In-Column Electrochemical Detection for Liquid Chromatography

This research focuses on the development of whole column detection (WCD) for liquid chromatography (LC). The WCD uses electrochemical techniques for detecting the analytes passing through the separation column. Electrode array for in-column electrochemical detection (ICED) is fabricated along the separation column to enable whole column separation monitoring and allow better understanding on the affinity of particular analyte to the stationary and mobile phases. Numerical models were built to understand the feasibility and differences of electrochemical detection within an unpacked and packed column. From the simulated results, the surface area of the electrode was not hindered by the presence of the particles in flow condition. An electrochemical microfluidic device has been successfully fabricated on PET (polyethylene terephthalate) substrate using the reverse imprinting technique. The photolithographically produced gold metal electrode lines were imprinted into the PET substrate using a blank mould and produced an inlaid electrode array with overall step residue within 40 nm. The semi-cured thermoset polyester channel was irreversibly thermal bonded on the PET substrate. The devices were able to tolerate pressure in excess of 90 bars. The PET column was packed with 5 μm C18 silica beads to perform reverse phase chromatography separation. The array was electrochemically characterised using standard redox probes in both stagnant conditions and under flow. Both numerical modelling and experimental data show improved sensitivity under flow and a limiting current which scaled linearly with cubic root of volume flow rate. Isocratic and gradient mode chromatographic separations of neurotransmitters and metabolites: serotonin, dopamine, adrenaline, 5- HIAA and DOPAC were conducted in the fabricated device. Separation progress was electrochemically detected at multiple locations along the column. Whole column assessment on separation efficiency and column packing efficiency monitoring were conducted using the ICED

Innovative system for deploying novel biosensors for water contaminant monitoring

The ability to remotely sense contaminants in surface and sub-surface waters can offer a deeper understanding of environmental conditions and an early warning system that will improve the efficiency of response to contaminant transport. The aim of this thesis was to develop an innovative system for field deployment of biosensors to facilitate environmental water quality monitoring. Two strands of work were addressed in developing themonitoring system (i) a flow cell in which to deploy the biosensor and (ii) integration of a uranyl ion (UO₂²⁺) biosensor. Three different flow cell designs were investigated using both a finite element computational model and a flow-fluorescence experimental technique. These comprised a flow channel featuring an expanded central region to accommodate the sensor, but the rates of channel expansion were varied between each design. A reduction in flow cell efficiency with increasing flow rate for all three cell designs was linked to the development of regions of flow recirculation (eddies). However, the most gradually expanding flow channel restricted eddy development to higher flow rates in comparison to the other designs. This more efficient design, and an optimised operational protocol, was thus identified as a recommended method of biosensor deployment. A biosensor for the detection of aqueous uranyl ions (UO₂²⁺) was developed from a sensor element created by Conroy (2012). Two different integration methods, screen-printed electrodes and solid gold electrodes for biosensor construction, were investigated. The biosensor response was linked to electrode nano-surface topography and electrode chemical composition, and was observed to be higher for the solid gold electrodes. The operational dynamic range of the integrated biosensor was improved by four orders of magnitude in comparison to the original laboratory proof of concept investigations for the sensor. Furthermore, recommendations for operational protocols were developed with respect to optimisation of integrated biosensor operation within a remotely controlled and automated water monitoring system

Electrochemical Biosensors for On-line Monitoring of Cell Culture Metabolism

Current research in the biotechnological field is hampered by the lack of available technologies dedicated to cell monitoring. While on the one hand physicochemical parameters, such as pH, temperature, cell density and adhesion, can be monitored quite easily with automated systems, on the other the variation of cell metabolism is still challenging. Indeed, the real-time detection of metabolites can noticeably extend the knowledge of the molecular biology for therapeutic purposes, as well as for the investigation of several types of diseases. Electrochem- ical biosensors are the ideal candidates for cell monitoring, since they can be integrated with the electronic portion of the system, leading to high-density arrays of biosensors with better performance in terms of signal-to-noise ratio, sensor response, and sample volumes. The present research covers the design, the fabrication, the characterization, and the valida- tion of a minimally-invasive system for the real-time monitoring of different metabolites in a cell culture. The electrochemical biosensor consists of an array of gold working electrodes accomplished by standard microfabrication processes. The deposition of carbon nanotubes and the selective modification with enzymes onto metallic electrodes is performed by adapt- ing an ultra-low volume dispensing system for DNA and protein drop cast. The biological sensing element ensures high selectivity for the target molecule to detect, while nanomate- rials confer superior performance (e.g. sensitivity) with respect to standard immobilization strategies. The on-line detection of glucose, lactate, and glutamate is achieved with an ad hoc fluidic system. The use of a microdialysis probe in direct contact with the cell culture avoids contamination problems and dilution steps for metabolite measurements. Carbon nanotube-based biosensors and the system for real-time measurements are validated on two cell lines under different experimental conditions. The electronic system for electrochemical measurements is also designed and realized with discrete components to be interfaced with the platform. The adopted architecture is able to optimally record the current ranges involved in the electrochemical cell, while the wireless communication between the electronic system and the remote station ensures minimally invasiveness and high portability of the device. Existing technologies and materials are used in an original manner to achieve the on-line monitoring of metabolites in stem cell-like cultures, paving the way for the development of miniaturized, high-sensitive, and inexpensive devices for continuous cell monitoring

Fluidic Dielectrophoresis: Electrokinetic Polarization and Manipulation of Electrical Liquid Interfaces for Biological and Sensing Applications

Development of rapid, sensitive and portable detection systems are important for effective detection of diseases in developing countries, biowarfare/anti-terrorism applications, environmental monitoring, and for basic biological research. One of the most specific and popular sensing platform is the enzyme-linked immunosorbent assay (ELISA) platform, which identifies the presence of a substance, specifically an antigen, in a liquid sample. Biosensing assays, like ELISA, offer sensitivity and selectivity, however, its assay time is long due to immobilization and detection through a secondary antibody. The assay also requires periodic rinsing steps to avoid non-specific binding and to remove excess proteins. Finally, the fluorescent detection instrumentation is required, and is still too bulky and costly for widespread daily laboratory, clinical and point-of-care use. The main challenge for producing low cost, portable, and easy to operate biosensing systems is then to miniaturize the sensing platform without any sophisticated instrumentation and complicated reagent protocols. In this work, we first explore a well-known electrokinetic phenomenon called dielectrophoresis (DEP), which traditionally has been studied in cells and particles, but here at a liquid-liquid interface, which we call fluidic dielectrophoresis (fDEP). The liquid-liquid interface with disparaging electrical properties - conductivity and permittivity - is shown to move when subjected to an alternating current (ac) electric field and the direction and magnitude is studied at varying applied frequencies and voltages. We found that when a biomolecular reaction occurs at the liquid-liquid interface it alters the electrical properties, which is transduced by interfacial displacement; we call this novel transduction method interfacial electrokinetic transduction (IET). We began with a model biomolecular reaction between biotin and avidin to validate our detection scheme. We then performed detection of hCG in human serum. Finally, we implemented impedance spectroscopy to non-optical monitor the position of the interface. Coupled with IET, the system non-optically monitored the position of the electrical interface in the presence of a biomolecular reaction. Collectively, we successfully developed the first, in solution, label-free non-optical biosensor. This novel biosensor was shown to detect biomarkers down to femtomolar concentration in human serum within minutes

Characterization and development towards electrochemical real-time LAMP detection in an integrated and portable device

Dissertação de mestrado integrado em Engenharia Biomédica (área de especialização em Eletrónica Médica)Nos últimos anos os biossensores eletroquímicos têm sido reportados como uma abordagem promis sora para a deteção de DNA. São atrativos para dispositivos point-of-care pela facilidade de miniaturização, compatibilidade com técnicas de microfabricação e facilidade de instrumentação. Contudo, a integração de uma fase de amplificação com a deteção de sinais continua a ser um desafio. A técnica LAMP (loop mediated isothermal amplification ) surgiu como solução, destacando-se pela sua robustez e sensibilidade. Uma plataforma portátil para deteção eletroquímica de DNA (Mobi-E) foi desenvolvida para incorporar as caraterísticas mencionadas. Neste trabalho foram caraterizados os principais componentes do disposi tivo: potencióstatos ASIC, controlo de temperatura e um chip fluídico que integra elétrodos e estruturas de aquecimento impressas. Seguidamente, efetuou-se uma pesquisa para funcionalização universal desses elétrodos utilizando MD LAMP (mediator displacement LAMP). O desempenho do potencióstato, especificamente a rotina de voltametria de onda quadrada e a leitura quase simultânea dos 6 elétrodos de trabalho de uma câmara, foi provado e comparado com um dispositivo comercial, obtendo-se resultados qualitativamente comparáveis. Foram ainda realizadas otimizações para estabilização do potencial de referência, através da conversão Ag/AgCl por FeCl3, e para precaver dificuldades de medição resultantes da limitada tensão de conformidade do potencióstato (1.8 V). Para tal, aumentou-se a área do elétrodo auxiliar, através da impressão de 8 camadas de Au (4 no elétrodo de trabalho), e efetuou-se, entre medições, o curto-circuito entre o elétrodo auxiliar e o de referência. O sistema de controlo de temperatura provou ser efetivo no aquecimento das câmaras até 65 ºC em 2.5 min e na monitorização assertiva das suas temperaturas. Não foi detetado cross-talk entre câmaras vizinhas, nem a formação severa de bolhas de ar. Como prova de conceito, foram testadas quatro abordagens para MD LAMP e foi identificada uma alternativa promissora (Stem-Loop ) ao standard, com a respetiva mudança relativa de sinal: 0.3 e 177.6. Esta dissertação culmina com a otimização da reação LAMP para uma libertação eficiente do medi ador. Duas promissoras combinações de concentrações de mediador, primer modificado e loop primer foram identificadas: 200 nM, 400 nM, 400 nM e 100 nM, 200 nM, 600 nM, respetivamente, obtendo-se uma relação sinal/ruído de 3.7 e 2.9. Os conhecimentos adquiridos viabilizam o avanço da investigação no sentido da obtenção de um dispositivo capaz de realizar LAMP eletroquímico em tempo real.Electrochemical biosensors have been reported in recent years as a promising approach for DNA detection. The ease of miniaturization, compatibility with microfabrication techniques and simple instru mentation make these sensors attractive for point-of-care (POC) devices. However, integrating an ampli fication stage with signal detection remains a challenge. Loop-mediated isothermal amplification (LAMP) has emerged as a robust and highly sensitive isothermal strategy. A novel portable platform for rapid electrochemical DNA detection (Mobi-E device) was developed to incorporate the aforementioned features. Within this work, an extensive characterization of the main components of the Mobi-E device, which comprises ASIC potentiostats, temperature control, and a fluidic chip with integrated inkjet-printed electrodes and heating structures, was performed, followed by a research towards target-independent electrode functionalization employing mediator displacement (MD) LAMP. The operation of the general functionalities of the ASIC potentiostat, specifically the square wave voltammetry (SWV) routine and the quasi-simultaneous read-out of all 6 working electrodes (WEs) of a chamber, was proven and its performance was benchmarked against a commercial device, achieving qualitatively comparable results. Inherent optimizations of the device were performed to stabilize the reference electrode (RE) potential, through Ag/AgCl conversion by FeCl3, and to address the criticality that the 1.8 V compliance voltage of the potentiostat brought to the measurements. For this issue, it is suggested to increase the counter electrode (CE) area by printing 8 Au layers (WE with 4 layers) and to short-circuit the CE and RE between measurements. The temperature control system proved to be effective in heating the chambers to 65 ºC in about 2.5 min and assertively monitoring their temperature. Furthermore, no cross-talk between neighbouring chambers and no severe bubble formation was detected. Four concepts for target-independent MD LAMP were tested as proof-of-concept and a promising alternative (”Stem-Loop universal reporter (UR)”) to the standard approach was identified. The following relative signal change was achieved: 0.3 and 177.6, respectively. This thesis culminates with the optimization of fluorescent LAMP reaction parameters towards an efficient mediator release. Two promising concentration combinations of mediator, modified primer (LB_Medc) and loop primer (LB) were identified: 200 nM, 400 nM, 400 nM and 100 nM, 200 nM, 600 nM, respectively. For these, the signal-to-noise ratio was 3.7 and 2.9. The knowledge acquired in this thesis allows moving forward towards a device able to perform electrochemical real-time LAMP readout

Detection of Pathogens in Water Using Micro and Nano-Technology

Detection of Pathogens in Water Using Micro and Nano-Technology aims to promote the uptake of innovative micro and nano-technological approaches towards the development of an integrated, cost-effective nano-biological sensor useful for security and environmental assays.  The book describes the concerted efforts of a large European research project and the achievements of additional leading research groups. The reported knowledge and expertise should support in the innovation and integration of often separated unitary processes. Sampling, cell lysis and DNA/RNA extraction, DNA hybridisation detection micro- and nanosensors, microfluidics, together also with computational modelling and risk assessment can be integrated in the framework of the current and evolving European regulations and needs. The development and uptake of molecular methods is revolutionizing the field of waterborne pathogens detection, commonly performed with time-consuming cultural methods. The molecular detection methods are enabling the development of integrated instruments based on biosensor that will ultimately automate the full pathway of the microbiological analysis of water

Detection of Pathogens in Water Using Micro and Nano-Technology

Detection of Pathogens in Water Using Micro and Nano-Technology aims to promote the uptake of innovative micro and nano-technological approaches towards the development of an integrated, cost-effective nano-biological sensor useful for security and environmental assays.  The book describes the concerted efforts of a large European research project and the achievements of additional leading research groups. The reported knowledge and expertise should support in the innovation and integration of often separated unitary processes. Sampling, cell lysis and DNA/RNA extraction, DNA hybridisation detection micro- and nanosensors, microfluidics, together also with computational modelling and risk assessment can be integrated in the framework of the current and evolving European regulations and needs. The development and uptake of molecular methods is revolutionizing the field of waterborne pathogens detection, commonly performed with time-consuming cultural methods. The molecular detection methods are enabling the development of integrated instruments based on biosensor that will ultimately automate the full pathway of the microbiological analysis of water

Mass transport aspects of polymer electrolyte fuel cells under two-phase flow conditions

Die Visualisierung und Quantifizierung von Flüssigwasseransammlungen in Polymerelektrolytmembran-Brennstoffzellen konnte mittels Neutronenradiographie erreicht werden. Dank dieser neuartigen diagnostischen Methode konnte erstmals die Flüssigwasseransammlung in den porösen Gasdiffusionsschichten direkt nachgewiesen und quantifiziert werden. Die Kombination von Neutronenradiographie mit ortsaufgelösten Stromdichtemessungen bzw. lokaler Impedanzspektroskopie erlaubte die Korrelation des inhomogenen Flüssigwasseranfalls mit dem lokalen elektrochemischen Leistungsverhalten. Systematische Untersuchungen an Polymerelektrolyt- und Direkt-Methanol-Brennstoffzellen verdeutlichen sowohl den Einfluss von Betriebsbedingungen als auch die Auswirkung von Materialeigenschaften auf die Ausbildung zweiphasiger Strömungen

Numerical and experimental study of flow and wall mass transfer rates in capillary driven flows in microfluidic channels

Micro-channels are believed to open up the prospect of precise control of fluid flow and chemical reactions. The capillary effect can be used to pump fluids in micro-channels and the flow generated can dissolve chemicals previously deposited on the walls of the channel. In this work, numerical and experimental approaches have been developed to investigate the wall mass transfer rate generated by capillary driven flows (CD-Flow). The purpose of this work is to analyze the wall mass transfer rates generated by a CD-Flow in a micro-channel. The results have implications in the optimization and design of devices for biological assays. The correlation for Sherwood number, Reynolds number, contact angle and time is reported. This correlation can be a useful tool for design purposes of microfluidic devices that work with fast heterogeneous reaction and have capillary driven flow as passive pumping system. The numerical results have been confirmed by the experimental results.La perspectiva del uso de micro-canales para el control preciso del flujo y de las reacciones químicas está ampliamente aceptada. Considerando que el efecto de las tensiones superficiales en la micro-escala es significativo, el bombeo pasivo basado en el uso de la tensión superficial para los Lab-on-a-chip resulta ser el método más eficaz.El propósito de este trabajo es analizar la transferencia de masa en la pared en un campo dinámico de un flujo impulsado por capilaridad. Los resultados permitirán mejorar el diseño y optimizar los dispositivos para ensayos biológicos. Se presenta una correlación entre el número de Sherwood, el número de Reynolds, el ángulo de contacto y el tiempo. La correlación puede ser una herramienta útil en el diseño de dispositivos microfluídicos que trabajen con una reacción rápida y heterogénea y usen el bombeo pasivo impulsado por el flujo capilar. Los resultados numéricos han sido confirmados por los resultados experimentales

Microfluidics and Nanofluidics Handbook

The Microfluidics and Nanofluidics Handbook: Two-Volume Set comprehensively captures the cross-disciplinary breadth of the fields of micro- and nanofluidics, which encompass the biological sciences, chemistry, physics and engineering applications. To fill the knowledge gap between engineering and the basic sciences, the editors pulled together key individuals, well known in their respective areas, to author chapters that help graduate students, scientists, and practicing engineers understand the overall area of microfluidics and nanofluidics. Topics covered include Finite Volume Method for Numerical Simulation Lattice Boltzmann Method and Its Applications in Microfluidics Microparticle and Nanoparticle Manipulation Methane Solubility Enhancement in Water Confined to Nanoscale Pores Volume Two: Fabrication, Implementation, and Applications focuses on topics related to experimental and numerical methods. It also covers fabrication and applications in a variety of areas, from aerospace to biological systems. Reflecting the inherent nature of microfluidics and nanofluidics, the book includes as much interdisciplinary knowledge as possible. It provides the fundamental science background for newcomers and advanced techniques and concepts for experienced researchers and professionals