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This thesis is a compilation of multiple techniques developed to characterise solutions of proteins, or proteins themselves. Proteins are the building blocks of life, and thus they are interesting to study for a deeper understanding of the mechanisms of life but also for developing diagnostic tools as well as medical therapies. The first chapter is a general overview of the thesis with an explanation of the different points developed further in the thesis such as proteins, protein aggregation, microfluidics, and optics. The second chapter describes a microfluidic platform for performing a 2D characterisation, separating the particles using capillary electrophoresis and diffusional sizing. This allowed to characterise the components of heterogenous solutions in function of their size and the mobility of the proteins. The third chapter is looking at existing well-known microfluidic techniques, like free-flow electrophoresis and diffusional sizing, combined with confocal microscopy. The single molecule detection allows to distinguish particles in function of their brightness, breaking the homogenous limitation of these microfluidic techniques under classical epifluorescence microscopy. The fourth chapter shows an elegantly simple way to characterise an exponential decrease of the residence time distribution within a nanocavity trap. The explanation is based only on a random walk assumption without requesting potential interactions. The fifth chapter describes a new method to size particles in solution based on interferometry scattering microscopy by looking at the correlation of the intensity of the interferometric term. The last chapter before the overall conclusion is based on a study of SARS-COV2 seroprevalence in the swiss canton of Zürich between December 2019 and January 2021. This study was conducted on two cohorts, the University Hospital patients, and the blood donation services of Zürich.

Over the twenty-year period between 1998 and 2018 the number of full-time farmers within the Northern Ireland (NI) agri-tech sector dropped from 22,524 to 16,964. As expected, this has also resulted in a commensurate loss in the total cattle livestock which has decreased from 1.77 million to 1.63 million. Despite this, NI beef and sheep meat production accounted for 15% of the total UK output and represents 27% of the gross agricultural turnover in 2017 or £1.3 billion in 2016, while animal by- products accounted for revenues worth £53 million. While the sector produces a number of high-quality food products, a significant amount of waste is generated during processing. Given increased global competition in the sector and new challenges including climate mitigation it is desired to enhance the overall value and image of the sector and hence valorisation of wastes is increasingly attractive. This project investigates valorisation of waste streams in the agri-food sector, particularly those which would have been traditionally sent for rendering or exported at low value to other countries for upgrading. The project specifically focuses on the production technologies for specialist chemical compounds from meat by-products and evaluation of the energy potential for plant-based wastes. Overall the data generated within this work aims to boost the agri-tech sector by creating additional revenue streams through diversification of the product portfolio. The main achievements of this project are associated with the production of traceable nutraceutical products and their intermediaries with proven analysis of the traceability system and the quality of the nutraceuticals exploited. These products will be processed by sustainable separation and extraction techniques that are efficient along with the understanding of global economics and market size of nutraceuticals. Furthermore, focussing upon the release of energy from specific biomass and kinetic modelling of such material in terms of reaction progress, rate of reaction and activation energy associated with each heating rate applied to the material. Note this thesis is heavily redacted for reasons owing to the commercial sensitivity of the subject matter. It would substantially prejudice the commercial interests of the author, the University or an external company if the entire thesis were made available in unredacted format. Note that pp 11-147 & 18- -307 are permanently redacted.

Aptamers are single stranded DNA or RNA sequences that bind with high affinity to a target molecule. The discovery of sequences that can bind to a target of interest starts with a mixture of random sequences which are refined through repeated incubation with the target followed by amplification of binding sequences in a process known as systematic evolution of ligands by exponential enrichment (SELEX). Aptamers produced by multiple SELEX rounds can assist bioanalytical scientific research through detecting molecules of interest in biosensors and have the potential to improve drug delivery to specific cells in humans. However, developing aptamers capable of useful application is difficult due to multiple unique challenges. Quantifying the ability for a pool of aptamers produced by SELEX to bind to a target has no single perfect method. In the field of aptamer research, scientists employ a variety of binding affinity tests which contain specific advantages and disadvantages. Reported values for binding affinity vary between different methods. When a pool does show evidence of most sequences binding, identifying the best sequences in a mixture of more than thousands of unique base pair arrangements provides a separate challenge. Finally, even after finding a sequence with evidence of strong binding to a desired target, there remains uncertainty if the aptamer will bind to the target as well in a real-world application as it does in a carefully controlled lab environment. This study examines aptamers at three distinct stages of development for separate capillary electrophoresis SELEX (CE-SELEX) experiments. For an aptamer already selected against transferrin receptor 1 (TfR1) with high affinity, a combination of flow cytometry and confocal microscopy provided evidence the aptamer did not significantly internalize into cancerous liver cells used to model human drug delivery. A separate aptamer pool, previously developed over 4 rounds of selection against low-density lipoprotein receptor (LDLR), is analyzed with preexisting bioinformatic tools as well as custom Python code on next generation sequencing (NGS) data, revealing a primer dimer sequence from polymerase chain reaction (PCR) which impacted the selection of aptamer length candidates but remained at a lower frequency than one possible aptamer candidate. After performing 4 rounds of CE-SELEX against mouse leptin, testing the binding affinity of rounds 1-3 with CE showed weak evidence of binding in early rounds which was lost in later rounds after correcting for an earlier false positive result. Documenting CE problems and optimization, even for an unsuccessful aptamer selection, may help improve future CE research, while detailing the experimental set up, discovery, and correction of the false positive result is important to help prevent future misleading conclusions.

Modern drug discovery relies on high-throughput screening for lead generation and protein target evaluation. There is a critical need to develop assays that not only have large-scale screening capabilities but provide insight of greater physiological relevance. It is in this space that biophysical methods are used to extract high information content datasets; however, throughput is often limited by sample introduction to the instrument. Droplet microfluidics represents an attractive approach for coupling high-throughput sampling with analytical and biophysical approaches. The aim of this thesis is to improve high-throughput workflows coupled to to capillary electrophoresis and ion mobility-mass spectrometry for the analysis of enzymatic activity and intact protein analysis. Optical assays are a fast and simple approach for screening small molecule activity against enzymatic reactions but can be prone to high false discovery rates. A capillary electrophoresis method was developed to screen against Sirtiuin-5 activity, where substrate and product peptides were separated and detected. Using a commercial capillary electrophoresis system, eight novel Sirtuin-5 inhibitors were confirmed through dose-response analysis. This assay was then coupled to a microchip capillary electrophoresis platform coupled to droplet sample introduction via an density-based oil drain. A sampling rate of 0.1 Hz, using only 4 nL sample volumes. This was applied to screen over 160 Sirtuin-5 samples. Future work is focused on developing a label-free mass spectrometry assay with on-line reagent addition. Native mass spectrometry coupled to ion mobility (IM-MS) has become an important tool for the investigation of protein structure and dynamics upon ligand binding. Additionally, collisional activation or collision induced unfolding (CIU) can further probe conformational changes induced by ligand binding. In this work we explore the high-throughput capabilities of CIU fingerprinting. Fingerprint collection times were reduced 10-fold over traditional data collections through the use of improved smoothing and interpolation algorithms. Fast-CIU was then coupled to a droplet sample introduction approach and applied to a 96-compound screen against Sirtuin-5. Over 20 novel Sirtuin-5 binders were identified, and it was found that Sirtuin-5 inhibitors will stabilize specific Sirtuin-5 gas-phase conformations. This work demonstrates that droplet-CIU can be implemented as a high-throughput biophysical characterization approach. Future work will focus on improving the throughput of this workflow and on automating data acquisition and analysis. Monoclonal antibodies (mAbs) are an important class of biotherapeutics, but the diversity and structural complexity of mAbs create an analytical challenge. Moreover, mAbs are produced through recombinant DNA technology and grown in mammalian cells, giving rise to complex background matrices and possible post-translational modification (PTM). A miniaturized MS-friendly mAb purification is developed and couped to droplet-MS. In-droplet calibrations were performed and an ultimate LOD of 0.15 mg/mL and LOQ of 2.6 mg/mL were achieved. This method was then applied to screen mAb production in 48 cell expression conditions with a rate of 0.04 Hz, identifying the most productive media conditions. This work was complemented by high resolution MS for PTM profiling, identifying a range of glycoforms on the intact mAb species. Future goals are aimed at coupling on-line sample preparation and droplet sample introduction to high resolution MS for high-throughput glycoform characterization.

Abstract Capillary Electrophoresis: Method Development and Application in Biomolecule Analysis Lindsay Veltri Biomolecule analysis is essential in disease diagnostics and the development of therapeutic agents. There is a vast array of methods available for biomolecule analysis, however, capillary electrophoresis is well-suited due to minimal sample consumption (~nanoliters of solution) and fast analysis times. In this report, capillary electrophoresis was used to analyze glycosylation of therapeutic proteins. Additionally, a capillary electrophoresis-mass spectrometry method is reported for the analysis of proteins. Post-translation protein modifications like glycosylation, play an important in protein structure and function. Modifications of protein glycosylation can lead to improved therapeutic efficacy. Therefore, significant efforts are made into the development of biosimilars via glycoengineering. A capillary electrophoresis method is reported for the determination of the branch specificity of a transferase enzyme used in glycoengineering, β-1,4-galactosyltransferase. Enzyme specificity was studied using both a native and non-native sugar as the donor molecule. Identification of positional isomers was enabled using a semi-permanent nanogel capillary coating. This enabled analysis of rate constants for each step in the reaction. Capillary electrophoresis-mass spectrometry is a powerful analytical tool for the analysis of biomolecules. However, coupling of the two techniques is not straightforward. Here, a novel, voltage-free interface for coupling capillary electrophoresis and mass spectrometry for the analysis of proteins is reported. Through the incorporation of a semi-permanent nanogel capillary coating, the limitations of bare-fused silica separations were overcome, and analyses of cationic proteins were performed at biological relevant pH values. The resulting charge state distributions determined proteins were not denatured, suggesting the method is an alternative method for native analyses.

The capillary zone electrophoresis-mass spectrometry (CZE-MS) platform, a well-developed analytical methodology, has been widely used for high-throughput nanoscale (e.g., < 5000 cells, single-cell sample) bioanalysis (e.g., proteomics and metabolomics). The sample size for a nanoscale study can be extremely limited in both volume (e.g., pL-nL) and amount (e.g., ng). Therefore, it is valuable to construct a high-throughput, user-friendly, and ultrasensitive CZE-MS platform for nanoscale analysis. This dissertation focuses on developing a novel quantitative sample handling device for ultralow-volume samples (e.g., pL-nL) and applying it for the online CZE-MS analysis of single cell samples. Firstly, a novel sample handling device, spray-capillary, was developed for quantitatively extracting pL-nL level samples (e.g., as low as 15 pL/s). Electrospray ionization (ESI) is an ionization approach that has been routinely applied to couple liquid phase separation with MS detection but not for sample handling. The spray-capillary device is constructed from a commercially available fused silica capillary with one end being treated as the sample inlet and the opposing end being treated as an MS end. The MS end will be fabricated to a sheathless interface for electrospray-assisted sampling handling. Our results show that a low-volume sample (e.g., pL range) can be quantitatively introduced into a bare capillary for a pressure-based elution and MS detection. In addition, the spray-capillary can serve as the CZE separation column for on-capillary CEZ-MS analysis, which largely reduced sample loss from the offline sample handling process. A standard peptides mixture was used for prove-of-concept for the quantitative nanoscale CZE-MS analysis. The sample loading process is reproducible and adjustable. Secondly, the original spray-capillary setup is manually operated, which makes the workflow low-throughput and prone to human error. Also, the usage of a bare capillary limits the performance of CZE-MS analysis (e.g., sample loss due to the adsorption on the capillary). To address these issues, we incorporated a commercially available CZE autosampler to perform fully automated spray-capillary analysis and utilized polyethylenimine (PEI) as capillary coating material to increase the separation efficiency. Thirdly, we redesigned the original spray-capillary device to perform the single-cell metabolomics analysis. The sample inlet end was laser pulled to a small tip (i.e., 15 µm o.d.) so it can be directly inserted into single onion cells for ultra-low volume microsampling. The extracted single-cell contents can be directly separated and analyzed in the modified spray-capillary using the online CZE-MS analysis, which significantly decreases sample loss by eliminating off-line sample handling processes. Spray-capillary-based direct infusion single-cell analysis identified 80 cross-verified onion metabolites, while spray-capillary–based CZE-MS single-cell analysis identified 160 cross-verified onion metabolites. Additional experiments include a relative migration time comparison with standard chemicals, and targeted MS/MS analysis were performed to confirm the identification of the metabolites. Overall, this work has demonstrated that spray-capillary provides a valuable solution for nanoscale microsampling and high-throughput omics analysis using the on-capillary CZE-MS analysis (e.g., single-cell analysis). The spray-capillary approach holds great potential for being incorporated into a variety of CZE-MS-based nanoscale analysis workflows for multi-omics study.

Short and medium-chain fatty acids (SMCFA) are known as essential metabolites found in gut microbiota that function as modulators in the development and progression of many inflammatory conditions as well as in the regulation of cell metabolism. Currently, there are few simple and low-cost analytical methods available for the determination of SMCFA. This work focuses on the identification and measurement of SMCFA in rat feces utilizing capillary electrophoresis with indirect photometric detection (CE-IPD). In chapter 2, several parameters are optimized for maximum resolution, efficiency, and signal-to-noise ratio of FAs. The developed CE-IPD method is next validated in chapter 3 to quantify the FAs in healthy rat feces. Application to a pilot study on the influences of developmental stages (adult vs. adolescent) and various drug treatments on fecal SMCFA concentration in rats demonstrates the proposed CE-IPD method is an efficient tool for investigations on biological functions of SMCFA in clinical laboratories.

Y-chromosome testing has become more prevalent in recent years as a means of identifying forensic samples using STRs or identifying biomarkers for disease or determining geographic origins of populations. Additionally, Y-chromosome analysis is especially useful in paternity testing as the Y chromosome is inherited paternally and the male-specific region of the Y chromosome does not undergo any recombination events, allowing the genotypic data of both the father and son to be identical. Though in most cases a father-son pair will have the same Y-allelic data, random mutations like allele insertions and deletions can occur, which can interfere and result in incorrect conclusions in regards to paternity testing, forensic analysis, or genealogy. Though the exact mechanism of Y loci mutability is unknown, postulations of factors that can cause mutations have been studied, as well as attempts to determine mutation rate specific to each locus. A multi-generational pedigree consisting of 9 males was analyzed using two different methodologies: capillary electrophoresis and next-generation sequencing. The samples were amplified using either a ForenSeq™ Signature DNA Prep Kit (Verogen, San Diego, CA) or a YFiler™ Plus PCR Amplification Kit (Thermo Fisher Scientific, Waltham, MA). Between the two methods, five Y-STR loci were identified as being discordant between a father-son pair. Next-generation sequencing identified an allele insertion at DYS385a/b, resulting in a potential tri-allelic locus, but was disproved after comparison with the capillary electrophoresis data of the sample. The capillary electrophoresis data identified four discordances between father-son pairs, one of which was an allele mutation with a gain of a repeat at DYS458. At DYS 389II, an allele insertion was identified, but was contradicted after comparison with the next-generation sequencing data. There was a potential null allele at DYS518 and either an OL variant allele or a 2 base pair deletion at DYS481. Following peak height ratio, stutter, and comparative analysis between the genotypic data of the two analysis methods, two of these discordances were proven to be errors, one was a definitive mutational event, and the other two could neither be confirmed nor denied due to differences in loci tested in each kit.

Secondary metabolites are organic compounds produced by an organism for reasons other than growth and development. In plants, secondary metabolites generally act as defense agents produced to deter predators and inhibit other competitive species. For humans, these compounds can often have a beneficial effect and are pursued and utilized as natural pharmaceuticals. The development of sensitive, high-throughput analytical screening methods for plant derived metabolites is crucial for natural pharmaceutical product discovery and plant metabolomic profiling. Here, metabolomic profiling methods were developed using a microfluidic capillary zone electrophoresis device and evaluated against traditional separation approaches. An alkaloid screening assay was constructed to analyze transgenic mutant plant extracts for novel metabolites. Putatively identified novel features were detected, elucidated, and then isolated and purified for pharmaceutical evaluation. Additionally, methods for the analysis of polyphenolic plant-derived secondary metabolites, such as cannabinoids, were also developed and evaluated. In this case, the occurrence of cross-instrumental variation was addressed, given the tight legal restrictions regarding commercialization the products in question. Lastly, the microfluidic CZE-MS methods were further applied for both primary and secondary metabolite profiling in a DMPK assay. This assay was developed to inclusively monitor metabolic changes as a response to varying concentrations of a therapeutic in circulation. The metabolomic methods developed and evaluated in this work displayed high sensitivity, efficiency, and accuracy and can be utilized across a wide variety of applications.

Positron emission tomography (PET) is a highly sensitive non-invasive imaging modality that uses small masses of radiolabeled “tracer” compounds to probe specific biochemical pathways within the whole body. PET is used extensively in clinical diagnostics, basic research, drug development, treatment monitoring, and in the new field of theranostics. To minimize radiation exposure to the radiochemist, the preparation (synthesis, purification and formulation) of PET tracers is performed using automated instruments known as radiosynthesizers. Because current systems are quite expensive, bulky (requiring operation in a specialized radiation-shielded hot cell) and consume tens to hundreds of times as much precursors and reagents compared to the amount of tracer needed for imaging, there has been intense interest in microfluidics in this field. A particular promising avenue has been the use of droplet-based radiochemistry platforms that can produce similar quantities as conventional systems, yet reduce volume and reagents by 100x, reduce synthesis times, and are small enough that they could be self-shielded and operated as a benchtop instrument in a normal laboratory. In addition to advantages for tracer production, this approach enables parallel syntheses by performing multiple reactions together on a single chip, paving the way to rapid, high-throughput radiochemistry for basic investigations, synthesis optimization, and tracer development. In one project, I designed a droplet chemistry heating platform involving a fast ceramic heater for accurate temperature control for a singular droplet synthesis. This initial platform was used in the development of the droplet synthesis of [18F]AMBF3-TATE, realizing an Isolated radiochemical yield (RCY) of 16�1% (n=5) with via isotopic exchange radiofluorination. I also developed a robotic system to perform fully-automated droplet reactions (up to 64 at a time). A Cartesian 3-axis gantry was built that could quickly move a fluidics head (for reagent dispensing and crude product collection) across a 20cm x 40cm workspace containing a set of four multi-reaction chips (each with 16 sites) each controlled by an independent heater, a pipette tip rack, microwell plates for reagents and reaction products, and a set of thin-layer chromatography (TLC) plates for reaction analysis. The system was extensively tested and characterized, and a scripting language was developed to allow high-throughput studies to be programmed as a series of intuitive operation sequences applied to sets of reaction sites. Consistency was assessed by performing a series of identical reactions (synthesis of [18F]Fallypride) which exhibited crude RCY of 79�5% (n=16). This was lower than the manual optimal conditions (90�1%, n=4), and with slightly higher variation. In a second project, I explored microfluidic technology related to the analysis of radiotracers. After the synthesis of a PET tracer, the resulting product must be purified, formulated in saline and finally must pass a number of quality control (QC) tests to ensure safety of the tracer and identity of the product. These tests are typically performed using equipment such as high-performance liquid chromatography (HPLC) which is an expensive instrument that requires significant table space for the device. We designed a “hybrid” capillary and PDMS microfluidic chip for the performance of these QC tests using capillary electrophoresis (CE). I helped develop a volumetric injection chip to inject a 4nL volume of analyte into the separation capillary in a highly repeatable manner. Using this device, injected sample peak relative standard deviation (RSD) was found from the UV absorbance detector electropherogram to be 1.5) of FLT from known synthesis impurities, and enabling impurity species to be quantified independently. The major limitation towards this device performing some of the required QC tests was the lack of a method for in situ radiation detection. Therefore, I added an avalanche photodiode (APD) to the previous device for the purpose of detecting radioactive species. This detector provides highly localized detection due to the short positron range and thus narrow peak widths in the radiation detector electropherogram. The radiation detector enabled confirmation of radiochemical identity, and radiochemical purity, and a limit of quantitation of 114 MBq/mL (3.1 mCi/mL), suitable for testing batches of tracers at higher levels of radioactivity concentration.

As a post translational modification protein glycosylation plays a crucial role in protein signaling, binding, kinetics and folding. In disease diagnosis and prognosis, monitoring glycosylation has been identified as a biomarker. Sialylation and sialic acid linkage in N-glycans are markers of cancers including liver, pancreatic and kidney cancer. Quantification of sialic acid linkage is analytically challenging because of the diverse linkages and the presence of heterogenous branching. A capillary electrophoresis method is reported that integrates a unique combination of enzymes and lectins to modify sialylated asparagine-linked glycans (N-glycans) in real time in the capillary so that N-glycan structures containing α2–6-linked sialic acid are easily separated, detected, and quantified. N-glycans were sequentially cleaved by the enzymes at the head of the separation capillary so that the presence of α2–6-linked sialic acids corresponded to a shift in the analyte migration time in a manner that enabled interpretation of the N-glycan structure. Complex N-glycans from α-1-acid glycoprotein were analyzed using this approach, revealing that a limited number of α2–6-linked sialic acids were present with biantennary, triantennary, and tetraantennary N-glycans of α-1-acid glycoprotein (AGP) generally containing 0 or 1 α2–6-linked sialic acid. The capillary electrophoresis method quantified the sialic acid linkages using nanoliter volumes of enzyme and lectins. Conversion with enzyme was in real time and incubation and separation occurred in less than 40 minutes. In biotherapeutics glycosylation of IgG is a critical attribute, and modification of the glycosylation will influence the IgG efficacy. Glycosyltransferases have been employed to modify this glycosylation. Capillary nanogel electrophoresis was utilized to create discrete regions for an online galactosyltransferase reaction and subsequent separation of substrate and product. The β1-4 galactosyltransferase enzyme, donor, and co-factor were patterned in the capillary. The substrate was driven through these zones and converted to galactosylated products which were separated and identified. The degree of glycosylation was discernable. The method was applied in establishing the Michaelis-Menten value, KM of the enzyme. Additionally, the method was adapted to transfer galactose residues to protein. The applicability of the method for real-time online modification of whole protein was demonstrated with the Herceptin glycoprotein. The method demonstrated the applicability of capillary electrophoresis to characterize glycosyltransferases. This method is compatible with low enzyme or substrate volumes and is fast and automated.

Forensic toxicology is a critical field in which scientific techniques are employed in order to establish the presence or absence of pharmacological substances and/or their metabolites within an individual. The results of such analyses can have legal implications, and toxicology has a number of important applications, including post-mortem investigations, workplace drug testing, therapeutic drug monitoring, and impaired driving studies. The focus of this specific body of work is on the use of toxicology in the detection and quantification of drugs of abuse –specifically opioids - in biological samples. In recent years, there has been a surge in opioid abuse and the need for forensic toxicology labs to process samples from such cases quickly and accurately continues to increase. As a result, it is imperative to research different techniques and technologies that can be applied in toxicology to improve efficiency of sample processing while still remaining sensitive and specific. Many toxicology laboratories today use immunoassay techniques for screening, and utilize a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for quantification. While these methods are established and reliable, the need to analyze an increasing number of samples in a more efficient time frame is essential, and with that, the need to develop and validate new analytical methods. This study sought to validate the use of Microchip Capillary Electrophoresis-Tandem Mass Spectrometry (CE-MS/MS) as a method for detecting and quantifying a panel of fourteen opioids. The experiments were run using a ZipChip (908 Devices, Boston, MA) as the separation scheme, which contains a small capillary where analytes are separated out by electrophoretic mobility - dictated largely by size and charge. These analytes were then ionized by electron spray ionization (ESI) at the end of the chip, and then detected, fragmented, and analyzed in a SCIEX 4500 Triple Quadrupole Mass Spectrometer (Framingham, MA). The analytical run time of the method evaluated was two and half minutes per sample. Calibration curves were run and the method was assessed for a number of validation parameters, including bias, precision, limit of detection, common analyte interferences, matrix interferences, and carryover, as recommended by the American Academy of Forensic Sciences Standards Board. The fourteen drugs and metabolites looked at in this study were 6-monoacetylmorphine, buprenorphine, codeine, dihydrocodeine, 2-ethylidene-1, 5-dimethyl-3, 3-diphenylpyrrolidine (EDDP), fentanyl, heroin, methadone, morphine, naloxone, norfentanyl, oxycodone, oxymorphone, and tramadol. All standards were ordered from Cerilliant (Round Rock, TX), as well as deuterated internal standards used for quantification purposes. This study showed that as the method currently stands, it can reliably detect this panel of opioids at limits of detection between 1 and 15 ng/mL, with the exception of buprenorphine and morphine, for which the method appeared less sensitive. While some applications desire higher sensitivity than this, this level of detection could be very useful as a screening technique that is quick and also far more specific than current immunoassay screening techniques, and provide the additional advantage of quantification for samples at slightly higher concentrations. Quality control samples at 100 ng/mL and 150 ng/mL generally showed consistent results and acceptable levels of bias and precision, indicating that the method can be used to reliably quantify this panel of opioids at those concentrations. In addition, interference signals detected during analysis of other common analytes often encountered with opioids were negligible, with the exception of heroin and norfentanyl. Analysis of ten lots of urine for blank matrix interferences also demonstrated low potential for interference, with the exception of heroin. Finally, there was no evidence of significant carryover between samples, or interference from the deuterated internal standards. While some potential instrumentation issues such as mass spectrometer calibration prompt further study, the method shows promise for future use as a high throughput analysis tool in forensic toxicology labs. CE-MS/MS has the added benefit of not only faster run times, but significantly less sample consumption per run, and additionally, less sample preparation. CE is a viable separation scheme for metabolites and forensic applications, and could make large impacts as an effective way to analyze toxicological samples.

The following is a comparison study of the Globalfiler™ PCR Amplification Kit analyzed on an ABI 3130 Genetic Analyzer Capillary Electrophoresis (CE) versus the ForenSeq™ DNA Signature Prep Kit analyzed on the MiSeq FGx™ System. The MiSeq FGx™ System measures results by Allele Read Count (ARC), while the CE measures results as Relative Fluorescent Units (RFU). Mixture samples were prepared in ratios of 1:1, 1:4, and 1:10 in replicates of four using a female major contributor and a male minor contributor, intended to represent some commonly seen mixture samples ratios in forensic cases [48]. Both systems performed equally well for the 1:1 mixture while the MiSeq FGx™ System had improved accuracy and precision for the 1:4 mixture compared to CE (4.033 + 1.506 ARC and 4.678 + 2.093 RFU, respectively). The MiSeq FGx™ System showed increased variation in the 1:10 mixture compared to CE (10.347 + 5.184 ARC and 9.311 + 3.363 RFU, respectively). Over the four replicates, the MiSeq FGx™ System had a total of 15 out of 528 possible alleles (2.84%) dropout compared to a total of 13 out of 384 possible alleles (3.39%) dropout on CE. The additional loci analyzed by the MiSeq FGx™ System results in a lower percentage of alleles lost due to dropout compared to CE. Isoalleles in sequence data may reveal the presence of minor contributor alleles that would otherwise be masked by the major contributor in length-based STR analysis. The presence of isoalleles are most helpful in mixture ratios greater than 1:1, where it is easier to assign alleles to a specific contributor. In certain cases, deconvolution of loci with shared alleles may not be improved by sequencing if intra-contributor isoalleles are present. Unless using known reference profiles, it is difficult to accurately assign alleles to contributors when intra-contributor isoalleles are present. Additionally, the sequencing data from the MiSeq FGx™ System provided information to aid the separation of stutter from true alleles. Previous studies report a significant increase in the amount of alleles present at some loci due to differences by nucleotide sequence, which may improve the discriminating power of those loci [31,52,53,54]. With all its potential, there is still much room for sequencing technology to improve before it becomes a standard analysis method in forensic laboratories.

Mass spectrometry (MS) has advantages as an analytical technique including label-free detection, high degrees of specificity and selectivity, capability for simultaneous monitoring/determination of numerous analytes, and rapid ionization/analysis, though throughput and sensitivity improvements are regularly sought. As a result, MS is commonly interfaced with different technologies including separations. Capillary electrophoresis (CE) is often selected over liquid chromatography(LC) for advantages including low volume requirements and faster separations, though online preconcentration is often necessary to improve limits of detection (LODs). In this work, a powerful preconcentration technique is paired with CE-MS to obtain LODs down to 10 pM, addressing the sensitivity limitation. Preconcentration here was shown to improve LODs by 5000-fold compared to a typical hydrodynamic injection for CE-MS. This method was applied for simultaneous determination of seven neurochemicals in biological samples with an excellent linear dynamic range (pM-µM). To improve throughputs, microfluidic sample introduction, especially droplet microfluidics, has shown promise for use with electrospray ionization (ESI)-MS, demonstrating sample introduction rates up to 30 Hz and droplet sizes down to 65 pL.1,2 In this dissertation, several droplet microfluidic assays are developed and paired with nanoelectrospray ionization (nESI) or ESI (droplet-nESI) for high throughput analyses. In one method, 5 nL droplets are generated at the end of a microdialysis probe to achieve temporal resolution near 10 s, substantially better than a standard LC-MS analysis with 5–20 min temporal resolution with microdialysis, offering much deeper insight into neurochemical dynamics. Using low flow nESI with MS/MS to overcome ionization suppression, we achieve low nM LODs (down to 2 nM) for simultaneous monitoring of seven neurochemicals, including trace neurotransmitters. This was applied to monitoring neurochemical dynamics in mouse brains in response to multiple drugs. In another method, droplet-nESI is used for high throughput screening in a directed enzyme evolution workflow. Directed evolution can provide much higher reaction yields, but screening can take weeks. Here, droplets are generated from well-plate reactions and infused into a sensitive nESI-MS/MS method to measure reaction yields with product LODs down to 12 nM. Over 1700 reactions (> 550 in triplicate) are screened in a single day, and over 1700 reactions (in triplicate) are screened in total. Seven enzymes with higher enzymatic activity are identified while achieving 10-fold higher throughput than LC-MS. Finally, a new mode of liquid-liquid extraction is developed called slug flow nanoextraction (SFNE) that uses only 5 nL of each phase per extraction, and partitioning equilibrium is reached within seconds while flowing in-line with a detector. This was applied for online sample clean-up of pharmaceuticals from biofluids in-line with MS/MS analysis, demonstrating nearly 20-fold-improvements in detection sensitivity with up to 60 extractions performed during a single infusion. Furthermore, an entirely online and automated system is developed for SFNE. This system is applied to screen 21 octanol-water partition coefficients (Kow) within 2 hr. Kows are an important physiochemical characteristic for understanding drug bioavailability, though current measurements are low-throughput. In a comparison, the developed method offers 10-fold higher throughput and 40-fold lower volume requirements than a typical workflow. The work described in this dissertation, though diverse, pushes forward what can be performed with mass spectrometry and bioanalysis. Each chapter introduces an analytically novel or innovative approach for the analysis of small molecules that advances important fields of research including physiochemical characterization, high throughput screening with mass spectrometry, and sensitive bioanalysis.

Bile salts are naturally occurring amphiphilic molecules that are synthesized in the liver and can solubilize hydrophobic molecules. Bile salts are of particular interest due to emerging applications including topical drug delivery, separations, and nanomaterials processing. Little is known about bile micelle formation processes including number of monomers per aggregate, the structure of the various micellar aggregates, and the mechanisms by which micelles bind chiral guest molecules. Bile salts are also distinguished from other amphiphiles in that they undergo multiple stepwise aggregation events. This work gains insight into the structure of cholate aggregates by monitoring changes in heteronuclear single quantum coherence (2D-HSQC) NMR signals with varied cholate concentrations in the absence and presence of a chiral guest molecule. Changes in the chemical shifts with cholate concentration (1-100 mM) reveal changing local environments around particular nuclei, revealing the extent to which particular regions of the structure participate in the dynamic processes of micelle formation and guest-host binding. In addition, fitting the chemical shift data from HSQC spectra to a phase transition model reveals stepwise, discrete critical micelle concentrations (CMCs), and corresponding differences in interaction surfaces as a function of stepwise aggregation, indicating which locations on the cholate structure “see” changes during each aggregation step. Several trends arise in the chemical shift perturbation data which illuminate the evolving landscape of the micelle structure with successive CMCs and 2 supports a stepwise model of aggregation. The chemical shift perturbations also allow for further understanding of the interaction and chiral selectivity of bile salts with chiral guest molecules.

Capillary electrophoresis has many advantages that make it a powerful technique for the analysis of proteins, which is challenging due to the natural heterogeneity of proteins. The small sample volume required for each analysis (nano- and pico-liter), fast separation times (min), and ability to couple the separation to mass spectrometry are all factors that make capillary electrophoresis an ideal technology for protein separations. Capillary zone electrophoresis and capillary gel electrophoresis are the two most commonly employed modes for protein analysis and allow for rapid, high resolution separations that can be automated. This makes capillary electrophoresis ideal for meeting the needs of protein analyses in the biopharmaceutical and clinical fields. Still, methods are continually improving in order to address the challenges of analyzing these samples with capillary electrophoresis, such as adsorption to the capillary wall, heterogeneity due to multiple isoforms, and maintaining native structure through analysis. The work presented utilized a coating and/or sieving matrix composed of the phospholipids 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and 1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) to confront these needs. The phospholipids lead to reduced adsorption and enhanced resolution that can be tuned with temperature. Furthermore, the phospholipids have been shown to support and enhance protein function and lifetime in-capillary, which makes the material promising for native analyses of proteins in the future. The phospholipids were the basis of a hybrid coating using cetyltrimethylammonium bromide (CTAB) that enabled simultaneous separation of acidic and basic proteins. The semi-permanent coating cost less than $0.10 to apply to a traditional silica capillary, and unlike previously published work, the ability to analyze both basic and acidic proteins using the phospholipid-cetyltrimethylammonium bromide (CTAB coating) without requiring the use of low pH buffers that lead to denaturing proteins. The phospholipid coating reduced protein adsorption compared to bare fused silica; however, it also suppressed and reversed the electroosmotic flow. This led to acidic proteins being analyzed in one polarity while basic proteins were analyzed in another polarity. Incorporating the cetyltrimethylammonium bromide (CTAB) into the phospholipid coat generated sufficient electroosmotic flow for simultaneous analysis of both acidic and basic proteins. The coating was stable through extensive flushing and exhibited no protein adsorption through six consecutive analyses with no flushing in between. The coating was applied to the analysis of abundant proteins in human serum and the accurate quantification further demonstrates the lack of interaction between the surface and the proteins. For the first time, the phospholipid nanogel was utilized to add a size-based component to the separation mechanism of proteins. The phospholipid nanogel cost $0.14 per 5 μL, which is less than commercial matrices currently available, and when used in-capillary as described in this paper, the matrix utilized for one sample is only $0.04. Furthermore, the thermally-responsive viscosity of the nanogel makes it a more user-friendly material than traditional gels since it can be inserted into the capillary similarly to buffers at temperatures below 24°C without the requirement of high pressures or chemical measures for cross-linking in-capillary that can lead to non-uniform matrices. The temperature is then raised to create a gel-like matrix for the separation. As demonstrated in prior work with DNA, the concentration of the nanogel can be utilized to tune the resolution to the size range of the analytes of interest, so the concentration used is determined by the mass range relevant to the analyte. Because the application of this material for the separation of proteins is novel, the separation mechanism is investigated with focus being placed on the impact of phospholipid concentration. A size-based component is superimposed on the preexisting charge-to-size ratio mechanism using the phospholipid. This leads to enhanced the resolution of proteins in human serum and allows for the accurate quantification of the proteins using external standards.

Capillary electrophoresis-mass spectrometry is a powerful technique for high-throughput and high efficiency separations combined with structural identification. Electrospray ionization is the primary interface used to couple capillary electrophoresis to mass analyzers; however, improved designs continue to be reported. A new interfacing method based on vibrating sharp-edge spray ionization is presented in this work to overcome the challenges of decoupling applied voltages and to enhance the compatibility with separations performed at near-neutral pH. The versatility and ease of use of this ionization source is demonstrated using β-blockers, peptides, and proteins. The cationic β-blocker pindolol was injected electrokinetically and detected at concentrations ranging from 10 nM to 5 μM, with an estimated detection limit of 2 nM. The vibrating sharp-edge spray ionization functions with flow rates from 70 to 200 nL/min and did not perturb the capillary electrophoresis separation electroosmotic flow as evidenced by the observation that most migration times differed less than 7% (n = 3) across a lab-built system interfaced to mass spectrometry and a commercial system that utilizes absorbance detection. For cationic βblockers the theoretical plates achieved in the capillary electrophoresis-mass spectrometry setup were 80% to 95% of that observed with a commercial capillary electrophoresis-UV absorbance detection system.

Capillary separation techniques offer fast separation for solutions of low detection volumeson the order of nL within seconds to minutes. However, detection efficiency is generallyreliant on high cost commercialized instrumentation. 3D printing is a relatively new andexciting field that has opened in the world of spectroscopic methods. The ability to quicklyfabricate devices have dramatically closed the gap between design and prototyping phasesof a project.In chapter 2, 3D printing technology will be used to design, develop, and optimize afluorescence detector that can be used as an alternative to commercial instruments. Thisdesign will follow exploration of integrated optics to further improve limits of detection(LOD). The detector offers a variety of applications including biological studies. Chapter 3will describe the detector’s use for fluorescence polarization measurements along with futuredirections to improve the design.

The use of short tandem repeats (STRs) for genotyping forensic case samples has long been an effective tool for human identification. However, interpretation of forensic STR mixture samples can be difficult and any additional information to aid in this process can be invaluable. Allele overlap and stutter during PCR can cause drop out of the minor contributor’s alleles and result in incorrect allele calling. The Scientific Working Group on DNA Analysis Methods (SWGDAM) provides a list of guidelines on how to interpret DNA typing results from forensic STRs and mixtures, but there is still a significant variation in the interpretation of mixture samples between analysts in the same laboratory and between laboratories. The Illumina MiSeq Forensic GenomicsTM system (Illumina Inc., San Diego, CA) is a massively parallel sequencing instrument that was developed specifically for the use in forensic DNA typing and which could provide sequence variations among on mixture samples. The ForenSeqTM DNA Signature Prep Kit is a kit that can be used with the MiSeq FGxTM platform. The DNA Primer Mix A (DPMA) included in the ForenSeqTM kit targets 27 autosomal STRs, 24 Y-STRs, 7 X-STRs and 94 identity single nucleotide polymorphisms (SNPs) on up to 32 or 96 samples, depending on the flow cell used. This study compares the STR performance on DNA mixtures of the MiSeq FGxTM and CE and evaluates its reliability and robustness. The MiSeq FGxTM provides data in read count and the CE in relative fluorescence units (RFU), so the two output data cannot be directly compared to one another. Instead, the ratio of two contributors was calculated at three mixture ratios (1:1, 1:4, and 1:9) to use as a mean of comparison. The mean contributor ratios calculated on the MiSeq FGxTM were 1.799, 7.595, and 13.524 for the 1:1, 1:4, and 1:9 mixtures, respectively. This was not significantly different from the CE mean contributor ratios of 1.818, 7.722, and 14.827, respectively. More allele dropouts occurred on the MiSeq FGxTM than the CE at both 1:4 and 1:9 mixture ratios, but sequencing provided the detection of six isoalleles based on sequence variants that could not be discerned by CE. Other studies have shown full profile generation at these ratios, indicating there could have been some issues during library preparation. Further studies should be performed to thoroughly validate the ForenSeqTM process and evaluate the sensitivity of the instrument. Until then, it is recommended that the ForenSeqTM kit and MiSeq FGxTM system be used at close to equal mixture ratios or in tandem with the CE to prevent genotypes miscalling.

Drugs of Abuse (DOAs) are among the single largest contributor to crime in the United States and present a high cost to society in terms of financial costs and physical/mental well-being of individuals. The forensic community requires a variety of validated methods to detect and analyze DOAs in a variety of different sample types, and most developed methods utilize a liquid or gas chromatography (GC or LC) separation system paired to a mass spectrometer (MS) detection detector. Capillary Electrophoresis (CE) based separation techniques have also been experimented with due to this technique’s high efficiency and speed, high resolving power, low sample consumption, and potentially lower cost when compared to GC or LC based techniques, even though the sensitivity of these systems is perceived to be weaker. The goal of this research to develop a CE-MS/MS method utilizing the ZipChipTM to demonstrate it can accurately and reliably detect and quantify DOAs. The DOAs analyzed for this method were opioids and benzodiazepines, and these were 6-monacetylmorphine, 7-aminoclonazepam, codeine, diazepam, dihydrocodeine, 2-Ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine fentanyl, heroin, hydrocodone, hydromorphone, meperidine, methadone, morphine, norfentanyl, oxycodone, and oxymorphone. Standard Practices for Method Validation in Forensic Toxicology guidelines from the Academy Standards Board (ASB) of Toxicology were used as the template for this validation; samples were prepared and analyzed as neat standards in diluent, blood and urine were assessed for interferences, ionization suppression/enhancement, and extraction recovery. The total runtime for the method was 3.5 minutes, with the retention time range being 1.4 to 2.9 minutes. All samples were prepared using compound standards diluted in metabolite diluent, which consisted of methanol, ammonium acetate, and water prior to injection. The calibration curves consisted of eight calibrator samples that ranged from 0.5 ng/ml to 200 ng/ml for all analytes, and a linear model was used for each compound. The minimum acceptable 2 value was set to >0.98, and each curve had a weighing factor of 12. Each curve for most of the compounds achieved the minimum requirement apart from two Codeine curves (0.9781 and 0.9785) and 7-aminoclonazepam (0.9791). Bias and precision were assessed at three concentrations- 5, 100, and 150 ng/ml. The minimum requirement for bias and precision for a compound was if the percent bias or coefficient of variation was within +/- 20%. Most compounds in this method exhibit acceptable levels of bias (except for Dihydrocodeine which had a bias of 24.58% at 100 ng/ml), and the only compounds to meet the minimum requirement for precision were 6-MAM, 7-aminoclonazepam, diazepam, fentanyl, methadone, and morphine. The limit of detection and limit of quantitation were both set at the lowest calibrator level of 0.5 ng/ml, and no carryover was observed in this method. No interferences occurred due to both deuterated internal standards and from common compounds such as benzylecogine, cocaine, and lidocaine, but blood cause signal interference with fentanyl and urine caused signal interference with methadone and norfentanyl. Ionization suppression and enhancement was observed for a majority of the compounds, and this observation will need to be assessed as to the effect it has on validation parameters in the future. The results collected suggest that accurate, reliable, and sensitive data may be collected if a compound has a specifically paired deuterated internal standard included in the sample. The speed of the suggested method and the minimal sample preparation could be desirable for forensic use. Further testing will need to be conducted to fully validate this method for blood and urine.

Exposomics is an emerging area of study that looks at how the environment around a person or persons affects their overall health. Biomarkers have emerged as useful tools to better understand how the exposome affects a person. In this work, we investigate two potential endogenous biomarkers, homocysteine and glutathione that have been previously implicated in a number of diseases that have been linked to environmental causes. We also investigated an environmental exposure, the fungicide Ziram, which epidemiologically has been linked to diseases. In our investigation, we utilized capillary electrophoresis and capillary electrophoresis-mass spectrometry to develop a method for homocysteine and identify a derivative to keep it from auto-oxidizing. We utilized mass spectrometry to identify the best ionization technique to detect Ziram and confirmation of where it binds on thiol-containing molecules. We also developed a method to extract glutathione-Ziram from serum and utilized liquid chromatography-mass spectrometry to begin a validation of glutathione-Ziram. Although future work is needed, we believe that this work was the beginning steps to understand biomarkers and their role in the exposome.

Proteins perform their functions as part of multi-protein complexes. These protein complexes are vital for carrying out and regulating cellular processes. As such, there is a need for tools to measure protein-protein interaction (PPI) affinity, stoichiometry, and inhibition in order to map interaction sites and screen for PPI modulators. Such measurements can be challenging because PPIs can span a wide range of affinities and stoichiometries. While many techniques exist for PPI analysis they often require large amounts of protein, have relatively low throughput, or have utility among a narrow range of PPIs. Capillary electrophoresis (CE) has demonstrated utility for determination of PPI affinity and for screening of PPI modulators. CE has a number of advantages in PPI analysis including low sample volume requirements, direct detection of complexes, and the potential for high-throughput. However, method development for analysis of PPIs by CE is often hampered by the need to maintain the native interaction during the separation and prevent protein adsorption to the capillary wall. Here protein cross-linking capillary electrophoresis (PXCE) is reported and described. Covalently cross-linking interacting proteins prior to electrophoresis eliminates the need to maintain the native interaction during the separation, facilitating method development. The PXCE method is demonstrated for an antibody-antigen interaction and heterodimer and homodimer heat shock protein complexes. Separation of free protein from protein complex is achieved either by using capillary zone electrophoresis or by capillary gel electrophoresis. PXCE is demonstrated to give quantitative results for PPI affinity and inhibition. Next, we expanded the utility of PXCE to access a wide range of PPIs including weak and multimeric oligomers. A short cross-linking reaction time of 10 s is found to have sufficient yields for a variety of complexes. Factors influencing non-specific cross-linking are also explored with concentrations of protein >20 µM yielding non-specific complexes. Apparent dissociation constants for seven different PPIs spanning from low nanomolar to low micromolar are presented. Good agreement was found to non-cross-linking methods. Assays of point mutations in the protein interaction site and nucleotide state dependence of association are also presented. Protein complexes less than about 250 kDa are accessible using the presented method. Separation time is also reduced to about 1 min/sample. Finally, a method for increasing the throughput of sample analysis is presented. Here, a microchip gel electrophoresis separation allowed for protein separation in 2.5 s. Further, a novel device for removing oil from segmented droplet flow based on density is demonstrated for coupling nanoliter-scale sample droplets to microchip separation for rapid and automated sample analysis. Throughputs of 10 s per sample are achieved with multiple injections made per sample. Utility of this device for application to PPI analysis and enzymatic reactions is presented. Specifically, Hsp70-Bag3 interaction and SIRT5 enzymatic reaction samples were assayed. The results suggest future utility of the device and PXCE method for screening of enzyme and PPI modulators.

Radiopharmaceuticals labeled with short-lived positron-emitting or gamma-emitting isotopes are injected into patients just prior to performing a positron emission tomography (PET) or single photon emission computed tomography (SPECT) scan. These imaging modalities are widely used in clinical care, as well as in the development and evaluation of new therapies via clinical trials. Unlike ordinary pharmaceuticals, the short lifetime of radiopharmaceuticals requires that they be produced in relatively small batches close to the geographical location where the patient is scanned. Since PET tracers are classified as drugs by regulatory agencies, they must pass stringent quality control (QC) tests after their production to ensure patient safety prior to injection. Performing and documenting these tests is cumbersome and time-consuming, and requires an array of expensive analytical chemistry equipment and significant dedicated lab space, and there is considerable interest in the development of automated and lower-cost approaches. By replacing conventional techniques with lab-on-a-chip technologies, it may be possible to achieve further reductions in the size, cost, and complexity of automated QC testing platforms. While some advances have been made for some of the many QC tests, high-resolution miniaturized methods suitable for assessment of chemical or radiochemical identity and purity are notably missing. We have been exploring microchip capillary electrophoresis (MCE) as a potential means to fill this gap. We have shown the potential to perform chemical identity and purity analysis by successful separation of the PET tracer [18F]FLT from all of its side products, with comparable limit of detection as HPLC. More recently, we demonstrated first-in-field work to add radiation detection to the MCE system, using a high-resolution positron detector to perform the radiochemical identity analysis of PET tracers. Microfluidic systems are also useful for the synthesis of radiopharmaceuticals such as radiolabeled peptides and antibody fragments, which provide a means to image disease-specific targets with extremely high specificity. To substantially reduce the high cost of radiolabeling, we have explored the feasibility of using a microdroplet reactor approach. As an example, a thiol-containing RGD peptide was labeled with fluorine-18 in a site-specific manner via the maleimide-based prosthetic group, [18F]FBEM on a microfluidic chip. We have also explored the site-specific radiofluorination of engineered antibody fragments (diabody) for ImmunoPET imaging via this microfluidic approach.These studies, and other work on small-molecule synthesis, have shown that there are advantages in performing radiochemistry in microdroplets. Active means of manipulating microdroplets, such as electrowetting-on-dielectric (EWOD) provides more flexibility of reaction implementation than passive methods, but EWOD devices are well known to suffer from a charging effect and the dielectric and hydrophobic layers have been found to suffer defects during harsh chemical reactions. Therefore, a novel droplet actuation mechanism, electro-dewetting, has been developed to address these challenges. Electro-dewetting uses an electric field formed inside a droplet to manipulate the adsorption of ionic surfactant molecules on the solid surface to change the contact angle. The underlying mechanism of this pheromone has been elucidated. We believe that all the work described here shows the potential for dramatic miniaturization of the complete PET tracer production process, and ultimately increasing the availability of diverse PET tracers for research use, further development, and clinical translation via lowering the cost and complexity of tracer production.

Proteins constitute a broad class of macromolecules which are abundant in all cells and are responsible for a wide variety of biological processes. Exploring the molecular mechanisms of these biological processes is pivotal, not only for understanding protein-related diseases and disorders, but also for the advancement of the biotechnological applications of proteins. Various techniques can be used for the structural characterization of proteins at both the atomic and molecular levels. Many proteins are structurally dynamic and thus may exhibit varying degrees of heterogeneity with regards to size, molar mass and molecular conformation. Currently, size-exclusion chromatography (SEC) coupled to multi-angle light scattering (MALS) is the most common technique used to study protein heterogeneity. Distributions of hydrodynamic volumes and molar masses can be obtained from SEC-MALS, however this technique faces several limitations such as high cost, poor reproducibility and adsorption, to name a few. Additionally, many proteins also undergo more subtle changes to their structures (such as domain rearrangements) which do not necessarily impact their overall size/molar mass. This project was therefore aimed at establishing a new method using free-solution capillary electrophoresis (CE) for the structural characterization of proteins, to be used complementarily to SEC. CE is a robust, cost- and time-effective technique that requires small sample amounts requiring minimal preparation. Additionally, the mechanism of separation enables distributions of electrophoretic mobilities to be obtained. CE in the critical conditions (CE-CC) allows for separation independent of molar mass, offering insight into the heterogeneity of a protein with regards to molecular conformations.

Protein glycosylation is one of the most prevalent post-translational modifications which plays an important role in determining the structure, stability, and function of proteins. Changes in glycosylation patterns are a hallmark of cancer and other diseases. Characterizing glycosylation is difficult because these structures vary in their linkage and monomer sequence, which gives rise to vast microheterogenity that could affect the function of the molecule. A novel method using capillary gel electrophoresis by integrating microscale enzymes and lectin reactions in the capillary was developed to verify glycan structure by analyzing monosaccharides. Capillary electrophoresis (CE) improves upon conventional methods of glycan structure characterization by reducing the amount of enzyme or lectins from milliliters to nanoliters and incubation times needed for enzyme reactions from hours to minutes. The significant advancement that was found is to use nanoliter volumes of enzymes costing about $ 0.01 for a single analysis and shorter incubation times. Characterizing glycosylation for complex glycans was demonstrated using enzymes alone or a combination of enzymes and lectins with aid of a nanogel to determine specific signatures in glycoproteins. Nanogels are used to increase enzyme stability, and non-covalently pattern enzymes in the capillary, enabling more cost-effective use of biological reagents that provide insight into glycan structures. This advancement allows for in-capillary electrophoretic mixing of a substrate and an enzyme to achieve similar rates irrespective of incubation times. These findings are important to study the Michaelis-Menten constants of two enzymes obtaining different catalysis rates. With the knowledge of rates, a cheaper enzyme was used instead of an expensive enzyme to obtain similar information, deciphering glycan linkage and composition in a cost-effective manner. The identification of type-1-LacNac signature in glycoproteins was demonstrated with the use of an enzyme and lectin combination. The enzymes and lectins can be used individually, or in combination, to identify the monomer and linkage information in glycans.

The development and validation of accurate and reliable analytical methods using techniques such as capillary electrophoresis, liquid chromatography, UV-spectrophotometry, spectrofluorimetry and mass spectrometry for separation and determination of some selected drugs in different matrices are presented in the first three chapters. The developed methods were applied to the determination of these compounds in their dosage forms and/or in biological fluids such as human plasma. The stability indicating characteristics including the analysis and separation of certain drugs from their alkaline, acidic, oxidative, thermal or photolytic degradation products along with the study of the degradation kinetics were also investigated. The first chapter focuses on the liquid chromatographic methods and the second chapter covers the capillary electrophoretic methods whereas the third chapter includes the UV-spectrophotometric and spectrofluorimetric methods. On the other hand, the fourth chapter shows how functionalized silica nanoparticles can be used in achieving enhanced electrophoretic separations of selected groups of acidic and basic drugs. Finally, the fifth chapter of this dissertation presents a library of fluorescently labeled-silica nanoparticles that can be used in certain hydrophobicity-based detection applications such as fingerprint detection.

Micro free-flow electrophoresis (μFFE) is a continuous separation technique in which analytes are streamed through a perpendicularly applied electric field in a planar separation channel. Analyte streams are deflected laterally based on their electrophoretic mobilities as they flow through the separation channel. The continuous nature of µFFE separations makes it uniquely suitable as the second dimension for multidimensional separations. The focus of this work is the development of coupling capillary electrophoresis (CE) to µFFE as a high speed two-dimensional (2D) separation platform, followed by an investigation of orthogonality of the two techniques, and finally a novel label-free detection method for µFFE separations. A new µFFE device was fabricated and coupled to CE via capillary inserted directly into the µFFE separation channel. High peak capacity separations of trypsin digested BSA and small molecule bioamines demonstrated the power of CE × µFFE. Since both methods rely on electrophoretic mobility to separate, an investigation on the orthogonality of the two techniques was carried out. µFFE can operate in many different separation modes to increase the orthogonality CE × µFFE. Lastly, fluorescent labeling of the analytes can cause the sample to lose its dimensionality affecting 2D separation peak capacity and coverage. A novel absorption detector was studied to demonstrate the first ever label free absorption detection on a µFFE device. A separation was performed on visible dyes and their detection limits quantified.

Significant work has been done studying the metabolism of adipose tissue in recent years, in an effort to more effectively combat the obesity epidemic. Adipose tissue has been found to participate in the metabolism signaling network, with BCAAs playing a vital role as messengers relating the amino acid content of high protein meals. BCAA plasma levels have also been found to be elevated in individuals with obesity or type 2 diabetes, with their increased levels often occurring prior to elevated resting glucose levels and insulin resistance. Their dynamics under various metabolic conditions must still be examined however, on a metabolically relevant time scale. Current methods are limited in their time response for in vivo studies, with time-points ranging from minutes to hours. Metabolic dynamics must be studied in near real time, in order to establish these bioamines as metabolic biomarkers. In this work, an in vivo platform is developed for monitoring bioamine metabolism dynamics in adipose tissue and skeletal muscle with 22 second temporal resolution. A high-speed online microdialysis-CE assay, capable of detecting BCAAs and their related metabolites, is utilized to monitor dynamics in near real time. Inguinal adipose tissue and quadriceps skeletal muscle serve as the sampling locations in C57BL6 mice. A systemic stimulation of insulin demonstrated our assay’s ability to detect induced metabolic dynamics. Stimulations of glucose, saccharin, and ace K provide further information regarding the metabolism of bioamines in adipose tissue.

We have developed a high-throughput assay for monitoring branched chain amino acid (BCAA) uptake and release dynamics in 3T3-L1 cells using microdialysis coupled to high speed capillary electrophoresis (CE). Isoleucine, leucine, and valine are important indicators of lipogenesis, as are alanine, glutamate, and glutamine, which are by-products produced through the catabolism of BCAAs. The major focus of this work will be on the development and optimization of a high-speed (high-throughput) CE assay for the separation of fluorescently labeled amino acids; particularly the BCAAs and their downstream metabolites. Development of this assay allows for us to ask important biological questions related to lipogenesis using an in vitro cell model. In combination with bulk culture, release and uptake rates of BCAAs were monitored and assessed as a function of various biological stimuli, including several concentrations of glucose, circulating concentrations of BCAAs, as well as insulin and artificial sweeteners. Changes in release profiles were also monitored as cells progressed through the differentiation cycle in order to see if development stage affects lipogenesis.

When considering the measurement of release events from cells, it can be done at levels as small as the single cell and performed in systems increasingly larger and more complex up to in vivo studies. Though in vitro systems lack the physiological relevance of in vivo, their simple and controlled environment is highly advantageous in preliminary mechanistic studies. In spite of this, there exists a serious gap in our ability to perform in vitro measurements on a wide array of analytes within a meaningful time frame. While electrochemical techniques are unparalleled in their ability to temporally resolve minute signals in biological systems, there is only a small class of targets which are suitable for this type of analysis. When analyzing non-electroactive analytes, measurements are often plagued by slow temporal responses (5+ minutes). Fluorescent imaging offers opportunities to monitor faster dynamics of non-electroactive analytes, but the target analyte must be either natively fluorescent or labeled, which can result in nonspecific binding and cytotoxicity. In both of these cases fast dynamics can be observed, but the array of analytes is small and only a few can be monitored simultaneously. In this work, a novel in vitro sampling platform is described which is capable of simultaneously monitoring approximately 15 non-electroactive analytes with 20 second temporal resolution. Cells were cultured on the surface of a microdialysis probe coupled with an analytical system for analysis. Small molecules released from the cells upon stimulation diffuse across the porous membrane because of the close proximity. A high-speed CE, built in house, enabled analysis of the collected dialysate. The ability of our platform to detect basal and stimulated release of amines was confirmed by transferring the probe between artificial cerebrospinal fluid (aCSF) and a potassium-spiked (100 mM K+-aCSF) stimulant solution. A variety of cell models were tested for compatibility with the in vitro-microdialysis platform, both single cell type and co-cultures were initiated. Adherence of viable cells was confirmed by labeling cells with either fluorescein diacetate (FDA) or specific antibody labelling, followed by imaging under a microscope. As a step towards continuously monitoring the change of non-electroactive analytes released from cultured cells, microdialysis was coupled directly to micro free flow electrophoresis (µFFE) device instead of the high-speed CE instrument.

According to recent literature, it is believed that the oligomeric form of amyloid beta (Aβ) is the leading cause of Alzheimer’s disease (1; 8; 10; 12-18). Additionally, recent studies have eluded to the impact of Alzheimer’s disease (AD) both economically and Socially in today’s society where an increase of about 71% of AD related deaths were recorded between 2000 and 2013 (7). Since the oligomeric forms of Aβ vary in size, shape and some believe conformation, it is vital to utilize a separation technique, such as capilllary electrophoresis (CE) to further understand Aβ aggregation. By understanding Aβ aggregation, treatment of AD or preventive care measurements could be additionally developed. Therefore in this study, field amplified sample stacking (FASS) technique on the CE was utilized to provide higher resolution in oligomeric Aβ1-42 detection without causing significant changes to the aggregation. It was observed that the FASS technique provided smaller peak widths and increased peak heights on the CE compared to the non-FASS conditions. Furthermore by conducting thioflavin-t (ThT) assays, it was observed changing the buffer concentrations in accordance to the FASS technique conditions did not effect the overall aggregation. Thioflavin-t (ThT) assays were also conducted in order to determine an agitation rate where the oligomers of Aβ1-42 were observed on the CE. The oligomeric species observed were believed to be less than or equal to 100 kDa. Additionally, Congo red and Orange G inhibition were conducted to confirm oligomeric Aβ1-42 species were observed on the CE. Both inhibition studies alongside TEM imaging proved the aggregates observed on the CE in the 27 hour aggregation were smaller than proto-fibrils. Future work on natural compound inhibition studies using CE are recommended to see how those inhibitors target Aβ1-42 species that are less than or equal to 100 kDa.

The accumulation of amyloid-beta (Aβ) oligomers is believed to be the driving force behind Alzheimer’s disease (AD) pathogenesis. Due to the metastable nature of Aβ oligomers, the knowledge of Aβ aggregation and accumulation is not well understood. Here, we use capillary electrophoresis (CE) and photo-induced crosslinking of unmodified proteins (PICUP) to learn about the aggregation of Aβ. First, we explore the effect of capillary coating on Aβ1-42 protein loss using CE. The dynamic coatings tested were PHEA and 50 kDa, 2000 kDa, 5000 kDa, and 8000 kDa PEO. The covalent coating tested was PVA. The results indicated that 2000 kDa PEO allowed for the best recovery of Aβ1-42 with 87%. This was better than the uncoated capillary recovery of 66%, but less than the optimal 90%. The biggest issue encountered was a protein recovery of greater than 100%, which is theoretically impossible. More procedure development should be done to solve this problem. Next, the PICUP reaction was optimized for analysis of Aβ1-42. Using a novel approach, the goal was to apply PICUP to capture Aβ1-42 oligomers and correlate bands via SDS-PAGE analysis to peaks via CE analysis. The results showed that reactants needed for successful PICUP application interfered with CE detection of Aβ1-42. We tested alternate reactants and several methods of removing reactants after PICUP application. We learned that dialysis was the most promising method of Aβ1-42 detection via CE after PICUP.

Abstract: Mutations in the mitochondrial genome (mtDNA) have been linked to a wide variety of disorders. mtDNA analyses require large starting samples and are complicated by the presence of nuclear mitochondrial pseudogenes (NUMTs), which can cause false positives in PCR-based approaches. Microfluidic chips (MFCs) allow assays, which are typically run at the laboratory bench, to be performed on small microchips, thus facilitating high-throughput analyses. We have used MFCs to analyze plasmid DNA (pDNA) and mtDNA, in an attempt to develop mtDNA diagnostic capabilities using small sample volumes. We were able to successfully manipulate 1 pg of pDNA, the approximate amount of mtDNA in 1500 fibroblasts, which is the lowest concentration of DNA to be successfully manipulated on MFCs that we are aware of. Based on this plasmid isolation we were also able to develop a MFC-based plasmid miniprep using 5 orders of magnitude fewer starting cells. Modification of these approaches resulted in the isolation of mtDNA from fibroblasts and leukocytes, albeit with some nuclear DNA (nDNA) contamination. Using the Supercoiled DNA (SC) ladder, we were able to develop a capillary electrophoresis (CE)-based plasmid separation that could resolve two plasmids with a 1.5 kb size difference. This resolution is sufficient to allow identification of the vast majority of reported mtDNA deletions. These experiments reported here therefore lay the groundwork for performing mtDNA diagnostics on MFCs.

The goal of this project was to reduce processing time for forensic DNA testing without incurring significant added costs and/or the need for new instrumentation, while still generating high quality profiles. This was accomplished by: 1) extraction normalization using the ChargeSwitch® Forensic DNA Purification Kit such that a small range of DNA concentrations was consistently obtained, eliminating the need for sample quantification and dilution; 2) developing fast PCR protocols for STR primer sets using shorter amplification methods, low volume reactions and non-fast thermal cyclers; and 3) developing a quicker 3130xl Genetic Analyzer detection method using an alternative polymer/array length combination. Extraction normalization was achieved through a reduction in bead quantity, thereby forcing an increase in bead binding efficiency. Four products (AmpliTaq Gold® Fast PCR Master Mix, KAPA2G™ Fast Multiplex PCR Kit, SpeedSTAR™ HS DNA Polymerase and Type-it Microsatellite PCR Kit) were evaluated for low volume (3μl) fast PCR on a 384-well Veriti® thermal cycler with the Identifiler primer set. KAPA2G™ was selected for 3μl fast PCR protocols using PowerPlex 16 HS and Identifiler Plus primer sets (42-51min), as well as 5μl and 6μl Identifiler fast reactions on a 9700 thermal cycler (51-60min). Alternative detection (POP-6™/22cm) achieved 24-28min run times, but with decreased resolution as compared to traditional POP-4®/36cm detection for alleles >200bp; however, 1bp resolution was still obtainable for alleles

Plasmonic nanoparticles are used in a wide variety of applications over a broad array of fields including medicine, energy, and environmental chemistry. The continued successful development of this class of materials requires the accurate characterization of nanoparticle stability for a variety of solution-based conditions. Although a wide array of methods exist, there is an absence of a unified, quantitative means for complete nanoparticle characterization. This work focuses on the challenges inherent with current methods through a comparative analysis of the current gold standard characterization methods. I propose using capillary electrophoresis and micro-capillary electrophoresis as powerful tools for better quantifying the inherent polydispersity and differences in surface functionalization within a nanoparticle sample. I present the Particle Instability Parameter (PIP) as a robust, quantitative, and generalizable characterization technique based upon UV-Vis absorbance spectroscopy to characterize colloidal instability. I validate PIP performance with both traditional and alternative characterization methods by measuring gold nanorod instability in response to different salt (NaCl) concentrations and as a function of solution pH, salt, and buffer type. I contextualize these methods within the literature on gold nanoparticle characterization to establish a standardized methodology for nanoparticle analysis. Finally, I present a concept for an integrated biodiagnostics platform using gold nanorods based upon an integrated microfluidic microspectrophotometry system for the detection of pathogens.

Abstract: Capillary electrophoresis (CE) is a fast and high efficiency separation technique based on the differential migration of charged species in an electric field. CE is useful for the separation of a wide range of analytes from small ions to large biomolecules. However, CE separations of proteins are challenging due to the adsorption of protein onto the capillary silica surface. Capillary coatings are the most common way to minimize this adsorption. This thesis focuses on the use of two-tailed cationic surfactant based coatings as means of preventing protein adsorption. Factors affecting the stability of two-tailed cationic surfactant coatings have been investigated. The impact of small i.d. capillaries (5-25) µm on enhanced stability of surfactant bilayer cationic coatings and on the efficiency of separation of basic proteins was studied. Using a dioctadecyldimethylammonium bromide (DODAB) coated 5 µm i.d. capillary, exceptional short term stability (210 consecutive runs) and long term stability (300 runs over a 30 day period) were achieved. The average separation efficiency of four basic model proteins was 1.4-2 millions plates/m. DODAB coatings were stable over a pH range of 3-8 as demonstrated by strong anodic magnitude of electroosmotic flow (EOF) and good EOF reproducibility. Surprisingly, at pH ≥ 9, EOF became less anodic and even became suppressed cathodic. The reason is unclear. Chemical degradation of DODAB at high pH was excluded. Increased vesicle size at high pH and/or accelerated desorption may be involved. A surfactant bilayer/diblock copolymer coating was developed to tune the EOF and prevent protein adsorption. The coating consisted of a DODAB bilayer which served as a strong anchor to the capillary wall and polyoxyethylene (POE) stearate to suppress the EOF. The coating has been applied successfully to the capillary zone electrophoretic separation of basic, acidic and histone proteins, and to capillary isoelectric focusing. The ability to tune the EOF enabled both single-step capillary isoelectric focusing (cIEF) and two-step cIEF to be performed. A strongly suppressed EOF coating provided a linear pH gradient and allowed for the separation of two hemoglobin variants HbA and HbS. Factors affecting the stability and EOF of the developed surfactant bilayer/diblock copolymer coating were studied. The magnitude of the anodic EOF can be tuned by varying the hydrophilic block POE chain length. The hydrophobic block of the diblock copolymer accounts for stability of the coating, with a longer (stearate) block giving the best stability. The sequential coating provided a stable and suppressed EOF over a broad range of pH 3.0-11.5. The EOF was suppressed and anodic at low pH. As the pH increases, the EOF was still suppressed but became cathodic. This reversal in EOF of the sequential coating is consistent with the reported applications of the sequential coating, and the behavior of the underlying DODAB bilayer. The sequential coating shows a good stability in buffers containing up to 20% v/v acetonitrile.

The interaction between nucleic acids and small molecule ligands is continuously generating significant interest due to their widespread biological and bioanalytical applications. We investigated the mechanical property of the binding between aptamers and small molecules. Using an ATP binding aptamer as an example, we observed that the mechanical stability of the aptamers that are bound with ATP is higher than those without a ligand. Therefore, a force-based sensor can be developed to detect small molecules using aptamers as a platform. We determined the dissociation constant, Kd, for aptamer-ligand interactions at the single-molecule level by applying a Hess-like cycle. Our experiments allow the Kd determination from only one ligand concentration which was further validated by our capillary electrophoresis (CE) method. By using only one ligand concentration, such a method not only saves time and material, but also is less susceptible to reduced reproducibility due to run-to-run fluctuations. G-quadruplex forming sequences which are wide spread in the genome particularly in telomeres and promoter regions have been shown to be therapeutic targets. G-quadruplex structures have been extensively studied using mostly conventional methods. However, the mechanical stability, thermodynamics, kinetics properties of these interactions at the single-molecule level, remains to be fully understood. While the formation of these G-quadruplex structures is highly dynamic, they are mechanically stabilized upon ligand binding which may affect their biological functions. Small molecules which bind to nucleic acid structures may interfere with vital cellular processes such as transcription and protein translation during cell division by acting as energy barriers or mechanical blockage. Therefore, understanding stability of nucleic structures from a mechanical stability stand point is critical to fully explore their therapeutic potentials. We have investigated the human telomeric repeat containing RNA (TERRA) G-quadruplex structures. By using a TERRA sequence that hosts only one G-quadruplex at the single-molecule level in a laser-tweezers instrument, we are able to investigate intramolecular G-quadruplex without implications from high order structures due to the tendency of the TERRA sequence to self-associate. Using a mechanical unfolding and refolding method, we revealed multiple TERRA G-quadruplexes in a solution mixture. One of the structures is consistent with the parallel conformation determined by NMR/X-ray techniques. In addition, we identified a partially folded structure that serves as an intermediate to the unfolding and refolding of the TERRA G-quadruplex. By applying a force-jump approach, the refolding kinetics of the TERRA G-quadruplex and the intermediate were measured and compared with those of human telomeric DNA (hTelo) G-quadruplex. We have found that formation of the TERRA G-quadruplex is slower than that of hTelo G-quadruplex. Together with the higher thermodynamic and mechanical stability of the TERRA with respect to the hTelo G-quadruplex, these results suggest different regulatory capacities of RNA and DNA G-quadruplexes for processes associated with human telomeres.Lastly, a quantitative assay was developed to investigate sphingosine kinase 2 (SphK2) activity both in vitro and with cell lysates, using capillary electrophoresis with laser-induced fluorescence (CE-LIF) and sphingosine fluorescein as the substrate. Sphingosine and sphingosine-1-phosphate are found in very minute quantities in cells and therefore require highly sensitive techniques for quantitative analysis.

Amylin (hIAPP) aggregates have been found in 90% of patients with type II diabetes at autopsy, and are suspected to play a role in the death of islet &beta-cells1. However, this aggregation process is not well understood. Here, we explore methods that utilize capillary electrophoresis (CE) as a means to better understand amylin's aggregation process. We examined the effects of solutions conditions: agitation, pH, salt, and temperature on amylin aggregation using Thioflavin T, dot blots, and capillary electrophoresis. Thiofalvin T was used to predict the lag time to &beta-sheet formation. Our results indicated all variables with the exception of agitation were feasible for study with CE. Capillary electrophoresis was then employed to observe the formation of oligomers with dot blots used as a confirmation technique. Overall, results showed all solutions conditions examined promote aggregation, however there was variance between time courses. Conflicting results suggest further study is needed to fully understand the observed amylin aggregation phenomena.

The focus of this dissertation centers on the development and applications of gas chromatography/mass spectrometry and capillary electrophoresis methodologies to quantify monomeric compositions of the β-O-4 linkages in lignin. Pretreatment is a required step in the utilization of lignocellulosic biomass for biofuels. Lignin is the target of pretreatment because it hinders the accessibility of enzymes and chemicals to cellulose. The effects of pretreatment are commonly assessed utilizing enzymatic saccharification and lignin assays. However, these techniques do not elucidate the effects of pretreatment on the monomeric make up of lignin. The overarching hypothesis of this dissertation is that changes in individual monolignol content upon pretreatment can be observed from quantification. To test the hypothesis, a pretreatment, solution phase Fenton chemistry, was conducted on various lignocellulosic biomass feedstocks. Enzymatic saccharification studies showed a significant increase in glucose production upon Fenton pretreatment, however, lignin assays did not show a significant decrease in lignin content. Project two of this dissertation aimed to synthesize analytical standards in order to develop a quantitative thioacidolysis technique. The successful synthesis of the three arylglycerols were conducted utilizing and epoxidation reaction scheme which was hypothesized to produce a single diastereomer, as supported by GC/MS and chiral CE analysis. Upon method development, a quantitative thioacidolysis GC/MS method was applied to untreated and Fenton treated biomass. Results from this project revealed there was no significant change in the three lignin monomers. To verify the method, quantitative thioacidolysis GC/MS method was applied to a pretreatment method known to degrade lignin, alkaline peroxide pretreatment. The results of this project showed a significant change in monolignol concentrations upon alkaline peroxide pretreatment. Analytical degradative techniques, such as thioacidolysis, has traditionally assessed lignin as monomeric ratios. However, as this dissertation showed, upon alkaline peroxide pretreatment, no significant change was seen in the monomeric ratios, but there was a significant difference in all three monolignol concentrations. These results support the overall hypothesis that changes in individual monolignol content upon pretreatment can be observed from quantification. The works of this dissertation provides an analytical method which contributes to the elucidation of lignin.

Bioethanol fermentation is an important process that is reducing the global demand on fossil fuels and remains a field of research for the foreseeable future. Carbohydrates are sourced from crops and hydrolyzed into simpler sugars then fermented into ethanol. Fermentation of sugars sourced from food crops presents a sustainability issue with a growing population. Fermentation of lignocellulosic material is more sustainable since it is sourced from non-food crops or wastes. It is comprised of a variety of both pentose and hexose sugars. The composition and ratio of these varies depending on the source of the material. Accurate analysis of the material is essential for both the monitoring of the hydrolysis and its fermentation to valuable end-products such as ethanol. Although there have been major advances in novel fermentation processes and the discovery and construction of novel microorganisms, development of methods for analysis of these complex substrates and their fermentation to ethanol has not advanced as rapidly. High Performance Liquid Chromatography (HPLC) is one of the most common techniques for the analysis of these complex substrates. The resolution of popular HPLC modes was compared and no mode was found to have complete separation of common fiber sugars. Free solution Capillary Electrophoresis / capillary zone electrophoresis (CE) is used and recognized in both research and industry as a viable technique for the separation of carbohydrates. Recent studies on the use of direct UV detection for determination of underivatized carbohydrates have shown great promise and, in this work, the technique was applied to lignocellulosic plant fiber analysis as well as monitoring its fermentation to ethanol and sugar alcohols. All resolution values with CE were higher than 0.5, in contrast to any HPLC mode investigated. The running cost of HPLC, for this application, is also much higher than CE. Determination of carbohydrates from lignocellulosic fiber by both HPLC on a cation exchange resin and by CE resulted in values 17-22 % higher with CE than HPLC. The influence of the counter-ion in the BackGround Electrolyte (BGE) was found to affect the resolution and time of the separation. 130 mM KOH was shown to be effective for a fast separation of simple mixtures and a mixture of 65 mM NaOH and 65 mM LiOH achieved a better resolution with more complex carbohydrate mixtures than the other BGE’s studied. In a quantitative study on fermentation samples, CE, HPLC and High Performance Anion Exchange Chromatography (HPAEC) closely agreed within experimental error (less than 7 % difference from the average total detected amount).

Lysosomes and endocytic organelles are intracellular bodies present in eukaryotic cells responsible for the degradation of endocytosed extracellular targets. Autophagosomes traffic proteins, organelles, and other intracellular components to lysosomes to facilitate degradation during the degradative process of autophagy. Multiple disorders have been connected to malfunctions in lysosomes (Nieman pick, galactosialidosis, Danon disease) and autophagosomes (Alzheimers, Parkinson's, Huntington's).Methods are needed to enrich organelles in order to study their properties without contamination from unwanted organelles. Current methods to enrich endocytic organelles do not result in highly enriched organelles (differential centrifugation), are time consuming and tedious (density gradient centrifugation), and can damage membranes. Methods are also needed to determine endocytic and autophagy organelle properties such as organelle molecular composition, organelle-specific biotransformation of anti-cancer drugs, individual organelle surface properties and marker protein levels, and pH. The work described in this thesis develops new techniques to improve our ability to enrich endocytic organelles and determine their properties. This work includes: (1) the magnetic enrichment of endocytic organelles and determination of pH by capillary cytometry, (2) the determination of the biotransformation of N-L-leucyldoxorubicin to doxorubicin, (3) the development of a workflow to determine preliminary identifications of enriched autophagosome samples and (4) determine temporal changes in individual autophagy organelle numbers, surface charge, and LC3-II levels from basal and rapamycin enhanced autophagy levels. These methods will improve our understanding of how lysosomes and autophagosomes contribute to disease, leading to better therapeutic strategies that may improve and lengthen people's lives. Endocytic organelle enrichment was done by trafficking dextran coated magnetic iron oxide nanoparticles to lysosomes and endocytic organelles prior to magnetic separation. No detectable enzymatic activity from mitochondria and peroxisomes were observed in the enriched endocytic organelle fractions suggesting that the enriched lysosomes were in high enrichment. A majority of enriched, individual endocytic organelles had an acidic pH as determined by capillary cytometry suggesting the enriched endocytic organelle fraction had intact membranes. Enriched endocytic organelle fractions were then used to determine the biotransformation of N-L-leucyldoxorubicin to doxorubicin. Previous reports had suggested endocytic organelles may be important for intracellular biotransformation. About 45% of the biotransformation from uterine sarcoma cell post nuclear fraction occurred in the enriched endocytic organelle fraction suggesting intracellular biotransformation may be more critical to prodrug activation than previously believed.Ultra high performance liquid chromatography coupled to near-simultaneous low- and high-collision energy mass spectrometry was used to determine preliminary identifications of compounds enriched or unique to enriched autophagosome fractions. A workflow was developed to detect and confirm features (unidentified compounds with a characteristic chromatographic tR and m/z value) in the enriched sample as well as making and confirming identifications from online databases. Multiple high-relevancy preliminary identifications were made that are relevant to autophagy as supported by literature searches. Following validation, these preliminary identifications could prove to be important to maintain autophagosome function and autophagy. Capillary electrophoresis coupled to laser induced fluorescence detection (CE-LIF) was used to determine temporal changes in the detected number of individual autophagy organelle events (phagophores, autophagosomes, amphisomes, and autolysosomes), of GFP-LC3-II levels, and of surface charge by CE-LIF. Pharmacological treatment with vinblastine was used to accumulate autophagosomes and phagophores from basal and rapamycin enhanced autophagy do detect temporal changes in autophagy organelles characteristic of the autophagy level and its autophagy flux. The dramatic contrast between time dependent changes in individual organelle properties between basal and rapamycin enhanced autophagy conditions demonstrates an anticipated complexity of autophagy flux which likely plays critical role in response to drug treatments, aging, and disease.

Micelle-forming bile salts have previously been shown to be effective pseudo-stationary phases for separating the chiral isomers of binaphthyl compounds with micellar electrokinetic capillary chromatography (MEKC). Here, cholate micelles are systematically investigated via electrophoretic separations and NMR using R, S-1, 1¿- binaphthyl- 2, 2¿-diylhydrogenphosphate (BNDHP) as a model chiral analyte. The pH, temperature, and concentration of BNDHP were systematically varied while monitoring the chiral resolution obtained with MEKC and the chemical shift of various protons in NMR. NMR data for each proton on BNDHP is monitored as a function of cholate concentration: as cholate monomers begin to aggregate and the analyte molecules begin to sample the micelle aggregate we observe changes in the cholate methyl and S-BNDHP proton chemical shifts. From such NMR data, the apparent CMC of cholate at pH 12 is found to be about 13-14 mM, but this value decreases at higher pH, suggesting that more extreme pHs may give rise to more effective separations. In general, CMCs increase with temperature indicating that one may be able to obtain better separations at lower temperatures. S-BNDHP concentrations ranging from 50 ¿M to 400 ¿M (pH 12.8) gave rise to apparent cholate CMC values from 10 mM to 8 mM, respectively, indicating that S-BNDHP, the chiral analyte molecule, may play an active role in stabilizing cholate aggregates. In all, these data show that NMR can be used to systematically investigate a complex multi-variable landscape of potential optimizations of chiral separations.

Protein Protein Pnteractions (PPIs) are key players in all aspects of cellular functions. In depth understanding of PPIs and discovery of PPI targeted therapeutics requires facile and user-friendly PPI analysis. Capillary Electrophoresis (CE) is a valuable tool to study affinity bindings including PPIs. Fast separation speed and nanoliter sample consumption suggest great potential for CE based High Throughput Screening (HTS). To explore CE as a PPI analysis and HTS tool, we have developed a CE based assay for Heat Shock Protein70 (Hsp70) and its co-chaperone Bcl2-Associated Anathogene 3 (Bag3). An Affinity Probe CE (APCE) method was developed for full length Hsp70 and Bag3 extracted from cell culture. In this assay, Hsp70 was labeled with Alexafluor® 488 and used as the affinity probe. With a surface modified capillary, the binding is detected as Hsp70 and complex peaks resolved and detected using Laser Induced Fluorescence (LIF). On a commercial instrument, the CE assay takes 6.5 min per run which allows a sample throughput of 220 tests per day. In a pilot screening, ~3300 small molecule library compounds were tested for novel inhibitor discovery. By comparing CE assay’s performance to an existing high throughput screening platform Flow Cytometry Protein Binding Assay (FCPIA), CE showed much better specificity as evidenced by lower hitting rate and higher confirmation rates in confirmatory tests. Fully realizing the low sample consumption benefit of CE based HTS requires low volume sample manipulation capability. Segmented flow microfluidics is an emerging technique that allows nanoliter to picoliter volume samples to be manipulated and coupled to CE analysis. In the second half of this thesis, unit operations of segmented flow microfluidics, namely splitting and reagent addition were developed to fulfill the need to aliquot and add reagent to nanoliter segmented flow samples. Based on these results, future work would be to integrate the CE separation with these sample manipulation techniques towards building a novel workflow for CE based HTS.

Polycyclic aromatic hydrocarbons (PAH) are a class of environmental pollutants consisting of a minimum of two fused aromatics rings originating from the incomplete combustion of organic matter and/or anthropogenic sources. Numerous possible anthropogenic and natural sources make the presence of PAH ubiquitous in the environment. The carcinogenic nature of some PAH and their ubiquitous presence makes their chemical analysis a topic of environmental and toxicological importance. Although environmental monitoring of PAH is an important step to prevent exposure to contaminated sites, it provides little information on the actual uptake and subsequent risks. Parent PAH are relatively inert and need metabolic activation to express their carcinogenicity. Covalent binding to DNA appears to be the first critical step in the initiation of the tumor formation process. To this end, the determination of short term biomarkers – such as monohydroxy-PAH metabolites (OH-PAH) - fills an important niche to interpret actual PAH exposure levels, prevent extreme body burdens and minimize cancer risk. One would certainly prefer an early warning parameter over a toxicological endpoint – such as DNA-adducts – indicating that extensive damage has already been done. Several methods have been developed to determine OH-PAH in specific tissue or excreta and food samples. The general trend for the analysis of OH-PAH follows the pattern of sample collection, sample clean-up and pre-concentration, chromatographic separation and quantification. Popular approaches for sample clean-up and preconcentration include liquid-liquid extraction (LLE) and solid-phase extraction (SPE). Chromatographic separation and quantification has been based on high-performance liquid iv chromatography-room temperature fluorescence detection (HPLC) and gas chromatographymass spectrometry (GC-MS). Although chromatographic techniques provide reliable results in the analysis of OHPAH, their experimental procedures are time consuming and expensive. Elution times of 30-60 minutes are typical and standards must be run periodically to verify retention times. If the concentrations of target species are found to lie outside the detector’s response range, the sample must be diluted and the process repeated. On the other end of the concentration range, many samples are “zeroes,” i.e. the concentrations are below detection limits. Additional problems arise when laboratory procedures are scaled up to handle thousands of samples under mass screening conditions. Under the prospective of a sustainable environment, the large usage of organic solvents is one of the main limitations of the current chromatographic methodology. This dissertation focuses on the development of a screening methodology for the analysis of OH-PAH in urine and milk samples. Screening techniques capable of providing a “yes or no” answer to OH-PAH contamination prevent unnecessary scrutiny of un-contaminated samples via conventional methods, reduce analysis cost and expedite the turnaround time for decision making purposes. The proposed methodology is based on capillary zone electrophoresis (CZE) and synchronous fluorescence spectroscopy (SFS). Metabolites extraction and pre-concentration is achieved with optimized SPE, LLE and/or QuEChERS (quick, easy, cheap, effective, rugged and safe) procedures. The small sample and extracting solvent volumes facilitate the simultaneous extraction of numerous samples via an environmentally friendly procedure, which is well-suited for routine monitoring of numerous samples. Sample stacking is successfully implemented to improve CZE limits of detection by two orders of magnitude. The unique electrophoretic pattern of positional isomers of OH-PAH demonstrates the potential of CZE for v the unambiguous determination of metabolites with similar chromatographic behaviors and virtually similar fragmentation patterns. The direct determination of OH-PAH without chromatographic separation is demonstrated via SFS. The non-destructive nature of SFS provides ample opportunity for further metabolite confirmation via chromatographic techniques