring laboratory-based sample preparation, assays, and analyses to the point of use via portable platforms. First, I characterize a loop-mediated isothermal amplification assay and combine it with lateral flow immunoassay for simple, colorimetric interpretation of results. Next, I optimize an ambient-temperature reagent storage method to eliminate cold-chain requirements and precision pipetting steps. I then incorporate loop-mediated isothermal amplification, lateral flow immunoassay, and reagent drying into two different integrated paperfluidic platforms and demonstrate their ability to separately detect bacteria and viruses in complex sample matrices. Finally, I couple loop-mediated isothermal amplification with particle diffusometry to optically determine pathogen presence by tracking the Brownian motion of particles added to an amplified sample. The combined loop-mediated isothermal amplification and particle diffusometry method is first characterized on a microscope and then translated to a smartphone-based platform. Each of these portable platforms are broadly applicable because they can be easily modified for identification of other pathogens at the point of use.</div>

<p dir="ltr">Since the beginning of the HIV/AIDS epidemic, 85.6 million people worldwide have become infected with HIV; more than half of whom have died from AIDS-related complications.[1] Sustained viral suppression below the clinically relevant threshold (1000 copies per mL) with highly active antiretroviral therapy (HAART) has proven effective at managing and prolonging the life expectancy of people living with HIV (PLHIV). However, in 2022, 11.3 million PLHIV had still not achieved viral suppression and may become susceptible to both HIV transmission and a variety of opportunistic infections. Of particular importance is the complex issue of patient non-compliance in global HIV management due to social, economic, behavioral, and healthcare access barriers, potentially disconnecting many PLHIV from the HIV care continuum. Therefore, to boost patient engagement in clinical care and to improve overall patient outcomes, new approaches to viral load monitoring practices need to be developed to increase access, particularly in regions of high HIV prevalence.</p><p dir="ltr">Nucleic acid amplification tests (NAATs) have emerged as potent tools for monitoring viral load, with reverse transcription quantitative polymerase chain reaction (RT-qPCR) being recognized as the benchmark due to its sensitivity and ability for real-time quantification enabled by fluorescence signal emission. Nevertheless, RT-qPCR is burdened by drawbacks including extended processing times, high operational costs, and the requirement for specialized laboratory facilities. In this study, we propose a novel method for HIV-1 viral load monitoring by integrating reverse-transcriptase loop-mediated isothermal amplification (RT-LAMP) with real-time particle diffusometry (PD). This approach allows for the continuous monitoring of changes in the diffusion of 400 nm fluorescent particles during RT-LAMP amplification, targeting the <i>p24</i> gene region of HIV-1 RNA. This enables the real-time detection of amplification curves, achieving a detection sensitivity in water samples as low as 25 virus particles per μL within a short duration of 30 minutes. Additionally, to address challenges related to amplification inhibition in complex human specimens, we developed a power-free sample processing system specifically designed for extracting HIV-1 RNA from both whole blood and plasma.Top of FormBottom of FormThis system modifies a commercially available spin-column protocol by integrating a syringe device and handheld bulb dryer, thus eliminating the requirement for a centrifuge. The adaptation allows for the completion of the entire extraction procedure, encompassing viral lysis, RNA capture, washing, and elution of purified HIV-1 RNA, within a timeframe of less than 16 minutes. Subsequent analyses, including RT-LAMP and RT-qPCR, demonstrate a limit of detection of 100 copies per μL and an average RNA recovery of 32% (for blood) and 70% (for plasma) in the elution fraction. Further investigations emphasize the significant presence of purified RNA in the spin column volume (termed as dead volume), and the cumulative recovered RNA copies align with those obtained using the gold standard centrifugation extraction method. Ultimately, we incorporated the real-time quantitative PD-RT-LAMP assay onto a field-compatible handheld portable platform suitable for field use, featuring built-in quality control measures. This platform enables sample-to-answer viral load testing near the point of care (POC). Subsequently, we undertook essential preparatory steps, such as reagent drying to obviate the need for cold storage, initial device calibration, and hands-on training of laboratory personnel regarding device operation, to validate device performance within a cohort of individuals living with HIV (PLHIV). These innovations facilitate quick and comprehensive viral load determination, offering promise for enhanced HIV management and patient care</p>