No description available.

Cette thèse concerne la détection des métaux lourds dans l’environnement et en particulier dans les eaux de surface et les sous-produits de désinfection de l’eau potable. Les deux catégories de contaminations ont des propriétés différentes de sorte que deux méthodes correspondantes ont été dévéloppées : l’une est basée sur des capteurs moléculaires fluorescents mis en oeuvre dans un micro-dispositif, l’autre est basée sur une détection électrochimique. Deux capteurs moléculaires fluorescents, Rhod-5N et DPPS-PEG, et plusieurs dispositifs microfluidiques ont été fabriqués et appliqués pour la détection des ions de métaux lourds, Cd (II) et Hg (II),dans les eaux de surface. Une nouveau circuit en PMMA est fabriqué par ablation laser femtoseconde et testé pour la détection de Cd2+ avec le Rhod-5N. De plus, des améliorations de la performance des circuits microfluidiques ont été faites. Une nouvelle méthode de détermination sensible de cinq acides haloacétiques (AHAs) dans les d'eaux a été développée. Elle est basée sur l'extraction électromembranaire (EME) avant électrophorèse capillaire avec détection de conductivité sans contact à couplage capacitif (CE-C4D). / This thesis is aimed at environmental contaminations detection, mainly heavy metal ions in surface water and disinfection by-products (DBPs) in drinking water. The two categories of contaminations have different properties so that two correspondent methods were developed: one is based on fluorescent molecular sensors in a microfabricated device, the other one is based on conductive detection. Two fluorescent molecular sensors, Rhod-5N and DPPS-PEG, and several microfluidic devices were developed and applied for heavy metal ions Cd (II) and Hg (II) detection in surface water. A new microchip made of PMMA was fabricated by femtosecond laser ablation and tested for Cd (II) sensing based on a fluorescent molecular sensor Rhod-5N. Further more, some improvements of the performance of microfluidic chips were made. A novel method for sensitive determination of five priority haloacetic acids (HAAs) in water systems has been developed based on electromembrane extraction (EME) prior to capillary electrophoresis with capacitively coupled contactless conductivity detection (CE-C4D).

Lab-on-a-chip (LOC) biological assays have the potential to fundamentally reform healthcare. The move away from centralized facilities to Point-of-Care (POC) testing of biological assays would improve the speed and accuracy of these, thereby improving patient care. Before LOC can be realized, a number of challenges must be addressed: the need for expert users must be abstracted away; the manufacturing cost of $5 per test threshold must be met; and the supporting infrastructure must be integrated down to an easily portable size. These challenges can be addressed with the deposition of microfluidics on CMOS chips. By designing application specific integrated circuits (ASICs) much of the automation and the supporting infrastructure needed to run these assays can be integrated into the chip. Additionally, CMOS fabrication is some of the most optimized manufacturing in industry today. One of the central challenges with LOC on ASIC is the signal acquisition from the microfluidics into the CMOS. Optical sensing of fluorescence is one form of sensing used for LOC assays. Despite a large literature, there has not been a strong demonstration of monolithic LOC fluorescence detection (FD) for low concentration samples. This work explores the limit-of-detection (LOD) for LOC FD through analysis of the signal and noise of a proposed acquisition channel. The proposed signal acquisition channel consists of an on chip photodiode and integrator based amplification circuits. A hand analysis of the signal propagation through the channel and the noise sources introduced by the circuitry, is performed. This analysis is used to establish relationships between different circuit parameters and the LOD of a hypothetical LOC device. The hand analysis is verified through simulation and the acquisition channel is implemented in: (i) the Austrian Microsystems 350nm CMOS process, (ii) discrete components. Testing of the CMOS chip revealed several issues not identified in extracted simulation; however, the discrete integrator demonstrated many of the trends predicted by the hand analysis and simulations and achieved a LOD of 7.2$\mu M$. This analysis provides insight into the engineering trade-offs required to improve the LOD, to enable more wide spread application of LOC FD.

Lab-on-a-chip (LOC) biological assays have the potential to fundamentally reform healthcare. The move away from centralized facilities to Point-of-Care (POC) testing of biological assays would improve the speed and accuracy of these, thereby improving patient care. Before LOC can be realized, a number of challenges must be addressed: the need for expert users must be abstracted away; the manufacturing cost of $5 per test threshold must be met; and the supporting infrastructure must be integrated down to an easily portable size. These challenges can be addressed with the deposition of microfluidics on CMOS chips. By designing application specific integrated circuits (ASICs) much of the automation and the supporting infrastructure needed to run these assays can be integrated into the chip. Additionally, CMOS fabrication is some of the most optimized manufacturing in industry today. One of the central challenges with LOC on ASIC is the signal acquisition from the microfluidics into the CMOS. Optical sensing of fluorescence is one form of sensing used for LOC assays. Despite a large literature, there has not been a strong demonstration of monolithic LOC fluorescence detection (FD) for low concentration samples. This work explores the limit-of-detection (LOD) for LOC FD through analysis of the signal and noise of a proposed acquisition channel. The proposed signal acquisition channel consists of an on chip photodiode and integrator based amplification circuits. A hand analysis of the signal propagation through the channel and the noise sources introduced by the circuitry, is performed. This analysis is used to establish relationships between different circuit parameters and the LOD of a hypothetical LOC device. The hand analysis is verified through simulation and the acquisition channel is implemented in: (i) the Austrian Microsystems 350nm CMOS process, (ii) discrete components. Testing of the CMOS chip revealed several issues not identified in extracted simulation; however, the discrete integrator demonstrated many of the trends predicted by the hand analysis and simulations and achieved a LOD of 7.2$\mu M$. This analysis provides insight into the engineering trade-offs required to improve the LOD, to enable more wide spread application of LOC FD.

This thesis focuses on the design of a versatile, portable, and cost-effective fluorescence detection system for LOC devices. Components that are widely available are used, such as LEDs for excitation and a microcontroller for processing. In addition, a photoresistor is tested for the feasibility of being used as a fluorescence detector, instead of the more commonly used photomultiplier tubes. The device also focuses on upgradeability and versatility, meaning that most of the major components can be replaced as long as power requirements remain unaffected. This allows for future additions to the device once they are available, as well as giving the user the power to choose which add-ons are needed since not all users may have the same requirements. The performance of the device after testing with fluorescein dyes and stained yeast cells indicate that it is capable of executing simple tasks, such as determining the presence and concentration of an analyte if given a sufficient amount. It also provided similar readings to commercial fluorescence analysers, which proves its ability to function as a fluorescence detector device. The thesis also proposes a MEMS diffraction grating that can be used for wavelength tuning. By being able to selectively measure across a range of wavelengths, the capability of the device is increased. Examples include being able to detect multiple fluorescent emissions, which will complement the multicoloured excitation LED nicely. In addition, the device will not be limited to a predetermined set of filters. This effectively allows more fluorescent dyes to be used with the device since any wavelength in the visible range can be selectively filtered for. Simulations of the proposed diffraction grating were performed in ANSYS to confirm the validity of the calculated values. In addition, tests were performed on a slide fabricated with diffraction gratings using values as close to the calculated values as possible. All of the results indicate that there is great promise in the proposed diffraction grating design and that it should be further investigated.

This thesis focuses on the design of a versatile, portable, and cost-effective fluorescence detection system for LOC devices. Components that are widely available are used, such as LEDs for excitation and a microcontroller for processing. In addition, a photoresistor is tested for the feasibility of being used as a fluorescence detector, instead of the more commonly used photomultiplier tubes. The device also focuses on upgradeability and versatility, meaning that most of the major components can be replaced as long as power requirements remain unaffected. This allows for future additions to the device once they are available, as well as giving the user the power to choose which add-ons are needed since not all users may have the same requirements. The performance of the device after testing with fluorescein dyes and stained yeast cells indicate that it is capable of executing simple tasks, such as determining the presence and concentration of an analyte if given a sufficient amount. It also provided similar readings to commercial fluorescence analysers, which proves its ability to function as a fluorescence detector device. The thesis also proposes a MEMS diffraction grating that can be used for wavelength tuning. By being able to selectively measure across a range of wavelengths, the capability of the device is increased. Examples include being able to detect multiple fluorescent emissions, which will complement the multicoloured excitation LED nicely. In addition, the device will not be limited to a predetermined set of filters. This effectively allows more fluorescent dyes to be used with the device since any wavelength in the visible range can be selectively filtered for. Simulations of the proposed diffraction grating were performed in ANSYS to confirm the validity of the calculated values. In addition, tests were performed on a slide fabricated with diffraction gratings using values as close to the calculated values as possible. All of the results indicate that there is great promise in the proposed diffraction grating design and that it should be further investigated.

Despite the ongoing “war on drugs” the seizure rates for phenethylamines and their analogues have been steadily increasing over the years. The illicit manufacture of these compounds has become big business all over the world making it all the more attractive to the inexperienced “cook”. However, as a result, the samples produced are more susceptible to contamination with reactionary byproducts and leftover reagents. These impurities are useful in the analysis of seized drugs as their identities can help to determine the synthetic pathway used to make these drugs and thus, the provenance of these analytes. In the present work two fluorescent dyes, 4-fluoro-7-nitrobenzofurazan and 5-(4,6-dichlorotriazinyl)aminofluorescein, were used to label several phenethylamine analogues for electrophoretic separation with laser-induced fluorescence detection. The large scale to which law enforcement is encountering these compounds has the potential to create a tremendous backlog. In order to combat this, a rapid, sensitive method capable of full automation is required. Through the utilization of the inline derivatization method developed whereby analytes are labeled within the capillary efficiently in a minimum span of time, this can be achieved. The derivatization and separation parameters were optimized on the basis of a variety of experimentally determined factors in order to give highly resolved peaks in the fluorescence spectrum with limits of detection in the low µg/mL range.

Doctor of Philosophy / Department of Chemistry / Stefan H. Bossmann / Cancer is a group of diseases that are characterized by uncontrolled growth and spread of cells. In order to treat cancer successfully, it is important to diagnose cancers in their early stages, because survival often depends on the stage of cancer detection. For that purpose, highly sensitive and selective methods must be developed, taking advantage of suitable biomarkers. The expression levels of proteases differ from one cancer type to the other, because different cancers arise from different cell types. According to the literature, there are significant differences between the protease expression levels of cancer patients and healthy people, because solid tumors rely on proteases for survival, angiogenesis and metastasis. Development of fluorescence-based nanobiosensors for the early detection of pancreatic cancer and non-small-cell lung cancer is discussed in this thesis. The nanobiosensors are capable of detecting protease/arginase activities in serum samples over a broad range. The functionality of the nanobiosensor is based on Förster resonance energy transfer and surface energy transfer mechanisms. The nanobiosensors for protease detection feature dopamine-coated Fe/Fe₃O₄ nanoparticles, consensus (cleavage) peptide sequences, meso-tetra(4-carboxyphenyl)porphine (TCPP), and cyanine 5.5. The consensus peptide sequences were synthesized by solid-supported peptide synthesis. In this thesis, improved consensus sequences were used, which permit faster synthesis and higher signal intensities. TCPP, which is the fluorophore of the nanoplatform, was connected to the N-terminal end of the oligopeptides while it was still on the resin. After the addition of TCPP, the TCPP-oligopeptide was cleaved off the resin and linked to the primary amine groups of Fe/Fe₃O₄-bound via a stable amide bond. In the presence of a particular protease, the consensus sequences attached to the nanoparticle can be cleaved and release TCPP to the aqueous medium. Upon releasing the dye, the emission intensity increases significantly and can be detected by fluorescence spectroscopy or, similarly, by using a fluorescence plate reader. In sensing of arginase, posttranslational modification of the peptide sequence will occur, transforming arginine to ornithine. This changes the conformational dynamics of the oligopeptide tether, leading to the increase of the TCPP signal. This is a highly selective technology, which has a very low limit of detection (LOD) of 1 x 10⁻¹⁶ molL⁻¹ for proteases and arginase. The potential of this nanobiosensor technology to detect early pancreatic and lung cancer was demonstrated by using serum samples, which were collected from patients who have been diagnosed with pancreatic cancer and non-small cell lung cancer at the South Eastern Nebraska Cancer Center (lung cancer) and the University of Kansas Cancer Center (pancreatic cancer). As controls, serum samples collected from healthy volunteers were analyzed. In pancreatic cancer detection, the protease/arginase signature for the detection of pancreatic adenocarcinomas in serum was identified. It comprises arginase, MMPs -1, - 3, and -9, cathepsins -B and -E, urokinase plasminogen activator, and neutrophil elastase. For lung cancer detection, the specificity and sensitivity of the nanobiosensors permit the accurate measurements of the activities of nine signature proteases in serum samples. Cathepsin -L and MMPs-1, -3, and -7 permit detecting non-small-cell lung-cancer at stage 1.

<p>Capillary electrophoresis (CE) has a wide range of applications in the field of analytical chemistry. In general the most expensive part in a CE system is the detector due to the fact that the detector must have a high sensitivity for small detection volumes and low concentrations. Building portable instruments is one way to make the instruments cheaper and has the advantage that they can be used virtually everywhere. However, downscaling of CE instruments puts some extra demands on the detector. This report describes the design and building of two homemade light-emitting diode (LED) based detectors; a LEDabsorbance photometric detector (LED-AP) and a LED-induced fluorescence (LED-IF) detector. The main goal was to install them inside a portable CE and make a simple separation. The performance of the two detectors had to be evaluated before the main goal could be achieved. p-Nitrophenol was used to create a sensitivity graph for the LED-AP detector, calculating the upper linearity to 5.6 mM when the sensitivity had dropped 10 % caused by non-linearity. The sensitivity graph also showed that the detector had an effective pathlength of 74.2 µm and a stray light of 4.5 % for a 75 µm i.d fused-silica capillary. The LED-IF detector was evaluated by determining the limit of detection (LOD) for fluorescein, at a signal to noise ratio of 3. The LOD was 0.72 µM ± 0.01 µM when immersion oil was used to limit the light scattering from the optic fibres in to the capillary and 0.58 µM ±0.02 µM when silicone oil was used. Without doing any improvements only the LED-AP detector could be used in the portable CE. As a common application area for portable CE instruments is environmental analysis, indirect detection using p-nitrophenol as a probe for separating anions was done to test the system. All analytes were eluted in less than 4 minutes.</p>

Capillary electrophoresis (CE) has a wide range of applications in the field of analytical chemistry. In general the most expensive part in a CE system is the detector due to the fact that the detector must have a high sensitivity for small detection volumes and low concentrations. Building portable instruments is one way to make the instruments cheaper and has the advantage that they can be used virtually everywhere. However, downscaling of CE instruments puts some extra demands on the detector. This report describes the design and building of two homemade light-emitting diode (LED) based detectors; a LEDabsorbance photometric detector (LED-AP) and a LED-induced fluorescence (LED-IF) detector. The main goal was to install them inside a portable CE and make a simple separation. The performance of the two detectors had to be evaluated before the main goal could be achieved. p-Nitrophenol was used to create a sensitivity graph for the LED-AP detector, calculating the upper linearity to 5.6 mM when the sensitivity had dropped 10 % caused by non-linearity. The sensitivity graph also showed that the detector had an effective pathlength of 74.2 µm and a stray light of 4.5 % for a 75 µm i.d fused-silica capillary. The LED-IF detector was evaluated by determining the limit of detection (LOD) for fluorescein, at a signal to noise ratio of 3. The LOD was 0.72 µM ± 0.01 µM when immersion oil was used to limit the light scattering from the optic fibres in to the capillary and 0.58 µM ±0.02 µM when silicone oil was used. Without doing any improvements only the LED-AP detector could be used in the portable CE. As a common application area for portable CE instruments is environmental analysis, indirect detection using p-nitrophenol as a probe for separating anions was done to test the system. All analytes were eluted in less than 4 minutes.