Browsing by Subject "Capillary electrophoresis"

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    Development of a microfluidic, segmented-flow, single molecule, enzyme activity assay and improvement of separation efficiency of basic proteins by application of a water- proofing agent as a coating in capillary electrophoresis
    (2012-08) Castro Barahona, Eric Rigoberto
    A novel, microfluidic platform for segmented flow assays has been developed using commercially available Teflon tubing and PEEK connectors. Such a system can be used to generate arrays of nano to pico liter sized droplets separated from each other by plugs of a fluourous solvent. Each droplet becomes an individual reaction vessel suitable for high-throughput applications. We have applied this method to the development of a single enzyme molecule fluorescence assay. Characterization of the droplet generation platform was done with the use of a 100 μm ID PEEK T-junction connector. When two immiscible streams, such as water and a fluorous solvent, meet at the T-junction an array of aqueous droplets separated by plugs of the solvent is generated inside the Teflon tubing. Experiments have shown that, like previous microfabricated segmented flow devices, our system can control the size of the droplets generated solely by changing the ratio of the flow rates of the two phases. Using this approach droplets can be produced with good reproducibility (better than 6% in all cases and better than 3% in most) over a wide range of flow rates. Rates of droplet generation of 10.37 ± 0.17 drops/s are easily achieved for good high-throughput potential. Fast on-line mixing of reagents and long term droplet stability of up to 7 days has also been demonstrated. The discovery of the heterogeneity of enzyme molecules with respect to activity has resulted in the development of a variety of single enzyme molecule assays, with the aim of investigating the prevalence and origin of this phenomenon. The segmented flow platform we have developed is well suited to the application of single enzyme assays. It has the advantage of high-throughput, as well as ease of fabrication compared to PDMS or silica based devices and elimination of exposure of the enzyme analyte to the walls of the channel or well. A segmented flow, single molecule assay has been developed for the enzyme alkaline phosphatase (AP). Single AP molecules were sequestered inside 100 pL droplets generated in a PEEK tee and stored in a length of 50 μm ID Teflon tubing. The droplet array was allowed to incubate for a suitable time period, during which the AP molecules converted the weakly fluorescent substrate AttoPhos® into a strongly fluorescent product. AP molecules were found, as in previous studies, to display heterogenous activity with up to a 9-fold difference between individual enzymes In the last section of this work we have used the commercial glass treatment Aquapel as a capillary wall coating agent to reduce protein absorption in capillary electrophoresis (CE). Due to their large number of potential sites for interactions with the fused-silica wall, protein separations with CE can often be difficult. For this reason, much effort is expended on the development on wall coating agents for the prevention of such interactions. Aquapel is a fluorous polymer used commercially to render glass surfaces hydrophobic. The efficacy of the coating was investigated using a suite of three basic proteins: lysozyme, cytochrome c and α-chymotrypsinogen. Separation efficiencies of up to 130,000 theoretical plates were achieved over a pH range of 4.0 to 7.0, a significant improvement over bare fused silica capillary. Electroosmotic flow (EOF) was reduced by the Aquapel coating but not entirely suppressed. The stability of the coating was also examined. 62 protein injections were performed over a two day period during which analyte migration times varied by less than 3.5%. Due to the ease of application and low cost, coating with Aquapel is an attractive alternative to available capillary coatings.
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    Development of capillary electrophoretic-based techniques to analyze doxorubicin in tissues, cells and subcellular fractions.
    (2010-09) Wang, Yaohua
    Doxorubicin (DOX) is a widely used anti-cancer drug. It is hypothesized that the efficacy and toxicity of DOX is related to its distribution and metabolism. Capillary electrophoresis (CE) including its variants such as micellar electrokinetic chromatography (MEKC) is becoming a popular method in bioanalysis due to its high sensitivity and separation efficiency, small sample size requirements, simple sample preparation procedures, versatility in sampling and short separation times. The goal of this thesis is to take advantage of these features and develop CE-based methods to investigate the metabolism, subcellular distribution and localization of DOX in biological samples after DOX treatments. A direct sampling technique was developed to quantify DOX at or near the tumor site in hepatocellular carcinoma tissues after chemoembolization. This technique allows for sampling small volume of tissues (<10 picoliters) selectively from adjacent tumor and non-tumor regions with high spatial resolution (100 micrometers) and reproducibility. Using this technique coupled with MEKC-LIF (laser-induced fluorescence detection), DOX was detected and quantified, in both tumor and non-tumor regions in resected human livers. A MEKC method was developed to monitor the incorporation, transformation and subcellular metabolism of a DOX prodrug, N-L-leucyl-doxorubicin (LeuDOX), which is expected to have higher efficacy and lower toxicity. It successfully separated LeuDOX from DOX and its major metabolite, doxorubicinol (DOXol). The metabolism of LeuDOX in four subcellular fractions of human uterine sarcoma cells suggested that LeuDOX is mainly activated to DOX in the lysosome-enriched fraction which contains hydrolytic enzymes. Metabolism of DOX in isolated subcellular fractions from young and old rat livers was investigated by MEKC-LIF, suggesting that this technique is adequate to investigate the effect of aging on the metabolism of DOX at the subcellular level. The study showed that the young rat liver is more metabolic active than that of the old rat. In subcellular drug analysis, the purity of the subcellular fractions limits the determination of the subcellular localization of metabolites. Organelles with high purity and biological function are necessary to refine the understanding of DOX metabolism in a specific organelle type. An immunoisolation method based on the use of an antibody specific to a peroxisomal membrane protein was developed to isolate peroxisomes with low levels of contaminating mitochondria and lysosomes. The metabolism of DOX and a model compound, BODIPY fatty acid analog, showed possible peroxisomal biotransformation of these xenobiotics. The techniques and methodologies developed in the dissertation work would be the basis of future developments including assessing the function of new prodrugs at the cellular and subcellular levels, profiling subcellular specific metabolism and monitoring drug distribution and metabolism in tissue cross-sections. These measurements are necessary to understand the efficacy and toxicity of chemotherapy drug treatments in preclinical and clinical studies.
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    Development of online high speed capillary electrophoresis fluorescence assays for 3’, 5’-cyclic guanosine monophosphate (cGMP) and pharmacokinetic monitoring of intranasal administration of drugs to the mammalian brain
    (2011-05) Mohns, Anne Pershing
    An innovative analytical system coupling online high speed microdialysis (MD) to capillary electrophoresis (CE) with laser induced fluorescence (LIF) detection has been developed for in vivo monitoring of chemical dynamics in the mammalian brain. Previously used for simultaneous in vivo monitoring and analysis of amino acid neurotransmitters; my research applied this powerful technique to the development of novel fluorescence assays for 1) the detection and monitoring of the intracellular messenger 3’, 5’ cyclic Guanosine Monophosphate (cGMP) and 2) monitoring pharmacokinetics of intranasal administration of drugs to the rat brain. cGMP plays a significant regulatory role in numerous biological processes, including modulation of protein phosphorylation. Of specific interest is cGMP’s proposed significance in the glutamate nitric oxide (NO) signaling pathway. As a downstream indicator of NO activity, the study of cGMP may lead to a greater understanding of the steps involved in reduction of oxidative stress and excitotoxic effects associated with human diseases and neurologically related disorders. Development of the cGMP assay was performed using a custom self built online, high speed MD-CE-LIF instrument. Derivatization was carried out in an online reaction with the guanine selective fluorogenic reagent phenylglyoxal (PGO). A flow gated interface was used to load the sample onto the separation capillary. Fluorescence was excited using the 364 nm line of an argon-ion laser in a sheath flow detector cell. Emission was collected at 90o, passed through a band-pass filter, and collected on a photomultiplier tube. Optimization and characterization of the cGMP assay was carried out to achieve maximum sensitivity, selectivity, resolution, and speed of analysis. Online detection limits of 14-19 nM (sample collected through an in-house manufactured microdialysis probe) have been achieved. cGMP was resolved at nanomolar concentrations with a temporal resolution on the order of 25-50 seconds. In vivo characterization of cGMP was performed in the rat striatum. Application of pharmacological agents via reverse microdialysis was performed to induce the stimulation of cGMP production. Experiments were carried out with high-K+ aCSF, glutamate, phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX), and nitric oxide donor 2-(N-N-Diethylamino)-diazenolate-2-oxide (DEANO). Studies to establish the pharmacology of cGMP are done for two reasons: 1) confirm the identity of the cGMP peak, and 2) test current hypotheses regarding cGMP’s function in vivo. Working in concert with the Alzheimer’s Research Center at Regions Hospital in St. Paul, we developed a novel fluorescence assay for the detection and monitoring of fluorescein in the rat brain following intranasal (i.n.) administration. A significant challenge in the development of clinical therapies for central nervous system (CNS) diseases and injuries is difficulty in delivering the agents directly to the target CNS regions due to blood brain and blood-cerebrospinal fluid barriers. The precise mechanisms underlying nose to brain transport post i.n. administration are not completely understood. MD-CE-LIF analysis of i.n. administered fluorescent compounds may elucidate possible pathways and mechanisms underlying transport from the nasal passages to the CNS and aid clinical development of drug therapies capable of directly targeting CNS regions associated with the pathology of neurological damage and neurodegenerative diseases. Development of the fluorescein assay was performed using the custom built online high speed MD-CE-LIF instrument. A flow gated interface was used to load the sample onto the separation capillary. Fluorescence was excited using a 488 nm solid state laser in a sheath flow detector cell. Emission was collected at 90o, passed through a band-pass filter, and collected on a photomultiplier tube. Optimization and characterization of the fluorescein assay was carried out to achieve maximum sensitivity, selectivity, resolution, and speed of analysis. The developed fluorescein assay achieved an online (sampled through an in house manufactured microdialysis probe) limit of detection (LOD) of 34 ± 4 pM with a temporal resolution on the order of 50 seconds. Electrophoretic separation of fluorescein was complete in 17-23 seconds at 23 kV. Animal protocol development and in vivo animal handling was carried out to characterize the fluorescein assay in vivo post intranasal administration of fluorescein. Fluorescein has been detected in the rat brain at picomolar to nanomolar concentrations and the first high temporal resolution pharmacokinetic profile of fluorescein in the rat olfactory bulb was achieved. The optimized fluorescein assay was used to study the effectiveness of chitosan and hydroxypropyl-β-cyclodextrin (HPβCD) as nasal absorption/nasal mucosal membrane permeability enhancers of intransally delivered fluorescein with brain pharmacokinetics measured in the rat olfactory bulb via MD-CE-LIF. Whole blood samples were drawn and analyzed via microdialysis to elucidate fluorescein’s likely nose to brain delivery pathways. Pharmacokinetic profiles of fluorescein in the olfactory bulb were attained with a temporal resolution not previously demonstrated or published in any intranasal drug delivery study. Initial chitosan experiments did not indicate increased absorption and detection of fluorescein in the rat olfactory bulb. HPβCD demonstrated significant fluorescein enhancement in the brain relative to intransally administered fluorescein without an absorption enhancer. The temporal resolution advantages associated with MD-CE-LIF allowed information to be measured on a time scale not possible in previous cGMP assays, and provided the first high temporal pharmacokinetic profile of fluorescein in the rat olfactory bulb. A high potential is indicated for application of MD-CE-LIF to brain pharmacokinetic study in the development of future intranasal drug therapies directly targeting the central nervous system.
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    Fabrication and characterization of a polydimethylsiloxane microfluidic pump for direct-sampling neuroscience experiments, with in-line capillary electrophoresis - laser-induced fluorescence chemical analysis
    (2011-05) Graf, Neil J.
    A polydimethylsiloxane (PDMS) peristaltic micropump was designed, fabricated, and characterized, for intended use within rodent brain direct-sampling neuroscience experiments, with capillary electrophoresis - laser-induced fluorescence (CE-LIF) chemical analysis. The micropump was fabricated in-part using replica molding (REM) and injection molding. The micropump channel was formed by bonding an open PDMS Gaussianshaped micromolded channel, to a featureless slab of PDMS. Two pieces of capillary tube interconnects were sealed within the closed-off microchannel, and used to make connections with the outside world. The micropump was actuated using piezoelectric cantilevers, with a precision machined microvalve attached to the tip of each cantilever actuator. Registration of the cantilevers and microvalves over the PDMS microchannel, was accomplished with the aid of in-house machined micropositioners. The micropump was thoroughly characterized, for use and application as a bio-analytical add-on attachment device, to an already existing CE-LIF instrument. The micropump was characterized for: various microchannel geometries; different microvalve sizes, tilt, positioning, and shutoff performance; micropositioner design and performance; and, flow rate, backpressure, and peristaltic signal analysis. A P-Q (or H-Q) plot was formed, to represent the performance of the micropump for maximum attainable backpressure (P), versus flow rate (Q). The linear plot was formed by experimentally collecting fourteen individual data points, each corresponding to a unique micropump, “state.” The P-Q plot as discussed within Chapter VIII, is very potent, in providing a 5-for-1 benefit ratio. The P-Q plot allows an experimentalist to obtain: 1) a means to understand how the micropump output performance for both flow rate and backpressure, can be optimized for any particular microfluidic application, 2) an experimentally characterized micropump performance curve/s, 3) an experimentally characterized system curve, 4) the maximum power output of the micropump, and 5) a means to acquire a quantitative measure of the suction lift requirements associated with rodent brain direct-sampling neuroscience experiments. A control volume analysis is provided, to additionally articulate and facilitate discussion of the direct-sampling methodology. Preliminary pilot study direct-sampling data is also provided, as a means to justify and prove viability of the direct-sampling technique, for future characterization and optimization direct-sampling CE-LIF neuroscience studies.
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    Microfluidic selection of DNA aptamers using capillary electrophoresis and micro free flow electrophoresis systematic evolution of ligands by exponential enrichment
    (2012-08) Jing, Meng
    SELEX is the process used to generate aptamers, which are ssDNA or RNA molecules that can bind specific targets with high affinity. Although aptamers show great potential in clinical applications, the generation process is currently tedious and inefficient, being a limiting step. Thus, understanding, developing, and applying advanced partitioning platform are pivotal. CE is an advanced separation method in SELEX and has been successfully used to generate aptamers toward multiple targets. However, there are still interesting questions unanswered, making our understanding in CE-SELEX lag behind its applications. We applied high-throughput sequencing on CE-SELEX selected pools against rhVEGF and obtained sequencing information of more than 104 sequences per pool, which allowed characterization on diversity of individual pools. This study revealed the coexistence of high diversity and fast enrichment rate of CE-SELEX. To further improve the separation platform, we integrated a μFFE device into the SELEX process. Using this device, 1014~1015 sequences were introduced and analyzed within 30 min, which was a 370 fold improvement compared to CE. As a proof of concept, four cycles of selection were performed to the target human IgE, and high affinity ligands were generated even after the first round of selection, proving the feasibility and high efficiency of μFFE in SELEX. Later, μFFE-SELEX was applied to generate aptamers for a membrane protein SERCA, whose selection has never been achieved in conventional SELEX due to the technical difficulty in target immobilization. High nM Kd pool was generated toward SERCA solubilized in C12E8 at the fifth round of selection, demonstrating that this separation strategy is more compatible with membrane proteins due to the free solution based separation, lower electric field, faster separation speed, and straightforward fraction collection. The success of this application opens a door for high-throughput generation of aptamers toward complex targets in the future. Besides SELEX, the function and activity of the ATPase SERCA were also explored in this thesis. It was discovered that nonspecific ssDNA sequences can bind to the endogenous regulator of SERCA, PLN, in a length dependent way. A highly sensitive and reproducible SERCA activity assay, which cut the use of SERCA by 2,000 folds, was developed to directly detect the product ADP via TR-FRET.