Self-aligned microchip device for automated measurement of quantal exocytosis

The entire thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file; a non-technical public abstract appears in the public.pdf file.Title from PDF of title page (University of Missouri--Columbia, viewed on February 28, 2011).Dissertation advisors: Dr. Shubhra Gangopadhyay and Dr. Kevin Gillis.Vita.Ph. D. University of Missouri--Columbia 2010.Here we describe a method to fabricate a multi-channel high-throughput microchip device for measurement of quantal transmitter release from individual cells. Instead of bringing carbon-fiber electrodes to cells, the device uses a self-aligning surface chemistry approach to bring cells to an array of electrochemical microelectrodes. The microelectrodes are small and "" in order to promote adhesion of a single cell whereas all other areas of the chip are covered with a thin "cytophobic" film to block cell attachement and facilitate movement of cells to electrodes. This film also insulates unused areas of the conductive film, thus the device is "self aligned." Amperometric spikes resulting from single-granule fusion events were recorded on the device and had amplitudes and kinetics similar to those measured using carbon-fiber microelectrodes. Use of this device will increase the pace of basic neuroscience research and may also find applications in drug discovery or validation.Includes bibliographical reference

Mesoporous iron oxide energetic composites with slow burn rate, sustained pressure and reduced ESD sensitivity for propellant applications

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file.Title from title screen of research.pdf file (viewed on May 7, 2009)Includes bibliographical references.Thesis (M.S.) University of Missouri-Columbia 2006.Dissertations, Academic -- University of Missouri--Columbia -- Electrical engineering.The objective of this thesis is to synthesize a slow burning nanoenergetic formulation of mesoporous iron oxide with sustainable pressure characteristics and reduced electrostatic discharge ignition sensitivity. The choice of iron oxide is made because of its redox reaction with Al-particles. We have attempted to reduce the combustion wave velocity by infiltrating polymers inside porous Fe₂O₃ and combining it with Al-nanoparticles. Furthermore, modifications with the polymers can reduce the electrostatic discharge (ESD) ignition sensitivity of nanoenergetic composites. The composites reported in this thesis will be useful for propellant applications because these propellants in general burn at a slow rate and provide sustained pressure in MPa range for few milliseconds. Propellant formulations that are currently being investigated contain metal oxide oxidizer and fuel nanoparticles that does not show sustained pressure characteristics. This thesis presents the results obtained with the nanoenergetic composites prepared using polymers, which exhibit the desired propellant characteristics

Self-Aligned Microchip Device for Automated Measurement of Quantal Exocytosis [abstract]

Biomedical Tissue Engineering, Biomaterials, & Medical Devices Poster SessionNeurons and endocrine cells secrete neurotransmitters and hormones as a method for cell-to-cell communication through the process of exocytosis. Disruption of exocytosis underlie neurological disorders such as Parkinson's disease and the accounts for the toxicity of clostridial neurotoxins. In order to study the regulation of exocytosis it is important to carry out studies at the level of single-cells and resolve single-vesicle release events. Carbon-fiber microelectrodes are commonly used to perform single-cell measurements but are slow and labor-intensive to use. Therefore we are developing microchip devices with arrays of electrochemical electrodes for high-throughput measurement of single-vesicle release events. One challenge in the development of these devices is automatically targeting individual cells to each recording electrode. Here we describe a microchip device that uses a self-aligning surface chemistry approach to target individual cells to each electrochemical microelectrode in an array. The microelectrodes are small and “cytophilic” in order to promote adhesion of a single cell whereas all other areas of the chip are covered with a thin “cytophobic” film to block cell attachement and facilitate movement of cells to electrodes. This cytophobic film also insulates unused areas of the conductive film. Amperometric spikes resulting from single-granule fusion events were recorded on the device and had amplitudes and kinetics similar to those measured using carbon-fiber microelectrodes. Use of this device will increase the pace of basic neuroscience research and may also find applications in assaying neurotoxins and development of pharmaceuticals

Ultrasensitive detection of lipoarabinomannan with plasmonic grating biosensors in clinical samples of HIV negative patients with tuberculosis.

BACKGROUND:Timely diagnosis of tuberculosis disease is critical for positive patient outcomes, yet potentially millions go undiagnosed or unreported each year. Sputum is widely used as the testing input, but limited by its complexity, heterogeneity, and sourcing problems. Finding methods to interrogate noninvasive, non-sputum clinical specimens is indispensable to improving access to tuberculosis diagnosis and care. In this work, economical plasmonic gratings were used to analyze tuberculosis biomarker lipoarabinomannan (LAM) from clinical urine samples by single molecule fluorescence assay (FLISA) and compared with gold standard sputum GeneXpert MTB/ RIF, culture, and reference ELISA testing results. METHODS AND FINDINGS:In this study, twenty sputum and urine sample sets were selected retrospectively from a repository of HIV-negative patient samples collected before initiation of anti-tuberculosis therapy. GeneXpert MTB/RIF and culture testing of patient sputum confirmed the presence or absence of pulmonary tuberculosis while all patient urines were reference ELISA LAM-negative. Plasmonic gratings produced by low-cost soft lithography were bound with anti-LAM capture antibody, incubated with patient urine samples, and biotinylated detection antibody. Fluorescently labeled streptavidin revealed single molecule emission by epifluorescence microscope. Using a 1 fg/mL baseline for limit of detection, single molecule FLISA demonstrated good qualitative agreement with gold standard tests on 19 of 20 patients, including accurately predicting the gold-standard-negative patients, while one gold-standard-positive patient produced no observable LAM in urine. CONCLUSIONS:Single molecule FLISA by plasmonic grating demonstrated the ability to quantify tuberculosis LAM from complex urine samples of patients from a high endemic setting with negligible interference from the complex media itself. Moreover, agreement with patient diagnoses by gold standard testing suggests that single molecule FLISA could be used as a highly sensitive test to diagnose tuberculosis noninvasively

Efficient and Rapid Detection of Salmonella Using Microfluidic Impedance Based Sensing

We present a low cost, easy to fabricate biosensor, which can quickly and accurately detect Salmonella typhimurium. This study also compares the advantages of the microfluidic biosensor over a nonmicrofluidic biosensor. High density interdigitated electrode array was used to detect Salmonella cells inside a microfluidic chip. Monoclonal anti-Salmonella antibodies were allowed to be immobilized on the surface of the electrode array for selective detection of Salmonella typhimurium. An impedance analyzer was used to measure and record the response signal from the biosensor. The biosensor provides qualitative and quantitative results in 3 hours without any enrichment steps. The microfluidic biosensor’s lower detection limit was found to be 3×103 CFU/mL compared to the 3×104 CFU/mL of the nonmicrofluidic biosensor, which shows that the microfluidic biosensor has 10-fold increased sensitivity. The impedance response of microfluidic biosensor was also significantly higher (2 to 2.9 times) compared to the nonmicrofluidic biosensor

Ultrasensitive detection of lipoarabinomannan with plasmonic grating biosensors in clinical samples of HIV negative patients with tuberculosis.

BACKGROUND:Timely diagnosis of tuberculosis disease is critical for positive patient outcomes, yet potentially millions go undiagnosed or unreported each year. Sputum is widely used as the testing input, but limited by its complexity, heterogeneity, and sourcing problems. Finding methods to interrogate noninvasive, non-sputum clinical specimens is indispensable to improving access to tuberculosis diagnosis and care. In this work, economical plasmonic gratings were used to analyze tuberculosis biomarker lipoarabinomannan (LAM) from clinical urine samples by single molecule fluorescence assay (FLISA) and compared with gold standard sputum GeneXpert MTB/ RIF, culture, and reference ELISA testing results. METHODS AND FINDINGS:In this study, twenty sputum and urine sample sets were selected retrospectively from a repository of HIV-negative patient samples collected before initiation of anti-tuberculosis therapy. GeneXpert MTB/RIF and culture testing of patient sputum confirmed the presence or absence of pulmonary tuberculosis while all patient urines were reference ELISA LAM-negative. Plasmonic gratings produced by low-cost soft lithography were bound with anti-LAM capture antibody, incubated with patient urine samples, and biotinylated detection antibody. Fluorescently labeled streptavidin revealed single molecule emission by epifluorescence microscope. Using a 1 fg/mL baseline for limit of detection, single molecule FLISA demonstrated good qualitative agreement with gold standard tests on 19 of 20 patients, including accurately predicting the gold-standard-negative patients, while one gold-standard-positive patient produced no observable LAM in urine. CONCLUSIONS:Single molecule FLISA by plasmonic grating demonstrated the ability to quantify tuberculosis LAM from complex urine samples of patients from a high endemic setting with negligible interference from the complex media itself. Moreover, agreement with patient diagnoses by gold standard testing suggests that single molecule FLISA could be used as a highly sensitive test to diagnose tuberculosis noninvasively