Life Sciences Commons^™

Development Of Electrochemical Aptamer-Based Sensors For Sensitive And Specific Detection Of Small-Molecule Targets, Yingzhu Liu

FIU Electronic Theses and Dissertations

Electrochemical aptamer-based (E-AB) sensors have been applied for diverse applications such as forensic science, pharmaceutical diagnostics, food safety, environmental monitoring, and personal medical care due to their rapid, accurate, and specific detection of analytes in complex samples. However, limited sensitivity and a lack of portable devices for point-of-care applications have greatly hindered the maturation of E-AB sensors from proof-of-concept designs to commercial systems. This dissertation describes several novel strategies to improve the sensitivities of E-AB sensors and fabricate portable paper-based devices to remedy these problems. We first detail the development of a novel approach to utilize a defined mixture of …

Exonuclease Digestion Assay For Streamlining Aptamer Characterization, Engineering, And Sensor Development, Juan Canoura

FIU Electronic Theses and Dissertations

Aptamers are DNA or RNA oligonucleotide-based bioreceptors isolated in vitro through the Systematic Evolution of Ligands by Exponential Enrichment. Given the ease with which a selection can be customized, aptamers can be evolved to function in nearly any chemical environment, making them tailormade for their final application. However, the post-SELEX characterization of the 100-1000’s aptamer candidates remains a significant bottleneck as there are no suitable techniques for high-throughput characterization of each candidate’s affinity/specificity. Moreover, the final aptamer must be engineered to possess signal reporting functionality; this is often done via trail-and-error truncation to yield a structure-switching aptamer. This dissertation describes …

The Utilization Of Volatile Organic Compounds And Human Leukocyte Antigen Genes For Ethnic-Specific Differentiation Within Target Populations, Alice Breia Boone

FIU Electronic Theses and Dissertations

Human scent has been previously defined as a complex mixture of volatile organic compounds (VOCs) detected in the headspace above a scent sample. Humans generate odor from several areas of the body including hair, mouth, hand, axillae, and foot. Due to the novelty of human scent research, human scent evidence has been undervalued in the court of law. However, this type of evidence has significant value when physical evidence is not available at crime scenes. To increase the individualization and differentiation power of human scent evidence, this study aimed to further investigate the identification of chemical signatures within axillae of …

An Investigation To Corroborate Volatile And Biological Profiles Of Human Odor For Forensic Subject Identification, Chantrell J.G. Frazier

FIU Electronic Theses and Dissertations

Hands are an integral part in transferring complex microbial communities to and from our surroundings. As a result, hands are significant in provenance investigations as specific microbiota can be deposited on everyday objects through touch interactions. Skin microbiome, including bacteria, fungi, and viruses, are unique to each person, and this ‘uniqueness’ can be exploited and applied to forensic identification. Skin microbiota and volatile organic compounds (VOCs) are closely related due to specific bacteria breaking down non-volatile organic compounds to volatile organic compounds that are characteristically present in human scent. However, analyses of microbiota from touch interactions have proven to be …

Aptamer-Based Voltammetric Biosensing For The Detection Of Codeine And Fentanyl In Sweat And Saliva, Rosa Lashantez Cromartie

FIU Electronic Theses and Dissertations

Despite the many governmental and medicinal restrictions created to combat the opioid epidemic in the United States, opioid abuse and overdose rates continue to rise. The development of an aptamer-based voltammetric sensor and biosensor is described in this dissertation. The aim was to develop a low-cost, sensitive, and specific aptamer-based sensor for on-site, label-free determination of codeine and fentanyl in biological fluids. To do this, the surfaces of screen-printed carbon electrodes (SPCE) were modified with gold nanoparticles (AuNPs), followed by the addition of single-stranded DNA aptamers. These were covalently bound to the electrode surface. Operations of the sensors were collected …

Evaluation Of Harvesting Time For Industrial Hemp (Cannabis Sativa L,) Pilot Project Varieties Grown In South Florida, Jordan W. Prats

FIU Electronic Theses and Dissertations

A field study was conducted to observe the natural development of cannabinoids in three daylength sensitive industrial hemp varieties Bubba Kush (BK), Emerald Flower (EF), and Golden Sunset (GS). Plants were configured in a randomized block design with 3 replications. Once 50% of the plants within a variety reached reproductive growth, plants were sampled weekly until senescence and analyzed through a HPLC-UV/DAD. The results from the study indicate that all three varieties of industrial hemp tested in field study reached reproductive growth within the first week of transplanting. This is due to a 12-hour day-length at the time and the …

Functionalized Aptamers For Detection Of Small-Molecule Targets, Haixiang Yu

FIU Electronic Theses and Dissertations

Aptamers have recently gained considerable attention for small-molecule detection in diverse applications such as drug identification, medical diagnostics, and environmental monitoring. However, the performance of aptamer-based sensors has been greatly limited by the low target affinity and responsiveness of small-molecule binding aptamers. This dissertation describes several novel aptamer engineering and isolation strategies to remedy this problem. Specifically, we first develop a generally applicable strategy to engineer split aptamers containing two binding domains termed cooperative-binding split aptamers (CBSAs). CBSAs exhibit higher target binding affinity and are far more responsive in terms of target-induced split aptamer assembly compared to single-domain parent split …

Aptamer-Based Assay For Detection Of Ochratoxin A, Amanda Nicole Bartley

FIU Electronic Theses and Dissertations

Ochratoxin A (OTA) is a potent mycotoxin found in a wide range of agricultural products that has been linked to mitochondrial damage and renal disease. The standard methods for OTA analysis currently rely on the use of high-performance liquid chromatography (HPLC) coupled to fluorescence detection or mass spectrometry. Toward a highthroughput analysis of OTA, a single-stranded DNA aptamer, modified with a fluorophore, coupled to a complementary sequence, modified with a FRET-based quencher that dissociates in the presence of the target toxin, is proposed. In order to integrate “target trapping,” aptamer immobilization methods were explored to mediate interference issues. Assays were …

The Use Of Elemental Databases In Forensic Science: Studies On Vehicle Glass Interpretation And Milk Powder Provenancing, Tricia Marie Hoffman

FIU Electronic Theses and Dissertations

The first study focuses on the development of a laser based method for the elemental analysis of solid milk powder. Milk powder samples originating from five different countries were analyzed to determine any geographic differences. A LA-ICP-MS method was developed and compared to k₀-INAA for several milk samples as well as a reference sample. Precision of 10% RSD or better and a bias of 10% was achieved for both techniques for most elements with LA-ICP-MS producing lower limits of detection (~ 1 ppm) for Sr. The comparison of LA-ICP-MS to k₀-INAA showed overlap of the 95% …

The Cyclodextrin-Perfluorinated Surfactant Host-Guest Complex: Fundamental Studies For Potential Environmental Remediation And Therapeutic Applications, Mary J. Errico

FIU Electronic Theses and Dissertations

Perfluoroalkyl substances (PFASs) are contaminants of emerging concern, and have been detected in drinking water, wildlife, humans, and the environment. Cyclodextrins (CDs), cyclic sugars composed of glucose monomers, are proposed as a potential remediation strategy. CDs can form host-guest complexes with hydrophobic molecules; this complexation could be capitalized on for PFAS removal and sequestration. These dissertation projects aim to study the fundamental host-guest interactions between a variety of PFASs and CDs for eventual applications in environmental and biological remediation. 1D and 2D Nuclear magnetic resonance (NMR) spectroscopic methods were employed to determine the strength, dynamics, and structure of the CD:PFAS …

Conformational Dynamics Of Biomolecules By Trapped Ion Mobility Spectrometry Dynamics, Juan Camilo Molano-Arévalo

FIU Electronic Theses and Dissertations

One of the main goals in structural biology is to understand the folding mechanisms and three-dimensional structure of biomolecules. Many biomolecular systems adopt multiple structures as a function of their microenvironment, which makes them difficult to be characterized by traditional structural biology tools (e.g., NMR, X-ray crystallography). As an alternative, complementary tools that can capture and sample multiple conformations needed to be developed. In the present work, we pioneered the application of a new variant of ion mobility spectrometry, trapped ion mobility spectrometry (TIMS), which provides high mobility resolving power and the possibility to study kinetically trapped intermediates as a …

The Optimization Of Pressure Cycling Technology (Pct) For Differential Extraction Of Sexual Assault Casework, Vanessa Martinez

FIU Electronic Theses and Dissertations

A two-step protocol has been devised as a rapid and selective alternative to conventional differential extraction techniques with an increased recovery of DNA. The protocol involves pressure cycling with the Barocycler^® NEP 2320 from Pressure Biosciences. Inc. in alkaline conditions for epithelial cell lysis and removal. This step is followed by alkaline lysis at 95^º C for extraction of sperm cell DNA. At 1:1 or 2:1 female to male cell ratios, high selectivity and complete separation can be achieved. But at higher ratios, male allelic dropout is observed. This protocol has been modified to generate a clean male …

Dna Aptamer Confirmation And Utilization For The Cyanotoxin, Cylindrospermopsin, Diane M. Catlin

FIU Electronic Theses and Dissertations

Cyanotoxins are posing an increasing threat to the health of humans and wildlife. Cylindrospermopsin is a cyanotoxin that occurs in warm climates and is harmful when ingested. The toxic effects of CYN can affect multiple organ systems. The effects, coupled with the evidence of a mass contamination of a water supply in Australia, prove that CYN needs to be investigated further.

Aptamers have become a desirable method for detection of CYN as a result of an aptamer’s high specificity and the ability to scale up experiments. Aptamers have been designed to bind with a variety of targets, including cyanotoxins. An …

Development Of A Maldi-Tof-Ms Method For The Analysis Of Cyanobacterial Neurotoxin Β-N-Methylamino-L-Alanine (Bmaa) In Search Of Bmaa Incorporation In Biological Samples, Laura M. Conklin

FIU Electronic Theses and Dissertations

Beta-N-methylamino-L-alanine (BMAA) is a non-protein amino acid produced by many cyanobacteria, and thought to induce neurotoxic effects through excitotoxicity, contributing to neurodegenerative diseases such as Amyotrophic Lateral Sclerosis/Parkinsonism-dementia complex (ALS-PDC) and Alzheimer’s. The ubiquitous nature of cyanobacteria, and evidence of biomagnification through our food web, creates a dire need for the development of an analytical platform that will provide accurate identification and quantification of BMAA amounts in our ecosystem and potential food supply. The present study evaluated the ability of a MALDI-ToF-MS method to detect and quantify BMAA in a variety of biological matrices. Through validation procedures, it …

Determination Of Extracellular Molecules Produced By Vibrio Harveyi Using Ms/Ms, Jose G. Roble

FIU Electronic Theses and Dissertations

Quorum sensing (QS) is a process that allows bacteria to sense the population density of cells around them by communicating with each other via autoinducer molecules. This cross-communication is crucial in the regulation of bacterial processes such as bioluminescence, virulence, and biofilm formation. Previous research by Milburn and Makemson on Vibrio harveyi suggested that in addition of the known biosynthesis of three well-characterized autoinducers, dozens of unknown molecules are also produced and released to the environment by V. harveyi. This study was performed using electrospray tandem mass spectrometry with the purpose of detection and characterization of the extracellular molecules …

Mangiferin As A Biomarker For Mango Anthracnose Resistance, Herma Pierre

FIU Electronic Theses and Dissertations

Mangos (Mangifera indica L.) are tropical/subtropical fruits belonging to the plant family Anacardiaceae. Anthracnose is the most deleterious disease of mango both in the field and during postharvest handling. It is most commonly caused by the Colletotrichum gloeosporioides complex. Mangiferin, a xanthanoid compound found in at least twelve plant families worldwide (Luo et al., 2012), is present in large amounts of the leaves and edible mangos. Even though this compound plays a pivotal role in the plant’s defense against biotic and abiotic stressors, no correlations been made between the compound and mango anthracnose resistance.

Mangos were collected, grouped …

The Effect Of Sample And Sample Matrix On Dna Processing: Mechanisms For The Detection And Management Of Inhibition In Forensic Samples, Lilliana I. Moreno

FIU Electronic Theses and Dissertations

The presence of inhibitory substances in biological forensic samples has, and continues to affect the quality of the data generated following DNA typing processes. Although the chemistries used during the procedures have been enhanced to mitigate the effects of these deleterious compounds, some challenges remain. Inhibitors can be components of the samples, the substrate where samples were deposited or chemical(s) associated to the DNA purification step. Therefore, a thorough understanding of the extraction processes and their ability to handle the various types of inhibitory substances can help define the best analytical processing for any given sample. A series of experiments …

Multimode Analysis Of Nanoscale Biomolecular Interactions, Purushottam Babu Tiwari

FIU Electronic Theses and Dissertations

Biomolecular interactions, including protein-protein, protein-DNA, and protein-ligand interactions, are of special importance in all biological systems. These interactions may occer during the loading of biomolecules to interfaces, the translocation of biomolecules through transmembrane protein pores, and the movement of biomolecules in a crowded intracellular environment. The molecular interaction of a protein with its binding partners is crucial in fundamental biological processes such as electron transfer, intracellular signal transmission and regulation, neuroprotective mechanisms, and regulation of DNA topology. In this dissertation, a customized surface plasmon resonance (SPR) has been optimized and new theoretical and label free experimental methods with related analytical …

Speciation, Metabolism, Toxicity, And Protein-Binding Of Different Arsenic Species In Human Cells, Szabina A. Stice

FIU Electronic Theses and Dissertations

Despite of its known toxicity and potential to cause cancer, arsenic has been proven to be a very important tool for the treatment of various refractory neoplasms. One of the promising arsenic-containing chemotherapeutic agents in clinical trials is Darinaparsin (dimethylarsinous glutathione, DMA^III(GS)). In order to understand its toxicity and therapeutic efficacy, the metabolism of Darinaparsin in human cancer cells was evaluated. With the aim of detecting all potential intermediates and final products of the biotransformation of Darinaparsin and other arsenicals, an analytical method employing high performance liquid chromatography inductively coupled mass spectrometry (HPLC-ICP-MS) was developed. This method was …

The Utilization Of Chiral Ion Mobility Spectrometry For The Detection Of Enantiomeric Mixtures And Thermally Labile Compounds, Howard K. Holness

FIU Electronic Theses and Dissertations

This dissertation utilized electrospray ion mobility mass spectrometry (ESI-IMS-MS) to develop methods necessary for the separation of chiral compounds of forensic interest. The compounds separated included ephedrines and pseudoephedrines, that occur as impurities in confiscated amphetamine type substances (ATS) in an effort to determine the origin of these substances. The ESI-IMS-MS technique proved to be faster and more cost effective than traditional chromatographic methods currently used to conduct chiral separations such as gas and liquid chromatography. Both mass spectrometric and computational analysis revealed the separation mechanism of these chiral interactions allowing for further development to separate other chiral compounds by …

Comprehensive Forensic Toxicological Analysis Of Designer Drugs, Madeleine Jean Swortwood

FIU Electronic Theses and Dissertations

New designer drugs are constantly emerging onto the illicit drug market and it is often difficult to validate and maintain comprehensive analytical methods for accurate detection of these compounds. Generally, toxicology laboratories utilize a screening method, such as immunoassay, for the presumptive identification of drugs of abuse. When a positive result occurs, confirmatory methods, such as gas chromatography (GC) or liquid chromatography (LC) coupled with mass spectrometry (MS), are required for more sensitive and specific analyses. In recent years, the need to study the activities of these compounds in screening assays as well as to develop confirmatory techniques to detect …

Assessment Of Submerged Vegetation As Indicators Of Irgarol Contamination, Melissa V. Fernandez

FIU Electronic Theses and Dissertations

Irgarol 1051 is a common antifoulant toxic to certain marine organisms. Submerged aquatic vegetation (SAV) are exposed to this herbicide when it leaches into the marine environment from painted structures, making SAVs ideal candidates to function as sentinel indicator of contamination. In the initial stage of this study, Coconut Grove and Key Largo Harbor were assessed for environmental exposure to Irgarol. Water, sediment and SAVs were collected, the latter two subject to automated solid phase extraction, and all samples analyzed by GC/MS-SIM for Irgarol and its metabolite, M1. Of the vegetation analyzed, Halodule and Syringodium had the highest capacity to …

Absolute Configuration And Biosynthesis Of Pahayokolide A From Lyngbya Sp. Strain 15-2 Of The Florida Everglades, Li Liu

FIU Electronic Theses and Dissertations

Pahayokolides A-D are cytotoxic cyclic polypeptides produced by the freshwater cyanobacterium Lyngbya sp. strain 15-2 that possess an unusual β-amino acid, 3-amino-2,5,7,8-tetrahydroxy-10-methylundecanoic acid (Athmu). The absolute configuration of pahayokolides A-D was determined using advanced Marfey’s method. It was also confirmed that a pendant N-acetyl-N-methyl leucine moiety in pahayokolide A was absent in pahayokolides B and pahayokolides C-D were conformers of pahayokolide A. Feeding experiments indicated that the biosynthesis of the Athmu sidechain arises from leucine or α-ketoisovalerate, however could not be further extended by three rounds of condensation with malonate units. Putative four peptide and one unique polyketide synthetases in …