Theses (Chemistry)

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    Microdroplets for drug discovery and delivery targeting neural models
    (2024-01-31) Forigua Coronado, Alejandro; Elvira, Katherine S.
    This dissertation explores the application of microdroplet technology in two pivotal areas of the pharmaceutical and biotechnological fields: drug discovery and drug delivery, with a focus on neural models. It presents a comprehensive study on the development and application of microfluidic devices for the fabrication of drug delivery particles and the modeling of cell membranes using Droplet Interface Bilayers (DIBs). The first part of this work, detailed in Chapters 2 and 3, describes the design and optimization of a Polydimethylsiloxane (PDMS) microfluidic platform for generating oil-in-water droplets. These droplets serve as precursors for polycaprolactone (PCL) microparticles, which have potential for controlled drug release. This platform showcases significant improvements in droplet generation and encapsulation efficiency compared to traditional batch processes and has been adopted for commercial-scale production. In the second part, Chapters 4 and 5 focus on the application of DIBs as a model for cell membranes. The research quantitatively analyzes the passive diffusion of memantine, an Alzheimer’s disease treating drug, and niacin, a common vitamin supplement used as a neuroprotective agent, and examines the impact of lipid formulation and droplet content on drug absorption and water transport. The findings highlight the advantages of using biomimetic lipid formulations for in vitro studies. This dissertation demonstrates the significant potential of microdroplet technology in enhancing the efficacy and precision of drug delivery systems and in providing more accurate models for cell membrane studies. The insights gained not only contribute to the academic understanding of drug interaction with cellular membranes but also pave the way for future innovations in neuropharmacology and biotechnology.
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    Quantitative Models for Accurate Reactivity Predictions and Mechanistic Elucidation
    (2024-01-05) Lu, Jingru; Leitch, David
    Accurate prediction of reaction outcomes is among the most important goals in chemical and pharmaceutical synthesis. In recent years, the ultrafast growth in computing power and the advancement of high-throughput experimental (HTE) technology have paved new ways to apply data-rich approaches in chemistry research. In organic synthesis, data-driven methods have found many successful applications in accelerating reaction condition optimization and developing machine learning models for reaction prediction. Despite all the impressive progress made in this area, accurate prediction of chemical reactivity remains challenging. This thesis describes development of quantitative reactivity models for accurate reaction prediction and mechanistic elucidation in organic synthesis. Starting with a minireview/perspective in Chapter 1, recent progress is discussed in data-rich approaches to reaction development and quantitative predictions for palladium-catalyzed reaction systems. In Chapter 2 and Chapter 3, quantitative predictive models are developed for two pharmaceutically important reaction systems: nucleophilic aromatic substitution (SNAr) and oxidative addition to palladium(0), a fundamental and usually the rate/selectivity determining step in palladium-catalyzed cross-coupling reactions. Both models focus on structure-reactivity relationships of the electrophiles. Diverse and reliable reaction rate data for training set was collected using high-throughput competition experimentation. These were used to construct multivariate linear regression models by quantitatively mapping a group of ground state molecular descriptors to the experimental reaction rates. Predictive accuracy is validated via a series of random train-test splits, as well as predicting outcomes for a wide variety of external reaction data. Following the procedures described above, generally applicable models for quantitative predictions on both the reaction rates and site-selectivity for both reaction systems have been realized. In addition to making quantitative reaction predictions, a structure-reactivity model constructed using high-quality data and mechanistically meaningful descriptors is also very useful in gaining mechanistic insights. This is demonstrated by the solvent effect study in Chapter 4 and the reaction mechanistic study in Chapter 5. From the quantitative reactivity scales constructed for oxidative addition to palladium(0) in different solvents, specific electrophiles were identified that exhibit significant solvent effects; the role of solvent was investigated case by case. These include the importance of solvent hydrogen-bond basicity as well as solvent polarity. Finally, the underlying mechanistic causes behind a series of systematic prediction outliers from our oxidative addition model were investigated. These reveal that the frontier orbital symmetry also plays an important role in determining reaction outcomes. Insights into these mechanistic aspects, which have a significant impact on both the reaction rate and site-selectivity in oxidative addition to palladium(0), enabled a refined quantitative model that incorporates frontier orbital descriptors.
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    Excitation Emission Matrix (EEM) Spectroscopy and Computational Evaluation of Excited States of Carbazole – Bromobenzothiadiazole (CBB)
    (2024-01-05) Joulaei Zonouz, Sara; Loock, Hans-Peter
    We examine the source of solvatochromism in a fluorescent organic dye using fluorescence Excitation Emission Matrix (EEM) spectroscopy, supported by ab initio calculations. The dye, carbazole–bromobenzothiadiazole (CBB), has a donor-, an acceptor-, and a bridging group connected by sigma bonds. The study of its fluorescence in liquid solutions shows a strong emission wavelength dependence on the solvent polarity but only a negligible dependence on solvent viscosity. In previous work, the polarity-induced solvatochromism was attributed to a twisted intermolecular charge transfer (TICT) state, where the excited state twists into a conformation in which a large dipole is generated between the carbazole (donor) group and the benzothiadiazole (acceptor) group. (1) Density Functional Theory (DFT) calculations are performed to map the excited state potential energy surface and the associated dipole moment in different solvent environments using the corrected linear response (cLR) solvent model (2). The calculations agree well with the observed energy differences and the solvatochromic shifts. To determine the configuration of the molecule in the excited state before emission, the dihedral angles between the three main groups of the molecule are investigated. Ab initio calculation indicates that the three dihedral angles between the donor-, bridging-, and acceptor-groups in the excited state change only by comparably small amounts between the Franck-Condon region and the potential minimum of the excited state, from which fluorescence is expected to occur. The molecular configuration in the excited state’s potential minimum is therefore close to the molecular structure in the Franck-Condon region, and not a “twisted” structure indicative of a TICT structure. Interestingly, we calculate a second local minimum on the excited state in the global excited state form when the third dihedral angle, θ_3 =90°. However, the energy in this twisted form is much higher than that of the first minimum configuration (θ_3= 0°). Since the second excited state minimum is not accessible upon excitation of the molecule emission is calculated to occur from the lowest excited state, which was, indeed, observed. Consequently, in this research, the solvatochromic shift is due to the charge separation around the acceptor domain, which is partially induced by the polarity of the environment. This work raises questions about the presence of TICT state in other molecules. We argue that the observation of solvatochromatic shifts, and the presence of donor and acceptor moieties alone are not sufficient to positively identify a TICT state. We found that TD-DFT calculation require a sophisticated solvent model to quantitatively model the solvatochromic shift. Also, we observed the natural transition orbitals are far more useful in identifying donor and acceptor moieties compared to the commonly used canonical orbitals. Excitation Emission Matrix (EEM) spectroscopy serves as a useful experimental tool to identify excitation and emission pathways.
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    Optimizing the encapsulation of SN-38 in PCL-b-PEG polymer nanoparticles for cancer therapy
    (2024-01-05) Silverman, Lisa; Moffitt, Matthew
    Background: In this study, we address challenges encapsulating the anticancer agent, SN-38, in polymer nanoparticles (PNPs) for improved cancer treatment. SN-38 is particularly difficult to encapsulate due to its poor solubility in water and many solvents. Methods: PNPs were synthesized using both bulk and microfluidic nanoprecipitation, and characterized for physico-chemical properties, release kinetics, and cytotoxicity and tumor penetration in 2D cell culture and tumor spheroids. Different formulations of PNPs, including different ratios of SN-38 and curcumin (CUR), and block co-polymers with different PEG terminal endgroups were compared to find the optimal formulation for encapsulating SN-38. Results: By co-encapsulating CUR with SN-38, we can achieve increased SN-38 encapsulation efficiencies in co-loaded SN-38/CUR-PNPs by up to ten-fold as compared to PNPs encapsulating SN-38 alone. Moreover, a two-phase microfluidic reactor demonstrates similar trends regarding SN-38 content with CUR co-encapsulation, compared to bulk nanoprecipitation methods. Our findings also reveal a decrease in PNP polydispersity from 0.34 to 0.07 as the initial CUR-to-polymer initial ratio increases from 0 to 10. Our first cytotoxicity studies show adding CUR does not significantly affect SN-38 potency. However, we observed significant differences in the potencies of SN-38/CUR-PNP formulations depending on formulation. An optimized formulation exhibited sub-nanomolar cytotoxicity against A204 cells, surpassing the potency of free SN-38 or PNPs containing only SN-38. We find that incorporating a thiol terminal end group onto the PEG in the PNP resulted in a doubling of SN-38 encapsulation efficiency from 10% in the reference SN-38/CUR-PNP-OCH3 to 21% in SN-38/CUR-PNP-SH, but that this increase is only seen when the SN-38/CUR drug mixture is used, and not when the drugs are encapsulated individually. Confocal microscopy shows encouraging results regarding PNP penetration throughout tumor spheroids, but EC50 cytotoxicity results in both 2D and 3D culture models show limited efficacy in cell killing of our formulations in 3D models, and although the SH-PNP formulation shows the best results in 2D models, the reference OCH3 formulation shows better performance in the 3D models. Conclusions: Our study presents a co-encapsulation strategy that significantly enhances SN-38 encapsulation efficiency within PNPs for improved cancer treatment strategies. These findings contribute to overcoming challenges associated with poor solubility of SN-38 and paves the way for the use of SN-38 in the clinic.  
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    The Development of Chemical Analytical Tools for Community Drug Checking
    (2024-01-03) Gozdzialski, Lea; Hore, Dennis
    Drugs have many uses from pleasure to pain relief, ceremony, and medicine. Drugs also carry risks, from uncomfortable side effects, to dependence, and even death. In the case of pharmaceuticals, most people are familiar with receiving instructional notes and warnings such as "take with food" or "let your pharmacist know about other medications that might have undesirable interactions." Strict quality control means that prescribed and regulated drug mixtures are known to be as safe as possible. In the case of the illicit drug market, such assurances of quality and support are not afforded. In response, this thesis focuses on advancing the technology required for drug checking, a grassroots harm reduction initiative that aims to provide a level of quality control to the illicit drug market using various analytical approaches. Due to the unprecedented and increasing number of overdose deaths, these services have been expanding throughout North America. Drug checking empowers people who use drugs with the knowledge of what they are consuming and further provides an avenue for education and support to communities about the local drug supply. However, implementation of drug checking faces many barriers not only systemically but analytically as well, in part due to the dynamic and unpredictable drug supply and demand for simple, cost-effective, and point-of-care techniques. This thesis explores several point-of-care analytical methods in their application to drug checking. These analytical methods include immunoassay test strips, infrared, Raman, and surface enhanced Raman spectroscopy, and gas chromatography–mass spectrometry. Notably, this research and development takes place while concurrently providing drug checking as a community harm reduction service. Most of the datasets used throughout this work are acquired at the service and reflect the local drug supply. A major focus of this research is on the detection of opioids and benzodiazepines in drug mixtures. Chemometric approaches are used to evaluate, compare, and improve the capability of multiple instruments in providing useful drug information for the local supply. This includes classification and quantification schemes using a wide range of methods such as partial least squares regression, local outlier factor, principal component analysis, random forest classifier, least angle squares regression, correlation analysis, k-nearest neighbours, multivariate curve resolution, and density-based spatial clustering. Performance metrics, such as true positive rates, false positive rates, F1 scores, and receiver operating curves for qualitative detection and root mean square error and accuracy profiles for quantification, are used for evaluation. Additionally, a custom analysis platform is developed and implemented using Python and Jupyter notebooks to allow for such developments to be actualized within the service. Beyond technical evaluation, discussion of the results of this research largely considers the practical requirements of point-of-care service delivery. Within this work, technical information regarding drug checking technologies and data analysis is contextualized within harm reduction and contributes to strengthening the body of drug checking literature and resources.
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    A Photophysical Investigation on the Role of Complexity on Controlling the Functions of Pluronic F127 and Sodium Deoxycholate Supramolecular Hydrogels
    (2023-09-29) Awasthi, Ankur; Bohne, Cornelia
    Colloidal states of matter, where a solid three-dimensional network entraps a liquid mobile phase are known as gels. Based on the nature of the interactions between the building blocks that constitute the solid matrix, gels can be classified into chemical or supramolecular gels. The covalent linkages involved in chemical gels make them irreversible and functionally rigid in nature. The non-covalent nature of the interactions between the building blocks in supramolecular gels makes them more reversible and susceptible to external stimuli. However, the involvement of non-covalent interactions in the formation of supramolecular gels, increases the complexity associated with such systems and makes it difficult to predict their function. The primary objective of this dissertation was to understand the essential theoretical concepts that could facilitate the prediction of function in supramolecular gels. With respect to hydrogels, function can have various meanings depending on the type of application intended. For example, the release kinetics of loaded drugs from hydrogels can be considered one kind of function for a hydrogel. Another kind of function for a hydrogel could be its mechanical strength that can impact the different kinds of application a particular gel can be employed for. An additional outcome the work done in this dissertation seeks to emphasize is the significance of increased complexity in the designing of functional systems. Complexity is defined by the interconnected relationships between the various molecules involved in functional systems. By investigating systems where the complexity is gradually increased, my aim is to show that this increased complexity can be harnessed to design better functional hydrogel systems. The first project involved using known theoretical concepts around the self-assembly of polymeric F127 hydrogels. The micellar microstructures of the hydrogel allow for guest localization in either the hydrophobic core or the hydrophilic corona. By choosing a water insoluble fluorophore like N, N′-bis(salicylidene)-(2-(3′,4′-diaminophenyl)benzothiazole (BTS), the hydrophobic effect was exploited to confine BTS to the core of the F127 micelles. This fluorophore confinement to the hydrophobic core of the micelles allowed us to generate an emissive hydrogel whose emission color was seen to be unaffected by changes in its environmental pH. This emission is seen to be a result of all three forms of BTS, i.e., the neutral, tautomer and dianionic forms, existing in the excited state. Additionally, by changing the iv concentration of BTS localized within the hydrogels, the emission color from F127 hydrogels can be modulated. The second project utilized the technique of fluorescence quenching to gain a detailed understanding of the microstructures present in sodium deoxycholate (NaDC) hydrogels. Pyrene was used as a polarity sensitive fluorophore to investigate this hydrogel system. The ratio between peak I and III (I/III) of pyrene steady-state emission spectra revealed details around the polarity of pyrene localization within the hydrogels. The accessibility of two different quenchers, an ionic quencher, iodide anion and a neutral quencher, nitromethane to excited pyrene was studied using steady-state and time-resolved fluorescence. Steady-state results indicate that pyrene in the hydrophilic parts of the gel is quenched more efficiently than in the hydrophobic parts of the gel, irrespective of the quencher used. However, time-resolved studies indicate that among the three possible microenvironments available for pyrene localization, there exists a microenvironment that allows access to nitromethane but not to iodide anions. These results indicate the availability of microstructures in the hydrogel that have a high negative charge density, which is responsible for the lack of access to iodide anions due to electrostatic repulsions. The addition of cucurbit[6]uril (CB[6]) to these hydrogels results in a relocation of pyrene from the hydrophilic region to the hydrophobic regions of the hydrogel, indicated by the lack of alteration to the I/III ratio with increasing quencher concentration as well as the decreased access of the quencher. Moreover, the time-resolved studies indicate an increased heterogeneity for pyrene, which is a result of its complexation to CB[6]. Using the information from this project and previous work in the group, a molecular image of the hydrogel is successfully created that can explain the difference in release kinetics of pyrene and rhodamine 6G from NaDC-CB[6] hydrogels. The last project was designed to investigate if the release kinetics of guests from a hydrogel system would be dependent on the binding kinetics between the building blocks of a multi component hydrogel. This involved the design and synthesis of a monourea derivative, and studying its gelation behavior and binding kinetics with CB[6]. The gelation studies involved the use of inversion vial tests to observe how the gelation behaviour of the urea is affected due to the presence of salts and CB[6]. The study of the binding kinetics showed the presence of both fast v (0–50 s) and slow kinetic processes (5-30 min). The information from the kinetic studies confirm the presence of binding interactions of the urea with NaCl and CB[6], which are possibly responsible for the changes in its gelation behaviour. Using the available information from the preliminary studies done in this work, prospective new functionalized ureas that may possess better gelation properties in aqueous solvents in presence of CB[6] are proposed.
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    Paper Spray Mass Spectrometry for the Quantitation of Drugs of Abuse in Biofluids and Street Drug Samples
    (2023-09-29) Borden, Scott A.; Gill, Chris; Brolo, Alexandre G.
    Paper spray mass spectrometry (PS-MS) has been developed as a tool for the analysis of drugs of abuse (DoA) in street drug samples, urine, and oral fluid. PS-MS is presented as a viable alternative to the traditional gas chromatography and liquid chromatography-mass spectrometry methods. PS-MS achieves sensitive and quantitative results in as little as 1-2 minutes with little to no sample preparation. Initial research presented in this thesis illustrates how PS-MS was developed for the analysis of fentanyl and related analogs of powdered drug slurries acting as a proxy for street drug samples, as a proof of concept for real world drug checking. Analysis of DoA in these pseudo-drug samples demonstrated the potential for both quantitative and qualitative analysis of fentanyl analogs. PS-MS was then demonstrated and evaluated for its effectiveness for real world drug checking applications during a world first demonstration of PS-MS for on-site drug checking in the Downtown Eastside of Vancouver, British Columbia, a recognized epicenter of the opioid overdose crisis. During the pilot test, 113 samples were submitted for analysis and successfully quantified using PS-MS, which targeted and quantified 49 different drug targets. Of these 113 drug samples, 44% of all samples were found to contain fentanyl, with a median concentration of 3.6% (w/w). The benzodiazepine etizolam was detected in 10 samples, none of the people who submitted these 10 samples expected a benzodiazepine to be present in their sample. It was later found that other drug checking technologies in use were underreporting the presence of etizolam or other benzodiazepines present in drug samples. These results, coupled with the quantitative capabilities and low levels of detection observed during the pilot test of PS-MS for drug checking demonstrated the efficacy of PS-MS and inspired further development of the application. PS-MS was then implemented by the Vancouver Island Drug Checking Project for the routine quantitative measurement of thousands of drug samples. During the span of this routine measurement, two unidentified compounds began appearing in carfentanil-containing drug samples. High resolution accurate mass (HRAM) mass spectrometry was used to determine the chemical composition of these two unknowns as C23H29N3O2 (m/z 380.2333) and C23H29N2O3 (m/z 381.2137). Further tandem mass spectrometry experiments were used for structural elucidation and the unknowns were putatively identified as desmethylcarfentanil amide and desmethylcarfentanil acid. LC-MS data on different drug samples containing the same compounds further supported the identification of these carfentanil structural analogs. µ-Opioid receptor binding modeling determined that the binding poses, and binding energies of these structural analogs were nearly identical to that of carfentanil, suggesting potentially similar activities/toxicities. PS-MS was further applied to the analysis of cannabinoids in urine and oral fluid samples. Due to the inherently low ionization efficiencies and sensitivity to cannabinoids observed with PS-MS, a reactive paper spray ionization method utilizing a diazonium salt as a on-paper derivatization reagent was developed. The derivatization scheme dramatically lowered the limits of detection for tetrahydrocannabinol (THC) in oral fluid and THC metabolite in urine, to levels able to meet forensic and clinical cutoff values (low parts per billion). The quantitative results were compared to a LC-MS results from a commercial clinical laboratory, demonstrating good agreement between the two methods. The results presented herein demonstrate the applicability and dramatic benefits of PS-MS for drug checking applications, as well as for cannabinoids in oral fluid and urine. High resolution mass spectrometry is demonstrated for the structural elucidation and identification of unknown drug compounds in an ever-changing street drug supply.
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    Using the surfaces of droplets formed using droplet-based microfluidic technologies to study biomolecular interactions
    (2023-09-01) McDonald, Alex R.; Elvira, Katherine S.
    This thesis explores droplet-based microfluidic technologies to fabricate bespoke emulsions, focusing on lipid- and protein-based interactions on the surface of the droplets. I introduce microfluidic technologies for droplet formation and factors that influence droplet shape and size. Different approaches to forming single and double emulsions using droplet-based microfluidic technologies are discussed along with considerations such as surface chemistry, droplet stability, and applications. First, I use double emulsions to produce biomimetic vesicles (liposomes) and explain why dewetting is a key step in liposome fabrication. I fabricated a lipid-based surface on aqueous droplets as a bottom-up cell membrane model using a novel combination of naturally-derived lipids in the aqueous phase and a simple plug-and-play microcapillary platform. These asymmetric liposomes remain stable in an asymmetric conformation for over 24 h and are within the size range of actual eukaryotic cells. I show that this cell membrane model is more biomimetic than other current models based on the lipid composition and conditions it is fabricated in. Second, I create a protein-based surface on oil droplets to explore the roles of proteins on droplet stability in beer. This was the first time a hop-oil-in-beer emulsion was made on a microfluidic device to explore the role that proteins have in long-term emulsion stability, which serves as the first step in understanding the unknown stabilization mechanism that keeps beer shelf-stable. By digesting gluten, a protein commonly found in beer during fermentation, with a gluten-specific enzyme, I show that hop-oil emulsion stability is influenced by the concentration of gluten present in solution. This thesis highlights the potential of droplet-based microfluidic technologies to create custom surfaces on emulsions and characterize their properties in two distinct applications: academic and industry.
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    Investigating the Influence of Chirality and Stable Free Radicals on the Optical and Electrical Properties of Charge Transfer Dyes
    (2023-08-31) Templeman-Vivian, Bennett; Leitch, David
    As the demand for advanced electronics continues, there is an increasing need for the development of technology and infrastructure to support these advancements. This thesis aims to address this need by exploring the synthesis of a series of chiral charge transfer dyes with hopes to investigate the chiral induced spin selectivity (CISS) effect. Furthermore, this thesis explores the effect that appending a stable radical to a well-known hole transport material has on the optoelectronic properties of the molecule. Chapter 1 provides an introduction to the topics of conjugation and charge transfer, laying the foundation for understanding the subsequent chapters. Additionally, the unique properties of organic charge transfer molecules are discussed and their applications are highlighted. In Chapter 2, previous work by Dr. Marc Junge is expanded on and three novel chiral charge transfer dyes are proposed to investigate the CISS. The three dyes are a donor-acceptor-chiral bridge-acceptor molecule (DA-(*)-A), a donor-acceptor-chiral bridge-acceptor-donor molecule (DA-(*)-AD), and an acceptor-chiral bridge-acceptor molecule (A-(*)-A). The synthesis of DA-(*)-AD was attempted using both the Stille and Suzuki Miyaura coupling reactions. Unfortunately, both methods were unsuccessful. The synthesis of DA-(*)-AD and A-(*)-A were successful through borylation and Suzuki Miyaura coupling. The optical properties of these dyes are explored using UV-Vis and fluorescence spectroscopy, with a particular focus on the fluorescence characteristics. These may indicate the formation of a twisted intramolecular charge transfer excited state. Chapter 3 focuses on observing the effect that appending a stable radical on a triphenylamine (TPA) core has on the optoelectric properties of the molecule. A monoverdazyl TPA, a diverdazyl TPA, and a triverdazyl TPA were synthesized. The dyes were characterized using EPR, high-resolution mass spectrometry (HRMS) and infrared spectroscopy. The optical properties were investigated through UV-Vis and fluorescence spectroscopy and the optical band gap was found. Cyclic voltammetry was employed to determine the oxidation and reduction potentials, enabling the identification of the energy levels of the singly occupied molecular orbital (SOMO) and the lowest unoccupied molecular orbital (LUMO). Diverdazyl TPA was further functionalized through a direct arylation to a thiophene derivative. Chapter 4 offers a summary of the findings and proposes further avenues of investigation. Furthermore, a preliminary study utilizing density functional theory (DFT) on the monoverdazyl dye is also presented. Overall, this thesis offers an exploration of the synthesis of organic chiral charge transfer dyes and the influence of stable radicals on optoelectronic properties of triphenylamines. The findings pave the way for future advancements in the field of advanced organic electronics.
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    Microfluidic Synthesis of Silver Loaded, pH Sensitive, Nanogels for Enhanced Radiotherapy
    (2023-08-31) Boulding, Kyra; Valente, Karolina; Brolo, Alexander G.
    Radiation therapy (RT) is an effective and commonly used course of treatment for cancer. However, RT lacks specificity resulting in sever acute and chronic side effects. Radiosensitizers, like silver nanoparticles (Ag NPs), possess properties that allow for the localized enhancement of RT effects. Nanoscale smart drug delivery systems proved the ability to impose specificity on inherently non-specific treatments. In this work, microfluidics was used to fabricate a pH-sensitive gelatin methacryloy (GelMA) nanogel for the smart delivery of radiosensitizers into the tumor microenvironment. Optimization of synthesis parameters was described, and transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used for characterization. Successful synthesis using a flow focusing microfluidic device produced low-polydispersity (PDI ~0.06), 123 ± 2 nm pH-sensitive GelMA nanogels, loaded with ~ 11 nm Ag NPs. DLS was then used to obtain preliminary evidence of the release of Ag NPs from the nanogels through the appearance, over time, of a second particle population. Additionally, a second synthesis method involving the in situ synthesis of Ag NPs during the process of nanogel crosslinking was described. This work provides support for the use of microfluidic devices to produce low-polydispersity nanoscale smart drug delivery systems, with applications in enhancing the efficacy of RT.
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    Application of Raman Spectroscopy for the characterization of carbon materials
    (2023-08-30) Guilherme da Fonseca, Bruno; Brolo, Alexandre G
    This dissertation discusses the applications of Raman spectroscopy for the characterization of carbon materials, through the investigation of heterogeneous hydrogenation, estimation of elemental and organic carbon from diesel emission particulate and automated classification. Carbon materials are present in several aspects of human life. Some illustrative examples come from technological use of graphite, diamond and glassy carbon and the impact of diesel emissions in human health. The general objective of the dissertation is to improve the characterization of carbon materials and show the potential and limitations of methods that combines Raman spectroscopy and chemometrics for carbon materials characterization. The literature is vast about the utility of Raman spectroscopy for characterizing carbon materials. However, some methods are either not clearly described in terms of reproducibility and implementation, or very specific to a particular type of material. Also, some studies describe their material based in a single spectrum instead of a spatial and statistical characterization. Chapter 2 demonstrates the usefulness of Raman maps for characterizing carbon films obtained chemically. The study investigated the heterogeneous hydrogenation in the samples and showed the effect of different parameters on the homogeneity of the material. Due to heterogeneous hydrogenation, some regions could be assigned to different types of carbon materials and could lead to different properties. This is highly relevant for potential use in industry as quality control tool. For the scientific community, this chapter showed the importance of the fluorescent background in the Raman spectrum for proper characterization of carbon materials. Organic carbon and elemental carbon are general classifications for particulate matter present in diesel emissions. These two species present potential harm to the health of workers, monitoring their presence is thus paramount for a safe workplace environment. The literature offers different approaches for estimating elemental carbon and the comparison with different techniques. Most of them ignore the organic fraction and don’t compare with the North American standard technique (NIOSH method). The fluorescent background and the correlation with hydrogenation of carbon materials that were observed in chapter 2 inspired the use of the background as source of information for estimating organic carbon content. The results from Raman and partial least squares in chapter 3 suggest the background as an important source of information for estimating organic carbon and total carbon. The predictions of total carbon agreed with the values obtained from the standard technique in the range between 60 and 600 µg. Although Raman spectroscopy is largely used for characterization of carbon materials, it is not rare to find misinterpretation of the spectra. Chapter 4 presents an automated prediction of carbon materials based on their Raman spectra and using principal component analysis followed by linear discriminant analysis. The Raman spectra used in for training the model were obtained from highly oriented pyrolytic graphite, glassy carbon, diamond-like carbon, hydrogenated graphite-like carbon and hydrogenated polymer-like carbon. The testing dataset was based on Raman spectra from the literature and from samples synthesized in the lab. The results showed accuracy of 97 % and some assignments found in the literature could be corrected by the model proposed in this chapter. Chapter 5 offers a summary of the research projects presented in this dissertation and discuss the possible future projects for developing the use of Raman spectroscopy and machine learning for carbon materials
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    Exploring Charge-Transfer Properties of Donor-Acceptor Functionalized Squaraine Dyes
    (2023-08-28) Stanley, Austin; Leitch, David
    In the realm of optoelectronics and solar technology, the demand for efficient and cost-effective materials has intensified. Squaraine dyes have emerged as a promising class of compounds for these applications, owing to their ability to absorb light and facilitate charge transport across different layers within materials. This thesis aims to present the synthesis and characterization of a novel donor-acceptor (DA) functionalized squaraine dye, featuring redox-active phenols that can be oxidized to phenoxyl radicals. Our objective is to investigate the charge-transfer (CT) properties of these compounds using spectroscopic techniques and computational modeling. Chapter 1 provides an introduction to organic DA compounds, the probing of intramolecular charge-transfer (ICT) in such systems, and a background on stable free radicals and squaraine dyes. The chapter sets the context for the application of these molecules in materials and outlines the goals of the thesis. Chapter 2 focuses on the synthetic methodology and nuclear magnetic resonance (NMR) spectroscopic characterization of a series of previously unreported DA compounds and squaraine dyes. The synthesis of compounds 2.14 and 2.15 is described using Suzuki-Miyaura chemistry, followed by the coupling of 2.14 to a known squaraine dye via Suzuki-Miyaura cross-coupling to form 2.25. The synthesis and NMR characterization of these compounds are presented, along with the proposal of a structural motif (2.31) and the discussion of attempts to synthesize it. Preliminary data supporting oxidative addition to form 2.30 are also provided. Chapter 3 focuses on the photophysical and redox properties of compounds 2.14, 2.15, and 2.25. UV-Visible and fluorescence spectrophotometry techniques are employed to demonstrate the CT characteristics of each compound. Cyclic voltammetry is used to investigate the oxidation potentials of the phenols in 2.14 and 2.25, while the attempt to isolate stable free radicals is discussed. Computational data using density functional theory (DFT) and time-dependent DFT (TD-DFT) are used to support the experimental findings, including the plotting of frontier molecular orbitals (FMOs) and natural transition orbitals (NTOs). Chapter 4 provides a comprehensive summary of the results presented in this thesis and suggests future experiments for further investigation. Additionally, preliminary computational results for compound 2.31 are briefly discussed. In conclusion, this thesis explores the CT properties of novel compounds, particularly focusing on the influence of donor character on the photophysical properties. This research contributes to the understanding and potential applications of DA functionalized squaraines in optoelectronics and solar technology.
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    Advances with Solid Substrate Spray Mass Spectrometry for Quantitative Illicit Drug Measurement
    (2023-08-28) Laxton, John-Clare; Gill, Chris
    The opioid crisis continues to be the leading cause of overdose and death among people who use drugs (PWUD). Harm reduction drug checking (HRDC) services aimed at preventing accidental overdose events do so by offering pre-consumption chemical measurements to help PWUD make informed decisions regarding a drug they intend to use. Conventional on-site drug checking technologies such as immunoassay test strips, colorimetry test strips, FT-IR, and portable Raman spectroscopy results do not possess adequate sensitivity for trace levels of toxic drugs, and/or the selectivity to detect newly emerging threats as they appear in the illicit drug supply. Chapter 1 of this thesis provides background information, as well as framework to why mass spectrometry (MS) is the ideal candidate for the analytical task of quantitative HRDC. Solid-substrate electrospray ionization (ESI) was developed as a rapid and direct ionization method for the qualitative and quantitative analysis of complex samples with little to no sample preparation. In this thesis, paper spray mass spectrometry (PS-MS) and paper capillary spray ionization (PCSI), two derivatives of solid-substrate ESI, were utilized for quantitative illicit drug measurements. Conventional (quantitative) chemical analysis requires samples to be sent to centralized laboratories with the requisite supporting infrastructure, however, innovations to the field of portable MS has revolutionized the paradigm of real-time chemical analysis by bringing the laboratory to the sample, answering chemical questions when and where they are needed. In Chapter 2 of this thesis, a simple, rapid, and quantitative ambient ionization tandem mass spectrometry method was developed and implemented for the analysis of real-world illicit drug samples utilizing a miniature mass spectrometer system. The performance, characterization, and improvement of a portable/miniature mass spectrometer (based on a rectilinear quadrupole ion trap) was initially characterized using legally exempt test kit drug standards. A novel spray solvent addition system was developed to improve inter- and intra-day reproducibility, calibration linearity, and analytical sensitivity. The system was then evaluated as a potential on-site, rapid, point-of-care chemical diagnostic system to identify and quantify illicit drug analytes. Target analytes were detected and quantified via PCSI-MS/MS for samples where conventional on-site drug checking technologies were not always effective. In Chapter 3 of this thesis, various internal standard (ISTD) utilization strategies were evaluated with the goal of simplifying HRDC PS-MS quantitative measurements while drastically reducing waste. This was achieved by depositing ISTDs on PS-MS strips, pre- and post-sample deposition, to evaluate its quantitative performance of illicit drug analytes. Pre-sample ISTD depositions reduce disruptions in (PS-MS) HRDC services due to ISTD supply/integrity and improves turnaround time for chemical analysis; all of which reduce analytical costs (i.e., ISTD consumables, labor costs, solvent) for real world samples. Post-sample ISTD depositions offer the ability to send in-field samples to off-site laboratories for appropriate ISTD deposition. A parallel study assessed the analytical performance of delivering 1 nanogram of ISTD per paper strip by one of two methods: 1) the conventional method by “Hand”, depositing volumes with a mechanical micropipette, and 2) by “Robot” through the assistance of a robotic liquid handling system. The goal of this thesis is to promote the feasibility and wide-spread adoption of solid-substrate spray-based MS technology as an on-site HRDC tool.
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    Modelling mechanisms of organometallic reactions from surface deposition to heterogeneous catalysis
    (2023-08-25) Donnecke, Sofia; Paci, Irina; McIndoe, J. Scott
    Theoretical and experimental methods are applied to study the elementary steps taking place in the gas phase and on the surface to help guide the rational design of new precursors and catalysts. The body of this work sets out to improve our fundamental understanding of the reactions taking place on various surfaces. A number of reaction pathways are modelled on the surface ranging from benchmarking the energetics of known reactions to modelling new systems and unexplored reaction pathways. Atomic layer deposition (ALD) of cobalt thin films is desirable in the fabrication of complex nanodevices and development of effective precursors requires a mechanistic understanding of the deposition pathways. The first two projects set out to understand the reactivity and deposition pathways of a number of existing and new Co precursors for ALD, and identify promising precursors to improve deposition. In a third project, the catalyzed oxygen reduction reaction (ORR) is studied to both benchmark computational methods and identify electronic trends across different metals and systems. DFT is shown to perform relatively well compared to CCSD(T) on difficult, high spin systems and modest model reduction in molecular catalysts is found to improve cost and convergence, while retaining the calculated chemical trends. A large number of reaction pathways are computed for known and new transition metal systems and the results are reported herein.
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    Efficient upcycling of low-functionality polymers using trifluoromethyl aryl diazirine chemistry
    (2023-08-14) Bi, Liting; Wulff, Jeremy Earle
    Crosslinking technologies are widely employed in our daily life: from silicone bakeware to epoxy adhesives and rubber tires. Traditionally, however, each type of commodity polymer substrate requires the use of a specific crosslinking method. Many desirable polymers—especially low-functionality polyolefins—cannot be crosslinked using these existing methods. To address this limitation, our group developed a family of bis-aryl-diazirine reagents that function through C−H, O–H or N–H insertions, and that can thereby act as universal crosslinkers for aliphatic polymers. Despite their increasing popularity as crosslinkers in a variety of fields (e.g. photopatterning, bioadhesives), little attention has been given to the synthesis of highly efficient reagents with reduced or eliminated side-products. In Chapter 2, structure–function relationships of mono-aryl-diazirines were studied, highlighting the fundamental role played by electronic properties in the thermal and photo-activation of the molecules, as well as their insertion efficiency. Building upon this study, Chapter 3 introduces a new generation of ether-linked trifluoromethyl bis-diazirine crosslinker, which is more than 10 times as effective as previous generations. The new reagent can also be activated using lower temperatures and longer wavelengths than earlier bis-diazirines—permitting the use of visible light for photopatterning. The efficacy of the electron-rich tether has been demonstrated at both the molecular and polymer level, showcasing the ability of the new linker to covalently adhere to low-surface energy materials and strengthen ultra-high molecular weight polyethylene fabrics. The covalent inter-chain crosslinks in thermoset materials make them difficult to reprocess and recycle. To address this issue, Chapter 4 introduces chemically cleavable groups (e.g. carbonates, oxalates, silyl ethers) into the bis-diazirine crosslinker, allowing for further reprocessing of commodity polymers after the initial crosslinking step. This new class of crosslinkers exhibits rapid reactions during both the crosslinking and decrosslinking steps across a wide range of substrates, including small-molecule models and low-functionality polymers (e.g. LDPE, aPP/LDPE, PEG, PDMS). By employing specific chemical uncoupling methods, these materials can be efficiently converted from thermoset to thermoplastic, presenting a new strategy for circularization of the polymer economy. Chapter 5 describes the synthesis of mono-aryl-diazirine reagents along with their successful use for the non-destructive surface modification of polydimethylsiloxane (PDMS) substrates by both thermal and ultraviolet activation. Bovine serum albumin (BSA) and immunoglobulin G (IgG) are immobilized as a model protein and antibody, respectively, and sensitive quantification of their amounts along with their stability on the surface is achieved by radiolabeling with iodine-125. Through both thermal and ultraviolet activation, two types of trifluoromethyl aryl diazirine reagents with electrophilic motifs were able to enhance the amount and stability of BSA and IgG immobilization on the surface. These techniques show great promise for multi-material modifications, patterning of biomolecules on surfaces and various other important biological and medical device applications. Author’s Note: In the context of this thesis, the term "low-functionality polymers" refers to materials like polyethylene and polypropylene that lack conventional organic functional groups such as alcohols, alkenes, aromatic rings, or halogens. Materials lacking these groups are intrinsically more challenging to crosslink, because one cannot make use of traditional organic reactions (e.g. alkylation, acylation, hydrosilylation, thiol-ene reactions, etc.) to form new covalent bonds.
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    Fundamental studies and potential optoelectronic applications of living crystallization-driven self-assembly
    (2023-08-14) Lei, Shixing; Manners, Ian
    Self-assembled nanostructures are receiving intense current attention because of their fascinating multifunctional properties and potential uses in the emerging field of nanoscience. Solution self-assembly of block copolymers (BCPs) is capable of generating a diverse range of colloidally stable core-shell micellar nanoparticles on the nano- and micro-scale. For BCPs containing a crystallizable core-forming block, living crystallization-driven self-assembly (CDSA) is of growing interest as a facile, ambient-temperature seeded growth route to colloidally stable 1D and 2D nanoparticles and more complex segmented and other hierarchical assemblies with low dispersity and predetermined dimensions. This field of research is growing rapidly with various emerging applications of these precisely controlled nanoparticles. The work presented in this thesis focuses on expanding the fundamental understanding and exploring the potential optoelectronic applications of living CDSA. Chapter 1 gives a general introduction by broadly discussing self-assembly examples in natural and synthetic systems, with a focus on BCP self-assembly, living CDSA and π-conjugated polymer nanoparticles (CPNPs). Chapter 2 involves studies of the size-dependent growth kinetics of living CDSA by investigating 1D seeded growth behavior. Chapter 3 describes an exploration of the preparation of aggregation-induced emission (AIE)-active, stimuli-responsive fluorescent 2D BCP nanoplatelets, and their proof-of-concept use for efficient and selective detection of mercury(II). Chapter 4 discusses the preparation and properties of tailored 2D organic semiconducting nanoplatelets from π-conjugated polymer amphiphiles with a crystallizable poly(di-n-hexylfluorene) (PDHF) core-forming block and a multiply-charged dendritic terminus. Chapter 5 involves studies of the preparation and resonant coupling of optical excitations in well-defined hybrid conjugates of semiconducting P3HT nanofiber–quantum dot. Finally, an outlook for this research area is discussed in Chapter 6 with the broad aim of preparing functional nanostructures with desirable properties for optoelectronic applications.
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    Development of Gold Nanoparticles for Targeting PD-L1-Presenting Breast Cancers
    (2023-07-31) Blevins, Derek James; Wulff, Jeremy Earle
    Our goal was to selectively target the immunosuppressive programmed cell death protein ligand 1 (PD-L1) presenting cancer cells with gold nanoparticles (AuNP). Initially, small molecules, described as antagonists for the PD-1/PD-L1 interaction, were considered as promising targeting agents for the gold platform. The competitive binding profiles of these molecules were evaluated using surface plasmon resonance (SPR). However, we found that none of the small molecule candidates were capable of disrupting the interaction, despite their apparent cell-based efficacy in other literature. Our findings indicate that the molecules were being mischaracterized as immunomodulators directly blockading the PD-1/PD-L1 interaction. In addition, we found no evidence of direct binding with the small molecules to either PD-1 or PD-L1, indicating they would not be suitable candidates as targeting agents. Instead, a commercially available monoclonal antibody (mAb, αPD-L1; from BioXCell) was found as a suitable alternative for targeting and blockading PD-L1 directly from PD-1. The antibody was conjugated with α-lipoic acid (αLA) through its NHS-ester such that the thiolated mAb may be grafted to functionalize the gold cores. The degree of functionalization on the gold core was quantified in vitro using SPR, where the relationship between valency and molecular weights of the gold core and unbound mAb binding to PD-L1 was studied. This SPR method was primarily used further to optimize the AuNP formulations until they were deemed suitable for cellular work. Gold cores with a surface area coated in 25% αLA-mAb and 75% PEG2000 had shown a near maximal response of binding relative to the unbound mAb, indicating a high degree of functionalized nanoparticles. This formulation was then moved forward into cellular work with naïve human white blood cells (Jurkat) that were stimulated with PHA (to produce PD-1), and the stimulation suppressed by the presence of PD-L1. In addition to AuNP formulation, we sought to investigate controlled release mechanisms indicative of the reducing character of the tumour microenvironment. We developed a series of disulfide tethers with a fluorogenic dye that induces turn-on fluorescence upon disulfide exchange, expected to show selectivity for the higher concentration of glutathione. Instead, we found that the common, but necessary, additive, fetal bovine serum (FBS), was triggering the premature release of our tethered fluorophore, disrupting our intended controlled-release studies. Through systematic investigation, we found that unwanted turn-on fluorescence from our dithiodiacid tethers was ultimately occurring due to esterase activity found in the FBS. This activity was shut down when methyl groups were installed at the α-position to the carbonyl carbon. Future work may use these bulkier dithiodiacid tethers for selective release in other AuNP formulations. Immunotherapeutic efficacy of the functionalized AuNPs was investigated by showing consistent stimulation in the presence of immunosuppressive PD-L1. Naïve Jurkat cells were stimulated with phytohemagglutinin (PHA), where the cell density would significantly increase relative to basal cell growth, and in the presence of solubilized PD-L1, show no change in density attributed to T cell exhaustion. When AuNPs were present, the cell density would reflect that of “uninhibited” stimulation control, regardless of PD-L1 presence, indicating the potential immunotherapeutic benefit of recovery from immune exhaustion.
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    Development of Stable, Active, and High-Throughput Experimentation Compatible Palladium Precatalysts
    (2023-07-27) Huang, Jingjun; Leitch, David
    Palladium-catalyzed cross-coupling has an approximately 50-year history, with seminal work in the 1970s leading to widespread use today, resulting in the 2010 Nobel Prize in Chemistry. The robustness of this chemistry, which enables constructions of C–C, C–H, C–N, C–O, and so on, has led to rapid growth and extensive applications of cross-coupling approaches in modern synthesis in the last decade. Alongside this, mechanistic studies of many Pd-catalyzed cross-coupling reactions are well-developed, with a wide range of catalytic systems established to maximize the reaction performance. Despite widespread acceptance of LPd0 species as key intermediates in the catalytic cycle, new developments with Pd0 precursors remain rare. Instead, PdII sources are the preferred precursor compounds; however, activation of these precatalysts involves reduction steps that can be complicated and challenging to elucidate. The works presented here describe the development of a series of new Pd0 precatalysts and one PdII precursor, which collectively resolve specific limitations of existing catalytic systems. The first palladium(0) precursor, DMPDAB–Pd–MAH, is an easily prepared, bench-stable, high-throughput screen compatible, and highly active precatalyst stabilized by an α-diimine ligand (N,N'-bis(2,6-dimethylphenyl)diazabutadiene, DMPDAB) and maleic anhydride (MAH). This precursor is an effective alternative to Pd2dba3•CHCl3 (the most commonly used palladium(0) source) for in situ catalyst formation. Furthermore, single-component phosphine-ligated palladium(0) precursors derived from DMPDAB–Pd–MAH exhibit superior performance in both catalytic cross-coupling reactions and stereoselective asymmetric allylic alkylations. Finally, to address limitations identified for systems based on DMPDAB–Pd–MAH, we report a new palladium(II) precursor, DMPDAB–Pd–(CH2TMS)2, for oxidative addition complex generation, high-throughput experimentation, and preparative-scale synthesis. This system takes advantage of straightforward PdII to Pd0 activation via reductive elimination of the alkyl ligands. Notably, DMPDAB–Pd–MAH is now commercially available at MilliporeSigma, and the other new precursors reported here are the subjects of filed patent applications. Thus, these new precursors have the potential for positive impact on improving reaction performance in industry-scale syntheses for complex organic molecules, including pharmaceuticals and agrochemicals.
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    Development of immunosensors and immunoassays using metallic nanostructures
    (2023-06-16) Tuckmantel Bido, Ariadne; Brolo, Alexandre Guimaraes
    Nanoplasmonic biosensors exploit the unique properties that arise from the interaction of light with free electrons of metallic nanostructures. They offer several advantages over the currently most employed methods of detection. These biosensors can be used for screening of infectious diseases, early diagnosis, management of chronic conditions, food quality and safety, among others. In this dissertation, the focus is on the development of surface-enhanced spectroscopy (SERS) and localized surface plasmon resonance (LSPR)- based immunosensors. A SERS-based immunoassay was developed for detection of IgG from human serum. A method for the sensor construction using poly(dimethylsiloxane) (PDMS) masks in 3D-printed platform, and extrinsic Raman labels (ERLs) were developed. SERS-based immunoassays are well-established in the research community. However, SERS-based sensors are questioned regarding their reproducibility and robustness. This is due to the large fluctuations in intensities in SERS measurements caused by inhomogeneous hotspots distributions in the sensing areas. The substantial local optical field variation within nanometers can affect reliability and reproducibility of these sensors. This effect particularly affects assays at low to ultralow concentrations (pM and lower) since there are less probed species per area in those conditions. Additionally, the common assumption that the analytical SERS intensities at all concentrations follow a Gaussian distribution can be detrimental to the reliable implementation of SERS-based assays. In this work, extensive SERS measurements were taken from the constructed sensors and the SERS intensities were shown to be log-normal distributed, particularly at low concentrations. Assessment of the number of measurements (sample size) per sensor at a particular concentration taken at different positions on the sensor surface were discussed. This study is a step forward towards the determination of best practices for a more reliable and robust employment of quantitative SERS-based immunoassays. A digital SERS protocol was also developed for the determination of SARS-CoV-2 (2019-nCoV) spike S1 + S2 ECD-His recombinant protein in saliva. In general, SERS-based immunoassays rely on a linear relationship between the average Raman intensity of a molecular reporter embedded in a SERS probe with the concentration of the analyte. As the concentration of the analyte decreases, the probability of a statistically significant number of SERS probes to be illuminated by the laser excitation also decreases. Additionally, there is large variation of intensities inherent to SERS and the inhomogeneous distribution of hotspots in the sensor being probed. These factors contribute to a loss in linearity of the assay at low concentrations of analyte and/or extrinsic Raman labels (ERLs). Here, an immunoassay was developed for determination of concentration of SARS-CoV-2 in saliva, and the conventional data treatment was compared to a digital protocol. The digital protocol generated a calibration curve with good linearity whereas the conventional approach did not. This approach is simple and can be employed in heterogeneous SERS immunoassays to improve both the limit-of-detection (LOD) and the dynamic working range. Finally, a low-cost localized surface plasmon resonance (LSPR)-based sensor was developed for SARS-CoV-2 screening in saliva. The sensor was built on plastic well plates for high throughput, but they can also be constructed in individual (discardable) plastic strips. The results were accessed using a plate reader, a ubiquitous equipment in laboratories and research centers. The sensor was challenged with 16 patient samples, being half COVID positive and half COVID negative. The sensor was developed in two modalities: 1) viral detection in saliva; and 2) antibody against COVID in saliva. Both sensors successfully classified all COVID positive patients (among hospitalized and non-hospitalized), and 7/8 COVID negative patients. This sensor is of low cost and of easy construction and is an alternative for SARS-CoV-2 screening in underserved communities. This sensor can be adapted to be used with other screening tests, by changing the element of recognition for other viral particles or antigens.
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    Monitoring Metabolic Alteration in Cancer Cells upon Radiation Therapy by Surface-Enhanced Raman Scattering (SERS)
    (2023-05-31) Chen, Xiangyu; Brolo, Alexandre Guimaraes
    Surface-Enhanced Raman Spectroscopy (SERS) is a highly selective and sensitive method that allows the tracking and identification of metabolites at low concentrations in cellular models. In this work, radiation-induced metabolism changes were investigated by SERS using the MCF-7 breast cancer cell line. SERS analysis was carried out on a time course (1, 4, 8, 12-days post exposure) of supernatant samples from MCF-7 cell line cultures previously exposed to either 5 or 20 Gy of ionizing radiation. A significant radiation biomarker, hypoxanthine, was identified through principal component analysis. The amount of hypoxanthine released into the extracellular environment increased over time after exposure to ionizing radiation. A potential mechanism for hypoxanthine production was suggested. Additionally, Caspase-3 inhibitor (Peptide Z-DEVD-FMK) treatment was employed to improve MCF-7 cell viability following exposure to ionizing radiation and to establish the relationship between the release of hypoxanthine and cell apoptosis. This study provides a valuable preliminary assessment of the application of SERS to study metabolic changes that can be extended in the future to human-derived samples.