UMD Theses and Dissertations
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Item DEVELOPMENT OF HORDEIN-PECTIN NANOPARTICLE COMPLEX FOR THE ENCAPSULATION OF BIOACTIVE COMPOUNDS FOR ENHANCED FUNCTIONALITIES(2023) Tarwa, Kevin; Wang, Qin; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Nanoparticle delivery systems composed of food polymers are a sustainable and eco-friendly approach to protect functional ingredients and promote healthier food options. In this research, a hordein-pectin nanoparticle complex (HP-NPC) was fabricated using an anti-solvent precipitation and electrostatic deposition (pH 4) method for the encapsulation of hydrophobic bioactive compounds to enhance their functionalities. First, hordein was extracted from whole barley grains to obtain a dried powder to synthesize hordein nanoparticles (HNP). Then pectin with a degree of esterification (DE) around 71% was applied as a coating material. The average particle size of the freshly prepared nanoparticle complex was relatively small (~246 ± 11 nm), and Fourier transform infrared spectroscopy (FTIR) indicated that cationic hordein interacted with anionic pectin mainly though newly formed hydrogen bonds and electrostatic interaction as indicated by their opposite surface charges. Scanning electron microscopy (SEM) revealed that the morphology of the nanoparticle complex was spherical with a smooth surface. The pectin coating was shown to have a protective effect against pH (3.0-9.0), heat (80 °C for 0-120 mins), and salt (0-100 µM) which are all factors known to degrade proteins. Second, lutein, a hydrophobic bioactive xanthophyll was encapsulated into HP-NPC to develop a lutein-hordein/pectin nanoparticle complex (L-HP-NPC). Since lutein has low water solubility and low bioavailability in the gastrointestinal tract (GIT), the effect of the encapsulation system on the functional properties of lutein was investigated. The loading capacity (LC%) and encapsulation efficiency (EE%) was around 15.5 and 82%, respectively. In vitro digestion resulted in a higher bioaccessibility of lutein for encapsulated HP-NPC (~22.3%), which is defined as the percentage of lutein accessible for absorption in the simulated intestinal fluid (SIF) compared to lutein encapsulated into HNP (~9%). The ability of pectin to produce gels in acidic media was shown to have a significant effect against gastric enzymes that can degrade both hordein and lutein. Also, lyophilization (an important step in food processing) had no significant effect on the stability of L-HP-NPC. This encapsulation system could potentially be used as a functional ingredient in the food industry to develop healthy and nutritious foods for consumers. Third, carvacrol, a phenolic monoterpene known for its antimicrobial properties was encapsulated into HP-NPC to develop a carvacrol-hordein/pectin nanoparticle complex (CA-HP-NPC). Special focus was on the solubility of encapsulate carvacrol due to its known low solubility in aqueous solutions. The antimicrobial effectiveness of the encapsulated nanoparticle complex was tested against non-pathogenic gram-positive L. innocua and gram-negative E. coli K12. CA-HP-NPC was still able to maintain a relatively small particle size (~207 ± 8 nm) after being dispersed into water post-lyophilization. Carvacrol was shown to be effective against the two bacteria, however, CA-HP-NPC did not show antimicrobial effectiveness. Although carvacrol was successfully encapsulated into the nanoparticle complex, further studies on their release properties need to be investigated to further understand their functional properties for food applications.Item DEVELOPMENT OF ANALYTICAL METHODS FOR CHARACTERIZATION OF NANOPARTICLES FOR BIO-MEDICAL AND ENVIRONMENTAL APPLICATION BY ION MOBILITY-ICP-MS(2017) Tan, Jiaojie; Zachariah, Michael R.; Chemical Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)The development of nanotechnology necessitates appropriate tools for nanoparticle characterization to assure product quality, evaluate safety and facilitate manufacturing. The properties of interest particularly relevant to nanomedicine and environmental ecotoxicology include size, shape, aggregation, concentration, dissolution, surface chemistry, and composition etc. Engineered nanoparticles in a complex matrix, at realistic concentration are two of the major challenges for analytical scientist. Potential transformation of pristine engineered nanomaterials when put in contact with either biological or environmental media further complicate the analytical task. In this dissertation, I aim to optimize and extend the application of novel hyphenated instruments consisting of differential mobility analysis (DMA) and inductively coupled plasma-mass spectrometry (ICP-MS) for real time size classification and elemental detection in biomedical and environmental fields. I have applied DMA-ICP-MS in quantitatively characterizing anti-tumor drug delivery platform to assist design and performance evaluation. Optimal balance among drug loading, stability and release performance was achieved and evaluated by DMA-ICP-MS. I have further developed a novel analytical methodology including DMA and ICP-MS operating in single particle mode (i.e. spICP-MS). I successfully demonstrated and validated the method for accurate and simultaneous size, mass and concentration measurement by NIST reference materials. DMA-spICP-MS was shown with the capability to characterize nanoparticle aggregation state and surface coating. In addition, this technique was shown to be useful for real-world samples with high ionic background due to its ability to remove dissolved ions yielding a cleaner background. Given this validated DMA-spICP-MS method, I applied it to quantifying the geometries of seven gold nanorod samples with different geometries. It was demonstrated that DMA-spICP-MS can achieve fast quantification of both length and diameter with accuracy comparable with TEM analysis. This method provided the capability to separate nanorods from spheres quantifying the geometry for each population. Finally, an interesting open and high-order rosette protein structure was investigated by electrospray-DMA. The staining procedure was optimized and effect of electrospray process on protein particle structure was evaluated. Protein particle after electrospray was largely maintained. Mobility simulation by MOBCAL showed close matches with experimental data and enabled peak assignment to various particle assembly structures.Item Evaluation of Optical Sensor Platforms for Multiplexed Detection of Proteins(2014) Spindel, Samantha; Sapsford-Medintz, Kim E; White, Ian; Bioengineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)This work investigated optical sensor platforms for protein multiplexing, the ability to analyze multiple analytes simultaneously. Multiplexing is becoming increasingly important for clinical needs because disease and therapeutic response often involve the interplay between a variety of complex biological networks involving multiple, rather than single, proteins. Moreover, one biomarker may be indicative of more than one disease, similar diseases can manifest with similar physical symptoms, and monitoring a disease requires the ability to detect subtle differences over time. Multiplexing is generally achieved through one of two routes, either through spatial separation on a surface (different wells or spots) or with the use of unique identifiers/labels (such as spectral separation - different colored dyes, or unique beads - size or color). We looked into combining both spatial separation and unique labels to further expand the multiplexing capabilities of surfaces. Our original research resulted in one of the few demonstrations of reactive semiconductor nanocrystal immunoassays for multiplexed analysis within a single well on a microtiter plate. Innovative planar surface fluorescent immunoassays were developed for both spatial and spectral multiplexing using Quantum Dots and prospective incorporation into a Point-of-Care (POC) device involving an evanescent wave scanner. These assays used standard microscope slides combined with flow cells and were designed to markedly reduce the amount of sample and reagents needed as compared to standard 96-well plate assays. The platform was optimized for detecting Chicken IgG and Staphylococcal Enterotoxin B (SEB); SEB is commonly used in the literature to characterize the performance of biosensor platforms. The planar surface fluorescent immunoassays were applied to a real-world public health need to detect renal injury. Two emerging novel biomarkers, Kidney Injury Marker-1 (KIM-1) and Neutrophil Gelatinase Associated Lipocalin (NGAL), were investigated for their potential to detect injury earlier and with more specificity than current methods using serum creatinine (SCr). Detecting these medically-relevant markers using planar surface fluorescence immunoassays could potentially allow for more rapid diagnosis of acute kidney injury (AKI), among other uses.Item MANIPULATION OF DNA TOPOLOGY USING AN ARTIFICIAL DNA-LOOPING PROTEIN(2012) Gowetski, Daniel; Kahn, Jason D; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)DNA loop formation, mediated by protein binding, plays a broad range of roles in cellular function from gene regulation to genome compaction. While DNA flexibility has been well investigated, there has been controversy in assessing the flexibility of very small loops. We have engineered a pair of artificial coiled-coil DNA looping proteins (LZD73 and LZD87), with minimal inherent flexibility, to better understand the nature of DNA behavior in loops of less than 460 bp. Ring closure experiments (DNA cyclization) were used to observe induced topological changes in DNA upon binding to and looping around the engineered proteins. The length of DNA required to form a loop in our artificially rigid system was found to be substantially longer than loops formed with natural proteins in vivo. This suggests the inherent flexibility of natural looping proteins plays a substantial role in stabilizing small loop formation. Additionally, by incrementally varying the binding site separation between 435 bp and 458 bp, it was observed that the LZD proteins could predictably manipulate the DNA topology. At the lengths evaluated, the distribution of topological products correlates to the helical repeat of the double helix (10.5 bp). The dependence on binding site periodicity is an unequivocal demonstration of DNA looping and represents the first application of a rigid artificial protein in this capacity. By constructing these DNA looping proteins, we have created a platform for addressing DNA flexibility in regards to DNA looping. Future applications for this technology include a vigorous study of the lower limits of DNA length during loop formation and the use of these proteins in assembling protein:DNA nanostructures.Item DEVELOPMENT OF TOOLS TO CHARACTERIZE PROTEIN-PROTEIN INTERACTIONS(2010) Jiang, Jiangsong; Li, Shuwei; Stewart, Richard; Cell Biology & Molecular Genetics; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Protein-protein interactions (PPIs) are crucial to most biological processes and activities. Large-scale PPI screening has been applied to model organisms as well as to human cells. Two approaches have been used extensively in high-throughput PPI studies: (i) the Yeast Two-Hybrid (Y2H) assay (a bottom-up method), and (ii) the tandem affinity purification (TAP) (a top-down method). However, a close examination of both techniques revealed issues that limit their effectiveness. Thus, it is important to develop new methods that can bridge the gap between the Y2H and the TAP. In this thesis, two approaches were developed to meet this need. The first approach was a photoaffinity labeling tool, which was based on a photo-caged reactive intermediate para-quinone methide (pQM) to study protein-peptide associations. This system was developed and optimized by using the interaction between catPTP1Bm and the EGFR peptide as a test case. Highly specific protein labeling was achieved, and mass spectrometry (MS) was used to identify the crosslinked site on the target protein. Interestingly, two peptides from catPTP1Bm detected by MS were found close to the enzyme-substrate binding interface in the three-dimensional structure of the complex, which demonstrated this method might be useful for the analysis of protein complex conformation. The second approach, named "PCA plus", took advantage of a technique referred to as "Protein-fragment Complementation Assay (PCA)". A hydrolysis-deficient mutant β-lactamase (E166N) was used, which enabled interacting protein labeling in live cells. With this modification, the PCA plus method realized live cell imaging with subcellular resolution. Fluorescent microscopy and flow cytometry analysis demonstrated its potential applications. In addition, a new β-lactamase substrate was developed for the PCA plus method and was applied to enable purification, from living cells, of prey protein interacting with a bait protein. The observed enrichment of interacting partners suggested the system could be used for high-throughput PPI screening. Moreover, this method could also be useful for the characterization of low affinity and transient PPIs because of its capacity on labeling interacting protein inside cells.Item Feasibility of Soluble Leaf Proteins as a Carrier for Vitamin D(2009) Cherian, Ansu Elizabeth; Lo, Y. Martin; Food Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Resurgence of vitamin D deficiencies in recent years has ascribed the need for expansion of fortification strategies in food. Alluding to the fat soluble and sensitive chemical nature of vitamin D, existing fortification strategies invariably require using a substantial amount of fat as carriers for vitamin D. Though milk proteins have demonstrated good binding properties with vitamin D; allergen issues, lactose intolerance, and the need to cater to vegan population deter its extensive use. In this study, soluble leaf proteins extracted from low-alkaloid tobacco leaves were investigated as a possible carrier. Crude tobacco leaf proteins were extracted by a high-throughput mechanistic process, followed by a freeze-drying process to encapsulate vitamin D. Up to 84.68% (w/w) of vitamin D was successfully retained by tobacco leaf proteins using the process developed, indicating that crude leaf protein recovered from tobacco could be employed as an effective carrier for vitamin D.Item Molecularly Imprinted Polymers for the Selective Recognition of Proteins(2009) Janiak, Daniel S.; Kofinas, Peter; Material Science and Engineering; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Molecular imprinting is a technique used to synthesize polymers that display selective recognition for a given template molecule of interest. In this study, the role of hydrogel electrostatic charge density on the recognition properties of protein-imprinted hydrogels was explored. Using 3-methacrylamidopropyl trimethylammonium chloride (MAPTAC) as a positively charged monomer and 2-acrylamido-2-methylpropane sulfonic acid (AMPS) as a negatively charged monomer, a number of acrylamide-based polyelectrolyte hydrogels with varying positive and negative charge densities were prepared. The imprinted hydrogels were synthesized in the presence of the target molecule bovine hemoglobin (Bhb). The ability of the hydrogels to selectively recognize Bhb was examined using a competitive template molecule, cytochrome c. The Bhb imprinted gels exhibited template recognition properties that were dependent on both the monomer charge density and on whether the chosen monomer carried a positive or negative charge. In addition to polyelectrolye hydrogels, polyampholyte hydrogels containing both positively and negatively charged monomers were also synthesized. The simultaneous presence of two oppositely charged monomers in the pre-polymerization mixture resulted in imprinted hydrogels with cavities that contain highly specific functional group orientation. The polyampholyte hydrogels exhibited decreased swelling when compared to their polyelectrolyte counterparts, due to the shielding of repulsive interactions between oppositely charge monomers. This decreased swelling resulted in greater template recognition, but lower selectivity, when compared to their polyelectrolyte counterparts. In addition, we found that common agents used in template extraction may be responsible for the specific and selective binding properties exhibited by molecularly imprinted polymers in many published studies, and the effect of variations of the template extraction protocol on the MIP recognition properties were also studied in depth.