Developments and applications of solid phase microextraction (SPME) coupled to direct analysis in real time (DART) and approaches towards small volume analysis

Solid phase microextraction (SPME) has been rapidly developing since its invention in 1990. Due to a flexible design and the ability to implement various extractive coatings, SPME has found use in numerous applications. One such application is the coupling of SPME to Direct Analysis in Real Time (DART), an ambient ionization mass spectrometry (AIMS) technique. AIMS techniques completely eliminate the use of chromatography, reducing lengthy workflows and improving time-efficiency. SPME coupled to DART-MS has been quite successful in achieving low detection limits and reproducible results in bioanalytical applications. These successes are mostly due to the excellent preconcentrating and interference resisting abilities of SPME coatings and detection capabilities of DART-MS. Certainly, as a novel technique, many aspects of SPME-DART-MS are yet to be explored. Hence, this thesis investigated fundamental aspects of SPME-DART-MS, in addition to exploring novel applications, such as detection of prohibited substances in small bio-volumes (i.e., ≤ 25 µL of oral fluid (OF) and human blood) and implementation of SPME-DART-MS as high-throughput technology for quantitation of opioids in urine and human plasma in less than 1.5 h for a set of 96 samples. Coupling of SPME to portable DART-MS instrumentation was investigated for on-site, in vivo drug detection in samples of OF as a potential tool of law enforcement, reporting detection limits that are below those proposed by regulatory agencies. Additionally, the use of biocompatible plastic materials such as polyetheretherketone (PEEK) was investigated as a potential alternative material for the manufacturing of SPME devices for DART-MS, which are usually made of stainless steel (SS). Performance of the PEEK devices was similar to those of SS, allowing for their use in quantification of drugs-of-abuse (DoAs) in OF and urine at low parts-per-billion levels (ppb). In addition, OF samples following coffee consumption from a female volunteer were used to monitor caffeine half-life. The results were in compliance with established half-life of caffeine reported in literature, and highlighted the potential of the PEEK devices for in vivo buccal swabbing testing. Another novel application of SPME described in this thesis was the development of miniaturized SPME devices “minitips” for analysis of small volumes. The analysis of small sample volumes (biofluids, tissues, and cells) has become more common within the scientific community in the last few years. For this type of analysis, very sensitive instrumental platforms and sample preparation methods are required to obtain qualitative and quantitative information. Hence, the SPME minitip format was introduced, and the tip apex was coated with 1 millimeter (mm) of polyacrylonitrile (PAN) and in-house-synthesized N-vinylpyrrolidone-co-divinylbenzene, also known as hydrophilic lipophilic balance (HLB) particles. Robustness, extraction efficiency, repeatability, reusability, and matrix effect (ME) assessment yielded good results. Benzodiazepines were extracted from 15 µL of phosphate buffer saline (PBS) and urine, and certain DoAs were extracted from 1 µL of OF using LC-MS/MS. Very good figures of merit were obtained, with limits of detection (LODs) and quantitation (LOQs) in the lower ppb range. DoAs were also extracted from 1 µL of blood by directly coupling the SPME minitips to nanoelectrospray ionization (nESI) devices. The SPME HLB minitips were also used for untargeted metabolomic profiling of caviar eggs via liquid chromatography high resolution mass spectrometry (LC/HRMS), thereby showing that despite introduced dilution, satisfactory results can be obtained. Indeed, good statistical separation of caviar eggs was observed, in addition to extracting well over a hundred significant features. Lastly, this thesis also details application of SPME for untargeted metabolomics profiling of patient affected with a genetic condition known as malignant hyperthermia (MH). Small human muscles obtained from Toronto General Hospital were sampled using SPME fibers and analysed via LC/HRMS using an untargeted metabolomics approach. Potential disease related compounds (i.e., biomarkers) were found, aiding in the elucidation of the biochemical pathways affected in patients with MH. The accepted diagnostic tool of MH is a highly invasive procedure which involves excision of a portion of the Gracilis muscle. Hence the prospect of using SPME as minimally invasive diagnostic tool was discussed. As this thesis shows, the opportunities of SPME are many; and its continuous development will most likely require efforts and collaboration from experts in numerous fields

Power extraction circuits for piezoelectric energy harvesters and time series data in water supply systems

This thesis investigates two fundamental technological challenges that prevent water utilities from deploying infrastructure monitoring apparatus with high spatial and temporal resolution: providing sufficient power for sensor nodes by increasing the power output from a vibration-driven energy harvester based on piezoelectric transduction, and the processing and storage of large volumes of data resulting from the increased level of pressure and flow rate monitoring. Piezoelectric energy harvesting from flow-induced vibrations within a water main represents a potential source of power to supply a sensor node capable of taking high- frequency measurements. A main factor limiting the amount of power from a piezoelectric device is the damping force that can be achieved. Electronic interface circuits can modify this damping in order to increase the power output to a reasonable level. A unified analytical framework was developed to compare circuits able to do this in terms of their power output. A new circuit is presented that out-performs existing circuits by a factor of 2, which is verified experimentally. The second problem concerns the management of large data sets arising from resolving challenges with the provision of power to sensor devices. The ability to process large data volumes is limited by the throughput of storage devices. For scientists to execute queries in a timely manner, query execution must be performant. The large volume of data that must be gathered to extract information from historic trends mandates a scalable approach. A scalable, durable storage and query execution framework is presented that is able to significantly improve the execution time of user-defined queries. A prototype database was implemented and validated on a cluster of commodity servers using live data gathered from a London pumping station and transmission mains. Benchmark results and reliability tests are included that demonstrate a significant improvement in performance over a traditional database architecture for a range of frequently-used operations, with many queries returning results near-instantaneously

Analytical techniques for biomass-restricted metabolomics: an overview of the state-of-the-art

Biomedical and clinical questions increasingly deal with biomass-restricted samples. To address these questions with a metabolomics approach, the development of new microscale analytical techniques and workflows is needed. Over the past few years, significant efforts have been made to improve the overall sensitivity of MSbased metabolomics workflows to enable the analysis of biological samples that are low in metabolite concentration or biomass. In this paper, factors that are crucial for the performance of biomass-restricted metabolomics studies are discussed, including sampling and sample preparation methods, separation techniques and ionization sources. Overviews of MS-based miniaturized metabolomics studies reported over the past five years are given in tables, with information provided on sample type, sample preparation volume, injection volume, separation techniques and MS analyzers. Finally, some general conclusions and perspectives are given.Analytical BioScience

DETECTION OF MYCOTOXINS USING SURFACE-ENHANCED RAMAN SPECTROSCOPY

Mycotoxins are toxic metabolites produced by fungus that can be parasites or saprophytes of crops or livestock forage. Consumer demand for plant-based foods and interest in animal-based foods originating from animals fed plant-based feed has been on the rise. Therefore, monitoring mycotoxins occurring in the food supply is more critical than ever. The goal of this project is to improve surface-enhanced Raman spectroscopy’s (SERS) ability to identify and detect mycotoxins using label-free SERS substrates. Two simple approaches were designed to enhance the detection of mycotoxins produced by the Aspergillus and Penicillium genera, ochratoxin A and aflatoxin B1. Ochratoxin A was successfully detected in wine samples spiked with the mycotoxin in a range of 0.01 to 1 ppm using a facile solvent-mediated extraction that showed the key role that the food matrix can play on the SERS substrate performance. The detection of aflatoxin B1’ SERS signals using bare gold nanoparticles was enhanced with the addition of human serum albumin (HSA) as a mediating molecule. A combination of the HSA-mediated protocol and a liquid-liquid extraction (LLE) method allows the detection of up to 2 ppb of AFB1 in compound feedstuff samples. Additionally, a simple SERS protocol applied to Aspergillus flavus grown in liquid and solid medium showed the technique’s capacity to classify between aflatoxigenic and non-aflatoxigenic species. Raman spectroscopy, SERS, Infrared Spectroscopy (IR) and surface-enhanced infrared absorption spectroscopy (SEIRAS) showed differences yet potential complementarity in their ability to identify mycotoxins produced by the Fusarium genus, deoxynivalenol and fumonisin B1

Marine organisms model species for the assessment of biological, environmental and economic impacts on marine aquaculture in Campania

Campanian region has an important mussel farming tradition, since the Cuman and Greek domination around 700 A.C. through the Bourbons until today. Mussels and the linked supply chain were always been present in the socio-economic scenario of the regional coastal area. Today mussel production in Campania represents almost all the aquaculture total production, resulting one of the most productive Italian regions in this sector. In order to evaluate biological, environmental and economic impacts on marine aquaculture, mussels of the genus Mytilus were chosen as model species. Mussels are perfect model organisms for their biological and physiological characteristics; they are bioengineering species, sedentary organisms, filter feeders, widespread all over the world, easy to keep in laboratories and important food source. Besides food source, thanks to their features mussels provide important ecosystem services as regulating the water column, supporting the food web and provisioning cultural services. Moreover, recently, are becoming source of important bioactive compounds, revealing to be organisms with a high potentiality in several applications. Different kind of stressors, anthropogenic as well as natural stressors, can impact, negatively or positively, on mussels and consequently on the ecosystem services they provide. Aims of the thesis are the evaluation of the effects of three different stressors on mussels supply chain: food frauds, ocean acidification and the grafting operation. Food frauds, the act of defrauding buyers of food and food ingredients for economic gain, can threaten the Campanian mussel supply chain. Unhealthy storage conditions, species substitutions, lack of label and informations on the product origin are all ordinary risks when buying mussels. The threats are both for human health than for the local economy of the honest mussel farmers. In order to investigate on the situation of the Campanian markets, Mytilus galloprovincialis mussels, the native species of the Mediterranean Sea, were sampled in different fish markets, local mussel farms and in their local natural environment. Mussels were genetically characterized in order to assess the species and their origins using the molecular markers COI, 16S, PAPM and 28S. The final aim was the attempt of the genetic identification of a local Campanian mussel, intended to officially recognize such local excellence product considering all its productions steps, from the seed recruitment to the marketing. Investigation on this topic has shown that Campanian local markets are not highly affected by the food frauds of species substitutions but is difficult define the situation for the other type of fraud, the origin declaration. Just using simple molecular biology tools was not possible identify different mussel populations and define the genetic characterization of the local Mytilus galloprovincialis species. Further investigations are essential in order to identify a simple, fast and cheap method for mussels origin identification. Mussels supply chain could be also affected by other kind of human impacts, not only in Campania region but on a global scale. Global changes can induce important vital alterations in aquatic systems, the increasing amounts of CO2 in the oceans, known as the phenomenon of Ocean Acidification, affect our life via influencing the environment and our economy. Ocean acidification and other anthropogenic stressors can were already proved to cause negatively or positively changes on coastal dynamics, in marine organisms and consequently to the ecosystems services that they provide. Ocean acidification effects were tested on Mytilus unguiculatus, a pacific mussel widely bread in the Chinese Sea with similar anatomy and physiology to M. galloprovincialis. Mytilus unguiculatus has been exposed to different pH values (7.4 and 7.8) and then were analyzed the respective physiological parameters (O2 consumption and NH4+ excretion), gene expression of NKA and NHE8 (both involved in the acid-base regulation mechanisms), free amino acids from mantle and gills with UPLC analysis, shell characteristics with AAS analysis, SEM pictures and X- ray testing. Mytilus unguiculatus easily survive in 7.8 pH conditions but further investigations (different life stages, longer exposure time, additional genes expression) are needed to better understand the effects of Ocean Acidification on this species and other species as the Mediterranean Mytilus galloprovincialis. However an anthropogenic stressor for certain organisms could have a positive impact on the linked aquaculture economy. It is the case of the induced grafting operation on the pearl molluscs, in order to produce a pearl. Since the first decade of 1900 pearls production is an industrialized process, there are some bivalves of the pacific area widely used in this manufacturing production. Molluscs are able to produce pearls in response to a natural stressors (the famous grain of sand, a small piece of broken shell, a parasite or a small animal) causing an injury in the mantle of the damaged mollusc. In the “classical pearl molluscs” pearl production is human surgically induced, imitating what happens in nature and using a nucleus of mother-of-pearl. In a similar way the Campanian mussel Mytilus galloprovincialis could react producing a pearl too. The local mussel could be a “new pearl molluscs” and pearls productions would be fully included in the well-established Campanian gemstone market. As for mussels farming activity, Campania region has an ancient coral manufacturing tradition, hosting in the southwestern coast of the Gulf of Naples the “the world’s capital of coral”. In conclusion, mussels have been further appreciated as model organisms in different application fields; food frauds can affect negatively the Campanian market and new investigations are needed in order to evaluate their origin; mussels seem to easily survive to the predicted future acidified oceans; finally, mussels have great potentials, among their innovative applications they could become “new pearl molluscs”. In order to carry on and improve the millennial Campanian mussel farming tradition, it is crucial enhance and promote Campanian mussels as excellent local products, promoting new productions and encourage buyers to make a conscious choice, both for enhancing local mussel farmers than for preserving the environment for the future generation

Sustainable Molecular Gelators: Beta-D-Glucoside Derived Structuring Agents and Their Material Application

Though molecular gelators may by synthesized and formulated into gels following a variety of methods, it should serve that the most valued methods may utilize renewable and waste resources and follow sustainable procedures. Molecular gelators are systems capable of structuring liquids into solid-like materials and they represent a class of surfactant and amphiphilic materials which posses the capability to be not only useful in their ability to form gels, but multifunctional in the ability to respond smartly to a variety of stimuli. Thus there is an interest in the development of sustainable molecular gelators capable of being applied to applications, which may have been previously linked to gels, or more interestingly those with which gels have not yet been applied. The use of naturally occurring starting materials in the form of renewable resources serves to inspire biomimetic species which allow for the simultaneous development of materials for applied research, while affording researchers the opportunity to study the importance of non-covalent intermolecular interaction and their roles in natural molecules. Deriving these gelators from the basic primary metabolites may help unveil the nature of these forces in their assembly, and most excitingly their function. In an effort to explore the functionality and utility of biobased gelled supramolecular systems, two gelators, one primarily structuring organic solvents, and the other a hydrogelator are discussed from a synthetic point of view and characterized for their non-covalent interactions. Enzymatic catalysis is performed to afford these species in high yields, allowing for the eventual potential commercialization of these gelators through benign means. In addition to the gelator characterization, the resultant composite gels are studied for their mechanical properties, but of most interest their ability to be defined as smart materials in their response to shear, heat and light. Lastly these materials are examined for their application to three different areas of current interest: healthful edible oil structuring, next-generation gelled fuels, and radiation sensing gels. This study should serve as a rigorous investigation not only in the sustainable development of functional value-added chemicals, but their formulation and processing into value-added materials

Stress and immune relevant proteins in lumpsucker (Cyclopterus lumpus L.) skin -establishing basic knowledge

Masteroppgave i havbruk - Nord universitet, 2017Sperret til 2022-10-1