Occurrence and transformation of organomercury in the Florida Everglades

In this study, a new method was developed based on aqueous phenylation, purge-and-trap preconcentration, gas chromatography (GC) separation, and detection by atomic fluorescence spectrometry (AFS) or inductively coupled plasma mass spectrometry (ICPMS). This technique is suitable for simultaneous determination of trace or ultratrace levels of CH3Hg+ and CH3CH2Hg+ in environmental samples. Method detection limits were 0.03 ng/L for both CH3Hg+ and CH3CH2Hg+ when AFS was used as the detector and 0.02 and 0.01 ng/L for CH3Hg+ and CH 3CH2Hg+ with ICPMS, respectively. The new method has the additional benefits of being free from interference by Cl - and dissolved organic matter. Using the method developed, both CH3Hg+ and CH3CH2Hg+ were detected in a number of soil and sediment samples collected from the Florida Everglades. The identity of CH3CH2Hg+ was verified by purge-and-trap-GC/MS analysis. The possibility of analytical artifact was excluded by using stable isotope tracer technique in combination with ICPMS detection. CH3CH 2Hg+ in the soil samples analyzed was at ng/g level, similar to that of CH3Hg+. The prevalence of CH 3CH2Hg+ in the soil of the Florida Everglades suggests that ethylation plays an important role in the geochemistry of Hg in this wetland. Soil incubation and sawgrass culture experiments using stable isotope tracers revealed that CH3Hg+ was mainly produced by microbial activities under anaerobic conditions, agreeing well with the general understanding of methylation mechanisms of Hg in the environment. Ethylation of Hg was not confirmed in these experiments, indicating that ethylation of Hg most probably follows different mechanisms in comparison to methylation. Further experiments revealed that trace levels of ethyllead species were able to transfer ethyl group to Hg in both deionized water and freshwater matrixes, producing CH3CH2Hg+. This might partially account for the occurrence of CH3CH2Hg+ in the relatively pristine environment of the Florida Everglades.

Elemental analysis of glass and ink by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and laser induced breakdown spectroscopy (LIBS)

The necessity of elemental analysis techniques to solve forensic problems continues to expand as the samples collected from crime scenes grow in complexity. Laser ablation ICP-MS (LA-ICP-MS) has been shown to provide a high degree of discrimination between samples that originate from different sources. In the first part of this research, two laser ablation ICP-MS systems were compared, one using a nanosecond laser and another a femtosecond laser source for the forensic analysis of glass. The results showed that femtosecond LA-ICP-MS did not provide significant improvements in terms of accuracy, precision and discrimination, however femtosecond LA-ICP-MS did provide lower detection limits. In addition, it was determined that even for femtosecond LA-ICP-MS an internal standard should be utilized to obtain accurate analytical results for glass analyses. In the second part, a method using laser induced breakdown spectroscopy (LIBS) for the forensic analysis of glass was shown to provide excellent discrimination for a glass set consisting of 41 automotive fragments. The discrimination power was compared to two of the leading elemental analysis techniques, μXRF and LA-ICP-MS, and the results were similar; all methods generated >99% discrimination and the pairs found indistinguishable were similar. An extensive data analysis approach for LIBS glass analyses was developed to minimize Type I and II errors en route to a recommendation of 10 ratios to be used for glass comparisons. Finally, a LA-ICP-MS method for the qualitative analysis and discrimination of gel ink sources was developed and tested for a set of ink samples. In the first discrimination study, qualitative analysis was used to obtain 95.6% discrimination for a blind study consisting of 45 black gel ink samples provided by the United States Secret Service. A 0.4% false exclusion (Type I) error rate and a 3.9% false inclusion (Type II) error rate was obtained for this discrimination study. In the second discrimination study, 99% discrimination power was achieved for a black gel ink pen set consisting of 24 self collected samples. The two pairs found to be indistinguishable came from the same source of origin (the same manufacturer and type of pen purchased in different locations). It was also found that gel ink from the same pen, regardless of the age, was indistinguishable as were gel ink pens (four pens) originating from the same pack.

Absolute configuration and biosynthesis of pahayokolide A from Lyngbya sp. Strain 15-2 of the Florida Everglades

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 Lyngbya sp. strain 15-2 were identified by using a PCR method and degenerate primers derived from conserved core sequences of known NRPSs and PKSs. Identification of one unique KS domain conflicted with the logic rule that the long side chain of Athmu was assembled by three rounds of ketide extensions if PKSs were involved. A gene cluster (pah) encoding a peptide synthetase putatively producing pahayokolide was cloned, partially sequenced and characterized. Seven modules of the non-ribosomal peptide synthetase (NRPS) were identified. Ten additional opening reading frames (ORFs) were found, responsible for peptide resistance, transport and degradation. Although the predicted substrate specificities of NRPS agreed with the structure of pahayokolide A partially, the disagreement could be explained. However, no PKS gene was found in the pah gene cluster.

The development of optimized high performance liquid chromatography systems for the analysis of improvised explosives

Existing instrumental techniques must be adaptable to the analysis of novel explosives if science is to keep up with the practices of terrorists and criminals. The focus of this work has been the development of analytical techniques for the analysis of two types of novel explosives: ascorbic acid-based propellants, and improvised mixtures of concentrated hydrogen peroxide/fuel. In recent years, the use of these explosives in improvised explosive devices (IEDs) has increased. It is therefore important to develop methods which permit the identification of the nature of the original explosive from post-blast residues. Ascorbic acid-based propellants are low explosives which employ an ascorbic acid fuel source with a nitrate/perchlorate oxidizer. A method which utilized ion chromatography with indirect photometric detection was optimized for the analysis of intact propellants. Post-burn and post-blast residues if these propellants were analyzed. It was determined that the ascorbic acid fuel and nitrate oxidizer could be detected in intact propellants, as well as in the post-burn and post-blast residues. Degradation products of the nitrate and perchlorate oxidizers were also detected. With a quadrupole time-of-flight mass spectrometer (QToFMS), exact mass measurements are possible. When an HPLC instrument is coupled to a QToFMS, the combination of retention time with accurate mass measurements, mass spectral fragmentation information, and isotopic abundance patterns allows for the unequivocal identification of a target analyte. An optimized HPLC-ESI-QToFMS method was applied to the analysis of ascorbic acid-based propellants. Exact mass measurements were collected for the fuel and oxidizer anions, and their degradation products. Ascorbic acid was detected in the intact samples and half of the propellants subjected to open burning; the intact fuel molecule was not detected in any of the post-blast residue. Two methods were optimized for the analysis of trace levels of hydrogen peroxide: HPLC with fluorescence detection (HPLC-FD), and HPLC with electrochemical detection (HPLC-ED). Both techniques were extremely selective for hydrogen peroxide. Both methods were applied to the analysis of post-blast debris from improvised mixtures of concentrated hydrogen peroxide/fuel; hydrogen peroxide was detected on variety of substrates. Hydrogen peroxide was detected in the post-blast residues of the improvised explosives TATP and HMTD.

Headspace analysis of smokeless powders: Development of mass calibration methods using microdrop printing for chromatographic and ion mobility spectrometric detection

Smokeless powder additives are usually detected by their extraction from post-blast residues or unburned powder particles followed by analysis using chromatographic techniques. This work presents the first comprehensive study of the detection of the volatile and semi-volatile additives of smokeless powders using solid phase microextraction (SPME) as a sampling and pre-concentration technique. Seventy smokeless powders were studied using laboratory based chromatography techniques and a field deployable ion mobility spectrometer (IMS). The detection of diphenylamine, ethyl and methyl centralite, 2,4-dinitrotoluene, diethyl and dibutyl phthalate by IMS to associate the presence of these compounds to smokeless powders is also reported for the first time. A previously reported SPME-IMS analytical approach facilitates rapid sub-nanogram detection of the vapor phase components of smokeless powders. A mass calibration procedure for the analytical techniques used in this study was developed. Precise and accurate mass delivery of analytes in picoliter volumes was achieved using a drop-on-demand inkjet printing method. Absolute mass detection limits determined using this method for the various analytes of interest ranged between 0.03–0.8 ng for the GC-MS and between 0.03–2 ng for the IMS. Mass response graphs generated for different detection techniques help in the determination of mass extracted from the headspace of each smokeless powder. The analyte mass present in the vapor phase was sufficient for a SPME fiber to extract most analytes at amounts above the detection limits of both chromatographic techniques and the ion mobility spectrometer. Analysis of the large number of smokeless powders revealed that diphenylamine was present in the headspace of 96% of the powders. Ethyl centralite was detected in 47% of the powders and 8% of the powders had methyl centralite available for detection from the headspace sampling of the powders by SPME. Nitroglycerin was the dominant peak present in the headspace of the double-based powders. 2,4-dinitrotoluene which is another important headspace component was detected in 44% of the powders. The powders therefore have more than one headspace component and the detection of a combination of these compounds is achievable by SPME-IMS leading to an association to the presence of smokeless powders.

Evaluation of contact and non-contact trapping efficiencies of human scent chemical profiles and their stabilities under different environmental conditions

Establishing an association between the scent a perpetrator left at a crime scene to the odor of the suspect of that crime is the basis for the use of human scent identification evidence in a court of law. Law enforcement agencies gather evidence through the collection of scent from the objects that a perpetrator may have handled during the execution of the criminal act. The collected scent evidence is consequently presented to the canines for identification line-up procedures with the apprehended suspects. Presently, canine scent identification is admitted as expert witness testimony, however, the accurate behavior of the dogs and the scent collection methods used are often challenged by the court system. The primary focus of this research project entailed an evaluation of contact and non-contact scent collection techniques with an emphasis on the optimization of collection materials of different fiber chemistries to evaluate the chemical odor profiles obtained using varying environment conditions to provide a better scientific understanding of human scent as a discriminative tool in the identification of suspects. The collection of hand odor from female and male subjects through both contact and non-contact sampling approaches yielded new insights into the types of VOCs collected when different materials are utilized, which had never been instrumentally performed. Furthermore, the collected scent mass was shown to be obtained in the highest amounts for both gender hand odor samples on cotton sorbent materials. Compared to non-contact sampling, the contact sampling methods yielded a higher number of volatiles, an enhancement of up to 3 times, as well as a higher scent mass than non-contact methods by more than an order of magnitude. The evaluation of the STU-100 as a non-contact methodology highlighted strong instrumental drawbacks that need to be targeted for enhanced scientific validation of current field practices. These results demonstrated that an individual's human scent components vary considerably depending on the method used to collect scent from the same body region. This study demonstrated the importance of collection medium selection as well as the collection method employed in providing a reproducible human scent sample that can be used to differentiate individuals.

Development of a dynamic headspace concentration technique for the non-contact sampling of human odor samples and the creation of canine training aids

Human scent and human remains detection canines are used to locate living or deceased humans under many circumstances. Human scent canines locate individual humans on the basis of their unique scent profile, while human remains detection canines locate the general scent of decomposing human remains. Scent evidence is often collected by law enforcement agencies using a Scent Transfer Unit, a dynamic headspace concentration device. The goals of this research were to evaluate the STU-100 for the collection of human scent samples, and to apply this method to the collection of living and deceased human samples, and to the creation of canine training aids. The airflow rate and collection material used with the STU-100 were evaluated using a novel scent delivery method. Controlled Odor Mimic Permeation Systems were created containing representative standard compounds delivered at known rates, improving the reproducibility of optimization experiments. Flow rates and collection materials were compared. Higher air flow rates usually yielded significantly less total volatile compounds due to compound breakthrough through the collection material. Collection from polymer and cellulose-based materials demonstrated that the molecular backbone of the material is a factor in the trapping and releasing of compounds. The weave of the material also affects compound collection, as those materials with a tighter weave demonstrated enhanced collection efficiencies. Using the optimized method, volatiles were efficiently collected from living and deceased humans. Replicates of the living human samples showed good reproducibility; however, the odor profiles from individuals were not always distinguishable from one another. Analysis of the human remains samples revealed similarity in the type and ratio of compounds. Two types of prototype training aids were developed utilizing combinations of pure compounds as well as volatiles from actual human samples concentrated onto sorbents, which were subsequently used in field tests. The pseudo scent aids had moderate success in field tests, and the Odor pad aids had significant success. This research demonstrates that the STU-100 is a valuable tool for dog handlers and as a field instrument; however, modifications are warranted in order to improve its performance as a method for instrumental detection.

Comparison of medical and forensic profiling potential of volatile biomarkers from different biological specimens from individuals and across populations

There is limited scientific knowledge on the composition of human odor from different biological specimens and the effect that physiological and psychological health conditions could have on them. There is currently no direct comparison of the volatile organic compounds (VOCs) emanating from different biological specimens collected from healthy individuals as well as individuals with certain diagnosed medical conditions. Therefore the question of matching VOCs present in human odor across various biological samples and across health statuses remains unanswered. The main purpose of this study was to use analytical instrumental methods to compare the VOCs from different biological specimens from the same individual and to compare the populations evaluated in this project. The goals of this study were to utilize headspace solid-phase microextraction gas chromatography mass spectrometry (HS-SPME-GC/MS) to evaluate its potential for profiling VOCs from specimens collected using standard forensic and medical methods over three different populations: healthy group with no diagnosed medical or psychological condition, one group with diagnosed type 2 diabetes, and one group with diagnosed major depressive disorder. The pre-treatment methods of collection materials developed for the study allowed for the removal of targeted VOCs from the sampling kits prior to sampling, extraction and analysis. Optimized SPME-GC/MS conditions has been demonstrated to be capable of sampling, identifying and differentiating the VOCs present in the five biological specimens collected from different subjects and yielded excellent detection limits for the VOCs from buccal swab, breath, blood, and urine with average limits of detection of 8.3 ng. Visual, Spearman rank correlation, and PCA comparisons of the most abundant and frequent VOCs from each specimen demonstrated that each specimen has characteristic VOCs that allow them to be differentiated for both healthy and diseased individuals. Preliminary comparisons of VOC profiles of healthy individuals, patients with type 2 diabetes, and patients with major depressive disorder revealed compounds that could be used as potential biomarkers to differentiate between healthy and diseased individuals. Finally, a human biological specimen compound database has been created compiling the volatile compounds present in the emanations of human hand odor, oral fluids, breath, blood, and urine.

Bilirubin: An animal pigment in the Zingiberales and diverse angiosperm orders

Strelitziaceae is a tropical monocot family comprising three genera and seven species: Ravenala Adans and Phenkospermum Endl., which are monotypic, and five species of Strelitzia Aiton. All species produce woody capsular fruits that contain vibrantly colored arillate seeds. Arils of the Strelitzia species are orange, those of Phenakospermum are red, and those of Ravenala are blue. Unlike most plant pigments, which degrade after cell death, aril pigments in the family persist for decades. Chemical properties of the compounds are unusual, and do not match those of known pigment classes (carotenoids, flavonoids, betalains, and the chlorophylls). I isolated the orange pigment from the arils of Strelitzia nicolai, and performed HPLC-ESMS, UV-visible, 1H NMR and 13C NMR analyses to determine its chemical structure. These data indicated the pigment was bilirubin-IX, an orange-yellow tetrapyrrole previously known only in mammals and some other vertebrates as the breakdown product of heme. Although related tetrapyrroles are ubiquitous throughout the plant kingdom and include vital biosynthetic products such as chlorophyll and phytochromobilin, this is the first report of bilirubin in a plant, and evidence of an additional biosynthetic pathway producing orange coloration in flowers and fruits. ^ Given the unexpected presence of bilirubin, Iexamined the fruits and flowers of twelve additional angiosperm species in diverse orders for the presence of bilirubin using HPLC and LC-MS. Bilirubin was present in ten species from the orders Zingiberales, Arecales, and Myrtales, indicating its wide distribution in the plant kingdom. Bilirubin was present in low concentrations in all species except those within Strelitziaceae. It was present in particularly high concentrations in S. nicolai, S. reginae and P. guyannense, and is thus responsible for producing color in these species. ^ No studies have examined the evolutionary relationship among all species in the family. Thus, I also constructed a molecular phylogeny of the family. This information, combined with further studies on the distribution and synthesis of bilirubin in plants, will provide a basis for understanding the evolutionary history of this pigment in the plant kingdom.^

An investigation of the relationship between antemortem and postmortem drug concentrations in blood

In the field of postmortem toxicology, principles from pharmacology and toxicology are combined in order to determine if exogenous substances contributed to ones death. In order to make this determination postmortem and (whenever available) antemortem blood samples may be analyzed. This project focused on evaluating the relationship between postmortem and antemortem blood drug levels, in order to better define an interpretive framework for postmortem toxicology. To do this, it was imperative to evaluate the differences in antemortem and postmortem drug concentrations, determine the role microbial activity and evaluate drug stability. Microbial studies determined that the bacteria Escherichia coli and Pseudomonas aeruginosa could use the carbon structures of drugs as a source of food. This would suggest prior to sample collection, microbial activity could potentially affect drug levels. This process however would stop before toxicologic evaluation, as at autopsy blood samples are stored in tubes containing the antimicrobial agent sodium fluoride. Analysis of preserved blood determined that under the current storage conditions sodium fluoride effectively inhibited microbial growth. Nonetheless, in many instances inconsistent drug concentrations were identified. When comparing antemortem to postmortem results, diphenhydramine, morphine, codeine and methadone, all showed significantly increased postmortem drug levels. In many instances, increased postmortem concentrations correlated with extended postmortem intervals. Other drugs, such as alprazolam, were likely to have concentration discrepancies when short antemortem to death intervals were coupled with extended postmortem intervals. While still others, such as midazolam followed the expected pattern of metabolism and elimination, which often resulted in decreased postmortem concentrations. The importance of drug stability was displayed when reviewing the clonazepam/ 7-aminoclonazepam data, as the parent drug commonly converted to its metabolite even when stored in the presence of a preservative. In instances of decreasing postmortem drug concentrations the effect of refrigerated storage could not be ruled out. A stability experiment, which contained codeine, produced data that indicated concentrations could continue to decline under the current storage conditions. The cumulative data gathered for this experiment was used to identify concentration trends, which subsequently aided in the development of interpretive considerations for the specific analytes examined in the study.

Characterization, sources, and transformations of dissolved organic matter (DOM) in Florida coastal Everglades (FCE)

Dissolved organic matter (DOM) is one of the largest carbon reservoirs on this planet and is present in aquatic environments as a highly complex mixture of organic compounds. The Florida coastal Everglades (FCE) is one of the largest wetlands in the world. DOM in this system is an important biogeochemical component as most of the nitrogen (N) and phosphorous (P) are in organic forms. Achieving a better understanding of DOM dynamics in large coastal wetlands is critical, and a particularly important issue in the context of Everglades restoration. In this work, the environmental dynamics of surface water DOM on spatial and temporal scales was investigated. In addition, photo- and bio-reactivity of this DOM was determined, surface-to-groundwater exchange of DOM was investigated, and the size distribution of freshwater DOM in Everglades was assessed. The data show that DOM dynamics in this ecosystem are controlled by both hydrological and ecological drivers and are clearly different on spatial scales and variable seasonally. The DOM reactivity data, modeled with a multi-pool first order degradation kinetics model, found that fluorescent DOM in FCE is generally photo-reactive and bio-refractory. Yet the sequential degradation proved a “priming effect” of sunlight on the bacterial uptake and reworking of this subtropical wetland DOM. Interestingly, specific PARAFAC components were found to have different photo- and bio-degradation rates, suggesting a highly heterogeneous nature of fluorophores associated with the DOM. Surface-to-groundwater exchange of DOM was observed in different regions of the system, and compositional differences were associated with source and photo-reactivity. Lastly, the high degree of heterogeneity of DOM associated fluorophores suggested based on the degradation studies was confirmed through the EEM-PARAFAC analysis of DOM along a molecular size continuum, suggesting that the fluorescence characteristics of DOM are highly controlled by different size fractions and as such can exhibit significant differences in reactivity.

Characterization of recombinant chloroperoxidase, and F103A and C29H/C79H/C87H mutants

Mechanistically and structurally chloroperoxidase (CPO) occupies a unique niche among heme containing enzymes. Chloroperoxidase catalyzes a broad range of reactions, such as oxidation of organic substrates, dismutation of hydrogen peroxide, and mono-oxygenation of organic molecules. To expand the synthetic utility of CPO and to appreciate the important interactions that lead to CPO’s exceptional properties, a site-directed mutagenesis study was undertaken. ^ Recombinant CPO and CPO mutants were heterologously expressed in Aspergillus niger. The overall protein structure was almost the same as that of wild type CPO, as determined by UV-vis, NMR and CD spectroscopies. Phenylalanine103, which was proposed to regulate substrate access to the active site by restricting the size of substrates and to control CPO’s enantioselectivity, was mutated to Ala. The ligand binding affinity and most importantly the catalytic activity of F103A was dramatically different from wild type CPO. The mutation essentially eliminated the chlorination and dismutation activities but enhanced, 4-10 fold, the epoxidation, peroxidation, and N-demethylation activities. As expected, the F103A mutant displayed dramatically improved epoxidation activity for larger, more branched styrene derivatives. Furthermore, F103A showed a distinctive enantioselectivity profile: losing enantioselectivity to styrene and cis-β-methylstyrene; having a different configuration preference on α-methylstyrene; showing higher enantioselectivites and conversion rates on larger, more branched substrates. Our results show that F103 acts as a switch box that controls the catalytic activity, substrate specificity, and product enantioselectivity of CPO. Given that no other mutant of CPO has displayed distinct properties, the results with F103A are dramatic. ^ The diverse catalytic activity of CPO has long been attributed to the presence of the proximal thiolate ligand. Surprisingly, a recent report on a C29H mutant suggested otherwise. A new CPO triple mutant C29H/C79H/C87H was prepared, in which all the cysteines were replaced by histidine to eliminate the possibility of cysteine coordinating to the heme. No active form protein was isolated, although, successful transformation and transcription was confirmed. The result suggests that Cys79 and Cys87 are critical to maintaining the structural scaffold of CPO. ^ In vitro biodegradation of nanotubes by CPO were examined by scanning electron microscope method, but little oxidation was observed. ^

Development of micro immunosensors to study genomic and proteomic biomarkers related to cancer and Alzheimer's disease

A report from the National Institutes of Health defines a disease biomarker as a “characteristic that is objectively measured and evaluated as an indicator of normal biologic processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention.” Early diagnosis is a crucial factor for incurable disease such as cancer and Alzheimer’s disease (AD). During the last decade researchers have discovered that biochemical changes caused by a disease can be detected considerably earlier as compared to physical manifestations/symptoms. In this dissertation electrochemical detection was utilized as the detection strategy as it offers high sensitivity/specificity, ease of operation, and capability of miniaturization and multiplexed detection. Electrochemical detection of biological analytes is an established field, and has matured at a rapid pace during the last 50 years and adapted itself to advances in micro/nanofabrication procedures. Carbon fiber microelectrodes were utilized as the platform sensor due to their high signal to noise ratio, ease and low-cost of fabrication, biocompatibility, and active carbon surface which allows conjugation with biorecognition moieties. This dissertation specifically focuses on the detection of 3 extensively validated biomarkers for cancer and AD. Firstly, vascular endothelial growth factor (VEGF) a cancer biomarker was detected using a one-step, reagentless immunosensing strategy. The immunosensing strategy allowed a rapid and sensitive means of VEGF detection with a detection limit of about 38 pg/mL with a linear dynamic range of 0–100 pg/mL. Direct detection of AD-related biomarker amyloid beta (Aβ) was achieved by exploiting its inherent electroactivity. The quantification of the ratio of Aβ1-40/42 (or Aβ ratio) has been established as a reliable test to diagnose AD through human clinical trials. Triple barrel carbon fiber microelectrodes were used to simultaneously detect Aβ1-40 and Aβ1-42 in cerebrospinal fluid from rats within a detection range of 100nM to 1.2μM and 400nM to 1μM respectively. In addition, the release of DNA damage/repair biomarker 8-hydroxydeoxyguanine (8-OHdG) under the influence of reactive oxidative stress from single lung endothelial cell was monitored using an activated carbon fiber microelectrode. The sensor was used to test the influence of nicotine, which is one of the most biologically active chemicals present in cigarette smoke and smokeless tobacco.

Mechanisms of chloroperoxidase-catalyzed enantioselective reactions as probed by site-directed mutagenesis and isotopic labeling

Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Chloroperoxidase contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes a variety of reactions, such as halogenation, peroxidation and epoxidation. The versatile catalytic activities of CPO coupled with the increasing demands for chiral synthesis have attracted an escalating interest in understanding the mechanistic and structural properties of this enzyme. In order to better understand the mechanisms of CPO-catalyzed enantioselective reactions and to fine-tune the catalytic properties of chloroperoxidase, asparagine 74 (N74) located in the narrow substrate access channel of CPO was replaced by a bulky, nonpolar valine and a polar glutamine using site-directed mutagenesis. The CPO N74 mutants displayed significantly enhanced activity toward nonpolar substrates compared to wild-type CPO as a result of changes in space and polarity of the heme distal environment. More interestingly, N74 mutants showed dramatically decreased chlorination and catalase activity but significantly enhanced epoxidation activity as a consequence of improved kinetic perfection introduced by the mutation as reflected by the favorable changes in k cat and kcat/KM of these reactions. It is also noted that the N74V mutant is capable of decomposing cyanide, the most notorious poison for many hemoproteins, as judged by the unique binding behavior of N74V with potassium cyanide. Histidine 105 (H105) was replaced by a nonpolar amino acid alanine using site-directed mutagenesis. The CPO H105 mutant (H105A) displayed dramatically decreased chlorination and catalase activity possibly because of the decreased polarity in the heme distal environment and loss of the hydrogen bonds between histidine 105 and glutamic acid 183. However, significantly increased enantioselectivity was observed for the epoxidation of bulky styrene derivatives. Furthermore, my study provides strong evidence for the proposed histidine/cysteine ligand switch in chloroperoxidase, providing experimental support for the structure of the 420-nm absorption maximum for a number of carbon monoxide complexes of heme-thiolate proteins. For the NMR study, [dCPO(heme)] was produced using 90% deuterated growth medium with excess heme precursors and [dCPO(Phe)] was grown in the same highly deuterated medium that had been supplemented with excess natural phenylalanine. To make complete heme proton assignments, NMR spectroscopy has been performed for high-resolution structural characterization of [dCPO(heme)] and [dCPO(Phe)] to achieve unambiguous and complete heme proton assignments, which also allows important amino acids close to the heme active center to be determined.

Functionalized carbon micro/nanostructures for biomolecular detection

Advancements in the micro-and nano-scale fabrication techniques have opened up new avenues for the development of portable, scalable and easier-to-use biosensors. Over the last few years, electrodes made of carbon have been widely used as sensing units in biosensors due to their attractive physiochemical properties. The aim of this research is to investigate different strategies to develop functionalized high surface carbon micro/nano-structures for electrochemical and biosensing devices. High aspect ratio three-dimensional carbon microarrays were fabricated via carbon microelectromechanical systems (C-MEMS) technique, which is based on pyrolyzing pre-patterned organic photoresist polymers. To further increase the surface area of the carbon microstructures, surface porosity was introduced by two strategies, i.e. (i) using F127 as porogen and (ii) oxygen reactive ion etch (RIE) treatment. Electrochemical characterization showed that porous carbon thin film electrodes prepared by using F127 as porogen had an effective surface area (Aeff 185%) compared to the conventional carbon electrode. To achieve enhanced electrochemical sensitivity for C-MEMS based functional devices, graphene was conformally coated onto high aspect ratio three-dimensional (3D) carbon micropillar arrays using electrostatic spray deposition (ESD) technique. The amperometric response of graphene/carbon micropillar electrode arrays exhibited higher electrochemical activity, improved charge transfer and a linear response towards H2O2 detection between 250&mgr;M to 5.5mM. Furthermore, carbon structures with dimensions from 50 nano-to micrometer level have been fabricated by pyrolyzing photo-nanoimprint lithography patterned organic resist polymer. Microstructure, elemental composition and resistivity characterization of the carbon nanostructures produced by this process were very similar to conventional photoresist derived carbon. Surface functionalization of the carbon nanostructures was performed using direct amination technique. Considering the need for requisite functional groups to covalently attach bioreceptors on the carbon surface for biomolecule detection, different oxidation techniques were compared to study the types of carbon-oxygen groups formed on the surface and their percentages with respect to different oxidation pretreatment times. Finally, a label-free detection strategy using signaling aptamer/protein binding complex for platelet-derived growth factor oncoprotein detection on functionalized three-dimensional carbon microarrays platform was demonstrated. The sensor showed near linear relationship between the relative fluorescence difference and protein concentration even in the sub-nanomolar range with an excellent detection limit of 5 pmol.