Microcystin-LR (MCLR) is hepatotoxic to animals and humans with disruption of liver structure causing cytoskeletal damage, necrosis and pooling of blood in the liver, leading to large increase in liver weight. It is also a strong liver tumor promoter and protein phosphatase inhibitor. Microcysin-LR binds protein phosphatases 1 and 2A, and influences regulation of cellular protein phosphorylation. In the present study, a colloidal gold based immunoassay test strip was developed for Microcystin-LR detection. The detection limit was found to be 1 ng/mL. 5 nm colloidal gold test strips exhibits more efficient for detection, compared with 20 nm colloidal gold test strips. The interaction between Microcystin-LR antibody (immunoglobulin G) and colloidal gold nanoparticles was investigated by various analytical methods, including Ultraviolet/Visible (UVNIS), Fourier Transform Infrared (FTIR) and Fluorescence spectroscopy as well as transmission electron microscopy (TEM).

The complexity of branching macromolecules around a core unit, such as poly(propylene)imine (PPI) dendrimers, has caught the attention of researchers for several years1,2,3,4,5. The structural framework of the core unit for these dendrimers is simply 1,4-diaminobutane. As the synthesis of higher-order generations of the macromolecule progresses, the molecules become very complex and tightly woven, but have predictable geometries and properties. The PPI dendrimers can be functionalized at the terminal ends of the branches in order to elicit different properties. The PPI dendrimer examined in this project has been terminated with a nitrile group rather than the usual amine group. Using mass spectrometry, the gas-phase fragmentation pathways under low energy collision conditions of the modified PPI dendrimer complexed with transition and alkali metals will be examined. The current project focuses on the fragmentation pathways of complexes consisting of alkali and transition metals with first and second-generation nitrile-terminated PPI dendrimers. The current project also utilizes a 15N-labeled nitrile-terminated first generation PPI dendrimer for comparison with the unlabeled first generation dendrimer fragmentation pathways. The dendrimers are synthesized in-house using 1,4-diaminobutane and acrylonitrile. Deuterium-labeled dendrimers are synthesized from d4-succinonitrile and d4-succinamide and reduced using LAD and LAH. The fragmentation pathways for the metal-dendrimer complexes tend to follow a similar pattern, with acetonitrile and acrylonitrile as the primary neutral losses. The complexes also utilize a radical mechanism for the loss of acetonitrile, with the nickel complex being an exception. The overall ease and efficiency of the elucidation of these fragmentation pathways makes mass spectrometry a very valuable method of analysis for these dendrimer-metal complexes.

Tandem mass spectrometry is widely used to characterize proteins in biological systems. Proteins are typically identified by sequence-specific mass spectra resulting from fragmentation of proteolytic peptides in the gas phase. Unfortunately, most conventional excitation processes generate limited numbers of fragments that are insufficient to de novo reconstruct peptide sequences. Photodissociation with 157 nm light provides a great alternative to conventional methods by generating abundant high-energy fragments with more than 90% sequence coverage. In this dissertation, photodissociation mechanisms are investigated and applications for biological sample analyses are also pursued. Photodissociation has first been implemented in a linear ion trap mass spectrometer to investigate photolytic processes. Peptides with N-terminal arginine yield abundant a＋1 radical ions, confirming that photodissociation is initiated by homolytic cleavage of peptide backbone C alpha-CO) bonds. The resulting radical fragments then undergo radical-driven fragmentation that is directed by amino acid side chains, leading to highly predictable secondary fragments. Since 157 nm photodissociation does not mobilize the ionizing proton in singly-charged arginine-containing peptide ions, it has been employed to probe protonation sites in singly-charged peptide fragment ions produced in collision-induced dissociation CID). The ionizing proton in these ions is shown to be located on the most basic residues), suggesting that the commonly used ion structures need to be revised. Photodissociation has also been implemented in a commercial tandem time-off-light TOF) mass spectrometer for peptide analysis. A peptide de novo sequencing algorithm has been developed to interpret peptide sequences directly from photodissociation spectra. These developments allow photodissociation to characterize peptide mixtures following liquid chromatography separation. In addition, photodissociation of glycopeptides in this apparatus enables both peptide sequences and glycan structures to be elucidated. With modest improvements, 157 nm photodissociation mass spectrometry could be applied to characterize complex biological samples.

In neurodegenerative diseases in general and in amyotrophic lateral sclerosis ALS) in particular, discoveries of disease-causing changes to genes have not often translated into an understanding of changes in protein function. For example, the nature of the “toxic gain of function” causing a familial form of ALS fALS) that results from over one hundred mutations in the gene encoding Cu/Zn superoxide dismutase SOD1) is still a matter of debate. Whether increased protein aggregation represents this gain of function in ALS, or in any neurodegenerative disease, is unknown. Recent studies have developed algorithms to predict how a given mutation affects protein aggregation propensity. I applied an aggregation rate predicting algorithm to fALS-causing SOD1 mutations and found that aggregation rate is related to ALS patient survival. In a second finding, loss of protein stability increased likelihood that a protein will unfold) is shown to be related to ALS patient survival. Thus I demonstrate herein that two synergistic properties, namely, increased protein aggregation propensity and decreased protein stability, are central to ALS etiology, and taken together account for 69% of the variability in mutant SOD1-linked fALS patient survival times Chapter 2). These results are used to rationalize aspects of neurodegenerative disease pathogenesis, including the selective vulnerability of particular neurons and late disease onset. For example, aggregation is a concentration-dependent process, and spinal cord motor neurons have higher concentrations of SOD1 than the surrounding cells. Protein aggregation, therefore, is expected to contribute to the selective vulnerability of motor neurons in fALS. Next, I investigated the effects of Cu and Zn binding on SOD1 stability using top-down mass spectrometric hydrogen/deuterium exchange HDX), which allows the simultaneous HDX analysis of multiple SOD1 isoforms [e.g with holo) and without metal ions apo)]. The bindings of metal ions were found to significantly affect SOD1 structure, and to an even greater extent than mutation per se. Calculations of the residual deuterium incorporation revealed extensive global disturbance of the apo SOD1 structure rather than limited disorder in metal-binding regions Chapter 3).

Nanomedicine deals with the development of nanoscale targeted drug delivery systems to improve the bioavailability of drugs. In order to deliver the nanocarriers and drugs to target cells in a tissue environment, it is critical to understand their targeting, cellular internalization and pharmacokinetics during systemic administration. My thesis work develops and employs advanced imaging techniques to provide useful information and deep understanding of the drug delivery via nanocarriers. The first part of my thesis focuses on the endocytosis of folate receptors FRs) which are overexpressed in over 1/3 human cancers. Real-time fluorescence imaging and single particle tracking SPT) analysis are used to study the intracellular trafficking of FR. FR endosomes migrating along microtubules is directly observed and bidirectional motions of FR endosomes are quantified by SPT. The role of microtubule-associated motor proteins, dynein and kinesin I, is demonstrated by microinjection with antibodies. Furthermore, our imaging tools allow the evaluation of the impact of cholesterol on intracellular transport of FR. It is found that cholesterol level regulates the FR trafficking by changing the endosomal distribution of Rab4 and Rab7 proteins which are adaptors for microtubule motors Biophys. J., 2008, 94:1508-1520, front cover story). Using Forster resonance energy transfer FRET) imaging, drug release from polymeric micelles, a superior nanocarrier for hydrophobic drugs, during cellular uptake and blood circulation is investigated. A FRET pair, DiIC 183) and DiOC183), is loaded into micelles to mimic hydrophobic drug molecules. By monitoring the FRET efficiency in live cells, the plasma membrane is demonstrated to be a temporal residence for micelle-released hydrophobic molecules before their delivery to target intracellular destinations Proc. Natl. Acad. Sci. USA, 2008, 105: 6596-6601). By monitoring the FRET efficiency in blood stream, DiIC18 and DiOC18 are found to quickly escape from micelles. FRET spectroscopy studies demonstrate that alpha- and beta-globulins are major factors for the observed fast release, whereas gamma-globulins, albumin, and red blood cells play minor roles. This information can help the development of micelle-based nanocarriers which can be resistant to disassembly in circulation Langmuir, 2008, 24:5213-17, front Cover Story). The same FRET technique is also used to study disulfide bond-reducing activity in endosomal compartments Proc. Natl. Acad. Sci. USA, 2006, 103: 13872-13877). To monitor nanocarriers, such as novel metal nanoparticles and nanorods, in tissue environment and to study the interactions between extracellular matrix and stroma, a multimodal multiphoton microscopy is developed. Because different modalities can be used to image different biological structures and nanocarriers, herein an easy-to-operate approach is presented to perform Coherent anti-Stokes Raman scattering CARS), two-photon excited fluorescence TPF), second harmonic generation SHG), and third harmonic generation THG) imaging using a single laser source composed of an 80 MHz fs laser, an optical parametric oscillator OPO), and a PPLN crystal for frequency doubling Optics express, 2009, 17: 1282-1290). It allows simultaneous imaging of different biological structures, e.g. vibrationally resonant CARS imaging of CH-rich myelin sheath in fresh spinal tissues and lipid bodies in live cells, SHG imaging of collagen fibers in liver tissue, THG imaging of gold nanorods in cell, tissue and animals after administration. This platform can provide complementary and important information for bioscience research.

This thesis reports on the design and development of a number of bio)sensors for the measurement of neurotransmitters, including nitric oxide NO), serotonin SR), dopamine DA), glutamate Gm) and glucose Glu). These chemicals are present in the central nervous system where they modulate a variety of physiological activities. SR is also present in the gastrointestinal tract where it regulates gastrointestinal motility. Glu is a well-known major source of energy in the physiological systems. The deficiency of which surfeit can be fatal. Monitoring these analytes in real-time is crucial for biomedical purposes including diagnosis and neurological mechanistic studies. However, their presence at very low concentrations and variable levels provides a challenge for their detection. Traditionally, neurotransmitters are monitored using microdialysis coupled with separation techniques such as chromatographic and capillary electrophoresis methods. Microdialysis is slow and cannot offer real-time analytical information. On the other hand, electrochemical sensors can provide real-time measurements in vivo； they are small and are easily implantable. However, obtaining sensor stability and selectivity in the complex biological environment is a major challenge. The main objective of this work was to develop bio)sensors for NO, SR, DA and Gm with high selectivity, stability and sensitivity. These were designed to provide linear range and detection limits in the physiological relevant concentrations and be able to function in real-time implantable conditions. Towards this goal, we first engineered the electrode surface with biocompatible materials and enzymes and miniaturized the sensors. We then characterized the microelectrodes and determined their analytical performance in vitro in standard laboratory conditions. Finally, we demonstrated the use of three of the most successful configurations to monitor levels of neurotransmitters in biological tissues in vitro and in vivo. 1) The NO microelectrode was used to measure NO in brain slices. 2) The DA biosensor was implanted in the intact brain of an anesthetised rat to measure DA during high frequency stimulation. 3) The SR sensor was implanted in the intestine of live embryos to quantify SR levels at multiple locations throughout the gastrointestinal system, and monitor changes of this chemical as a result of pharmaceutical interventions. The analytical performance characteristics of all the sensor configurations were thoroughly investigated. After a general overview and introduction of bio)sensor technology Chapter 1), the research work and accomplishments are presented systematically in seven chapters. These include both fundamental and practical aspects in the fabrication of these devices. Chapter 2 focuses on the development and characterization of an NO sensor and its application on brain slices. Chapter 3 describes a sensor for dual detection of NO and Gm. Chapters 4 and 5 present optimization and in vivo implementation of an enzymatic biosensor for DA. Chapter 6 describes the fabrication of a carbon fiber microelectrode and its application for in vivo monitoring of SR in live zebrafish embryo. Chapters 7 and 8 describe synthesis and characterization of gold-polypyrrole composites for use in the immobilization of enzymes and biosensors fabrication. The research results of this work have been published in 10 peer-reviewed research articles six published, one in revision and one submitted), one topical review and one book chapter.

The need for in situ chemical analysis in fields such as environmental or security analysis has lead to the development of detection methodologies using lab scale instrumentation. Because of the excellent analytical capabilities of mass spectrometry in terms of speed, sensitivity, specificity, and versatility, this technique has been investigated for achieving needed solutions. The solutions developed using traditional instruments have proven useful on a limited scale due to large instrument size and limitations in sample introduction. The purpose of this thesis is to demonstrate the analytical capabilities of multiple versions of home-built mass spectrometers by analysis of environmentally relevant analytes. The instruments investigated are characterized not only by their portability but by their use of atmospheric pressure inlets, allowing the introduction of externally generated ions. The instruments used are fully functional as indicated by mass ranges up to m/z 450, unit or better) resolution, tandem mass spectrometric capabilities, and limits of detection in the low to sub-ppb range. The instruments also demonstrated the capability for further improvements in analytical parameters and versatility by physical and electronic modifications to the instrument. Ambient ionization methods developed in lab, such as desorption electrospray ionization DESI) and low temperature plasma LTP) ionization have allowed rapid, direct analysis of analyte from an untreated surface. Many highly relevant applications have been developed using these ionization methods on lab scale and handheld instruments. Modification of a handheld instrument has allowed the investigation of security screening experiments, namely the detection of explosives on a large area using ambient ionization methods.

The goal of this research is to develop protein sensing devices from artificial conical nanopores. In the first part of this work, single conical nanotubes are used as resistive-pulse sensing devices. A key challenge for this sensing paradigm is building selectivity into the protocol so that the current pulses for the target analyte can be distinguished from current pulses for other species that might be present in the sample. It is demonstrated here that this can be accomplished with a protein analyte by adding to the solution an antibody that selectively binds the protein. Because the complex formed upon binding of the antibody to the protein is larger than the free protein molecule, the current-pulse signature for the complex can be easily distinguished from the free protein. The second part of the research also involves resistive-pulse sensing of protein analytes. Proteins of various sizes were detected with a conical nanotube sensor. The effect of protein size on translocation through a narrow nanotube tip was examined. The size of the protein was found to have a dramatic effect on current-pulse duration. The current-pulse frequency was also affected by the protein size and nanotube tip opening diameter. These studies are important towards the optimization of protein resistive-pulse sensors. In the third part, a new method for optimizing protein resistive-pulse sensing is investigated. Previously, all resistive-pulse sensing work has been done at potentials &le； ＋/-1 V. In this work, proteins were sensed at much higher potentials i.e., up to 4 V). High potential sensing results in a significant decrease in the standard deviation of current-pulse duration for protein analytes. Decreasing the standard deviation in duration allows for better discrimination of analytes, and allowed for two proteins in a mixture to be distinguished. In the last part of this work, a new type of sensor is developed from single conical nanopores. Protein molecules are immobilized on the surface of the nanopore walls and the isoelectric point of the immobilized proteins are determined from current-voltage curves. Isoelectric point determination is made based on the ion current rectification phenomenon exhibited by conical nanopores. At pHs above and below the isoelectric point of the immobilized proteins, the nanopore surface will carry a charge, and therefore current-voltage curves will show ion current rectification. However, at the pH corresponding to the isoelectric point of the immobilized proteins, there will be no surface charge and the current-voltage curves will show no ion current rectification.

Robust modeling methods were implemented for the transfer of near-infrared calibration models in intra and inter-brand situations. A network of four instruments from two brands (Foss Infratecs and Bruins OmegAnalyzerGs) was used to implement spectral pretreatment methods, local and variable selection techniques, and orthogonal methods to transfer protein, oil, and linolenic acid models across instruments of the same brand and across instruments of different brands. A total of fifty seven techniques were implemented among which spectral filtering methods based on the smoothing of high frequency components of Fourier and wavelet transforms. A new approach to local similarity was introduced. Results showed that the effectiveness of the various methods was instrument, parameter, and validation set dependent. In some situations, no differences could be observed between master and secondary unit predictions. Local methods appeared to be the weakest methods, most likely due to a problem of over-fitting (specialization) of the calibration set. The transfer of calibrations across brands was possible with performances similar, or better, than in intra-brand calibration transfer.

Spectroelectrochemical sensors uniquely employ electrochemistry, spectroscopy, and a chemically selective film in a single device. The advantage to combining these three components is to achieve a higher level of selectivity. The three “modes of selectivity” exclude interferences from the sensor response based on the analytes electrochemical, optical, and ionic characteristics. Furthermore, the implementation of the selective film also helps to achieve a lower level of sensitivity due to its ability to concentrate the analyte at the sensing surface. To be detected, the analyte must: 1) partition into the film, 2) be electrochemically active in the selected potential window, and 3) either the analyte or its electrolysis product must absorb or emit light at the monitored wavelength. Analyte detection is based upon a change in optical response due to the conversion of the analyte between two oxidation states that results from the stepping or cycling of the applied potential. The Chemical Sensors research group at the University of Cincinnati has been working to develop and improve this sensing method. This dissertation presents the results of research completed on two aspects of this project. The first study includes the characterization and performance of a novel selective film material, sulfonated polystyrene-block-polyethylene-ran-butylene)-block-polystyrene SSEBS). The ability of the film to preconcentrate analytes, as well as exclude interferences, was examined. Furthermore, the diffusion coefficient of a redox probe Rubpy)32＋) in the film was calculated using chronoamperometry and cyclic voltammetry. In the second study, a novel method is demonstrated for using the spectroelectrochemical sensor for multiple analyte detection. Prior to this study, the spectroelectrochemical sensor has detected non-ideal analytes in one of two ways: 1) weakly absorbing metal ions bind with an organic ligand in-situ to form a complex that has the desired characteristics, or 2) weakly absorbing metal ions are deposited onto, and then stripped from the bare electrode using anoidic stripping voltammetry, which also results in the desired change in optical response. The novel method demonstrated in this dissertation combines these two approaches to the detection of non-ideal analytes into a single experiment. Potential limitations of this method, such as ASV at a chemically modified electrode and metal ion competition for the complexing ligand and ion-exchange sites in the selective film, were also investigated.