published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

The electrochemical detector provides a promising detection mode for capillary electrophoresis (CE) due to its excellent sensitivity, good portability, high selectivity, easy miniaturization, low capital and running cost. To widen its scope for determining trace analytes in complex samples, three dual-electrode detectors were fabricated to enable the determination of electro-inactive analytes, to assess co-eluted peaks and to give a large enhancement of the detection sensitivity by modifying electrode surface using multi-walled carbon nanotubes (MWNTs). To determine trace non-electroactive amino acids present in human tears, a serial dual-electrode detector was developed using an upstream on-capillary Pt film electrode to oxidize bromide to bromine at +1.0 V and a downstream Pt disk electrode to detect the residual bromine at +0.2 V after their reaction with amino acids eluted out from the separation capillary. The bromide reagent was introduced after CE separation by a newly designed coaxial post-column reactor fabricated onto the PMMA chip. Using optimized CE buffer containing 20 mM borate, 20 mM SDS at pH 9.8, L-glutamine, L-alanine and taurine were baseline separated with detection limits ranging from 0.56-0.65 μM and a working range of 2-200 μM for L-glutamine and of 2-300 μM for both L-alanine and taurine. Method reliability was established by close to 100% recoveries for spiked amino acids and good agreement between the measured and the literature reported amino acid concentrations in tears. For the determination of polyphenols in wine, a microchip-CE device was fabricated with a dual-opposite carbon fiber microelectrode operated in a parallel mode to assess peak purity. Under optimized conditions, (+)-catechin, trans-resveratrol, quercetin, (-)-epicatechin and gallic acid were baseline separated within 16 min with detection limits ranging from 0.031- 0.21 mg/L and repeatability of 2.0-3.3 % (n=5). The use of an opposite dual-electrode enables the simultaneous determination of peaks and measurement of their current ratios at +0.8 V and +1.0 V vs Ag/AgCl. The capability of using current ratio to identify the presence of co-migrating impurities was demonstrated in a mixed standard solution with overlapping (+)-catechin and (-)-epicatechin peaks and in a commercial red wine with interfering impurities. Matching of both the migration time and the current ratio reduce false positive and validate polyphenol quantitation in red wine. Lastly, a dual-opposite MWNTs modified carbon fiber microelectrode (CFME) was developed to determine the biomarkers (4-nitrophenol, 4-nitrophenyl-glucuronide and 4-nitrophenyl-sulfate) needed to assess exposure to methyl parathion. Use of the MWNTs modified CFME showed a much higher sensitivity than bare CFME, with a detection limit of 0.46 μM for 4-nitrophenol. Baseline separation of all three biomarkers was obtained within 31 min by a 45 cm long capillary under 12 kV in a 20 mM phosphate buffer at pH 7.0. The method developed was successfully utilized to determine low levels of biomarkers in human urine without using complex pretreatment steps and delivered recoveries ranging from 95.3 - 97.3% and RSDs within 5.8% (n=3). Using a parallel dual-electrode detector was shown to deliver reliable results with matching current ratios and comparable migration time to those obtained from biomarker standards. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

published_or_final_version / Chemistry / Master / Master of Philosophy

published_or_final_version / Obstetrics and Gynaecology / Master / Master of Philosophy

The neurochemical problem (1) and the directive of the neuroanalytical chemist (2) can be stated as follows: (1) The chemical space of the nervous system is populated by hundreds of neuroactive species linked through extensive biological circuits which are dynamically changing in time and space in response to myriad inputs. (2) Neurochemical analysis techniques should therefore have the appropriate temporal, spatial, and chemical resolution to study these systems, while perturbing them so minimally as to allow unfettered in vivo measurements. New tools and concepts for analytical measurements of neurotransmitters, neuropeptides, and synaptic vesicles are developed and presented in this dissertation on analytical measurements for addressing the neurochemical problem. The introduction gives a broad overview of chemical neuroscience and introduces quantitative visualization of the multidimensional resolution paradigm for analytical chemists seeking to design effective experiments. Chapters two through four detail advancements in data processing and instrument design which decrease detection limits and allow for improved spatial, temporal, chemical resolution in capillary electrophoresis measurements of neurotransmitters, metabolites, and synaptic vesicles. Chapter five discusses the development of fast-scan controlled-adsorption voltammetry which has dramatically increases the spatial and temporal resolution of basal dopamine measurement in vivo. Chapter six introduces online-preservation microdialysis as a way to overcome enzymatic degradation of endogenous opioid neuropeptides during in vivo sample collection. Because of this discovery of the secretory behavior these neuropeptides is reported in the anterior cingulate cortex (ACC), a region of the brain deeply associated with pain signaling. The advancement of peptide drugs particularly glycosylated neuropeptide analogs through new methods of mass spectrometry analysis for rapid feedback in drug development are presented in chapter 7. Chapter eight concludes this work with future directions pointing towards single-cell electrochemical and mass spectrometry measurements, shotgun-microdialysis for high-throughput screening of neurotherapeutics, preliminary data on the effect of chronic pain of endogenous opioids in the ACC, and the beginnings of in vivo neuroproteomics analysis in rodent pain models.

No description available.

Single-molecule fluorescence spectroscopy techniques such as Fluorescence Correlation Spectroscopy (FCS) and single-molecule Förster Resonance Energy Transfer (smFRET) not only possess an unprecedented high sensitivity but also have high temporal and spatial resolution. Therefore, they have an immense potential both in investigation of fundamental biological principles and in clinical applications. FCS analyses are based on both theoretical approximations of the beam geometry and assumptions of the underlying molecular processes. To address the accuracy of analysis, firstly the experimental conditions that should be fulfilled in order to obtain reliable physical parameters are discussed and the input parameters are carefully controlled accordingly to demonstrate the performance of FCS measurements on our home-built confocal multiparameter photon-counting microscope in several in vitro and in-vivo applications. Secondly, we performed a comprehensive FCS analysis of rhodamine family of dyes to evaluate the validity of assigning the correlation relaxation times to the time constant of conformational dynamics of biomolecules. While it is the common approach in literature our data suggests that conformational dynamics mainly appear in the correlation curve via modulation of the dark states of the fluorophores. The size and shape of the folded, unfolded and chemically-denatured states of the N-terminal Src-homology-3 of downstream of receptor kinases (DrkN SH3) were investigated by FCS and smFRET burst experiments. Based on the data, we conclude that a considerable sub-population of the denatured protein is in a closed loop state which is most likely formed by cooperative hydrogen bonds, salt bridges and nonpolar contacts. As a clinical application, we developed and characterized an ultrasensitive capillary electrophoresis method on our multiparameter confocal microscope. This allowed us to perform Direct Quantitative Analysis of Multiple microRNAs (DQAMmiR) with about 500 times better sensivity than a commercial instrument. Quite remarkably, we were able to analyze samples of cell lysate down to the contents of a single cell.

Single-molecule fluorescence spectroscopy techniques such as Fluorescence Correlation Spectroscopy (FCS) and single-molecule Förster Resonance Energy Transfer (smFRET) not only possess an unprecedented high sensitivity but also have high temporal and spatial resolution. Therefore, they have an immense potential both in investigation of fundamental biological principles and in clinical applications. FCS analyses are based on both theoretical approximations of the beam geometry and assumptions of the underlying molecular processes. To address the accuracy of analysis, firstly the experimental conditions that should be fulfilled in order to obtain reliable physical parameters are discussed and the input parameters are carefully controlled accordingly to demonstrate the performance of FCS measurements on our home-built confocal multiparameter photon-counting microscope in several in vitro and in-vivo applications. Secondly, we performed a comprehensive FCS analysis of rhodamine family of dyes to evaluate the validity of assigning the correlation relaxation times to the time constant of conformational dynamics of biomolecules. While it is the common approach in literature our data suggests that conformational dynamics mainly appear in the correlation curve via modulation of the dark states of the fluorophores. The size and shape of the folded, unfolded and chemically-denatured states of the N-terminal Src-homology-3 of downstream of receptor kinases (DrkN SH3) were investigated by FCS and smFRET burst experiments. Based on the data, we conclude that a considerable sub-population of the denatured protein is in a closed loop state which is most likely formed by cooperative hydrogen bonds, salt bridges and nonpolar contacts. As a clinical application, we developed and characterized an ultrasensitive capillary electrophoresis method on our multiparameter confocal microscope. This allowed us to perform Direct Quantitative Analysis of Multiple microRNAs (DQAMmiR) with about 500 times better sensivity than a commercial instrument. Quite remarkably, we were able to analyze samples of cell lysate down to the contents of a single cell.

Diss. (sammanfattning) Uppsala : Univ., 2003. / Härtill 6 uppsatser.

Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 5 uppsatser.