The promoter region of the katG gene of Escherichia coli has been fused to two reporter genes GFPuv, encoding green fluorescent protein derived from Aequorea victoria and luxCDABE, encoding bacterial luciferase, from Photorhadbus luminescens to compare the qualities of these two reporters in microbial biosensor applications. In Escherichia coli both reporter systems produce stable signals. The lux construct was more sensitive at lower concentrations of hydrogen peroxide and the response time was shorter when compared with GFPuv. The latter, however, was better able to sense oxidative stress at concentrations that impaired signal output in the E. coli lux system. Low level non-induced bioluminescence was observed using the P. luminescens reporter system and this was utilised to measure EC50. As many compounds produce an increase in luminescence when incubated with this system, there is no means of specifically identifying any oxidative pollutants in the unknown sample. The system is limited to compounds that produce oxidative stress. Here we describe a system to add specificity to the stress-response whole-cell biosensor using glucose oxidase, which produces from glucose, hydrogen peroxide and gluconate. On incubation of these two adjuncts, glucose and glucose oxidase, with the pkatGlux whole cell biosensor, we found that the system was specific for glucose and had a range of sensitivity from 2 to 12 mM glucose. We propose that by adding glucose oxidase to the oxidative stress whole cell biosensor the specificity of the oxidative stress response can be increased, and by adding other oxidase enzymes the range of compounds that can be detected is expanded. There are enzymes of which the products of metabolism include glucose, beta-galactosidase converts lactose into glucose and galactose. The enzymes, beta-amylase and beta-amlyglucosidase digest starch to produce glucose and cellulases that act on cellulose to liberate glucose. Glucose oxidase then converts glucose to hydrogen peroxide and gluconate, the latter of which induces an increase in luminescence from the E. coli lux system. Thus it is possible to further develop the theme of adding in specificity to the stress response whole cell biosensor in the use of dual enzyme systems, where the first enzyme acts on the first substrate to yield glucose on to which glucose oxidase can metabolise, to yield hydrogen peroxide. If pkatGlux is incubated with a dual enzyme system then the number of compounds that can be biosensed can be increased and a greater specificity introduced. Samples may originate from lake, river or soil samples. These will not be 'clean'; they may contain organic debris, dirt and other bacteria that could interfere with the biosensing process. To this end lake and soil samples were spiked with substrates to see if direct sensing is possible, without the need for sample preparation. It was indicated that biosensing could take place in samples that originated from an aqueous environment. Where there were high levels of soil present, luminescence signal was quenched, which was restored on extraction of the substrate with appropriate solvent.

As a self-assembled mimetic structure of biological membranes, polydiacetylene liposomes have been studied for the development of platforms for various applications including nano-containers, nano-transporters, and nano-delivery systems for biological-, life- and materials-science applications. Liposomes incorporating amphiphilic polymer poly(10, 12 pentacosadiynoic acid) (PDA) was used as a building block for investigations mimicking cellular reaction and processes at the membrane cell. Changes in local membrane micro-organization and packing as a result of biomolecular and bioparticle reactions and processes at the liposomal membrane were investigated through the use of colorimetric and emission responses of PDA liposomes in solution phase. My dissertation comprises of six chapters. I provide brief overview of each chapter in the following paragraphs: Chapter 1: Introduction. In this chapter, an introduction is given on structure and function of lipid bilayer and multilayer of liposomes from a perspective of shared features with biological membranes. Amphiphilic molecules along with natural lipids at (or higher) critical micelle concentration self-assemble in aqueous medium, thereby, forming a lipid bilayer or multilayer to reduce the free energy of the system. When one of the components of the lipid bilayer is a polymerizable monomer, micelles/liposomes with enhanced mechanical and chemical stability are achieved. The lipid bilayer of liposomes is a boundary that includes at least three different regions: inside aqueous cavity, hydrophobic membrane zone, and membrane-aqueous interfaces. The membrane surface is available for further functionalization. In general, all three regions of the liposomes are utilized for both fundamental and applied studies. For example, the PDA liposomes have been employed for biosensing, drug/protein/nucleic acid transport and delivery and target release, and various probing cellular-like reactions and processes at the membranes. Here, in this chapter, literature on PDA was reviewed for a time period of 2008-2015. Furthermore, emphasis was given to application of PDA liposomes as (bio) sensing elements utilizing colorimetric, fluorescence, and FRET mechanisms. Chapter 2. Polydiacetylene (PDA) liposomes have been accepted as attractive colorimetric bionanosensors. The molecular recognition elements, either embedded within the liposomal membrane or covalent bound at the membrane surface, are available for interaction with biological and chemical analytes. Usually, PDA liposomes perform transduction activity through perturbation of the conjugated polymer backbone, which provides a colorimetric change in solution or solid-state phase. Here, we report that trapping self-quenched fluorescent specie within inner cavity of the liposomes is a simple and effective analytical tool for evaluating biomolecular binding events at the membrane surface. The release of fluorophores in response to the membrane binding event led to amplified emission signal which was utilized for probing reactions at the membrane surface that mimics reactions occurring at the cellular membrane surface. Specifically, a covalent binding on enzyme-substrate reaction resulted in a change of membrane fluidity, thereby releasing inner fluorophore content of the PDA liposomes. Fluorescent markers were loaded at or higher self-quenched concentration in the cavity of the liposome. Amplification of the fluorescence intensity was positively correlated with the concentration of protein added in the solution. The bilayer fluidity alteration also appears to depend on the molecular weight of the protein bound at the membrane. Overall, binding of protein with membrane promoted changes in the local PDA membrane organization and packing that enhanced the membrane permeability. The encapsulated content therefore leaked through “transient pores” formed in the membrane yielding substantial emission amplification. Chapter 3. Inspired by stability of the PDA liposomes, surface functionalization with a variety of molecules and loading within bilayer and inner cavity of the liposomes, we utilized liposomes as biocatalytical nanoreactors. Removable template molecules were embedded in the lipid bilayer and active protein encapsulated in the internal cavity was used for studying the transport properties of liposomes through substrate-enzyme reactions. Detergent Triton X-100 was used to remove a small portion of lipid and template molecules embedded in the membrane. The removal of lipid/template molecules not only affected the membrane fluidity but also provided transient pores in the membrane, allowing transport of substrate for enzymatic oxidation of glucose and 2-deoxy-glucose. Three important biological-relevant properties of cellular membrane: transport, bioavailability, and bio-reactivity of enzyme and substrate were studied. We found that enzyme molecules retained their reactivity when encapsulated within the aqueous inner cavity of the PDA liposomes, and that their activity was comparable to that in the bulk solution. Chapter 4. This chapter introduces studies on (at least partially) answering important questions how and if anchored enzyme activity at the liposome surface is affected through limited diffusion and spatial constraints. A further crucial question was investigated what effect of protein binding at the surface of the liposomes to enzymatic activity was. These relevant questions were important for increasing our fundamental knowledge related to reactions, interactions, and transport processes in biological cellular systems. A functionalized liposome system containing enzyme (Trypsin) covalently attached at the PDA liposome surface was synthesized. Using PDA liposomes as an immobilization scaffold, we evaluated and compared the cleavage behaviors of Trypsin in either immobilized at the membrane surface or in the free form. The covalent binding interaction and tryptic cleavage at the membrane-water interface was monitored by UV-vis and fluorescent spectroscopy, fluorescent anisotropy and spectro-micro-imaging. Trypsin binding at the membrane appeared to be significantly affected the enzymatic activity of the bound enzyme as seen from colorimetric response of the PDA liposomes. Chapter 5. Hierarchical structures support structures with new functionalities, therefore, advances in fabrication and characterization of biomimetic systems based on biological building blocks may present substantial potential rewards in material science. We take advantage of non-covalent forces known in biology for creating spatial organization by assembly tobacco mosaic virus-liposome polymeric hierarchical systems through biotin-streptavidin linkages. The advantage of using the biological thin rods such as TMV is that it can span the whole liposomal membrane allowing us to create microscopic hinge structures that connected liposomes. Our findings through electron and fluorescence microscopy confirmed that SA-TMV motif was able to stay inserted within the lipid bilayer of liposomes which yielded hierarchical structures after binding with Bt-liposomes. These hierarchical structures may find potential applications in targeted load (drug/protein/DNA) delivery, investigations involving virus-cell interactions, and sensing of virus particles. Chapter 6. Conclusions and Future work The present work in this dissertation utilized exploitation of biological self-assembly of small lipid molecules and larger biological-like motifs for enhancing our understanding of reactions and processes occurring at the cellular membrane surface. Overall the following four major studies were accomplished; 1. Sensing through amplified delivery, 2. Triggering an encapsulated bioreactor system at nanometric size, 3. Holding active biological elements when liposomes perform an attachment matrix, 4. Formation of hierarchical structures promoted by self-assembling of biological motifs with mimickers of cell membrane From our findings by mimicking the lipid bilayer of cell structures through liposomal membrane future work holds different ways to contribute in enhancing fundamental understanding of biological behavior. Active transport is an important function of all natural cells, playing important roles in intercellular communication. Liposomes composed of natural and polymerizable lipids may allow investigation involving exocytosis, formation of filopodia, vesicle fusion, budding and reproduction of neural synapses. Our liposome system may also mediate a broader range of highly selective and sensitive detection and sensing of cellular reactions and processes in physiological condition. I hope that this work in collaboration with multiple PIs will contribute to the fields at the interface of biology and material science.

Yang, Kar Lai Alice. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2013. / Includes bibliographical references. / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts also in Chinese.

Biosensors are instrumental in the detection of analytes in a wide range of areas including enzyme kinetics and disease diagnosis. A proof-of-principle upconversion nanophosphor (UCNP) based biosensor based on luminescence energy transfer between UCNPs, acting as the energy transfer donor, and enzymes and biologically relevant proteins, the energy transfer acceptor is reported here. Analyte detection has been performed by ratiometric sensing by monitoring the change in the multiple emission bands of the UCNPs. Chapter 1 is an introduction into the emerging field of UCNPs as biosensing agents. These nanoparticles offer numerous advantages over current biosensing agents (namely organic dyes and quantum dots) including resistance to photobleaching and photoblinking, long emissive lifetimes, a large anti-Stokes' shift and near infrared (nIR) excitation to eliminate autofluoresence, and multiple characteristic emission bands for sensing multiple analytes. Chapter 2 describes the synthesis and characterisation of Yb3+/Tm3+ and Yb3+/Er3+ co-doped UCNPs via a range of different preparative methods; thermal decomposition, microwave irradiation and a one-step solvothermal process to produce hydrophilic UNCPs. In addition, commercial UCNPs, kindly donated by Phosphor Technology, were also characterised and used as a benchmark for characterisation of the newly synthesised UCNPs. Chapter 3 describes the detection of the enzyme pentaerythritol tetranitrate reductase (PETNR), through energy transfer between the commercial Yb3+/Tm3+ doped UCNPs and the enzyme using ratiometric sensing. These proof-of-principle results were published in Dalton Transactions. In addition, ratiometric change of the UCNP emission bands was able to monitor the enzyme-substrate turnover in a two electron redox reaction. Chapter 4 describes techniques for increasing the scope and sensitivity of the proof-of-principle UCNP-enzyme biosensing system. Small, hydrophilic Yb3+/Tm3+ and Yb3+/Er3+ doped UCNPs, synthesised in chapter 2, were able to detect glucose oxidase and cytochrome c, in addition to PETNR. Covalent attachment of PETNR to Yb3+/Tm3+ doped UCNPs was additionally achieved. Chapter 5 describes the incorporation of UCNPs into optical ring resonators (ORRs) in order to develop a lost cost, label-free, rapid response biosensor. Drop casting and inkjet printing methods for the deposition of UCNPs onto these devices were investigated and emission of UCNPs was achieved, for the first time, by ORR excitation.

Thesis (Ph. D.)--University of Washington, 2000. / Vita. Includes bibliographical references (leaves 291-335).

Diss. (sammanfattning) Linköping : Univ., 2001. / Härtill 4 uppsatser.

Diss. (sammanfattning) Linköping : Univ., 2002. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaf 259).

Three reference interferometry techniques which overcome the effects of laser jitter noise in sensitive nanodetection experiments are presented. Experiments performed with a Mach-Zehnder interferometer in parallel with an ultrahigh-Q microresonator for nanodetection of a record polystyrene particle size down to 12.5 nm radius are described. The first interferometry technique employed in this work sees the implementation of a Mach-Zehnder interferometer in parallel with a microsphere to show the versatility of these devices across detection systems. Using a least squares fitting method on simulated results, it is shown that the parallel Mach-Zehnder can detect resonant wavelength shifts of the microcavity down to hundreds of attometers, provided sufficient system stability. Furthermore, a cavity resonant wavelength shift detection sensitivity of 0.14 femtometers is observed experimentally with a loaded microsphere Q of 2.0x10^8 in a buffer solution. For experiments which require high optical intensities, splitting off part of the optical power for use in an interferometer may reduce the dynamic range of power sensitive measurements. To rectify this problem, two in-line systems are investigated: the serial connected Mach-Zehnder and Fabry-Perot interferometers. According to simulation, the use of a Mach-Zehnder interferometer is not suitable for serial interferometry. In light of this problem, a serial Fabry-Perot interferometer is proposed. It is shown that with a least squares fitting method to fit experimental data the inline Fabry-Perot interferometer can measure resonant wavelength shifts down to a few femtometers, again provided sufficient system stability. Experimental results show a cavity resonant wavelength shift detection sensitivity of 0.5 femtometers observed with a microsphere Q of 2.1x10^7 in a buffer solution. / Graduate

<p>The first part of this study details the formation and characterization of octadecanethiol (ODT) self-assembled monolayers (SAMs) on GaAs (100) substrates from solution and vapor phases. The liquid-phase-deposited monolayers were prepared by immersing the substrate in an ethanolic solution, while vapor-deposited monolayers were prepared by the vapor phase transport of ODT in an ultrahigh vacuum (UHV) environment. The structural and optical properties of the resulting SAMs were examined with contact angle (CA) analysis, photoluminescence (PL) spectroscopy, atomic force microscopy (AFM), high-resolution x-ray photoelectron spectroscopy (HRXPS), and spectroscopic ellipsometry. Although well-ordered films were formed by both preparation routes, PL, AFM, CA analysis, HRXPS, and ellipsometry measurements revealed that the overall quality, structure, and durability of the mono layers depend on the deposition technique. Collectively, the results suggested that more robust monolayers exhibiting greater surface coverage and therefore, increased passivation and stability characteristics are assembled from vapor phase. </P> <P> The second part of this work describes the development of a hybrid GaAsaptamer biosensor for the label-free detection of analytes. The implemented sensmg strategy relies on the use of functional alkanethiol SAMs as biorecognition elements as well as the sensitivity of the GaAs PL emission to the local environment at its surface. Specifically, GaAs substrates were modified with thiol-derivatized aptamers and exposed to the target biomolecules. The resulting modification in the PL intensity is attributed to a specific aptamer-target biorecognition interaction and the accompanying ligand-induced structural change in the aptamer conformation. Modeling the performance data by means of Poisson-Boltzmann statistics in combination with the dead layer model indicates a good correlation between the structural conformation of the aptamers and the GaAs PL yield. The results demonstrate the potential of the prospective luminescence-based GaAs-SAM biosensor in real-time sensing assays requiring a simple and effective means of direct analytical detection. </p> / Thesis / Doctor of Philosophy (PhD)