Analytical chemistry

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by progressive loss in function of motor neurons. Elevated levels of biologically important metal ions, such as copper (II) (Cu(II)), zinc (II) (Zn(II)) or iron (III) (Fe(III)), may contribute to the disease. Moreover, Cu(II) interactions with other proteins associated with ALS have been investigated; however, the effects of metallation on TAR DNA-binding protein of 43 kDa (TDP-43) are less known. The aim of the thesis was to evaluate interactions between full-length TDP-43 and metal ions, and gain insight into the mechanisms of these interactions. In Chapter 2, electrochemical methods were used to evaluate the coordination of Cu(II) ions to immobilized TDP-43. Cu(II)-TDP-43 binding was favourable at a neutral pH. Surface characterization confirmed protein immobilization and Cu(II)-TDP-43 coordination. Competitive Zn(II) ion binding studies revealed Zn(II) increases Cu(II) binding. In Chapter 3, Fe(III) ion binding studies revealed that Fe(III) reduces Cu(II) binding when co-exposed to the TDP-43-Au surface. Data shows significant uptake of Cu(II) by TDP-43 protein which may have important implications in normal and diseased states of TDP-43, indicating surface bioelectrochemistry is a viable tool for fundamental exploration of proteins and metals, and their interactions, as they inform disease mechanisms, disease detection and drug screening.

Author Keywords: Amyotrophic Lateral Sclerosis, bioelectrochemistry, electrochemistry, metalloprotein, surface characterization, TDP-43

The Wabigoon River is known for an historic mercury (Hg) pollution source, caused by a chlor-alkali facility operating in the 1960s. As legacy Hg contamination continues to cause serious adverse health effects to the local communities living in the Wabigoon River region, it is imperative to undertake additional research to understand the deposition and transport of historical mercury in this system and more importantly, its conversion into methylmercury (MMHg) which renders it bioavailable for ongoing bioaccumulation. The aim of this dissertation was to evaluate the transport and accumulation of Hg species by doing a spatial and temporal analysis of concentrations of mercury and methylmercury along the Wabigoon River, as well as assessing rates of methylation and demethylation, identifying areas of higher methylmercury production. Results show that locations downstream from the pollution source still show elevated mercury concentrations, with levels at least five times higher in water and up to 134 times higher in sediments compared to background levels. Among selected study sites, the Hydroelectric dam, the Wabigoon Rapids wetland and Clay Lake were identified to have high capacity for methylmercury production in the system, with notably Clay Lake presenting a higher potential for methylmercury accumulation due to the observed lower methylmercury demethylation rate. Furthermore, the impact of wetting and drying cycles on Hg methylation in riverbed and wetland locations within the Wabigoon River system was investigated through a laboratory simulation. Findings indicated increased susceptibility of riverbed locations to wetting and drying cycles.

Author Keywords: Demethylation, Mercury, Methylation, Methylmercury, Wabigoon River

During the COVID-19 pandemic, nucleic acid and antibody-based testing methods were heavily relied upon, but can be costly, time-consuming and exhibit high false -negative and -positive rates. Thus, alternative strategies are needed. Viral antigens such as the SARS-CoV-2 spike (S) glycoprotein are critical in the function of the virus and useful as diagnostic biomarkers for viral infections. For biosensing applications, aptamers are suitable high-affinity and cost-effective binding partners for their specific targets. Using localized surface plasmon resonance (LSPR), real-time, rapid acquisition of results can be achieved, essential for improving the efficacy of a sensor. Herein, LSPR aptamer sensors were fabricated for the detection of the SARS-CoV-2 protein. Data indicate that the best performing aptasensor was the streptavidin-biotin sensor, while the current gold aptasensor exhibited lower sensitivity and the fabrication of the carboxyl aptasensor was unsuccessful. The S1 aptamer selectively bound the S1 protein with high binding affinity. Excellent shelf-life stability, reusability, and high recovery in complex matrices was also maintained. Additionally, a receptor binding domain (RBD) functionalized sensor was fabricated to examine the interactions with angiotensin converting enzyme 2 (ACE2), for future assessment of inhibitors used in drug therapies. Overall, LSPR has been demonstrated as a viable tool for measuring SARS-CoV-2 related aptamer-protein and protein-protein interactions, and this strategy may be applied to other viral or non-viral antigen targets.

Author Keywords: Antigen-based Detection, Coronavirus, COVID-19, Inhibition, Localized Surface Plasmon Resonance, SARS-CoV-2

Phenolic compounds are used in industry, such as agriculture and biotechnology, and inevitably end up in our environment. These compounds may serve as a phenolic precursor to produce raw materials for a wide range of applications. Chemical oxidation has been the common synthetic pathway to oxidize phenols and related compounds. However, traditional chemical approaches suffer from use of harsh chemicals, waste generation, and lack of reaction selectivity. Electrochemical synthesis has emerged as an alternative method to mitigate common challenges associated with organic synthesis. Herein, electrochemical oxidation of 2,6-diphenylphenol (DPP) and 2,2-dihydroxybiphenol (DHBP) was carried out and compared to traditional chemical oxidation. Contrasted with chemical oxidation, cyclic voltammetry of DPP resulted in a range of products based on the specific potential ranges used, whereas chemical oxidation of DHBP yield a dark-coloured polymeric product. The electrooxidation and chemical oxidation of DPP and DHBP resulted in a solution colour change, indicative of the formation of new, but different products monitored by UV-vis, and characterized by nuclear magnetic spectroscopy (NMR), X-ray single crystal diffraction, IR spectroscopy, transmission electron microscopy (TEM), and gas chromatography-mass spectrometry (GC-MS). The data indicate that the synthetic outcomes are dependent on the synthetic methodology employed, and that electrooxidation and chemical oxidation can form products unique to the pathway utilized.

Author Keywords: chemoselectivity, electrochemistry, phenols, radical, synthesis

Amino acids make up proteins, which are the building blocks of life. A balance of amino acids is needed to maintain a healthy state. Tyrosine (Tyr) is synthesized from the metabolism of phenylalanine, which is an essential amino acid, meaning it can only be obtained from the diet. It is related to many metabolic and neurodegenerative diseases. Tyr can undergo post-translational modifications such as phosphorylation and nitration, which are implicated in cancer and nitrative stress, respectively. Although there are many methods to detect Tyr and its analogues, phosphotyrosine (pTyr) and nitrotyrosine (nTyr), these methods are time-consuming, involve expensive instruments and involve tedious process. This research proposes a new type of nanomaterials, carbon dots (CDs), to detect these amnio acids. Data indicate that CDs can be used to detect nTyr with a limit of detection of 34 μM in the linear range of 20 - 105 μM. The amenability of CD-nTyr assay was also tested in various biological matrices and biological molecules and was shown to be sensitive to nTyr. Nitration of Tyr was carried out in the presence of sodium nitrite and hydrogen peroxide catalyzed by either Cu(II) or Fe(III) to mimic biological reactions and CDs were tested as both inhibitors and indicators of Tyr nitration. Although CDs did not inhibit the nitration reaction of Tyr, they did not serve as indicators of Tyr nitration due to the quenching of CDs by the nitrating agents. This shows the importance of using CDs to detect nTyr and further use it for biological applications to detect diseased states.

Author Keywords: amino acids, carbon dots, nanomaterials, sensor, spectroscopy, tyrosine

Euglena gracilis is an enigmatic and adaptable organism that has great bioremediationpotential and is best known for its metabolic flexibility. The research done in this dissertation addresses (1) how growth conditions impact cellular composition, and (2) how chemometric approaches (such as statistical design of experiments and artificial neural networks) are viable alternatives to the conventional biosorption models for process optimization. Using high-resolution mass spectrometry for biosorbent characterization is a powerful way to assess the chemical characteristics of lyophilized and fractionated cells with high precision, especially to screen for compound classes that may have potentiality for rare earth element removal. Growth conditions impacted cellular composition and separated size fractions of cells yielded different molecular/chemical properties as described by compositional abundances, thus different biosorptive potential. Untargeted analysis demonstrated that exponential dark-grown cells with glucose supplementation were abundant in polyphenolic- and carbohydrate-like compounds, molecular species highly involved in rare earth element binding. Light grown cells had more heterogeneity and the highest molecular weighted fractions from light grown cells (fraction D) had the most abundances of polyphenolic- and protein-like structures. Chemometric modeling used identified the best and worst conditions for iii dysprosium sorption and showed that pH had the most significant influence on bioremoval. Bioremoval ranged from 37% at pH 8 to 91% at pH 3 at Dy concentration ranging from 1 to 100 μg L-1. The work presented in the PhD dissertation will aid in understanding the chemical characteristics of biosorbents by using a Van krevelen analysis of elemental ratios whether algal cells are grown in different environmental growth conditions, or when algal cell are size fractionated. This is especially applied for the screening for metal binding potentiality to Dysprosium. Chemometric methods provide an alternative method for the investigating factors for bioremoval, and applications for process optimization and for real-world applications. This dissertation will aid in understanding chemical characteristics when a biosorbent is grown in a given condition and which factors are important for rare earth element (REE) bioremoval. The significance of this work aims to look for alternate ways to screen biosorbents and using a more efficient experimental design for REE bioremoval.

Author Keywords: bioremoval, biosorption, chemometrics, dysprosium, euglena, mass spectrometry

A novel passive sampler for mercury monitoring was prepared using organosilica sol-gel materials. It comprises a binding layer with thiol groups for mercury complexation and a porous diffusive layer through which mercury can diffuse and arrive at the binding layer. Our study demonstrated that this new sampler follows the principle of passive sampling. The mass of mercury accumulated in the binding layer depends linearly on the mercury concentration in solution, the sampling rate and the exposure time. A typical sol-gel sampler is characterized by a diffusive layer of 1.2 &mum, in which mercury ions diffuse with a coefficient of D = 0.09~10-6 cm2/s. The capacity for mercury uptake is approximately 0.64 &mug/cm2. Mercury diffusion and binding in the passive sampler are independent of the type of mercury-chloride complex. Its sampling rate increases with increasing water turbulence and decreases with increasing DOM amount. The field trial of sol-gel sampler in Miller Creek shows the concentration gained from the sol-gel passive sampler is slightly lower than that from the spot sampling.

The efficacy of the diffusive gradients in thin films (DGT) passive samplers to provide accurate measurements of free metal ions and those complexed with dissolved organic matter (DOM) was investigated. DOM controls the diffusive properties of DOM-complexed metal species in natural systems. Knowing the diffusion coeiffiecent (D) for DOM of different molecular weights (MW) and the major environmental variables influencing D is critical in developing the use of DGT passive samplers and understanding labile species. D and MW were determined for natural and standard DOM. No noticeable changes in DOM MW were observed during the diffusion process, suggesting that DOM remains intact following diffusion across the diffusive gel. Data analysis revealed that MW had the greatest influence on D, with a negative relationship between D and MW, except in tidal areas where ionic strength influence on D was significant. This study provides further characterization of the variables influencing D using the DGT technique.

Author Keywords: Diffusion coefficient, Diffusive gradients in thin films, Dissolved organic matter, Flow field-flow fractionation, Principal Component Analysis, UV-Vis Spectroscopy

Selenium is an essential trace nutrient to many organisms, yet in high concentrations it is toxic. Organic selenium is more bioavailable to aquatic biota than inorganic selenium, but is usually found in much lower concentrations. Algae are known to biotransform inorganic selenium into several organo-selenium compounds, but it is unknown whether any of these bioaccumulate in the food chain. In this study, selenium was incorporated into the methionine residues of an algal photosynthetic protein, c-phycocyanin from Spirulina spp. The extent of selenium incorporation was quantified by inductively coupled plasma-mass spectrometry (ICP-MS), and the protein was identified using electrospray mass spectrometry (ES-MS).

C-phycocyanin was isolated and purified from Spirulina with a final recovery of 20-30 % of the total c-phycocyanin present. Selenomethionine replaced 92.8% ± 1.22 of the methionine residues in c-phycocyanin when grown in 2.5 ppm sodium selenite. ES-MS was used to obtain protein spectra, and pure c-phycocyanin was identified. Data of full scans provided estimated masses of both protein subunits--α-chain measured at 18,036 Da; β-chain measured at 19,250 Da--close to the theoretical masses. Protein fragmentation by collision-induced dissociation and electron capture dissociation provided approximately 52 % amino acid sequence match with c-phycocyanin from Spirulina platensis. This study demonstrates the incorporation of selenium into an algal protein, and the identification of c-phycocyanin using electrospray ionization-mass spectrometry.

Silver nanoparticles (nAg) are the largest and fastest growing class of nanomaterials, and are a concern when released into aquatic environments even at low μg L-1+). Diffusive gradient in thin films (DGT) with a thiol-modified resin were used to detect labile silver and carbon nanotubes (CNT-sampler) were used to measure nAg. Laboratory uptake experiments in lake water provided an Ag+ DGT diffusion coefficient of 3.09 x 10 -7 cm2s-1 and CNT sampling rates of 24.73, 5.63, 7.31 mL day-1, for Ag+, citrate-nAg and PVP-nAg, respectively. The optimized passive samplers were deployed in mesocosms dosed with nAg. DGT samplers provided estimated Ag+ concentrations ranging from 0.15 to 0.98 μg L-1 and CNT-samplers provided nAg concentrations that closely matched measured concentrations in water filtered at 0.22 μm.

Author Keywords: ICP-MS, mesocosms, nanoparticles, nanosilver, passive sampling