Spelling suggestions: "subject:"solid phase"" "subject:"solid chase""
151 |
In Vivo Detection of Trace Organic Contaminants in Fish Using Solid Phase MicroextractionWang, Shuang 18 October 2010 (has links)
The feasibility of using solid phase micro-extraction (SPME) as an in vivo sampling tool for analysis of trace environmental contaminants in fish exposed to municipal wastewater effluents (MWWEs) was validated using controlled laboratory and field experiments. SPME was compared with traditional extraction techniques, including solid phase extraction (SPE) in water and solid-liquid extraction (SLE) in fish tissues to assess relative efficiencies. All three techniques were used to quantify the presence of eight compounds of interest in fish exposed to MWWEs in the laboratory, as well as in wild and field caged fish upstream and downstream of three wastewater treatment plants in the Grand River watershed. Atrazine, carbamazepine, naproxen, diclofenac, gemfibrozil, bisphenol A, fluoxetine and ibuprofen were selected as target compounds due to their diverse chemical characteristics and frequent detection in surface waters and sediments around the world. The distribution coefficients between various sample matrices (water, fish) and extraction phases (SPME fibers) were compared, as were extraction profiles and bioconcentration factors of target analytes in muscle of fish exposed to MWWEs under laboratory conditions, during field caging studies, or collected (wild) from the Grand River. Poly(dimethylsiloxane) (PDMS) medical grade tubing was utilized as the SPME extraction phase, which when kinetically calibrated, were effective at extracting and quantifying the target analytes from both water and fish tissue relative to traditional techniques. Caged and in wild fish exposed to MWWEs from all three municipal treatment plants bio-accumulated detectable levels of several of the target chemicals. All target analytes (except for fluoxetine) were identified in the MWWEs and exposed fish by SPME at low concentrations (ng/L). The presence and concentration of the targeted analytes in both water and wild fish living in the Grand River watershed varied with season and proximity to the wastewater outfalls. Results demonstrate that properly applied SPME can detect and quantify selected contaminants in fish tissues, surface water, and wastewater effluents. In vivo SPME allows for non-lethal sampling of fish, which creates the opportunity for monitoring contaminant exposure in receiving environments influenced by MWWEs or non-point-source runoff while minimizing the impact on the organisms.
|
152 |
Chemical Engineering of Small Affinity ProteinsLindgren, Joel January 2014 (has links)
Small robust affinity proteins have shown great potential for use in therapy, in vivo diagnostics, and various biotechnological applications. However, the affinity proteins often need to be modified or functionalized to be successful in many of these applications. The use of chemical synthesis for the production of the proteins can allow for site-directed functionalization not achievable by recombinant routes, including incorporation of unnatural building blocks. This thesis focuses on chemical engineering of Affibody molecules and an albumin binding domain (ABD), which both are three-helix bundle proteins of 58 and 46 amino acids, respectively, possible to synthesize using solid phase peptide synthesis (SPPS). In the first project, an alternative synthetic route for Affibody molecules using a fragment condensation approach was investigated. This was achieved by using native chemical ligation (NCL) for the condensation reaction, yielding a native peptide bond at the site of ligation. The constant third helix of Affibody molecules enables a combinatorial approach for the preparation of a panel of different Affibody molecules, demonstrated by the synthesis of three different Affibody molecules using the same helix 3 (paper I). In the next two projects, an Affibody molecule targeting the amyloid-beta peptide, involved in Alzheimer’s disease, was engineered. Initially the N-terminus of the Affibody molecule was shortened resulting in a considerably higher synthetic yield and higher binding affinity to the target peptide (paper II). This improved variant of the Affibody molecule was then further engineered in the next project, where a fluorescently silent variant was developed and successfully used as a tool to lock the amyloid-beta peptide in a β-hairpin conformation during studies of copper binding using fluorescence spectroscopy (paper III). In the last two projects, synthetic variants of ABD, interesting for use as in vivo half-life extending partners to therapeutic proteins, were engineered. In the first project the possibility to covalently link a bioactive peptide, GLP-1, to the domain was investigated. This was achieved by site-specific thioether bridge-mediated cross-linking of the molecules via a polyethylene glycol (PEG)-based spacer. The conjugate showed retained high binding affinity to human serum albumin (HSA) and a biological activity comparable to a reference GLP-1 peptide (paper IV). In the last project, the possibility to increase the proteolytic stability of ABD through intramolecular cross-linking, to facilitate its use in e.g. oral drug delivery applications, was investigated. A tethered variant of ABD showed increased thermal stability and a considerably higher proteolytic stability towards pepsin, trypsin and chymotrypsin, three important proteases found in the gastrointestinal (GI) tract (paper V). Taken together, the work presented in this thesis illustrates the potential of using chemical synthesis approaches in protein engineering. / <p>QC 20140207</p>
|
153 |
ISOPRENOID ANALOGS AS CHEMICAL GENETIC TOOLS TO PROVIDE INSIGHTS INTO FARNESYL TRANSFERASE TARGET SELECTION AND CELLULAR ACTIVITYTroutman, Jerry 01 January 2006 (has links)
Protein farnesylation is an essential post-translational modification required for the function of numerous cellular proteins including the oncoprotein Ras. The farnesyl transferase (FTase) catalyzed reaction is unique because farnesyl diphosphate (FPP), the farnesyl group donor for the reaction, forms a significant portion of a target protein binding site. The major goal of this research was to exploit this unique property of the FTase reaction and determine if changing the structure of the farnesyl donor group would affect FTase protein targeting. A small library of structural analogues of FPP was synthesized. Michelis-Menten steady-state kinetic analyses and competition reactions were used to determine the effect of these structural modifications on FTase targeting. We found that the analogues did affect FTase protein selectivity and that this could be exploited to induce unnatural target selectivity into the enzyme.
The second goal of this research was to determine the effect of FPP analogues on the function of FTase target proteins. To test the effect of these analogues we determined whether the unnatural lipid could ablate oncogenic H-Ras biological function in a Xenopus laevis model system. Several analogues were able to disrupt oncogenic H-Ras function while others mimicked the activity of FPP. These results indicated that some of the FPP analogues may act a prenyl group function inhibitors that could lead to an important new class of anti-cancer therapeutics.
Another major goal of this research was to use the FPP analogues as unnatural probes for the endogenous cellular activity of FTase target proteins. We developed antibodies to two of the unnatural FPP analogues to study their activity in cell cultureUtilizing these antibodies we found that alcohol prodrugs of the FPP analogues could be incorporated into cellular proteins in an FTase dependent manner. The ability of cell permeant analogues to be incorporated into live cells enhances the chances that such a molecule could be used to modify oncogenic cellular proteins with a prenyl group function inhibitor.
|
154 |
DEVELOPMENT OF AN EXTRACTION METHOD FOR THE MASS SPECTRAL ANALYSIS OF ORGANIC GUNSHOT RESIDUE FROM CLOTHINGCasper, Brent 01 January 2015 (has links)
This dissertation will focus on the extraction of volatile organic compounds associated with gunshot residue from articles of clothing, followed by analysis with mass spectrometry. During the discharge of a weapon, a cloud of volatile organic gunshot residue (OGSR) is dispersed around a firearm. This will create a high probability of transfer between the OGSR and the clothing of individuals who are near a discharged weapon.
The first part of this dissertation will be the development of a method for removal of volatile OGSR from articles of clothing. Extraction of OGSR will be completed by solid phase microextraction (SPME), followed by separation and analysis by gas chromatography-mass spectrometry (GC/MS). Many parameters will require optimization for proper extraction of OGSR from articles of clothing. Following development of a SPME procedure, figures of merit were determined such as linearity and limits of detection/quantification, obtaining levels of detection of 0.206 ng/cm2 on a 100 cm2 cotton cloth. Applications of this extraction method were investigated including the determination of the distance OGSR travels from a discharged weapon and the extraction of OGSR with different clothing materials by SPME.
The second part of this dissertation will focus on the development of an on-line solvent extraction method for removal of OGSR from articles of clothing, followed by analysis with paper spray mass spectrometry. Issues using SPME of certain types of clothing materials required the development of an alternative method for removal of OGSR from articles of clothing. Use of an on-line solvent extraction technique of OGSR from articles of clothing followed by analysis with paper spray mass spectrometry allowed for detection of OGSR at comparable levels to a headspace SPME procedure. Use of paper spray with an ion trap mass spectrometer permitted the soft ionization of OGSR compounds followed by tandem mass spectrometry to obtain structural information.
Extraction of OGSR from articles of clothing has potential to determine if an individual was present during the discharge of a firearm. Extraction of OGSR from articles of clothing will provide an alternative to traditional methods of gunshot residue analysis currently in use.
|
155 |
Site-specific labeling of affinity molecules for in vitro and in vivo studiesPerols, Anna January 2014 (has links)
The thesis is focused on site-specific labeling of affinity molecules for different applications where two types of binding proteins, Affibody molecules and antibodies, have been used. For the purpose of improving the properties of Affibody molecules for in vivo imaging, novel bi-functional chelators for radiolabeling using the radionuclide 111In were evaluated. In a first study, two chelators denoted NOTA and DOTA, respectively, were separately conjugated via maleimide chemistry to a C-terminal cysteine residue in a HER2-binding Affibody molecule (ZHER2:2395). In vivo evaluation using mice with prostate carcinoma cell line xenografts showed that the 111In-NOTA-MMA-ZHER2:2395 tracer exhibited faster clearance from blood than the 111In-DOTA-MMA-ZHER2:2395 counterpart,resulting in improved tumor-to-organ ratios. In a second study the in vivo imaging properties of a third tracer, 111In-NODAGA-MMA-ZHER2:2395, was investigated in tumor-bearing mice. While the tumor uptake was lower than seen for the 111In-DOTA-MMA-ZHER2:2395 tracer, a low uptake in non-targeted organs and a fast clearance from blood resulted in higher tumor-to-organ ratios for 111In-NODAGA-MMA-ZHER2:2395 compared to the DOTA variant. In a following study, a synthetically produced HER2-targeting affibody variant, denoted ZHER2:S1, was used where NODAGA, NOTA and DOTA chelators instead were conjugated via an amide bond to the N-terminus. In vivo evaluation in mice showed an unfavorable uptake in liver for 111In-NOTA-ZHER2:S1, resulting in a discontinuation. The study showed faster clearance of 111In-NODAGA-ZHER2:S1 from blood, but also an increased uptake in bone in comparison to 111In-DOTA-ZHER2:S1. As bone is a common metastatic site in prostate cancer, the favorable tumor-to-bone ratio for 111In-DOTA-ZHER2:S1 suggests it as the tracer of choice for prostate cancer. Further, the DOTA chelator was also evaluated as conjugated to either N- or C-terminus or to the back of helix 3 via an amide bond, where the in vivo evaluation showed that that C-terminal conjugation resulted in the highest contrast. Site specificity is also of great importance for labeling antibodies, as conjugation in the antigen-binding regions might influence the affinity. A method for site-specific labeling of antibodies using an IgG-binding domain that becomes covalently attached to the Fc-region of an antibody by photoconjugation was optimized. By investigation of positions most suitable for incorporation of the photoreactive probe, the conjugation efficiencies were increased for antibody subclasses important for both diagnostic and therapeutic applications. In addition, optimized variants were used in combination with an incorporated click-reactive handle for selective labeling of the antibody with a detection molecule. / <p>QC 20140929</p>
|
156 |
DEVELOPMENT OF FLUOROUS SOLID-PHASE EXTRACTION (FSPE) ON A MICROCHIP AND ITS APPLICATION TO PROTEOMICSXU, ZHENPO 20 November 2013 (has links)
The origin of fluorous interaction was explored and experimentally examined based on both HPLC and CEC data in this project. It was found that the selective fluorous interaction is a kind of reduced instantaneous or induced dipole interaction compared to the hydrophobic interaction.
A series of FPPM preparation parameters were optimized. The optimized FPPM column can resolve the components in a manner that was otherwise not possible with its non-fluorous (hydrocarbon) counterpart.
Following, the CEC separation of fluorous analytes on FPPM stationary phase based upon fluorous-fluorous interaction was realized for the first time. It was also found that, quantitatively, hydrophobic stationary phases have better methylene selectivity (〖 α〗_(-CH_2-)), while fluorous stationary phases have better perfluoromethylene selectivity (〖 α〗_(-CF_2-)). Thermodynamically, ∆G_(-CF_2- → -CF_2-)^° : ∆G_(-CH_2- → -CH_2-)^° (Gibbs free energy change of transferring a –CF2– unit to pure fluorous stationary phase versus Gibbs free energy change of transferring a –CH2– unit to pure hydrophobic stationary phase) is approximately equal to 8:1.
A new concept, hypothetical water percentage (HWP) based on the comparison of 〖 α〗_(-CH_2-) and〖 α〗_(-CF_2-) was proposed for the first time to quantitatively evaluate the hydrophobicity/fluorophilicity of a stationary phase. A stationary phase can be classified as fluorous stationary phase when the HWP is less than 0 (more negative indicates more fluorous), or as a hydrophobic stationary phase when the HWP is larger than 100. For the range between 0 and 100, the stationary phase can be treated as either fluorous or hydrophobic due to the similar values of〖 α〗_(-CH_2-) and〖 α〗_(-CF_2-).
Fluorous tagged peptides and proteins (up to 5800 Da) were effectively separated from their non-fluorous counterparts on the FPPM stationary phase in capillary-based columns and detected both on-line with ESI-MS and off-line with MALDI-MS. Finally, the FPPM solid-phase extraction (SPE) stationary phase was transplanted from the capillary to a microchip format. This microchip exhibits the merits of both selective fluorous interaction and micro total analysis system (µTAS). / Thesis (Ph.D, Chemistry) -- Queen's University, 2013-11-19 23:11:16.636
|
157 |
Towards more selective sorbents for extraction of drugs and biomarkers from biological fluids using molecularly imprinted polymersMoein, Mohammad Mahdi January 2014 (has links)
Sample preparation has a critical role as a first step in analytical processes, especially in bioanalysis and environmental analysis. A good sample preparation technique should be robust and stable, regardless of the sample matrix. The aim of this thesis is to design and synthesize molecularly imprinted polymers that can be used in various sample preparation techniques, such as on-line MEPS, on-line SPE and on-line monolithic pre-columns used for the extraction of drugs, hormones, and cancer biomarkers from human plasma and urine samples. Additional aim was to provide full automation, on-line coupling, short sample preparation time and high-throughput. In this thesis MIP in MEPS was used on-line with liquid chromatography-tandem mass spectrometry (LC/MS/MS) for the determination of sarcosine in human urine and plasma samples. The method was fully automated and the packed sorbent could be used for about hundred extractions. In additional work a coated needle with MIP-Sol-Gel as thin layer was prepared and used for the microextraction of bilirubin from human plasma and urine. Small sample volumes could be handled and the validation of the method showed that the method was robust and selective. In a further work MIP-SPE on-line with HPLC was used for the extraction and determination of dextromethorphan in human plasma samples. MIP-SPE showed a good selectivity and high recovery (87% - 92%). On-line MIP monolithic pre-column was prepared and used in a coupled system for the extraction of tramadol in human plasma and urine samples. The MIP monolithic pre-column showed good selectivity and high extraction recovery was obtained (91-96%). The extraction and analysis of human insulin in plasma and pharmaceutical formulation solutions were carried out using MIP-SPE on-line with HPLC. The validation of the method showed that the method was accurate and robust. / <p>At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 5: Submitted.</p>
|
158 |
The application of ICP-MS to high matrix samples such as those found in the ceramics industryLandon, Mark R. January 2007 (has links)
Although the benefits of ICP-MS are well documented, the determination of analytes at low levels in high concentrations of matrix elements has proved difficult. As ICP-MS is a 'flow into' instrument the deposition of salts throughout the system is a common cause of significant loss of signal. The application of desolvation of aluminosilicate samples, to aid in the production of more robust plasma conditions, was investigated to increase the efficiency of the ICP in processing the sample. The performance of the ICP-MS was monitored with different cone arrangements and by running the skimmer cones at elevated temperatures. An alternative to modification of the instrument is to employ chemical modification of the sample and hence the separation of Au and Pt from an aluminosilicate matrix via the use of solid phase extraction (SPE) columns were investigated as a means of dealing with high levels of dissolved solids. OVB based SPE columns were found to give high retentions of Au and Pt when chelated with ammonium pyrrolidinediethylcarbamate (APOC). A second alternative that avoided digestion of the aluminosilicate matrices, was to carry out the analysis using laser ablation (LA). LA-ICP-MS is becoming increasingly used for trace elemental analysis but as yet no universal calibration method is available. The general problems associated with matrix matched standards are inherent as the ablation mechanism and plasma conditions can differ dramatically with very small changes in matrix composition. Hence the addition of chromophores was employed to increase the absorption of the laser energy. The use of vanillic, nicotinic and pyrazinoic acid were used to improve the ablation of pressed powder discs at the laser wavelength of 213 nm. Synthetic aluminosilicate discs and standard additions were both employed for the calibration and determination of Ti.
|
159 |
Optimization of a Needle Trap DeviceZhan, Weiqiang 09 1900 (has links)
Various needle trap devices (NTDs) with different designs for different applications have been developed during the past decade. A theoretical model on the fundamentals of the NTD was recently proposed, which employed the theory of frontal (gas-solid) chromatography to describe the sampling process, where a gaseous sample was continuously introduced into the sorbent bed. In this investigation, different types of sorbent particles with different dimensions were packed into the needle as adsorbents. The effect of particle dimension, which would affect the packing density and consequently the capacity, the extraction efficiency, and desorption efficiency of the NTD were experimentally investigated and the proposed theory was validated. The results demonstrated that NTDs packed with small particles possess higher extraction capacity and efficiency but much higher resistance to flow as well. The higher resistance did not necessarily result in poor desorption efficiency, because desorption efficiency was affected by both the sorbent bed structure and the desorption gas flow. The relationships observed among those physical parameters provide valuable guidance on how to design an NTD with high performance potential for future applications. For particulate sampling, it was found that NTDs packed with different particles presented high collection efficiency of the particulates being investigated, and the collection efficiency was dominated by the pore size and distribution of the sorbent bed packed inside the needle. Collection efficiency also increased with increase in solidity of the sorbent bed; the increase in humidity of the aerosol sample; and the decrease of sampling rate. The results also provide valuable guidance on the optimisation of needle trap for particulate collection.
|
160 |
High-throughput analysis of biological fluids using 96-blade (thin-film) solid phase microextraction systemMirnaghi, Fatemeh Sadat January 2012 (has links)
The initial research of this thesis involves the evaluation of different strategies for developing diverse chemistries of highly stable coatings for the automated 96-blade (thin-film) solid phase microextraction (SPME) system. Thin-film geometry increases the volume of extractive phase, and consequently improves the sensitivity of the analysis. Sol-gel technology was used for the preparation of octadecyl (C18)-silica gel thin-film coating. The evaluation of the C18-silica gel SPME extractive phase resulted in stable physical and chemical characteristics and long-term reusability with a high degree of reproducibility.
Biocompatible polyacrylonitrile (PAN) polymer was used for the preparation of particle-based extractive phases in order to improve the biocompatible characteristics of SPME coatings for the extraction from biological samples.
Three different immobilization strategies were evaluated for developing highly stable coatings for the automated 96-blade SPME system. The spraying was found to be the optimal method in terms of stability and reusability for long-term use.
The optimized C18-PAN coating demonstrated improved biocompatibility, stability, and reusability for the extraction of benzodiazepines from human plasma in comparison with those of C18-silica gel coating.
To improve the biocompatible properties of the C18-PAN SPME coating for long-term direct analysis from whole blood, different modification strategies were studied and evaluated. The modification of the coating with an extra layer of biocompatible polyacrylonitrile resulted in significant improvement in the blood compatibility in long-term use.
‘Extracted blood spot’ (EBS) sampling was introduced as a novel approach to overcome the limitations of dried blood spot sampling. EBS includes the application of a biocompatible SPME coating for spot sampling of blood or other biofluids. The compatibility of EBS sampling with different analytical methods was demonstrated. The utilization of EBS as a fast sampling and sample preparation method resulted in a significant reduction of matrix effects through efficient sample clean-up.
Modified polystyrene-divinylbenzene (PS-DVB)-PAN and phenylboronic acid (PBA)-PAN 96-blade SPME coatings were developed and evaluated for the extraction of analytes in a wide range of polarity. These coatings demonstrated efficient extraction recovery for both polar and non-polar groups of compounds, and presented chemical and mechanical stabilities and reproducible extraction efficiencies for more than 100 usages in biological sample.
|
Page generated in 0.0487 seconds