Matching Items (23)
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- All Subjects: Chemistry
- Creators: Ros, Alexandra
Description
Bioanalytes such as protein, cells, and viruses provide vital information but are inherently challenging to measure with selective and sensitive detection. Gradient separation technologies can provide solutions to these challenges by enabling the selective isolation and pre-concentration of bioanalytes for improved detection and monitoring. Some fundamental aspects of two of these techniques, isoelectric focusing and dielectrophoresis, are examined and novel developments are presented. A reproducible and automatable method for coupling capillary isoelectric focusing (cIEF) and matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) based on syringe pump mobilization is found. Results show high resolution is maintained during mobilization and &beta-lactoglobulin; protein isoforms differing by two amino acids are resolved. Subsequently, the instrumental advantages of this approach are utilized to clarify the microheterogeneity of serum amyloid P component. Comprehensive, quantitative results support a relatively uniform glycoprotein model, contrary to inconsistent and equivocal observations in several gel isoelectric focusing studies. Fundamental studies of MALDI-MS on novel superhydrophobic substrates yield unique insights towards an optimal interface between cIEF and MALDI-MS. Finally, the fundamentals of isoelectric focusing in an open drop are explored. Findings suggest this could be a robust sample preparation technique for droplet-based microfluidic systems. Fundamental advancements in dielectrophoresis are also presented. Microfluidic channels for dielectrophoretic mobility characterization are designed which enable particle standardization, new insights to be deduced, and future devices to be intelligently designed. Dielectrophoretic mobilities are obtained for 1 µm polystyrene particles and red blood cells under select conditions. Employing velocimetry techniques allows models of particle motion to be improved which in turn improves the experimental methodology. Together this work contributes a quantitative framework which improves dielectrophoretic particle separation and analysis.
ContributorsWeiss, Noah Graham (Author) / Hayes, Mark A. (Thesis advisor) / Garcia, Antonio (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2011
Description
Water-soluble, adenosine triphosphate (ATP)-stabilized palladium nanoparticles have been synthesized by reduction of palladium salt in the presence of excess ATP. They have been characterized by electron microscopy, energy dispersive X-ray spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, and X-ray diffraction in order to determine particle size, shape, composition and crystal structure. The particles were then subsequently attached to a glassy carbon electrode (GCE) in order to explore their electrochemical properties with regard to hydrogen insertion in 1 M sodium hydroxide. The particles were found to be in the size range 2.5 to 4 nm with good size dispersion. The ATP capping ligand allowed the particles to be air-stable and re-dissolved without agglomeration. It was found that the NPs could be firmly attached to the working electrode via cycling the voltage repeatedly in a NP/phosphate solution. Further electrochemical experiments were conducted to investigate the adsorption and absorption of hydrogen in the NPs in 1 M sodium hydroxide. Results for cyclic voltammetry experiments were consistent with those for nanostructured and thin-film palladium in basic solution. Absorbed hydrogen content was analyzed as a function of potential. The maximum hydrogen:Pd ratio was found to be ~0.7, close the theoretical maximum value for β phase palladium hydride.
ContributorsLamb, Timothy (Author) / Buttry, Daniel A (Thesis advisor) / Yarger, Jeffery (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2013
Description
Complex samples, such as those from biological sources, contain valuable information indicative of the state of human health. These samples, though incredibly valuable, are difficult to analyze. Separation science is often used as the first step when studying these samples. Electrophoretic exclusion is a novel separations technique that differentiates species in bulk solution. Due to its ability to isolate species in bulk solution, it is uniquely suited to array-based separations for complex sample analysis. This work provides proof of principle experimental results and resolving capabilities of the novel technique. Electrophoretic exclusion is demonstrated at a single interface on both benchtop and microscale device designs. The benchtop instrument recorded absorbance measurements in a 365 μL reservoir near a channel entrance. Results demonstrated the successful exclusion of a positively-charged dye, methyl violet, with various durations of applied potential (30 - 60 s). This was the first example of measuring absorbance at the exclusion location. A planar, hybrid glass/PDMS microscale device was also constructed. One set of experiments employed electrophoretic exclusion to isolate small dye molecules (rhodamine 123) in a 250 nL reservoir, while another set isolated particles (modified polystyrene microspheres). Separation of rhodamine 123 from carboxylate-modified polystyrene spheres was also shown. These microscale results demonstrated the first example of the direct observation of exclusion behavior. Furthermore, these results showed that electrophoretic exclusion can be applicable to a wide range of analytes. The theoretical resolving capabilities of electrophoretic exclusion were also developed. Theory indicates that species with electrophoretic mobilities as similar as 10-9 cm2/Vs can be separated using electrophoretic exclusion. These results are comparable to those of capillary electrophoresis, but on a very different format. This format, capable of isolating species in bulk solution, coupled with the resolving capabilities, makes the technique ideal for use in a separations-based array.
ContributorsKenyon, Stacy Marie (Author) / Hayes, Mark A. (Thesis advisor) / Ros, Alexandra (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2012
Description
This work demonstrated a novel microfluidic device based on direct current (DC) insulator based dielectrophoresis (iDEP) for trapping individual mammalian cells in a microfluidic device. The novel device is also applicable for selective trapping of weakly metastatic mammalian breast cancer cells (MCF-7) from mixtures with mammalian Peripheral Blood Mononuclear Cells (PBMC) and highly metastatic mammalian breast cancer cells, MDA-MB-231. The advantage of this approach is the ease of integration of iDEP structures in microfliudic channels using soft lithography, the use of DC electric fields, the addressability of the single cell traps for downstream analysis and the straightforward multiplexing for single cell trapping. These microfluidic devices are targeted for capturing of single cells based on their DEP behavior. The numerical simulations point out the trapping regions in which single cell DEP trapping occurs. This work also demonstrates the cell conductivity values of different cell types, calculated using the single-shell model. Low conductivity buffers are used for trapping experiments. These low conductivity buffers help reduce the Joule heating. Viability of the cells in the buffer system was studied in detail with a population size of approximately 100 cells for each study. The work also demonstrates the development of the parallelized single cell trap device with optimized traps. This device is also capable of being coupled detection of target protein using MALDI-MS.
ContributorsBhattacharya, Sanchari (Author) / Ros, Alexandra (Committee member) / Ros, Robert (Committee member) / Buttry, Daniel (Committee member) / Arizona State University (Publisher)
Created2013
Description
Polydimethyl siloxane is a commonly used fabrication material for microfluidic devices. However, its hydrophobic nature and protein adsorption on the surface restricts its use for microfluidic applications. Also, it is critical to control the electroosmotic flow for electrophoretic and dielectrophoretic manipulations. Therefore, surface modification of PDMS is essential to make it well suited for bioanalytical applications. In this project, the role of polyethylene oxide copolymers F108 and PLL-PEG has been investigated to modify the surface properties of PDMS using physisorption method. Measuring electroosmotic flow and adsorption studies tested the quality and the long-term stability of the modified PDMS surface. Static and dynamic coating strategies were used to modify the PDMS surface. In static coating, the PDMS surface was incubated with the coating agent prior to the measurements. For dynamic coating, the coating agent was always present in the solution throughout the experiment. F108 and PLL-PEG were equally effective to prevent the protein adsorption under both strategies. However, dynamic coating was more time saving. Furthermore, effective reduction of EOF was observed with F108 coating agent under dynamic conditions and with PLL-PEG coating agent under static conditions. Moreover, PLL-PEG dynamic coatings exhibited reversal of EOF. These important findings could be used to manipulate EOF and suggest optimal coating agent and strategies for PDMS surface treatment by the physisorption method.
ContributorsManchanda, Shikha (Author) / Ros, Alexandra (Thesis advisor) / Hayes, Mark (Committee member) / Liu, Yan (Committee member) / Arizona State University (Publisher)
Created2012
Description
The human gastrin receptor (CCKBR or CCK2R) is a class A G protein-coupled receptor (GPCR) found throughout the central nervous system, stomach, and a variety of cancer cells. CCK2R is implicated in the regulation of biological processes, including anxiety, satiety, arousal, analgesia, psychosis, and cancer cell growth and proliferation. While CCK2R is an attractive drug target, few drugs have managed to effectively target the receptor, and none have been brought to market. Contributory to this is the lack of high-resolution crystal structure capable of elucidating the binding regions of CCK2R to streamlining drug screening. While GPCRs are not amenable to traditional structural analysis methodologies, the advent of lipidic cubic phase (LCP) crystallography and serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs), has extended the applicability of X-ray crystallography to these integral membrane proteins. LCP-SFX depends on optimizing the protein of interest for extraction, purification, and crystallization. Here we report our findings regarding the optimization of CCK2R suggesting the synergistic relationship between N-terminal truncations and the insertion of a fusion protein along ICL3, in addition to a 30-residue truncation of the C-terminus. Samples were expressed in Sf9 insect cells using a Bac-to-Bac baculovirus expression system, extracted using n-Dodecyl-β-D-Maltoside detergent, and purified via TALON immobilized metal-ion affinity chromatography. The constructs were characterized via SDS-PAGE, Western blot, and size exclusion chromatography. These findings demonstrate the improvements to CCK2R’s crystallographic amenability upon these modifications, however significant improvements must be made prior to crystallization trials. Future work will involve screening C-terminal truncations, thermostabilizing point mutations, and co-crystallizing ligands. Ideally this investigation will serve as a model for future CCK2R structural analysis and contribute to a heightened interest in CCK2R as a therapeutic target.
ContributorsStevens, Alexander Wade (Author) / Liu, Wei (Thesis director) / Chiu, Po-Lin (Committee member) / Mills, Jeremy (Committee member) / School of Human Evolution & Social Change (Contributor) / School of Molecular Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Osteocalcin (Oc) is the most abundant non-collagen protein found in the bone, but its precise function is still not completely understood. Three glutamic acid (Glu) residues within its sequence are sites for vitamin K-dependent post-translational modification, replacing a hydrogen with a carboxylate located at the γ-carbon position, converting these to γ-carboxyglutamic acid (Gla) residues. This modification confers increased binding of Oc to Ca2+ and hydroxyapatite matrix. Presented here, novel metal binding partners Mn2+, Fe3+, and Cr3+ of human Oc were determined, while the previously identified binders to (generally) non-human Oc, Ca2+, Mg2+, Pb2+ and Al3+ were validated as binders to human Oc by direct infusion mass spectrometry with all metals binding with higher affinity to the post-translationally modified form (Gla-Oc) compared to the unmodified form (Glu-Oc). Oc was also found to form pentamer (Gla-Oc) and pentamer and tetramer (Glu-Oc) homomeric self-assemblies in the absence of NaCl, which disassembled to monomers in the presence of near physiological Na+ concentrations. Additionally, Oc was found to form filamentous structures in vitro by negative stain TEM in the presence of increased Ca2+ titrations in a Gla- and pH-dependent manner. Finally, by combining circular dichroism spectroscopy to determine the fraction of Gla-Oc bound, and inductively-coupled plasma mass spectrometry to quantify total Al concentrations, the data were fit to a single-site binding model and the equilibrium dissociation constant for Al3+ binding to human Gla-Oc was determined (Kd = 1.0 ± 0.12 nM). Including citrate, a known competitive binder of Al3+, maintained Al in solution and enabled calculation of free Al3+ concentrations using a Matlab script to solve the complex set of linear equations. To further improve Al solubility limits, the pH of the system was lowered to 4.5, the pH during bone resorption. Complementary binding experiments with Glu-Oc were not possible due to the observed precipitation of Glu-Oc at pH 4.5, although qualitatively if Glu-Oc binds Al3+, it is with much lower affinity compared to Gla-Oc. Taken together, the results presented here further support the importance of post-translational modification, and thus adequate nutritional intake of vitamin K, on the binding and self-assembly properties of human Oc.
ContributorsThibert, Stephanie (Author) / Borges, Chad R (Thesis advisor) / LaBaer, Joshua (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021
Description
Insulator-based dielectrophoresis (iDEP) has attracted considerable attention due to its ability to precisely capture and manipulate nanoparticles and biomolecules. A distinctive approach for effective manipulation of nanometer-sized proteins employing iDEP technique by generating higher electric field (E) and gradient (??2) in the iDEP microfluidic devices is delineated. Strategies to generate higher ??2 in the iDEP devices were outlined using numerical simulations. Intriguingly, the numerical simulation results demonstrated that by decreasing the post-to-post gap in the iDEP microfluidic devices, the ??2 was increased by ⁓12 fold. Furthermore, the inclusion of channel constrictions, such as rectangular constriction or curved constriction into the straight channel iDEP microfluidic device led to a significant increase in ??2. In addition, the inclusion of rectangular constrictions in the straight channel iDEP microfluidic device resulted in a greater increase in ??2 compared to the incorporation of curved constrictions in the same device. Moreover, the straight channel device with horizontal post-to-post gap of 20 μm and vertical post-to-post gap of 10 μm generated the lowest ??2 and the ??2 was uniform across the device. The rectangular constriction device with horizontal and vertical post-to-post gap of 5 μm generated the highest ??2 and the ??2 was non-uniform across the device. Subsequently, suitable candidate devices were fabricated using soft lithography as well as high resolution 3D printing and the DEP behavior of ferritin examined under various experimental conditions. Positive streaming DEP could be observed for ferritin at low frequency in the device generating the lowest ??2, whereas at higher frequency of 10 kHz no DEP trapping characteristics were apparent in the same device. Importantly, in the device geometry resulting in the highest ??2 at 10 kHz, labeled ferritin exhibited pDEPtrapping characteristics. This is an indication that the DEP force superseded diffusion and became the dominant force.
ContributorsMAHMUD, SAMIRA (Author) / Ros, Alexandra (Thesis advisor) / Borges, Chad (Committee member) / Mills, Jeremy (Committee member) / Arizona State University (Publisher)
Created2024
Description
Without a doubt, protein is the most crucial biomolecule performing life and biological functions of any living cell. Profiling various protein expression in individual cells has raised a great interest for scientist and researchers over decades in attempts to reveal cell-to-cell variation, which used to be masked in many previous population average measurement methods. Immunofluorescence (IF) has been a well-established single cell protein analysis technique as for its fast and high-resolution detection and localization, simple and adaptable workflows, and affordable instrumentation. However, inadequate detection sensitivity and multiplexing capability are the two limitation of this platform that remain incompletely addressed in many decades. In this work, several improvements have been proposed and demonstrated to improve existing drawbacks of conventional immunofluorescence. An azide-based linker featured in the novel fluorescent probes synthesis has enable iterative protein staining on the same tissue sample, which subsequently increase the multiplex capacity of IF. Additionally, the multiple fluorophore introduction to the proteins target via either layer by layer biotin-cleavable fluorescent streptavidin or tyramide signal amplification (TSA) have significantly increase the detection sensitivity of the platform. With these advances, IF has the potential to detect, image and quantify up to 100 protein targets in single cell in the tissue sample. In addition of desirable features of IF, these improvements have further turned the technique into a powerful proteomic study platform for not only research setting but also clinical study setting. It is anticipated this highly sensitive and multiplexed, renovated IF method will soon be translated into biomedical studies.
ContributorsPham, Thai Huy (Author) / Guo, Jia (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2023
Description
The kinetic behavior of acidified aqueous reaction systems based on the oxidation of sodium oxalate by potassium permanganate was investigated by UV-Vis absorbance spectrophotometry as part of a wider project to develop time-temperature indicators based on such systems. Reaction systems in various eutectic salt solutions as well as salt-free aqueous solutions were prepared and measured to establish the precision and accuracy of four different reaction systems designed to run for specified periods of time at 25°C. Reaction progress was also monitored in reaction systems previously subjected to prolonged periods of storage at −80°C, as well as systems subjected to repeated freezing and thawing, to determine the effect of preliminary freeze time or multiple freeze-thaw cycles on reaction run time. Eutectic-free aqueous reaction systems showed a noticeable decrease in run time from freshly frozen to 2 week frozen, but minimal to no decrease with increasing preliminary freeze time. Perchlorate-based reaction systems showed minimal to no decrease in reaction time with increasing preliminary freeze time, and were found to be capable of tolerating up to three <5-minute excursions into room temperature before reaction time was significantly affected. It was also found that all reaction systems studied were capable of reaching their target run times within the intended ±10% limit of accuracy, as average run times for each system ranged from 0.4% to 6.9% off from the target run time. These systems were found to be reproducible with considerable precision as well, with the average coefficient of variation for each system ranging from 2.6 to 5.5.
ContributorsUy, Aaron Gabriel Buenconsejo (Author) / Borges, Chad R. (Thesis advisor) / Ros, Alexandra (Committee member) / Herckes, Pierre (Committee member) / Arizona State University (Publisher)
Created2024