The construction and optimization of a surface plasmon resonance sensor for the studies of bio-molecular interactions

Bio-molecular interactions exist ubiquitously in all biological systems. This dissertation project was to construct a powerful surface plasmon resonance (SPR) sensor. The SPR system is used to study bio-molecular interactions in real time and without labeling. Surface plasmon is the oscillation of free electrons in metals coupled with surface electromagnetic waves. These surface electromagnetic waves provide a sensitive probe to study bio-molecular interactions on metal surfaces. This project resulted in the successful construction and optimization of a homemade SPR sensor and the development of several new powerful protocols to study bio-molecular interactions. It was discovered through this project that the limitations of earlier SPR sensors are related not only to the instrumentation design and operating procedures, but also to the complex behaviors of bio-molecules on sensor surfaces that were very different from that in solution. Based on these discoveries the instrumentation design and operating procedures were fully optimized. A set of existing sensor surface treatment protocols were tested and evaluated and new protocols were developed in this project. The new protocols have demonstrated excellent performance to study biomolecular interactions. The optimized home-made SPR sensor was used to study protein-surface interactions. These protein-surface interactions are responsible for many complex organic cell activities. The co-existence of different driving forces and their correlation with the structure of the protein and the surface make the understanding of the fundamental mechanism of protein-surface interactions a very challenging task. Using the improved SPR sensor, the electrostatic interaction and hydrophobic interaction were studied separately. The results of this project directly confirmed the theoretical predictions for electrostatic force between the protein and surface. In addition, this project demonstrated that the strength of the protein-surface hydrophobic interaction does not solely depend on the hydrophobicity as reported earlier. Surface structure also plays a significant role.

Role of Vinculin in Regulating Force-Sensitive Dynamics of Adherens Junctions During Collective Cell Migration

Dynamic processes such as morphogenesis and tissue patterning require the precise control of many cellular processes, especially cell migration. Historically, these processes are thought to be mediated by genetic and biochemical signaling pathways. However, recent advances have unraveled a previously unappreciated role of mechanical forces in regulating these homeostatic processes in of multicellular systems. In multicellular systems cells adhere to both deformable extracellular matrix (ECM) and other cells, which are sources of applied forces and means of mechanical support. Cells detect and respond to these mechanical signals through a poorly understood process called mechanotransduction, which can have profound effects on processes such as cell migration. These effects are largely mediated by the sub cellular structures that link cells to the ECM, called focal adhesions (FAs), or cells to other cells, termed adherens junctions (AJs).

Overall this thesis is comprised of my work on identifying a novel force dependent function of vinculin, a protein which resides in both FAs and AJs - in dynamic process of collective migration. Using a collective migration assay as a model for collective cell behavior and a fluorescence resonance energy transfer (FRET) based molecular tension sensor for vinculin I demonstrated a spatial gradient of tension across vinculin in the direction of migration. To define this novel force-dependent role of vinculin in collective migration I took advantage of previously established shRNA based vinculin knock down Marin-Darby Canine Kidney (MDCK) epithelial cells.

The first part of my thesis comprises of my work demonstrating the mechanosensitive role of vinculin at AJ’s in collectively migrating cells. Using vinculin knockdown cells and vinculin mutants, which specifically disrupt vinculin’s ability to bind actin (VinI997A) or disrupt its ability to localize to AJs without affecting its localization at FAs (VinY822F), I establish a role of force across vinculin in E-cadherin internalization and clipping. Furthermore by measuring E-cadherin dynamics using fluorescence recovery after bleaching (FRAP) analysis I show that vinculin inhibition affects the turnover of E-cadherin at AJs. Together these data reveal a novel mechanosensitive role of vinculin in E-cadherin internalization and turnover in a migrating cell layer, which is contrary to the previously identified role of vinculin in potentiating E-cadherin junctions in a static monolayer.

For the last part of my thesis I designed a novel tension sensor to probe tension across N-cadherin (NTS). N-cadherin plays a critical role in cardiomyocytes, vascular smooth muscle cells, neurons and neural crest cells. Similar to E-cadherin, N-cadherin is also believed to bear tension and play a role in mechanotransduction pathways. To identify the role of tension across N-cadherin I designed a novel FRET-based molecular tension sensor for N-cadherin. I tested the ability of NTS to sense molecular tension in vascular smooth muscle cells, cardiomyocytes and cancer cells. Finally in collaboration with the Horwitz lab we have been able to show a role of tension across N-cadherin in synaptogenesis of neurons.

Footbridge system identification using wireless inertial measurement units for force and response measurements

With the main focus on safety, design of structures for vibration serviceability is often overlooked or mismanaged, resulting in some high profile structures failing publicly to perform adequately under human dynamic loading due to walking, running or jumping. A standard tool to inform better design, prove fitness for purpose before entering service and design retrofits is modal testing, a procedure that typically involves acceleration measurements using an array of wired sensors and force generation using a mechanical shaker. A critical but often overlooked aspect is using input (force) to output (response) relationships to enable estimation of modal mass, which is a key parameter directly controlling vibration levels in service.

This paper describes the use of wireless inertial measurement units (IMUs), designed for biomechanics motion capture applications, for the modal testing of a 109 m footbridge. IMUs were first used for an output-only vibration survey to identify mode frequencies, shapes and damping ratios, then for simultaneous measurement of body accelerations of a human subject jumping to excite specific vibrations modes and build up bridge deck accelerations at the jumping location. Using the mode shapes and the vertical acceleration data from a suitable body landmark scaled by body mass, thus providing jumping force data, it was possible to create frequency response functions and estimate modal masses.

The modal mass estimates for this bridge were checked against estimates obtained using an instrumented hammer and known mass distributions, showing consistency among the experimental estimates. Finally, the method was used in an applied research application on a short span footbridge where the benefits of logistical and operational simplicity afforded by the highly portable and easy to use IMUs proved extremely useful for an efficient evaluation of vibration serviceability, including estimation of modal masses.

Sensor inteligente para medição de cargas mecânicas

A medição precisa da força é necessária para muitas aplicações, nomeadamente, para a determinação da resistência mecânica dos materiais, controlo de qualidade durante a produção, pesagem e segurança de pessoas. Dada a grande necessidade de medição de forças, têm-se desenvolvido, ao longo do tempo, várias técnicas e instrumentos para esse fim. Entre os vários instrumentos utilizados, destacam-se os sensores de força, também designadas por células de carga, pela sua simplicidade, precisão e versatilidade. O exemplo mais comum é baseado em extensómetros elétricos do tipo resistivo, que aliados a uma estrutura formam uma célula de carga. Este tipo de sensores possui sensibilidades baixas e em repouso, presença de offset diferente de zero, o que torna complexo o seu condicionamento de sinal. Este trabalho apresenta uma solução para o condicionamento e aquisição de dados para células de carga que, tanto quanto foi investigado, é inovador. Este dispositivo permite efetuar o condicionamento de sinal, digitalização e comunicação numa estrutura atómica. A ideia vai de encontro ao paradigma dos sensores inteligentes onde um único dispositivo eletrónico, associado a uma célula de carga, executa um conjunto de operações de processamento de sinal e transmissão de dados. Em particular permite a criação de uma rede ad-hoc utilizando o protocolo de comunicação IIC. O sistema é destinado a ser introduzido numa plataforma de carga, desenvolvida na Escola Superior de Tecnologia e Gestão de Bragança, local destinado à sua implementação. Devido à sua estratégia de conceção para a leitura de forças em três eixos, contém quatro células de carga, com duas saídas cada, totalizando oito saídas. O hardware para condicionamento de sinal já existente é analógico, e necessita de uma placa de dimensões consideráveis por cada saída. Do ponto de vista funcional, apresenta vários problemas, nomeadamente o ajuste de ganho e offset ser feito manualmente, tornando-se essencial um circuito com melhor desempenho no que respeita a lidar com um array de sensores deste tipo.

MEMS SENSOR PLATFORMS FOR IN SITU CHARACTERIZATION OF LI-ION BATTERY ELECTRODES

Lithium-ion batteries provide high energy density while being compact and light-weight and are the most pervasive energy storage technology powering portable electronic devices such as smartphones, laptops, and tablet PCs. Considerable efforts have been made to develop new electrode materials with ever higher capacity, while being able to maintain long cycle life. A key challenge in those efforts has been characterizing and understanding these materials during battery operation. While it is generally accepted that the repeated strain/stress cycles play a role in long-term battery degradation, the detailed mechanisms creating these mechanical effects and the damage they create still remain unclear. Therefore, development of techniques which are capable of capturing in real time the microstructural changes and the associated stress during operation are crucial for unravelling lithium-ion battery degradation mechanisms and further improving lithium-ion battery performance. This dissertation presents the development of two microelectromechanical systems sensor platforms for in situ characterization of stress and microstructural changes in thin film lithium-ion battery electrodes, which can be leveraged as a characterization platform for advancing battery performance. First, a Fabry-Perot microelectromechanical systems sensor based in situ characterization platform is developed which allows simultaneous measurement of microstructural changes using Raman spectroscopy in parallel with qualitative stress changes via optical interferometry. Evolutions in the microstructure creating a Raman shift from 145 cm−1 to 154 cm−1 and stress in the various crystal phases in the LixV2O5 system are observed, including both reversible and irreversible phase transitions. Also, a unique way of controlling electrochemically-driven stress and stress gradient in lithium-ion battery electrodes is demonstrated using the Fabry-Perot microelectromechanical systems sensor integrated with an optical measurement setup. By stacking alternately stressed layers, the average stress in the stacked electrode is greatly reduced by 75% compared to an unmodified electrode. After 2,000 discharge-charge cycles, the stacked electrodes retain only 83% of their maximum capacity while unmodified electrodes retain 91%, illuminating the importance of the stress gradient within the electrode. Second, a buckled membrane microelectromechanical systems sensor is developed to enable in situ characterization of quantitative stress and microstructure evolutions in a V2O5 lithium-ion battery cathode by integrating atomic force microscopy and Raman spectroscopy. Using dual-mode measurements in the voltage range of the voltage range of 2.8V – 3.5V, both the induced stress (~ 40 MPa) and Raman intensity changes due to lithium cycling are observed. Upon lithium insertion, tensile stress in the V2O5 increases gradually until the α- to ε-phase and ε- to δ-phase transitions occur. The Raman intensity change at 148 cm−1 shows that the level of disorder increases during lithium insertion and progressively recovers the V2O5 lattice during lithium extraction. Results are in good agreement with the expected mechanical behavior and disorder change in V2O5, highlighting the potential of microelectromechanical systems as enabling tools for advanced scientific investigations. The work presented here will be eventually utilized for optimization of thin film battery electrode performance by achieving fundamental understanding of how stress and microstructural changes are correlated, which will also provide valuable insight into a battery performance degradation mechanism.

Rapid and sensitive ethanol sensor based on hollow Au/V2O5 nanotubes via emulsion-electrospinning route

Hollow electrospun V2O5 and Au/V2O5 nanotubes have been successfully synthesized by combining emulsion electrospinning (EE) and post calcination treatment. Immiscible polyvinyl pyrrolidone (PVP)/metal salts/dimethylformamide (DMF) solution and polystyrene (PS)/DMF solution are chosen for the EE to form PS PVP/metal salts) core-sheath nanofibers, in which PS nanorods were formed and encapsulated within in the PVP/metal salts nanofibers owing to the stretching forces and de-emulsified force during the electrospinning. Excellent sensitivity and rapid response-recovery behaviors against ethanol have been successfully achieved based on our hollow ceramic (V2O5 and Au/V2O5) nanotubes.

Graphene quantum dots and Nafion composite as an ultrasensitive electrochemical sensor for the detection of dopamine

A novel electrochemical sensor for highly sensitive and selective detection of dopamine (DA) was developed based on a graphene quantum dots (GQDs) and Nafion composite modified glassy carbon electrode (GCE). GQDs were synthesized by a hydrothermal approach for cutting graphene sheets into GQDs and characterized by TEM, UV-vis, photoluminescence, and FT-IR spectra. The GQDs had carboxyl groups with a negative charge, which not only provided good stability, but also enabled interaction with amine functional groups in DA through electrostatic interaction to enhance the specificity of DA. The interaction and electron communication between GQDs and DA can be further strengthened via π-π stacking force. Nafion was used as an anchoring agent to increase the robustness of GQDs on the electrode surface and sensor stability and reproducibility. The GQDs-Nafion composite exhibits a good linear range of 5 nM to 100 μM and a limit of detection as low as 0.45 nM in the detection of DA. The proposed electrochemical sensor also displays good selectivity and high stability and could be used for the determination of DA in real samples with satisfactory results. The present study provides a powerful avenue for the design of an ultrasensitive detection method for clinical application.

A forward and backward secure key management in wireless sensor networks for PCS/SCADA

Process Control Systems (PCSs) or Supervisory Control and Data Acquisition (SCADA) systems have recently been added to the already wide collection of wireless sensor networks applications. The PCS/SCADA environment is somewhat more amenable to the use of heavy cryptographic mechanisms such as public key cryptography than other sensor application environments. The sensor nodes in the environment, however, are still open to devastating attacks such as node capture, which makes designing a secure key management challenging. In this paper, a key management scheme is proposed to defeat node capture attack by offering both forward and backward secrecies. Our scheme overcomes the pitfalls which Nilsson et al.'s scheme suffers from, and is not more expensive than their scheme.

Fusion of in-vehicle sensor data to develop Intelligent Driver Training System (IDTS)

The over represented number of novice drivers involved in crashes is alarming. Driver training is one of the interventions aimed at mitigating the number of crashes that involve young drivers. To our knowledge, Advanced Driver Assistance Systems (ADAS) have never been comprehensively used in designing an intelligent driver training system. Currently, there is a need to develop and evaluate ADAS that could assess driving competencies. The aim is to develop an unsupervised system called Intelligent Driver Training System (IDTS) that analyzes crash risks in a given driving situation. In order to design a comprehensive IDTS, data is collected from the Driver, Vehicle and Environment (DVE), synchronized and analyzed. The first implementation phase of this intelligent driver training system deals with synchronizing multiple variables acquired from DVE. RTMaps is used to collect and synchronize data like GPS, vehicle dynamics and driver head movement. After the data synchronization, maneuvers are segmented out as right turn, left turn and overtake. Each maneuver is composed of several individual tasks that are necessary to be performed in a sequential manner. This paper focuses on turn maneuvers. Some of the tasks required in the analysis of ‘turn’ maneuver are: detect the start and end of the turn, detect the indicator status change, check if the indicator was turned on within a safe distance and check the lane keeping during the turn maneuver. This paper proposes a fusion and analysis of heterogeneous data, mainly involved in driving, to determine the risk factor of particular maneuvers within the drive. It also explains the segmentation and risk analysis of the turn maneuver in a drive.

Towards continuous surveillance of fruit flies using sensor networks and machine vision

In Australia, the Queensland fruit fly (B. tryoni), is the most destructive insect pest of horticulture, attacking nearly all fruit and vegetable crops. This project has researched and prototyped a system for monitoring fruit flies so that authorities can be alerted when a fly enters a crop in a more efficient manner than is currently used. This paper presents the idea of our sensor platform design as well as the fruit fly detection and recognition algorithm by using machine vision techniques. Our experiments showed that the designed trap and sensor platform is capable to capture quality fly images, the invasive flies can be successfully detected and the average precision of the Queensland fruit fly recognition is 80% from our experiment.

Biomechanical force displacement analysis of strength of fixation of tracker holding devices to bone in computer aided joint replacement

Computer aided joint replacement surgery has become very popular during recent years and is being done in increasing numbers all over the world. The accuracy of the system depends to a major extent, on accurate registration and immobility of the tracker attachment devices to the bone. This study was designed to asses the forces needed to displace the tracker attachment devices in the bone simulators. Bone simulators were used to maintain the uniformity of the bone structure during the study. The fixation devices tested were 3mm diameter self drilling, self tapping threaded pin, 4mm diameter self tapping cortical threaded pin, 5mm diameter self tapping cancellous threaded pin and a triplanar fixation device ‘ortholock’ used with three 3mm pins. All the devices were tested for pull out, translational and rotational forces in unicortical and bicortical fixation modes. Also tested was the normal bang strength and forces generated by leaning on the devices. The forces required to produce translation increased with the increasing diameter of the pins. These were 105N, 185N, and 225N for the unicortical fixations and 130N, 200N, 225N for the bicortical fixations for 3mm, 4mm and 5mm diameter pins respectively. The forces required to pull out the pins were 1475N, 1650N, 2050N for the unicortical, 1020N, 3044N and 3042N for the bicortical fixated 3mm, 4mm and 5mm diameter pins. The ortholock translational and pull out strength was tested to 900N and 920N respectively and still it did not fail. Rotatory forces required to displace the tracker on pins was to the magnitude of 30N before failure. The ortholock device had rotational forces applied up to 135N and still did not fail. The manual leaning forces and the sudden bang forces generated were of the magnitude of 210N and 150N respectively. The strength of the fixation pins increases with increasing diameter from three to five mm for the translational forces. There is no significant difference in pull out forces of four mm and five mm diameter pins though it is more that the three mm diameter pins. This is because of the failure of material at that stage rather than the fixation device. The rotatory forces required to displace the tracker are very small and much less that that can be produced by the surgeon or assistants in single pins. Although the ortholock device was tested to 135N in rotation without failing, one has to be very careful not to put any forces during the operation on the tracker devices to ensure the accuracy of the procedure.