1,721,048 research outputs found
Development of a micro and nano cavity microfluidic-SERS platform
Get PDFSubstrates for Surface Enhanced Raman Spectroscopy (SERS) that combine high enhancement with low-cost, reproducible fabrication are an important challenge. In particular substrates that enable multimodal methods such as SERS with Fluorescence Correlation Spectroscopy (FCS) or Electrochemical Impedance Spectroscopy (EIS) and amenable to microfluidic assembly are attractive but also challenging to implement.
This thesis explored novel methods for reproducible fabrication of pore array substrates for application in fluorescence correlation and in particular, for surface enhanced spectroscopy, with the underlying aim of developing microfluidic platforms that can ultimately be applied to study of microcavity supported lipid bilayers.
Chapter 2 describes two new ways of hot embossing 3 μm diameter cavity arrays onto an optically transparent PMMA substrate for implementation in a microfluidic device, using positive embossing masters made of hardened silicone and
silica beads chemically bonded onto an Au-Si wafer.
Chapter 3 optimizes sphere lithography methods to achieve outstanding pore array
packing over 1 cm2 gold surface. This was accomplished by correlating electrodeposition i-t curve with electron imaging data to identify a reproducible point at which deposition has reached the equator of the sphere template, independent of
the sphere/electrode dimensions. These advances dramatically improve the electroactive area variability between batches of cavity arrays used as electrodes and SERS reproducibility.
Finite-Difference Time Domain (FDTD) simulations and experiments designed to study
the angle dependence of incident light on the SERS signal from the improved arrays confirmed that distribution and intensity of the field at the cavity surface could be tuned by tilting the substrate over controlled angles.
Chapter 4 describes novel methods to confine and improve the electric field distribution at the bottom of the cavity by nano sub-structuring four different diameter voids ranging from 510 nm to 3 μm diameter using oxygen plasma etching. A robust
and efficient fabrication technique provided plasmonic nano sub-structured arrays which showed consistently higher intensities of SERS and Metal Enhanced Fluorescence (MEF) signal than their unstructured equivalents and better variability of results intra and inter samples, particularly for small size cavities.
Finally, chapter 5 describes implementation of the optimized arrays into a PET/PMMA-based two-channel microfluidic device designed to host up to four different lipid membrane compositions
Microcavity supported lipid bilayers; biomimetic models of the cell membrane
Get PDFBiomimetic models of the cell membrane are sought after as they have the potential to provide a realistic representation of an organism’s lipid bilayer. They can be used to understand lipid dynamics, signalling, drug permeability and membrane protein diffusion in an environment that is away from the complexity of the real living cell. This thesis examines the application of a new type of lipid membrane model, the micro-cavity supported lipid bilayer (MSLB), to study drug-membrane interactions and glycolipid containing bilayers using electrochemical impedance spectroscopy (EIS). Chapter 1 outlines the structure and function of the cell membrane and describes current models used to replicate the functions of the cellular bilayer. The limits of these models are also discussed particularly in the context of stability, lipid fluidity and addressability of both sides of the bilayer. The biomimetic MSLB system is then explored as a viable alternative in this thesis and is described in Chapter 2. 2.80 ± 0.04 μm diameter gold arrays were used and their surfaces were chemically modified to render them hydrophilic which aided the assembly of lipid bilayers using Langmuir Blodgett to form the initial monolayer and vesicle disruption to create the final bilayer structure.
This model is applied in Chapter 3 as a means of assessing drug plasma membrane interactions of two representative non-steroidal anti-inflammatory drugs; ibuprofen and diclofenac. These drugs were chosen as their log P values are well established and their interactions with membranes have been characterized by other methods. Their impact on the cavity array supported lipid membrane was investigated using EIS. Chapter 4 uses the MSLB model to study the interactions between the ganglioside, GM1, and disease relevant lectins by fabricating asymmetric GM1 containing lipid bilayer membranes. The influence of lipid/sterol composition on GM1-lectin recognition and aggregation was also considered.
Overall, this work demonstrates that, using EIS as the interrogation method, it is possible to sensitively explore interactions between external molecules and the lipid bilayer using these MSLBs. The MSLBs are a significant advance on current lipid membrane models as they permit accurate representations of cell membrane in elements of composition, fluidity, asymmetry and deep aqueous well on either side of the membrane
Integrin αIIbβ3: from platelet membrane to biomimetic models
Get PDFThe cell membrane is complex mixture of phospholipids, sphingolipids, sterols and proteins that combine to provide a semi-permeable barrier between the extracellular and intracellular environments, while also providing a functional role in cell-signalling, cell adhesion, and membrane transport. This complexity means that it is often better to study both the lipid and protein constituents of the cell membrane using artificial membrane models. Here, the transmembrane protein integrin αIIbβ3 was used as a model protein to be reconstituted into physiologically-relevant artificial lipid systems. αIIbβ3 is an integral membrane protein found in platelets and is a key mediator of thrombosis. Upon activation αIIbβ3 undergoes significant
conformational rearrangement, clustering, and ligand-binding to enable complex bidirectional signalling. It is this structural rearrangement, aggregation and ligand
binding that was the key focus of this project.
In chapter 2, and before reconstitution into artificial lipid models, αIIbβ3 was first studied in its native environment, the platelet. Here, DTT and Mn2+ were used to induce the activated form of αIIbβ3. It was found that both activators lead to structural changes in the integrin protein and varying degrees of platelet
aggregation, without the full range of response normally associated with physiological agonists. Chapter 3 focused on the production of αIIbβ3-reconstituted liposomes. It was found that integrin-ligand binding lead to a reduction in αIIbβ3 mobility, as well as integrin clustering. αIIbβ3 was also found to preferentially
excluded from cholesterol rich regions of lipid vesicles. Chapter 4 focused on the insertion of αIIbβ3 into a novel, cavity-spanning, lipid bilayer. Here, it was possible to determine αIIbβ3 diffusion co-efficients and induce protein aggregation. Finally, in chapter 5, cytoskeletal mimics were incorporated alongside a supported lipid bilayer in order to better imitate the conditions encountered by the cell membrane
The application of Ru(II) polypyridyl complexes to cellular imaging and sensing
Get PDFFluorescent microscopy is the key bio-imaging tool that is used to study live cells. Luminescent transition metal complex have been explored extensively for many years
across a range of applications from solar energy to molecular therapeutics, but it is only over the last decade that they have been seriously considered as cellular imaging probes. Their unique photophysical properties including large Stokes shift, red emission
wavelengths, good photostability, and sensitivity to molecular oxygen mean they are more than just contrast agents, making them invaluable in diagnostics and theranostics.
A key aim of this thesis was to drive forward the demonstrated applications of luminescent Ru(II) and Os(II) probes conjugated to cell penetrating peptides in cellular imaging and sensing using confocal microscopy, and the relatively new phenomenon of super resolution microscopy, specifically, STimulated Emission Depletion (STED) microscopy.
Chapter 1 overviews the general photophysical and optical of ruthenium and osmium polypyridyl complexes, examining the current status of their application in live cellular
imaging and sensing. Microscopy techniques such as confocal laser scanning microscopy, stimulated emission depletion (STED) microscopy and fluorescence lifetime imaging microscopy (FLIM) are discussed and how they are used alongside luminescent probes for cellular sensing.
Despite its NIR emission and high photostability Os(II) complexes have not been reported as general imaging probes before this report. Chapter 2 describes a novel Os(II) complex conjugated to the octa-arginine (R8) cell-penetrating peptide sequence as a cellular imaging probe, and compares it to its Ru(II) analogue. The photophysical
properties of [Os(bpy)2(pic-arg8)]10+ and [Ru(bpy)2(pic-arg8)]10+ are examined and compared, as well as its ability to be taken up by live CHO and Sp2 cells, examined by confocal microscopy.
Chapter 3 outlines a novel Ru(II) oxygen sensing probe, conjugated to the mitochondrial localising peptide sequence FrFkFrFk, in order to direct the complex to the mitochondria of live HeLa cells using confocal microscopy to confirm location. Fluorescent lifetime imaging microscopy (FLIM) allowed [(Ru(bpy)2phen-Ar)2-FrFKFrFK]7+ to measure oxygen concentrations and reactive oxygen species (ROS) from within the mitochondria of live HeLa cells.
Chapter 4 focuses on ratiometric oxygen sensing in live cells. Here, two probes are examined – a ratiometric molecule and a ratiometric core-shell nanoparticle. In both cases, the oxygen-sensitive probe consists of a Ru(II) complex, while the oxygeninsensitive reference probe is a BODIPY complex.
Finally, Chapter 5 describes the application of Ru(II) probes to STimulated Emission Depletion (STED) microscopy. Here, signal peptides conjugated to two novel Ru(II) probes directs them to the endoplasmic reticulum (ER) and nucleus of HeLa cells. The probes performance under confocal microscopy and STED is compared in the improved image resolution achieved as well as their photostability under the intense STED xii depletion laser. High resolution images of the ER and nuclear DNA during the stages of mitosis are captured using STED microscopy
Fluorescent probes for lipid droplet and lipid membrane imaging in cells and models
Get PDFCell membranes are believed to be laterally ordered into micro and nano-domains comprising of more fluid liquid-disordered (Ld) and more viscous liquid-ordered (Lo) phases. The latter subphases contain high concentrations of cholesterol and glycosphingolipids. These so call lipid rafts are experimentally distinguishable on the basis of their resistance to detergent solubilisation and are believed to play important roles in membrane function including in protein trafficking and signalling as they can drive protein-protein interactions through sequestering of proteins to these domains. Membrane domains in living cells are difficult to interrogate as they are dynamic and at sub-microscopic length scales they are outside the range of most conventional microscopies. However, they can potentially be imaged using recently developed super-resolution methods and as they are dynamic structures their diffusion can be measured using correlation methods. Therefore, new fluorescent probes are needed that can (a) partition selectively to membranous regions, (b) target the Lo and Ld phases selectively (c) that have appropriate photophysical properties compatible with the above techniques. These include large Stokes shift, high selectivity, excellent photostability, high molecular brightness, low cytotoxicity and high quantum yields. A key aim of this thesis was to design and synthesize new fluorescent probes that sequester specifically to lipid rich regions of cells or models and can distinguish Lo/Ld regions or lipid droplets, using confocal microscopy, fluorescence correlation spectroscopy (FCS), fluorescent lifetime imaging (FLIM) and the relatively new technique of super resolution microscopy, specifically, STimulated Emission Depletion (STED) microscopy
Microcavity PDMS and gold substrates for supported lipid bilayers
No full textCell membranes surround all living cells and are comprised of a complex matrix of phospholipids and proteins. The proteins embedded in or bound to the exterior of the membrane are responsible for a wide range of processes, for example cell signalling, and transport of material in and out of the cell. Understanding how transmembrane proteins behave within the lipid membrane system will allow for a better understanding of molecular mechanisms of diseases as well as the development more targeted therapeutics. However, due to the complex nature of the cell membrane environment, it is difficult to selectively study single protein species within the whole cell system. This has driven the development of model membrane systems, which allow for the sub-division of these complex systems into simpler forms and allow for the study of individual membrane proteins. Solid supported lipid bilayers have been widely used as model systems, however they have multiple limitations, the most important being the influence of the underlying substrate on the bilayer. This can impede lipid fluidity and is particularly detrimental to mobility of reconstituted proteins as substrate-protein interactions can impede motion and even cause protein to denature.
This thesis attempts to address this by developing substrates for studying membrane proteins in a biomimetic environment where such interactions are minimized. The initial substrates, designed for optical measurements, comprise of a microcavity array substrate formed in Polydimethylsiloxane (PDMS). A method for spanning bilayers over these PDMS microcavity arrays was developed and lipid diffusion dynamics over the cavities was assessed using Fluorescence Lifetime Correlation Spectroscopy (FLCS). Importantly, diffusion coefficients for lipids over these cavities are 2 to 3 times faster than on flat PDMS, and are more akin to diffusion rates normally observed in liposomes, indicating that the bilayer is minimally influenced by the underlying substrate.
In the second part of this thesis an analogous substrate and bilayer deposition method was developed using gold substrates with the objective of using electrochemical methods to address the bilayer or trigger events within the cavity.
Firstly lipid bilayers are spanned in a similar manner as developed for PDMS and the bilayer modified gold was characterised by electrochemical impedance spectroscopy (EIS). Incorporation of ion transporting molecules into the supported bilayers is also investigated by EIS. Finally a novel means of inducing electrically controlled release of reagent from inside the gold cavities to a lipid bilayer suspended across the cavity was developed using a ferrocene/cyclodextrin complex. To demonstrate this Streptavidin was released to a biotinylated lipid bilayer and its interaction with the bilayer was monitored using electrochemical impedance spectroscopy
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Peptide-directed metal complex luminophores: candidates for photodynamic therapeutics
Get PDFDespite their potential to overcome critical limitations of conventional organic dyes, metal complex
luminophores have yet to be truly accepted as probes for cellular imaging and phototherapy. Longlived
and reactive luminophore excited states grant a sensitivity not currently achievable by organic
probes and offer the ability to efficiently photosensitise cellular toxicity. A barrier to their exploitation
to date has been their relatively poor uptake and unpredictable localisation, especially to important
theranostic targets like DNA. However, signal peptides are a powerful strategy towards achieving
precision-targeting of key organelles and were previously successfully implemented to deliver metal
complexes to the nucleus and mitochondria - two locales where cellular DNA resides. The
overarching aim of this thesis was therefore: to explore the candidacy of peptide targeted Ru(II)
luminophores for imaging and photo-destruction of DNA in live cells.
Two prominent Ru(II) complexes were established as candidate complexes to derivatise under the
scope of this work. The first was [Ru(bpy)2(dppz)]2+ - a molecular light switch for DNA that is nonluminescent
in water but switches on upon intercalating DNA. The second was [Ru(tap)2(bpy)]2+
- a
complex which possesses an excited state reduction potential sufficiently positive to photo-oxidise
and damage DNA. Chapter 3 explored efficient synthesis routes to conjugatable derivatives of Ru(II)
luminophores with a highlight being the development of a novel protocol to prepare tris-heteroleptic
Ru(II) complexes in unprecedented yield. Chapter 4 investigated the interaction of Ru-dppz
conjugates with DNA in vitro and in live cells, where remarkably, both nuclear and mitochondrial
DNA were successfully targeted permitting high resolution imaging of structure and cellular phase.
Phototoxicity was induced at higher irradiation intensities leading to cellular apoptosis. Chapter 5
investigated the photo-reactivity of a nuclear-targeted Ru-tap conjugate in live cells where singlet
oxygen independent photo-oxidation of DNA led to photosensitised destruction of HeLa cells with
spatiotemporal control. Finally, Chapter 6 explored additional imaging and biophysical applications
of Ru(II) luminophores
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