Studies of exocytosis at single cells

Intercellular communication via chemical signaling is vital to the healthy functioning of multicellular organisms. In exocytosis, intracellular vesicles undergo Ca2+-triggered fusion with the cell plasma membrane, releasing their chemical messengers into the extracellular space. As exocytosis serves as the primary mechanism of communication at neuronal synapses, great emphasis has been placed on understanding the complex cellular regulation of this process. This dissertation focused on the use of amperometry and fast scan cyclic voltammetry at carbon-fiber microelectrodes to monitor exocytosis in real-time at both isolated neurons and chromaffin cells, well-characterized model cells for neuronal exocytosis. These techniques provide the necessary temporal resolution and sensitivity required to detect the chemical signals resulting from individual vesicular release events. Amperometric recordings at midbrain dopamine neurons showed that somatodendritic dopamine release is exocytotic, with a bimodal distribution of vesicular events. A combinatorial approach was used to demonstrate alterations in biogenic amine exocytosis in mice lacking the mitochondrial uncoupling protein UCP2 or the hormone leptin. Conversely, a mouse model of fragile X syndrome revealed no deficiencies in vesicular release mechanisms. Electrochemical methodologies were developed to distinguish catecholamine transmitters from the L-tyrosine-derived trace amines. Application of these methods revealed poor vesicular accumulation of trace amines precludes their function as false transmitters. Finally, vesicular quantal size in chromaffin cells was shown to be resistant to exogenous application of catecholamine precursors

Electrochemical Carbon Nanoprobes for Biological and Chemical System Studies

The progress in the field of nanoelectrochemistry requires preparation and characterization of nanometer scale electrochemical probes. The focus of my Ph.D. research was on development of carbon nanopipette-based electrodes with versatile and controllable geometry and their applications to nanoscale studies of chemical and biological systems. Carbon nanopipette (CNP) electrodes offer important advantages, including high sensitivity and improved analytical selectivity. They can serve as nanoreactors for sampling ultra-small solution volumes and studies of individual nanoparticles. CNPs were prepared by chemical vapor deposition (CVD) of carbon into the pre-pulled quartz capillaries. By changing pulling parameters and CVD conditions, we fabricated several types of carbon nanoprobes suitable for different experiments described in this thesis. After discussing the fabrication and characterization of carbon nanoprobes in Chapter 1, Open CNPs with the simplest geometry will be presented first (Chapter 2). Open CNPs can be used as multi-functional probes based on simultaneous recording of the ion current through the pipette and electronic current produced by oxidation/reduction of molecules at the carbon nanoring. They were employed as resistive-pulse sensors to detect gold nanoparticles (NPs) and NPs modified with antibodies and antigens. Open CNPs can also work as nanosensors for biological analytes. Both open CNPs and cavity carbon nanopipettes were applied in electroanalysis of dopamine and other neurotransmitters (Chapter 3). By depositing Pt into the nanocavity, we produced nanoelectrodes with a high surface area and increased catalytic activity for measurement of reactive oxygen and nitrogen species (ROS and RNS) in biological cells (Chapters 4, 5 and 6). These electrodes were employed as scanning electrochemical microscopy (SECM) tips for spatially resolved electrochemical experiments inside single biological cells and subcellular compartments. Disk-type nanoprobes were produced by filling the CNP cavity with carbon and served as a substrate for attaching single Au NPs and studying their electrocatalytic properties (Chapter 7). After polishing or focused ion beam (FIB) milling to obtain well-defined geometry, carbon disk electrodes became useful for quantitative SECM studies of surface reactions and electrochemical imaging

Développement des nanoélectrodes et utilisation de la microscopie électrochimique à balayage pour la détection du peroxyde d'hydrogène

La réaction électrochimique de réduction d'oxygène (RRO) peut impliquer des processus qui se déroulent à la surface d'un catalyseur. Peu de méthodes électrochimiques permettent d'étudier ces processus localement. La microscopie électrochimique à balayage (SECM) est un outil qui permet d'étudier des réactions électrochimiques dans un espace très restreint de dimension micrométrique ou même nanométrique. La résolution de la SECM dépend de la taille de l'électrode utilisée. Dans ce mémoire, une méthode reproductible de fabrication de microélectrodes de géométrie disque et de diamètre entre 50 nm et 1 um a été développée. La procédure de fabrication implique l'utilisation d'une étireuse de pipette pour produire des microélectrodes en 4 étapes, suivie d'un polissage mécanique. Les microélectrodes ainsi obtenues ont été caractérisées par microscopie optique, microscopie électronique à balayage, microscopie électrochimique à balayage et voltampérométrie cyclique. Ces microélectrodes ont été utilisées pour l'étude de l'activité catalytique de la porphyrine de cobalt déposée sur l'or et le carbone vitreux par l'intermédiaire de l'aminothiophénol en utilisant la microscopie électrochimique à balayage (SECM) en mode substrat génération/tip collection. Dans cette expérience, le H202 a été généré sur le substrat par réduction de O2 à différents potentiels. L'utilisation d'une microélectrode nanométrique a permis de déterminer la cinétique de la catalyse de la RRO par des porphyrines en utilisant un modèle de simulation numérique. \ud ______________________________________________________________________________ \ud MOTS-CLÉS DE L’AUTEUR : réduction d'oxygène, microscopie électrochimique à balayage, métalloporphyrines, thiols autoassemblés sur l'or, microélectrodes

In silico study on in vitro experiments to determine the electric membrane properties of a realistic cochlear model for electric field simulations on cochlear implants

To further develop and optimise the design of cochlear implants, a numerical model with precise material properties and authentic geometry is required. Since simulation results strongly depend on the accuracy of the estimates of the electrical properties of cochlear membranes, it is important to have a reliable in vivo method for measuring electrical impedance changes in the cochlear compartments. This work is a preliminary attempt to model, simulate and analyse the behaviour of a novel in-vitro experimental system for conducting plausible in-vivo measurements on mammalian cochlea membranes.Zur Weiterentwicklung und Optimierung des Designs von Cochlea-Implantaten ist ein detailliertes numerisches Modell der Cochlea erforderlich. Da die Simulationsergebnisse stark von den elektrischen Eigenschaften der Cochlea-Membranen abhängen, ist es wichtig, ein zuverlässiges In-vivo-Verfahren zur Messung des elektrischen Impedanzverlaufs zu haben. Diese Arbeit ist eine vorbereitende Studie, das Verhalten eines neuartigen In-vitro-Versuchssystems zur Durchführung plausibler In-vivo-Messungen an Cochlea-Membranen von Säugetieren zu modellieren, zu simulieren und zu analysieren

Surface-enhanced Raman Spectroscopy for Single Molecule Analysis and Biological Application

Surface-enhanced Raman spectroscopy (SERS) is a surface analytical technique, which enhances the Raman signal based on the localized surface plasmon resonance (LSPR) phenomenon. It has been successfully used for single molecule (SM) detection and has extended SERS to numerous applications in biomolecular detection. However, SM detection by SERS is still challenging especially with traditional SERS substrates and detection methods. In addition, the fundamental understanding of the SERS enhancement mechanism is still elusive. Furthermore, the application of SERS in biological field is still in the early stage. To address these challenges, there are two main aspects of SERS studied in my dissertation: (a) fundamental aspects through systematic experimental studies combined with simulations, which focus on SM detection, Raman enhancement mechanisms, and (b) the development and optimization of the SERS-based nanoprobe for biomarkers detection from fluidic devices to a single cell. In my dissertation, the following studies have been investigated. First, the sensitivity of a home-made SERS instrument was tested. SM detection was realized by utilizing a highly curved nanoelectrode (NE) to limit the number of attached nanoparticle (NP), which will allow us to have even a single NP on NE (NPoNE) junction in the SERS detection area. The molecule number in a single NPoNE junction which contributes to SERS can be hundreds or even SM. In this first study, we also conducted a correlation study between electrochemical current and SERS to monitor the dynamic formation of the plasmonic junctions. Second, we investigate electromagnetic and chemical enhancement factor tuning by the electrode potential with the assistant of Au@Ag core-shell NPs. The electrode potential induced electromagnetic enhancement (EME) tuning in the Au@Ag NPoNE structure has been confirmed by 3D Finite-difference time-domain (FDTD) simulations. Last is the design of a SERS-based nanoprobe for biomarkers detection and the effort towards single cell analysis. Finally, several SERS-active substrates were examined for biomarkers (H+, glucose, and H2O2) detection, including gold NPs (AuNPs) colloid and AuNPs decorated glass nanopipette. In summary, my dissertation presents the fabrication and development of gold tip nanoelectrode for chemical detection, which can achieve SM sensitivity. SM SERS can be used to improve the fundamental understanding and provide more in-depth insight into mechanisms of SERS and the chemical behaviors of SM on surfaces and in plasmonic cavities. Second, the fabrication and optimization of SERS-active, flexible nanopipette for biological applications. The flexible nanopipette probe provides a platform for reliable detection and quantitative analysis of biomarkers at a single cell level, which is critical and vital for detecting diseases earlier and understanding the fundamental biological process better

Scanning electrochemical microscopy for the characterisation of surfaces modified with biological molecules

This thesis describes a novel fabrication procedure for microelectrodes to be used with the scanning electrochemical microscope (SECM), the characterisation of a variety of novel impedance based immunosensors, and the characterisation of a novel oligonucleotide biosensor. The thesis firstly describes the development of a protocol for the fabrication of reproducible microelectrodes characterised to identify suitability in use with the SECM. The thesis then describes the use of SECM in feedback mode to interrogate a variety of antibody-polyelectrolyte films determining whether the changes observed by impedance were detectable by SECM. A screen printed carbon ink surface was patterned with an array of biotinylated polyethyleneimine (PEI) which was exposed to Neutravidin and then the biotinylated antibody of interest. Using ferrocenecarboxylic acid as the redox couple, the array was interrogated by SECM, scanning before and following exposure to a series of concentrations of the complementary antigen and a non-complementary antigen. Upon the exposure of the PEI/Neutravidin/biotinylated antibody array to the antigen, the feedback current over the functionalised region was observed to change. The change observed increased as the concentration of the antigen exposed to the array was increased showing linear correlation. On exposure of the array to a non-complementary antigen, only a small change in the feedback current was observed. NSE, PSA, Ciprofloxacin and NTx were all investigated with limits of detection of 0.5 pg ml-1, 1 pg ml-1, 0.1 ng ml-1 and 1 nM respectively. Finally using a similar method as employed above, SECM was utilised in the detection of binding events of short oligonucleotides. Once again scans were conducted before and after exposure to both complementary and non-complementary oligonucleotide sequences and by subtraction absolute changes in feedback current were determined. On exposure to the complementary oligonucleotide sequence a change in feedback was observed when the array was exposed to the non-complementary oligonucleotide sequence, as with the antibody/antigen array, only a small change in the feedback current was observed.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Monitoring single heart cell biology using lab-on-a- chip technologies

Abstract There has been considerable interest in developing microsensors integrated within lab-on-a-chip structures for the analysis of single cells; however, substantially less work has focused on developing "active" assays, where the cell‘s metabolic and physiological function is itself controlled on-chip. The heart attack is considered the largest cause of mortality and morbidity in the western world. Dynamic information during metabolism from a single heart cell is difficult to obtain. There is a demand for the development of a robust and sensitive analytical system that will enable us to study dynamic metabolism at single-cell level to provide intracellular information on a single-cell scale in different metabolic conditions (such as healthy or simulated unhealthy conditions). The system would also provide medics and clinicians with a better understanding of heart disease, and even help to find new therapeutic compounds. Towards this objective, we have developed a novel platform based on five individually addressable microelectrodes, fully integrated within a microfluidic system, where the cell is electrically stimulated at pre-determined rates and real-time ionic and metabolic fluxes from active, beating single heart cells are measured. The device is comprised of one pair of pacing microelectrodes, used for field-stimulation of the cell, and three other microelectrodes, configured as an enzyme-modified lactate microbiosensor, used to measure the amounts of lactate produced by the heart cell. The device also enables simultaneous in-situ microscopy, allowing optical measurements of single-cell contractility and fluorescence measurements of extracellular pH and cellular Ca2+ from the single beating heart cell at the same time, providing details of its electrical and metabolic state. Further, we have developed a robust microfluidic array, wherein a sensor array is integrated within an array of polydimethylsiloxane (PDMS) chambers, enabling the efficient manipulation of single heart cells and real-time analysis without the need to regenerate either working electrodes or reference electrodes fouled by any extracellular constituents. This sensor array also enables simultaneous electrochemical and optical measurements of single heart cells by integrating an enzyme-immobilized microsensor. Using this device, the fluorescence measurements of intracellular pH were obtained from a single beating heart cell whose electrical and metabolic states were controlled. The mechanism of released intracellular [H+] was investigated to examine extracellular pH change during contraction. In an attempt to measure lactate released from the electrically stimulated contracting cell, the cause of intracellular pH change is discussed. The preliminary investigation was made on the underlying relationship between intracellular pH and lactate from single heart cells in controlled metabolic states

Microiontophoresis as a technique to investigate Spike Timing Dependent Plasticity

Spike timing dependent plasticity (STDP) is a form of synaptic plasticity that depends on the relative time of activation of a presynaptic neuron and its postsynaptic neuron. STDP in the synapses made by Schaffer collateral afferents onto hippocampal CA1 pyramidal neurons (CA3-CA1 synapses) is NMDA receptor dependent. The objective of the current study was to develop and test a technique of glutamate iontophoresis that could replace the role of presynaptic neurotransmitter release at the CA3-CA1 synapse, so that the postsynaptic mechanisms involved in the induction of STDP could be isolated for study. Therefore, this document describes: (1) fabrication of electrodes that could be used for millisecond-level microiontophoresis in acute slice preparations of the juvenile rat hippocampus; (2) characterization of the properties and limitations of microiontophoresis in slice tissue, specifically for activation of postsynaptic ionotropic glutamate receptors at the CA3-CA1 synapse; (3) induction of STDP by pairing microiontophoresis with postsynaptic depolarization; (4) characterization of the properties and limitations of microiontophoretically induced STDP. It was determined that microiontophoresis is a viable technique to study the postsynaptic mechanisms of STDP at the CA3-CA1 synapse. My results also show that microiontophoretically induced STDP exhibits many of the same general properties as STDP induced either synaptically or by exogenously applied agonists. Microiontophoretically induced STDP also exhibits other features that will need to be considered during the design and interpretation of further experiments

Rationally Designed DNA Origami Carriers for Quantitative Single Molecule Detection with Nanopipettes

The ability to detect small concentrations of biomarkers in patient samples is one of the cornerstones of modern healthcare. In general, biosensing approaches employed to address this need are based on measuring signals resulting from the interaction of a large ensemble of molecules with the sensor. Here, a biosensor platform using DNA origami, featuring a central cavity with a target–specific DNA aptamer, as carriers for translocation through nanopores which enables individual biomarkers to be identified and counted to compile a sensing signal is reported. It is shown that the modulation of the ion current through the nanopore upon the DNA origami translocation strongly depends on the presence and in fact the size of a central cavity. While DNA origami without a central cavity cause a single peak in the ion current, DNA origami of the same dimensions but featuring a central cavity lead to double peaks in the ion current. This is also true for DNA origami (with and without central cavities) made of similar sized DNA but of different dimensions. It is also observed that the peak characteristics, peak amplitude and the dwell time, are different depending on the presence or absence of a central cavity. This work exploits these parameters to generate a biosensing platform capable of detecting human C–reactive protein (CRP) in clinically relevant fluids. DNA origami frames with cavities large enough to lead to clear ion current double peaks were designed and a CRP–specific DNA aptamer was introduced into the cavity. Also, upon binding of CRP, the ion current peak changes to a single peak and the peak characteristics change. Using this three–parameter classification, CRP–occupied and unoccupied carriers can be distinguished when they translocate through the nanopore. Thus CRP biosensing by computing the ratio of occupied vs total number of frames with a limit of detection of 3 nM is successfully demonstrated