Computer simulation of radio-frequency methane/hydrogen plasmas and their interaction with GaAs Surfaces

The following thesis describes the computer modelling of radio frequency capacitively coupled methane/hydrogen plasmas and the consequences for the reactive ion etching of (100) GaAs surfaces. In addition a range of etching experiments was undertaken over a matrix of pressure, power and methane concentration. The resulting surfaces were investigated using X-ray photoelectron spectroscopy and the results were discussed in terms of physical and chemical models of particle/surface interactions in addition to the predictions for energies, angles and relative fluxes to the substrate of the various plasma species. The model consisted of a Monte Carlo code which followed electrons and ions through the plasma and sheath potentials whilst taking account of collisions with background neutral gas molecules. The ionisation profile output from the electron module was used as input for the ionic module. Momentum scattering interactions of ions with gas molecules were investigated via different models and compared against results given by quantum mechanical code. The interactions were treated as central potential scattering events and the resulting neutral cascades were followed. The resulting predictions for ion energies at the cathode compared well to experimental ion energy distributions and this verified the particular form of the electrical potentials used and their applicability in the particular geometry plasma cell used in the etching experiments. The final code was used to investigate the effect of external plasma parameters on the mass distribution, energy and angles of all species impingent on the electrodes. Comparisons of electron energies in the plasma also agreed favourably with measurements made using a Langmuir electric probe. The surface analysis showed the surfaces all to be depleted in arsenic due to its preferential removal and the resultant Ga:As ratio in the surface was found to be directly linked to the etch rate. The etch rate was determined by the methane flux which was predicted by the code.

Electrochemical RNA genosensors for toxic algal species: enhancing selectivity and sensitivity

Harmful algal blooms (HABs) are becoming more frequent as climate changes, with tropical species moving northward. Monitoring programs detecting the presence of toxic algae before they bloom are of paramount importance to protect aquatic ecosystems, aquaculture, human health and local economies. Rapid and reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention over the past decade as an alternative to the impractical standard microscopic counting-based techniques. This work reports on a PCR amplification-free electrochemical genosensor for the enhanced selective and sensitive detection of RNA from multiple Mediterranean toxic algal species. For a sandwich hybridization (SHA), we designed longer capture and signal probes for more specific target discrimination against a single base-pair mismatch from closely related species and for reproducible signals. We optimized experimental conditions, viz., minimal probe concentration in the SHA on a screen-printed gold electrode and selected the best electrochemical mediator. Probes from 13 Mediterranean dinoflagellate species were tested under optimized conditions and the format further tested for quantification of RNA from environmental samples. We not only enhanced the selectivity and sensitivity of the state-of-the-art toxic algal genosensors but also increased the repertoire of toxic algal biosensors in the Mediterranean, towards an integral and automatic monitoring system.

Electrochemical RNA genosensors for toxic algal species: enhancing selectivity and sensitivity

Harmful algal blooms (HABs) are becoming more frequent as climate changes, with tropical species moving northward. Monitoring programs detecting the presence of toxic algae before they bloom are of paramount importance to protect aquatic ecosystems, aquaculture, human health and local economies. Rapid and reliable species identification methods using molecular barcodes coupled to biosensor detection tools have received increasing attention over the past decade as an alternative to the impractical standard microscopic counting-based techniques. This work reports on a PCR amplification-free electrochemical genosensor for the enhanced selective and sensitive detection of RNA from multiple Mediterranean toxic algal species. For a sandwich hybridization (SHA), we designed longer capture and signal probes for more specific target discrimination against a single base-pair mismatch from closely related species and for reproducible signals. We optimized experimental conditions, viz., minimal probe concentration in the SHA on a screen-printed gold electrode and selected the best electrochemical mediator. Probes from 13 Mediterranean dinoflagellate species were tested under optimized conditions and the format further tested for quantification of RNA from environmental samples. We not only enhanced the selectivity and sensitivity of the state-of-the-art toxic algal genosensors but also increased the repertoire of toxic algal biosensors in the Mediterranean, towards an integral and automatic monitoring system.

Environmental Dynamics of Dissolved Black Carbon in Aquatic Ecosystems

Black carbon (BC), the incomplete combustion product from biomass and fossil fuel burning, is ubiquitously found in soils, sediments, ice, water and atmosphere. Because of its polyaromatic molecular characteristic, BC is believed to contribute significantly to the global carbon budget as a slow-cycling, refractory carbon pool. However, the mass balance between global BC generation and accumulation does not match, suggesting a removal mechanism of BC to the active carbon pool, most probable in a dissolved form. The presence of BC in waters as part of the dissolved organic matter (DOM) pool was recently confirmed via ultrahigh resolution mass spectrometry, and dissolved black carbon (DBC), a degradation product of charcoal, was found in marine and coastal environments. However, information on the loadings of DBC in freshwater environments and its global riverine flux from terrestrial systems to the oceans remained unclear. The main objectives of this study were to quantify DBC in diverse aquatic ecosystems and to determine its environmental dynamics. Surface water samples were collected from aquatic environments with a spatially significant global distribution, and DBC concentrations were determined by a chemical oxidation method coupled with HPLC detection. While it was clear that biomass burning was the main sources of BC, the translocation mechanism of BC to the dissolved phase was not well understood. Data from the regional studies and the developed global model revealed a strong positive correlation between DBC and dissolved organic carbon (DOC) dynamics, indicating a co-generation and co-translocation between soil OC and BC. In addition, a DOC-assistant DBC translocation mechanism was identified. Taking advantage of the DOC-DBC correlation model, a global riverine DBC flux to oceans on the order of 26.5 Mt C yr-1 (1 Mt = 1012 g) was determined, accounting for 10.6% of the global DOC flux. The results not only indicated that DOC was an important environmental intermediate for BC transfer and storage, but also provided an estimate of a major missing link in the global BC budget. The ever increasing DBC export caused by global warming will change the marine DOM quality and may have important consequences for carbon cycling in marine ecosystem.

Drug of abuse analysis: new methods in herbal and biological matrices for medicinal chemistry and forensic investigation

In this Ph.D. project, original and innovative approaches for the quali-quantitative analysis of abuse substances, as well as therapeutic agents with abuse potential and related compounds were designed, developed and validated for application to different fields such as forensics, clinical and pharmaceutical. All the parameters involved in the developed analytical workflows were properly and accurately optimised, from sample collection to sample pretreatment up to the instrumental analysis. Advanced dried blood microsampling technologies have been developed, able of bringing several advantages to the method as a whole, such as significant reduction of solvent use, feasible storage and transportation conditions and enhancement of analyte stability. At the same time, the use of capillary blood allows to increase subject compliance and overall method applicability by exploiting such innovative technologies. Both biological and non-biological samples involved in this project were subjected to optimised pretreatment techniques developed ad-hoc for each target analyte, making also use of advanced microextraction techniques. Finally, original and advanced instrumental analytical methods have been developed based on high and ultra-high performance liquid chromatography (HPLC,UHPLC) coupled to different detection means (mainly mass spectrometry, but also electrochemical, and spectrophotometric detection for screening purpose), and on attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) for solid-state analysis. Each method has been designed to obtain highly selective, sensitive yet sustainable systems and has been validated according to international guidelines. All the methods developed herein proved to be suitable for the analysis of the compounds under investigation and may be useful tools in medicinal chemistry, pharmaceutical analysis, within clinical studies and forensic investigations.

Chemiluminescence – based bioanalytical devices for astrobiological, food safety and clinical applications

At the intersection of biology, chemistry, and engineering, biosensors are a multidisciplinary innovation that provide a cost-effective alternative to traditional laboratory techniques. Due to their advantages, biosensors are used in medical diagnostics, environmental monitoring, food safety and many other fields. The first part of the thesis is concerned with learning the state of the art of paper-based immunosensors with bioluminescent (BL) and chemiluminescent (CL) detection. The use of biospecific assays combined with CL detection and paper-based technology offers an optimal approach to creating analytical tools for on-site applications and we have focused on the specific areas that need to be considered more in order to ensure a future practical implementation of these methods in routine analyses. The subsequent part of the thesis addresses the development of an autonomous lab-on-chip platform for performing chemiluminescent-based bioassays in space environment, exploiting a CubeSat platform for astrobiological investigations. An origami-inspired microfluidic paper-based analytical device has been developed with the purpose of assesses its performance in space and to evaluate its functionality and the resilience of the (bio)molecules when exposed to a radiation-rich environment. Subsequently, we designed a paper-based assay to detect traces of ovalbumin in food samples, creating a user-friendly immunosensing platform. To this purpose, we developed an origami device that exploits a competitive immunoassay coupled with chemiluminescence detection and magnetic microbeads used to immobilize ovalbumin on paper. Finally, with the aim of exploring the use of biomimetic materials, an hydrogel-based chemiluminescence biosensor for the detection of H2O2 and glucose was developed. A guanosine hydrogel was prepared and loaded with luminol and hemin, miming a DNAzyme activity. Subsequently, the hydrogel was modified by incorporating glucose oxidase enzyme to enable glucose biosensing. The emitted photons were detected using a portable device equipped with a smartphone's CMOS (complementary metal oxide semiconductor) camera for CL emission detection.

Methods for detection of anatoxin-a(s) by liquid chromatography coupled to electrospray ionization-tandem mass spectrometry

Anatoxin-a(s) is a potent irreversible inhibitor of the enzyme acetylcholinesterase with a unique N-hydroxyguanidine methylphosphate ester chemical structure. Determination of this toxin in environmental samples is hampered by the lack of specific methods for its detection. Using the toxic strain of Anabaena lemmermani PH-160 B as positive control, the fragmentation characteristics of anatoxin-a(s) under collision-induced dissociation conditions have been investigated and new LC-MS/MS methods proposed. Recommended ion transitions for correct detection of this toxin are 253 > 58, 253 > 159, 235 > 98 and 235 > 96. Chromatographic separation is better achieved under HILIC conditions employing a ZIC-HILIC column. This method was used to confirm for the first time the production of anatoxin-a(s) by strains of Anabaena oumiana ITEP-025 and ITEP-026. Considering no standard solutions are commercially available, our results will be of significant use for the correct identification of this toxin by LC-MS/MS. (C) 2009 Elsevier Ltd. All rights reserved.