No description available.

O presente trabalho abordou o sistema de Tomografia por Coerência Óptica (OCT) e sua aplicação à área de microfluídica. Para tanto, foram realizados testes de caracterização física de circuitos microfluídicos, utilizando modelos 3D (tridimensionais) construídos a partir de imagens de OCT destes circuitos. A técnica foi, assim, avaliada como potencial ferramenta de auxílio na aferição de microcanais. Indo além, este trabalho estuda e desenvolve técnicas de análise para fluxos microfluídicos, em especial técnicas baseadas no padrão de speckle. Em primeiro momento, métodos já existentes foram estudados e aprimorados, como o Speckle Variance OCT, em que foi obtido um ganho de 31% em tempo de processamento. Outros métodos, como o LASCA (Laser speckle Contrast Analysis), baseados na autocorrelação de speckle, são adaptados às imagens de OCT. Derivado do LASCA, o método de análise desenvolvido baseado na autocorrelação de intensidade motivou o desenvolvimento de um arranjo de OCT próprio e software de aquisição customizado, com taxa de amostragem da ordem de 8 kHz. O método proposto foi, então, capaz de distinguir fluxos volumétricos variados, e seus limites de detecção foram testados, comprovando sua viabilidade de aplicação para análise de movimento browniano e fluxos volumétricos abaixo de 10 μl/min. / This work discusses the Optical Coherence Tomography system (OCT) and its application to the microfluidics area. To this end, physical characterization of microfluidic circuits were performed using 3D (three-dimensional) models constructed from OCT images of such circuits. The technique was thus evaluated as a potential tool to aid in the inspection of microchannels. Going further, this workpaper studies and develops analytical techniques for microfluidic flow, in particular techniques based on speckle pattern. In the first instance, existing methods were studied and improved, such as Speckle Variance - OCT, where a gain of 31% was obtained in processing time. Other methods, such as LASCA (Laser Speckle Contrast Analysis), based on speckle autocorrelation, are adapted to OCT images. Derived from LASCA, the developed analysis technique based on intensity autocorrelation motivated the development of a custom OCT system as well as an optimized acquisition software, with a sampling rate of 8 kHz. The proposed method was, then, able to distinguish different flow rates, and limits of detection were tested, proving its feasibility for implementation on Brownian motion analysis and flow rates below 10 μl/min.

O presente trabalho abordou o sistema de Tomografia por Coerência Óptica (OCT) e sua aplicação à área de microfluídica. Para tanto, foram realizados testes de caracterização física de circuitos microfluídicos, utilizando modelos 3D (tridimensionais) construídos a partir de imagens de OCT destes circuitos. A técnica foi, assim, avaliada como potencial ferramenta de auxílio na aferição de microcanais. Indo além, este trabalho estuda e desenvolve técnicas de análise para fluxos microfluídicos, em especial técnicas baseadas no padrão de speckle. Em primeiro momento, métodos já existentes foram estudados e aprimorados, como o Speckle Variance OCT, em que foi obtido um ganho de 31% em tempo de processamento. Outros métodos, como o LASCA (Laser speckle Contrast Analysis), baseados na autocorrelação de speckle, são adaptados às imagens de OCT. Derivado do LASCA, o método de análise desenvolvido baseado na autocorrelação de intensidade motivou o desenvolvimento de um arranjo de OCT próprio e software de aquisição customizado, com taxa de amostragem da ordem de 8 kHz. O método proposto foi, então, capaz de distinguir fluxos volumétricos variados, e seus limites de detecção foram testados, comprovando sua viabilidade de aplicação para análise de movimento browniano e fluxos volumétricos abaixo de 10 μl/min. / This work discusses the Optical Coherence Tomography system (OCT) and its application to the microfluidics area. To this end, physical characterization of microfluidic circuits were performed using 3D (three-dimensional) models constructed from OCT images of such circuits. The technique was thus evaluated as a potential tool to aid in the inspection of microchannels. Going further, this workpaper studies and develops analytical techniques for microfluidic flow, in particular techniques based on speckle pattern. In the first instance, existing methods were studied and improved, such as Speckle Variance - OCT, where a gain of 31% was obtained in processing time. Other methods, such as LASCA (Laser Speckle Contrast Analysis), based on speckle autocorrelation, are adapted to OCT images. Derived from LASCA, the developed analysis technique based on intensity autocorrelation motivated the development of a custom OCT system as well as an optimized acquisition software, with a sampling rate of 8 kHz. The proposed method was, then, able to distinguish different flow rates, and limits of detection were tested, proving its feasibility for implementation on Brownian motion analysis and flow rates below 10 μl/min.

The focus of this work is to present in detail the implementation of a three dimensional direct simulation Monte Carlo methodology on unstructured Delaunay meshes (U-DSMC). The validation and verification of the implementation are shown using a series of fundamental flow cases. The numerical error associated with the implementation is also studied using a fundamental flow configuration. Gas expansion from microtubes is studied using the U-DSMC code for tube diameters ranging from 100Æ’ÃÂ�m down to 100nm. Simulations are carried out for a range of inlet Knudsen numbers and the effect of aspect ratio and inlet Reynolds number on the plume structure is investigated. The effect of scaling the geometry is also examined. Gas expansion from a conical nozzle is studied using the U-DSMC code for throat diameters ranging from 250 Æ’ÃÂ�m down to 250 nm. Simulations are carried out for a range of inlet Knudsen numbers and the effect of inlet speed ratio and inlet Reynolds number on the plume structure is investigated. The effect of scaling the geometry is examined. Results of a numerical study using the U-DSMC code are employed to guide the design of a micropitot probe intended for use in analyzing rarefied gaseous microjet flow. The flow conditions considered correspond to anticipated experimental test cases for a probe that is currently under development. The expansion of nitrogen from an orifice with a diameter of 100Æ’ÃÂ�m is modeled using U-DSMC. From these results, local ¡¥free stream¡¦ conditions are obtained for use in U-DSMC simulations of the flow in the vicinity of the micropitot probe. Predictions of the pressure within the probe are made for a number of locations in the orifice plume. The predictions from the U-DSMC simulations are used for evaluating the geometrical design of the probe as well as aiding in pressure sensor selection. The effect of scale on the statistical fluctuation of the U-DSMC data is studied using Poiseuille flow. The error in the predicted velocity profile is calculated with respect to both first and second-order slip formulations. Simulations are carried out for a range of channel heights and the error between the U-DSMC predictions and theory are calculated for each case. From this error, a functional dependence is shown between the scale-induced statistical fluctuations and the decreasing channel height.

Cyanobacteria, also known as blue-green algae for a long time, are the most ancient and problematic bloom-forming phylum on earth. An alert levels framework has been established by World Health Organization(WHO) to prevent the potential harmful cyanobacterial blooms. Normally, low cyanobacteria levels are found in surface water. 2000 cyanobacterial cells/mL and 100,000 cyanobacterial cells/mL are established for WHO Alert Level 1 and 2, respectively. However, eutrophication, climate change and other factors may promote the spread of cyanobacteria and increase the occurrence of harmful cyanobacterial blooms in water on a global scale. Hence, a rapid real time cyanobacteiral monitoring system is required to protect public health from the cyanotoxins produced by toxic cyanobacterial species. Current methods to control or prevent the development of harmful cyanobacterial blooms are either expensive, time consuming or not effective in the long term. The best method to control the blooms is to prevent the formation of the blooms at the very beginning. Although emerging advanced autofluorescence-based sensors, imaging flow cytometry applications, and remote sensing have been utilized for rapid real-time enumeration and classification of cyanobacteria, the need to accurately monitor low-level cyanobacterial species in water remains unsolved. Microflow cytometry has been employed as a functional cell analysis technique in past decades, and it can provide real-time, accurate results. The autofluorescence of cyanobacterial pigments can be used for determination and counting of cyanobacterial density in water. A pre-concentration system of an automated cyanobacterial concentration and recovery system (ACCRS) based on tangential flow filtration and back-flushing technique was applied to reduce the sample assay volume and increase the concentration of target cells for further cell capture and detection. In this project, a microflow cytometry platform with a microfluidic device and an automated pre-concentration system was established to monitor cyanobacteria and provide early warning alerts for potential harmful blooms. In this work, quantification of low-level cyanobacterial samples (∼ 5 cyanobacterial cells/mL) in water has been achieved by using a microflow cytometer together with a pre-concentration system (ACCRS). Meanwhile, this platform can also provide early warning alerts for potential harmful cyanobacterial blooms at least 15 days earlier before reaching WHO Alert Level 1. Results have shown that this platform can be applied for rapid determination of cyanobacteria and early warning alerts can be triggered for authorities to protect the public and the environment. / Thesis / Doctor of Engineering (DEng) / Harmful cyanobacterial blooms have been a rising risk to the public heath across the world in recent decades. Alert levels of cyanobacteria in water has also been established. In this case, a rapid on-side monitoring system for cyanobacteria is required. In this thesis, a microflow cytometer platform combined with a bacterial concentration and recovery system was built to quickly monitor the relatively low level of cyanobacteria for early warning alerts. A pre-enrichment system based on tangential flow filtration and back-flushing technique was applied to increase the concentration levels of microbial samples and a microfluidic device capable of collecting phycocyanin fluorescence was designed to count cyanobacterial cells. The limit of quantification for cyanobacterial concentration based on the microflow cytometry platform was as low as ∼ 5 cells/mL. We can claim that the microflow cytometry platform can provide useful early warning alerts for the decision-makers to control the potential harmful cyanobacterial blooms at the very early stage and protect the aquatic animals and public health.

No description available.

The conventional battery for genotoxicity testing is not well-suited to assessing the large number of chemicals needing evaluation. Traditional in vitro tests lack throughput capacity, provide little mechanistic information, and have poor specificity in predicting in vivo genotoxicity. The Health Canada GeneTox21 research program is developing a multi-endpoint platform for modernized in vitro genotoxicity assessment. The GeneTox21 assays include the TGx-DDI transcriptomic biomarker (i.e., 64-gene expression signature to identify DNA damage-inducing (DDI) substances), the MicroFlow® assay (i.e., a flow cytometry-based micronucleus (MN) test), and the MultiFlow® assay (i.e., a multiplexed flow cytometry-based reporter assay that yields mechanism-of-action (MoA) information). As part of GeneTox21 development, the objective of this study was to investigate the utility of the TGx-DDI transcriptomic biomarker, multiplexed with the MicroFlow® and MultiFlow® assays, as an integrated testing strategy for screening data-poor substances prioritized by Health Canada’s New Substances Assessment and Control Bureau. Human lymphoblastoid TK6 cells were exposed to 3 control and 10 data-poor substances, using a 6-point concentration range. Cells were exposed for 4 hours with or without exogenous metabolic activation. Gene expression profiling was conducted using the targeted TempO-SeqTM assay, and the TGx-DDI classifier was applied to the dataset. Classifications were compared with those based on the MicroFlow® and MultiFlow® assays. Benchmark Concentration (BMC) modeling was used for potency ranking. The results of the integrated hazard calls indicate that five data-poor compounds are genotoxic in vitro, causing DNA damage via a clastogenic MoA, and one is positive via a pan-genotoxic MoA. Two compounds are likely irrelevant positives in the MN test; two are considered possibly genotoxic causing DNA damage via an ambiguous MoA. From quantitative analyses of concentration-response data, we observed nearly identical potency rankings for each assay with two main potency groups being observed. This ranking was maintained when all endpoint BMCs were converted into a single score using the Toxicological Prioritization (ToxPi) approach. Overall, this study contributes to the establishment of a modernized approach for effective genotoxicity assessment and chemical prioritization for further regulatory scrutiny. We conclude that integration of the TGx-DDI biomarker with other GeneTox21 assays is an effective NAM-based strategy for genotoxicity assessment of data-poor compounds.

No description available.

Mass spectrometry is being increasingly used in biomarker research mainly due to its ability to achieve high selectivity coupled with high sensitivity. This dissertation focuses on quantitative mass spectrometric investigations of two protein biomarkers i.e. serum thymidine kinase 1 (TK1) and human osteopontin (OPN). First part of this research was focused on developing a liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) method for measuring the activity of TK1 in serum by monitoring the conversion of a TK1 specific exogenous substrate, 3’-deoxy-3’-fluorothymidine (FLT), to its mono-phosphorylated form 3’-deoxy-3’-fluorothymidine monophosphate (FLT-MP). A method to quantify FLT-MP on LC-MS/MS was developed and validated. The method was linear over the range of 2.5-2000 ng/mL with a mean correlation coefficient of 0.9935. Using the developed method, serum TK1 activity was measured in serum from hepatocellular carcinoma (HCC) patients and age-matched controls under standardized conditions. A sub-population of the HCC patient samples showed an almost 20-fold enhanced TK1 activity compared to the controls. A method was developed and validated for quantifying human osteopontin from plasma using immunoaffinity isolations coupled with microflow liquid chromatography and tandem mass spectrometry (MFLC-MS/MS). A biologically relevant tryptic peptide ‘GDSVVYGLR’ which is unique to hOPN was identified and used as a signature peptide for this method. The method was validated over a range of 25-600 ng/mL. The performance of the method was compliant with USFDA validation guidance. In addition, a stable isotope labeled (SIL) peptide GDSVVYGLR* and an extended SIL peptide TYDGRGDSVV*YGLRSKSKKF’ were evaluated as internal standards (IS) to account for signature peptide digestion instability and variability. In the digestion variability studies, the use of extended SIL peptide as internal standard limited the total variability within ±30%. Alternatively, when SIL peptide was used as internal standard the variability ranged from -67.4% to +50.6 %. The applicability of the validated method was demonstrated by analyzing plasma samples obtained from 10 healthy individuals and 10 breast cancer patients. More than 9-fold increase in the mean plasma hOPN concentration was seen in 30% of the breast cancer patient samples (n=10) in comparison to the healthy volunteer samples. In a proof of concept investigation, a stable isotope labeled signature peptide was evaluated as an internal standard to compensate for immunocapture variability during quantification of human osteopontin (hOPN) by immunoaffinity coupled LC-MS/MS. Immunocapture variability was induced by varying the antibody amount per well. The immunocapture variability ranged from -80.9 % to +77.0 % when the IS was added after immunocapture and from -37.5% to +20.3% when the IS was added before immunocapture. The lower variability demonstrates the ability of SIL-IS peptide to compensate for variation during immunocapture.

Fluid suspended biological particles (bioparticles) flowing through a non-uniform electric field are actuated by the induced dielectrophoretic (DEP) force, known to be dependent upon the bioparticles’ dielectric phenotypes. In this work: a 10-1000 kHz DEP actuation potential applied to a co-planar microelectrode array (MEA) induces a DEP force, altering passing bioparticle trajectories as monitored using: (1) an optical assay, in which the lateral bioparticle velocities are estimated from digital video; and (2) a capacitive cytometer, in which a 1.478 GHz capacitance sensor measures the MEA capacitance perturbations induced by passing bioparticles, which is sensitive to the bioparticles’ elevations. The experimentally observed and simulated lateral velocity profiles of actuated polystyrene microspheres (PSS) and viable and heat shocked Saccharomyces cerevisiae cells verify that the bioparticles’ dielectric phenotypes can be inferred from the resultant trajectories due to the balance between the DEP force and the viscous fluid drag force.