Biofunctionalized polymer interfaces for capture, isolation, and characterization of bacteria

Doctor of PhilosophyDepartment of Chemical EngineeringRyan R. HansenThe goal of this research is to develop bio-functional interfaces, designed using polymeric materials, for improved separation and isolation of bacteria for detection and characterization. Microbes impact many aspects of our society, from health to environment to industrial processes. In most cases, microbes exist in complex environments, where thousands of other organisms may also be present. Thus, detecting and characterizing specific microbial targets often necessitates that they are first isolated. Polymeric materials hold several advantages for this type of separation. They can be modified with biomolecules to capture specific microorganisms and can be designed to release captured organisms on-demand using an environmental stimulus. This thesis will explore each of these concepts, beginning with (1) the design of patterned polymer interfaces to tailor the surface reactivity towards biomolecules, (2) bio-functionalization of surface polymers with lectin molecules for bacteria capture, and (3) bio-functional, photodegradable hydrogels for dissection of microbes from membrane surfaces during early-stage biofouling events. The first portion of this thesis aims at fabricating micro/nano-structured patterns of the novel block co-polymer, poly(glycidyl methacrylate)–block–poly(vinyl dimethyl azlactone) (PGMA₅₆-b-PVDMA₁₇₅) onto silicon slides. These polymers use azlactone-based reactions to covalently couple biomolecules to the surface. Bottom-up and top-down chemical co-patterning methods, including microcontact printing, parylene lift-off, and interface directed assembly are investigated for formation of reproducible, brush-like and crosslinked polymers on the substrates. The second portion of this thesis uses these polymer interfaces to capture microbial contaminants from solution using lectin-based binding. Lectin-functionalized interfaces are promising for affinity-based microorganism capture and isolation of bacteria from samples such as blood, urine, and wastewater. However, the equilibrium dissociation constants (K[subscript]D) of lectin-carbohydrate interactions, 2-3 orders of magnitude higher than antibody-antigen binding constants, results in poor cell capture efficiency. To address this limitation, surfaces are designed to combine reactive polymer coatings that generate high lectin surface densities with nanoscale surface structures, ultimately improving cell capture. Both detection sensitivity and bactericidal impact of these optimized surfaces are characterized. Finally, the competing effects on capture due to lectin surface density and due to exopolysaccharide expression levels on the bacteria cell surface is compared. The final portion of this thesis focuses on the use of lectin-functionalized, photodegradable hydrogels to separate and isolate microbes that attach to membrane surfaces during early-stage biofouling, an approach termed polymer surface dissection (PSD). Photo-responsive, biofunctional polyethylene glycol (PEG)-based hydrogels are developed to detach targeted biofilm flocs or cells adhered onto PVDF membranes. A patterned illumination tool then delivers light to the hydrogel in a spatiotemporally controlled manner to release an extracted floc without damage. Microbes can then be sequenced to identify the composition of biofilm flocs at different stages of aggregation. The PSD approach can be used to characterize biofouling in many membrane-based bioseparation processes, here it has been developed to investigate membrane biofouling in anaerobic membrane bioreactors. Understanding the initial stages of biofouling from a mechanistic standpoint could help understand the critical microorganisms in wastewater communities that initiate the biofouling process, information that can inform novel techniques to mitigate biofilm formation

Metabolic investigation of dietary impact on colorectal cancer risk

Colorectal cancer (CRC) is the third most commonly diagnosed cancer and the second leading cause of cancer death worldwide. Yet there is large geographical variation in incidence, with CRC often referred to as a ‘Westernised’ disease. A large body of both epidemiological and experimental evidence has linked the consumption of a diet high in fat and biological protein to increased CRC risk, whereas an inverse association has been demonstrated for high levels of fibre consumption. Moreover, it is theorised that diet’s impact on CRC risk is mediated through colonic microbial metabolism and the subsequent production of either pro- or anti-carcinogenic metabolites such as secondary bile acids and short chain fatty acids (SCFA), respectively. In the second and third chapters of this thesis, ultra high-performance mass-spectrometry (UPLC-MS) was used to investigate diet associated changes to faecal and urinary metabolites of participants in a Dietary Exchange Study. African American participants, who typically eat a high-fat, low-fibre, Western diet swapped diets for two weeks with rural African participants, who typically eat a high-fibre, low-fat diet, and vice versa. Previously published data demonstrated that Westernisation of the diet led to a striking increase in biomarkers of CRC risk in rural Africans, yet Africanisation of the diet led to decreased CRC biomarkers in African Americans. In Chapter 2, global profiling revealed six metabolites associated with changes to diet and CRC risk. Acylcarnitines increased in both faecal and urinary samples in response to the adoption of a Western diet and with further mechanistic evidence, could serve as biomarkers of diet associated increase in CRC risk. In Chapter 3, analysis of faecal samples using an UPLC-MS bile acid profiling method demonstrated increases in secondary bile acids associated with the consumption of a high-fat, low-fibre diet. However, in contrast to this trend, 3-ketocholanic acid (3-KCA) a derivative of carcinogenic lithocholic acid (LCA), increased with the consumption of a high-fibre, low-fat diet. This highlighted a potential LCA detoxification mechanism. In Chapter 4, this was investigated further. Firstly, both faecal microbiota and selected microbial species were shown to have the capacity to produce 3-KCA. This was a novel finding. Secondly, the carcinogenic potential of 3-KCA compared to that of LCA was investigated using a HCT116 cell line. 3-KCA was shown to be significantly less cytotoxic than LCA, and preliminary results also showed a trend towards reduced genotoxicity. Although further experiments, using additional cell lines and animal models, will be required to validate these results, these initial data imply that 3-KCA may be less carcinogenic than LCA. Taken together, these data shine a spotlight on the potential of synbiotic intervention, harnessing both probiotic species with 3-KCA producing capacity and the substrates which sustain them, in the detoxification of colonic LCA, and thereby in the reduction of CRC risk in high-risk populations. Accordingly, Chapter 5 set out to explore the potential of prebiotic intervention for CRC risk reduction in Alaska Native peoples, who suffer the highest incidence rates of CRC globally. Urinary and faecal samples from the first 20 participants of the ongoing ‘Fibre to reduce colon cancer in Alaska Native peoples’ study were analysed using Nuclear Magnetic Resonance (NMR) analysis. Although global faecal and urinary metabolic profiles were not significantly changed by the intervention, it remains to be seen whether this will remain the case when the remaining samples are analysed on study completion. Despite this, these data demonstrate the potential of NMR-based profiling in the non-invasive and accurate detection of CRC biomarkers. Collectively, the results presented in this thesis highlight how microbiota manipulation can potentially be harnessed to reduced CRC risk and how NMR and UPLC-MS can be used to assess associated metabolomic changes in a non-invasive manner.Open Acces

Development of novel scaffold systems for modulating biotic activity

Composite scaffolds are the core of tissue engineering therapeutics that are being used to revolutionise modern medicine. Microbial interactions with scaffolds have become an important area of study as infection has been a major problem for these devices. This study explores means of modulating biotic activity through the use of differentiated coatings on a biocompatible polymer matrix. Both antibacterial and pro-bacterial scaffold systems were developed for chronic skin wounds and ethylene-mediated spoilage respectively. Chronic skin wounds present a major problem for the health sector. Wound dressings and other treatments have many shortfalls. Tissue engineered skin grafts are recognised as having great potential, but as yet these have failed to address wound sepsis. An acellular wound healing scaffold is proposed: The bi-phasic scaffold is comprised of a bioresorbable aliphatic polyester supporting a polyvinyl alcohol (PVA) hydrogel webbing capable of delivering erythromycin antibiotic and bioactive factors. Erythromycin was loaded into the PVA employing ethanol as a carrier and sustained release assays were performed showing that Staphylococcus aureus could be inhibited for up to 5 days. A novel in vitro co-culture was used to validate the scaffold which proved it could simultaneously prevent bacterial biofilms while allowing for fibroblast adhesion and proliferation. The price of fresh food is on the rise and spoilage is a key inefficiency in the fresh food value chain. In post-harvest storage facilities ethylene can build up and cause the food to spoil before it reaches the consumer. Chemical scrubbers are used to prevent this but their active agents require regular replacement. Biofilters using live microbes have dramatically longer working lifetimes. An ethylene biofilter was designed using tissue engineering principles offering control of biofiltration properties. Mycobacterium strain NBB4 cells were immobilised to a porous matrix in an agar coating. 0.4% w/v agar was found to be the optimal concentration for ethylene removal. The biofilter was able to degrade ethylene efficiently for > 85 days and had a shelf life up to > 60 days when in humidified packaging. The biofilters prevented bananas from rotting for up to 1 month compared to controls that spoiled in 2 weeks

Development of novel scaffold systems for modulating biotic activity

Composite scaffolds are the core of tissue engineering therapeutics that are being used to revolutionise modern medicine. Microbial interactions with scaffolds have become an important area of study as infection has been a major problem for these devices. This study explores means of modulating biotic activity through the use of differentiated coatings on a biocompatible polymer matrix. Both antibacterial and pro-bacterial scaffold systems were developed for chronic skin wounds and ethylene-mediated spoilage respectively. Chronic skin wounds present a major problem for the health sector. Wound dressings and other treatments have many shortfalls. Tissue engineered skin grafts are recognised as having great potential, but as yet these have failed to address wound sepsis. An acellular wound healing scaffold is proposed: The bi-phasic scaffold is comprised of a bioresorbable aliphatic polyester supporting a polyvinyl alcohol (PVA) hydrogel webbing capable of delivering erythromycin antibiotic and bioactive factors. Erythromycin was loaded into the PVA employing ethanol as a carrier and sustained release assays were performed showing that Staphylococcus aureus could be inhibited for up to 5 days. A novel in vitro co-culture was used to validate the scaffold which proved it could simultaneously prevent bacterial biofilms while allowing for fibroblast adhesion and proliferation. The price of fresh food is on the rise and spoilage is a key inefficiency in the fresh food value chain. In post-harvest storage facilities ethylene can build up and cause the food to spoil before it reaches the consumer. Chemical scrubbers are used to prevent this but their active agents require regular replacement. Biofilters using live microbes have dramatically longer working lifetimes. An ethylene biofilter was designed using tissue engineering principles offering control of biofiltration properties. Mycobacterium strain NBB4 cells were immobilised to a porous matrix in an agar coating. 0.4% w/v agar was found to be the optimal concentration for ethylene removal. The biofilter was able to degrade ethylene efficiently for > 85 days and had a shelf life up to > 60 days when in humidified packaging. The biofilters prevented bananas from rotting for up to 1 month compared to controls that spoiled in 2 weeks

Polymer Blends and Compatibilization

The market is continuously looking for substitutes for expensive polymers or tailor made polymers for specific applications. Therefore, polymer blends are gaining more interest since they possess a great potential to fulfill these needs. Blending not only results in better final properties, but can also improve the processing behavior and reduce costs. In the field of polymer blends, there are numerous parameters that influence the morphology, e.g., viscosity ratio, blend composition, shear conditions, and blend ratio. There is still a great deal of potential to scientifically exploit the possibilities of blend technology, which is necessary to obtain a foundation based on science, engineering, technology, and applications in order to make it possible to tailor polymer blends as desired. However, combining two or more different polymers to receive favorable properties by blending often results in immiscible polymer blends. This immiscibility goes hand-in-hand with phase separation leading to weak mechanical properties. The high interfacial tension causing this can be reduced by compatibilization of polymer blends. There are different methods to achieve this, such as adding block and graft copolymers, reactive polymers to form block and graft copolymers, nanoparticles or organic molecules. Using suitable compatibilizers, not only is the interfacial adhesion between matrix and its blends reduced, but also the dispersion of the dispersed phase is improved, the adhesion between the phases is enhanced and the morphology is stabilized. This can lead to improved mechanical and morphological properties. Designing new polymer blends or improving the properties of immiscible polymer blends by compatibilization is very challenging, but an excellent way to exploit the full potential of polymers for applications and their varied needs. This Special Issue is a source of information on all recent aspects of polymer blend technology

Advances and Applications of Nano-antimicrobial Treatments

Nowadays, great concerns are associated with the resistance demonstrated by many microorganisms towards the conventional antibiotic therapies. The failure of traditional antimicrobials, and the increasing healthcare costs, have encouraged scientific research and the development of novel antimicrobial agents. Particularly, there is a great deal of interest in nanotechnologies and in antibacterial products obtained through the incorporation of antibacterial agents or the deposition of antibacterial coatings for prevention of biofilm-associated infections. The main focus of the forthcoming Special Issue is, therefore, to present the most recent efforts in scientific research in the development of advanced antimicrobial materials, with special attention to nature-inspired antimicrobial agents and antimicrobials nanomaterials and nanocoatings. For this purpose, we intend to collect original research articles and reviews on the synthesis and characterization of antimicrobial agents, as well as on the development of antimicrobial products for different applications

Functional Nanomaterials and Polymer Nanocomposites: Current Uses and Potential Applications

This book covers a broad range of subjects, from smart nanoparticles and polymer nanocomposite synthesis and the study of their fundamental properties to the fabrication and characterization of devices and emerging technologies with smart nanoparticles and polymer integration

Micro/Nano-Chip Electrokinetics

Micro/nanofluidic chips have found increasing applications in the analysis of chemical and biological samples over the past two decades. Electrokinetics has become the method of choice in these micro/nano-chips for transporting, manipulating and sensing ions, (bio)molecules, fluids and (bio)particles, etc., due to the high maneuverability, scalability, sensitivity, and integrability. The involved phenomena, which cover electroosmosis, electrophoresis, dielectrophoresis, electrohydrodynamics, electrothermal flow, diffusioosmosis, diffusiophoresis, streaming potential, current, etc., arise from either the inherent or the induced surface charge on the solid-liquid interface under DC and/or AC electric fields. To review the state-of-the-art of micro/nanochip electrokinetics, we welcome, in this Special Issue of Micromachines, all original research or review articles on the fundamentals and applications of the variety of electrokinetic phenomena in both microfluidic and nanofluidic devices