Extending the uses of lipid-membrane coated electrodes: Next generation of lipid membrane biosensors and smart implantable cell-electrode devices

University of Technology Sydney. Faculty of Science.The ability to combine both a functional sensing and signalling membrane-electrode interface system is crucial for developing new technologies that can directly connect the living biosphere with electrical devices. However, there is a considerable distinction between both the chemical and biomechanical properties of live cell membranes versus synthetic electrical prostheses, thus there remain significant challenges that must be overcome in order to establish stable and functionally predictable interactions between these different components. The sparsely tethered bilayer lipid membrane possesses the necessary skeleton onto which novel chemistries can be added in order to succeed in the first iteration of correctly integrating electronic coupling with biological tissue. This dissertation presents an investigation into controlling the ionic and the electronic interface and then detecting ion fluxes arising from nearby biologically active cells at the nanometer scale, by using the detectable electrical signals derived from interfacing of membranes with a gold electrode. In it, the feasibility of implementing tBLMs as either an interface between biological systems and electrical devices or for continual sensing in real-time or for diagnostic purposes is investigated. Commencing is a comprehensive review of variant artificial lipid membrane models and the impedance spectroscopy approach (Chapter 1). A demonstration of the intimate nanoscale contacts of cells with the surface of the electrode is presented in Chapter 2. The aim of this study was to examine the feasibility of applying tBLMs in bio-implantable devices to offer specific transmission of electrical signals to individual target neurons to improve signal fidelity. This was to be achieved by reducing leakage pathways, thereby minimizing electrophoretic ion currents being lost into the surrounding interstitial medium. Chapter 3 describes how, instead of using the lipid membrane-covered electrodes to signal to cells, the electrode might be used to as a nano-biosensor for cell detection. Various approaches to increase sensitivity were explored to enhance this capability. The necessity for detection at the nanometer scale is explored in Chapter 4, recording in real-time the laser-generated heat pulses arising from laser-illuminated gold nanoparticles. Detection of these heat pulses required attachment of the gold nanoparticles to the membrane surface, while non-specific binding of gold nanoparticles failed to elicit a measurable response. Conclusions and perspectives are presented in Chapter 5, sum up of the significant achievements presented in this dissertation, which has focused on extending our understanding of cell membrane interactions and exploring the feasibility of using these across a range of applications

<i>In Vitro</i> Enzymatic Studies Reveal pH and Temperature Sensitive Properties of the CLIC Proteins

Chloride intracellular ion channel (CLIC) proteins exist as both soluble and integral membrane proteins, with CLIC1 capable of shifting between two distinct structural conformations. New evidence has emerged indicating that members of the CLIC family act as moonlighting proteins, referring to the ability of a single protein to carry out multiple functions. In addition to their ion channel activity, CLIC family members possess oxidoreductase enzymatic activity and share significant structural and sequence homology, along with varying overlaps in their tissue distribution and cellular localization. In this study, the 2-hydroxyethyl disulfide (HEDS) assay system was used to characterize kinetic properties, as well as the temperature and pH profiles of three CLIC protein family members (CLIC1, CLIC3, CLIC4). We also assessed the effects of the drugs rapamycin and amphotericin B, on the three CLIC proteins’ enzymatic activity in the HEDS assay. Our results demonstrate CLIC1 to be highly heat-sensitive, with optimal enzymatic activity observed at neutral pH7 and at a temperature of 37 °C, while CLIC3 had higher oxidoreductase activity in more acidic pH5 and was found to be relatively heat stable. CLIC4, like CLIC1, was temperature sensitive with optimal enzymatic activity observed at 37 °C; however, it showed optimal activity in more alkaline conditions of pH8. Our current study demonstrates individual differences in the enzymatic activity between the three CLIC proteins, suggesting each CLIC protein is likely regulated in discrete ways, involving changes in the subcellular milieu and microenvironment

A rationally designed synthetic antimicrobial peptide against Pseudomonas-associated corneal keratitis: Structure-function correlation

Contact lens wearers are at an increased risk of developing Pseudomonas-associated corneal keratitis, which can lead to a host of serious ocular complications. Despite the use of topical antibiotics, ocular infections remain a major clinical problem, and a strategy to avoid Pseudomonas-associated microbial keratitis is urgently required. The hybrid peptide VR18 (VARGWGRKCPLFGKNKSR) was designed to have enhanced antimicrobial properties in the fight against Pseudomonas-induced microbial keratitis, including contact lens-related keratitis. In this paper, VR18\u27s modes of action against Pseudomonas membranes were shown by live cell Raman spectroscopy, live cell NMR, live-cell fluorescence microscopy and measures taken using sparsely tethered bilayer lipid membrane bacterial models to be via a bacterial-specific membrane disruption mechanism. The high affinity and selectivity of the peptide were then demonstrated using in vivo, in vitro and ex vivo models of Pseudomonas infection. The extensive data presented in this work suggests that topical employment of the VR18 peptide would be a potent therapeutic agent for the prevention or remedy of Pseudomonas-associated microbial keratitis

Cholic Acid-Based Antimicrobial Peptide Mimics as Antibacterial Agents

There is a significant and urgent need for the development of novel antibacterial agents to tackle the increasing incidence of antibiotic resistance. Cholic acid-based small molecular antimicrobial peptide mimics are reported as potential new leads to treat bacterial infection. Here, we describe the design, synthesis and biological evaluation of cholic acid-based small molecular antimicrobial peptide mimics. The synthesis of cholic acid analogues involves the attachment of a hydrophobic moiety at the carboxyl terminal of the cholic acid scaffold, followed by the installation of one to three amino acid residues on the hydroxyl groups present on the cholic acid scaffold. Structure&ndash;activity relationship studies suggest that the tryptophan moiety is important for high antibacterial activity. Moreover, a minimum of +2 charge is also important for antimicrobial activity. In particular, analogues containing lysine-like residues showed the highest antibacterial potency against Gram-positive S. aureus. All di-substituted analogues possess high antimicrobial activity against both Gram-positive S. aureus as well as Gram-negative E. coli and P. aeruginosa. Analogues 17c and 17d with a combination of these features were found to be the most potent in this study. These compounds were able to depolarise the bacterial membrane, suggesting that they are potential antimicrobial pore forming agents

A rationally designed synthetic antimicrobial peptide against Pseudomonas-associated corneal keratitis: Structure-function correlation

Contact lens wearers are at an increased risk of developing Pseudomonas-associated corneal keratitis, which can lead to a host of serious ocular complications. Despite the use of topical antibiotics, ocular infections remain a major clinical problem, and a strategy to avoid Pseudomonas-associated microbial keratitis is urgently required. The hybrid peptide VR18 (VARGWGRKCPLFGKNKSR) was designed to have enhanced antimicrobial properties in the fight against Pseudomonas-induced microbial keratitis, including contact lens-related keratitis. In this paper, VR18's modes of action against Pseudomonas membranes were shown by live cell Raman spectroscopy, live cell NMR, live-cell fluorescence microscopy and measures taken using sparsely tethered bilayer lipid membrane bacterial models to be via a bacterial-specific membrane disruption mechanism. The high affinity and selectivity of the peptide were then demonstrated using in vivo, in vitro and ex vivo models of Pseudomonas infection. The extensive data presented in this work suggests that topical employment of the VR18 peptide would be a potent therapeutic agent for the prevention or remedy of Pseudomonas-associated microbial keratitis