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dc.contributor.advisorJongyoon Han.en_US
dc.contributor.authorSarkar, Aniruddhen_US
dc.contributor.otherMassachusetts Institute of Technology. Department of Electrical Engineering and Computer Science.en_US
dc.date.accessioned2013-06-17T19:56:21Z
dc.date.available2013-06-17T19:56:21Z
dc.date.copyright2013en_US
dc.date.issued2013en_US
dc.identifier.urihttp://hdl.handle.net/1721.1/79325
dc.descriptionThesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Electrical Engineering and Computer Science, 2013.en_US
dc.descriptionCataloged from PDF version of thesis.en_US
dc.descriptionIncludes bibliographical references.en_US
dc.description.abstractCells sense stimuli, process information and respond using signaling networks regulated by enzymatic activity of various proteins. Aberrations in signaling are associated with diseases such as cancer. Most current methods lack the sensitivity to measure enzymatic activity in single cells and instead measure the average of large cell populations. Cellular heterogeneity, overlooked in these methods, is widespread and relevant. Microfabricated tools are uniquely suited to single cell analysis due to the match in size scale which enables high sensitivity, high throughput measurements. In this thesis we develop a microfluidic platform for the direct measurement of enzyme activities from selected single cells without disrupting their extracellular context. We develop modules to: enhance enzyme assay sensitivity by microfluidic confinement, interface microfluidic devices with selected single cells, enable multiplexing and then integrate these modules to perform single cell assays. We first investigate electrokinetic trapping of charged biomolecules in a nanofluidic concentrator for enhancing enzyme assay sensitivity by simultaneously accumulating enzyme and substrate into a reaction plug. Non-linear enhancement of reaction kinetics in this device is predicted by a mathematical model and experimentally verified. A linear enhancement mode is developed where only the enzyme is accumulated and is reacted with substrate later in an enclosed volume defined by integrated pneumatic valves or by micro-droplets formed using an integrated droplet generator. This device is then used to perform high-throughput measurement of secreted cellular proteases. We then develop a nicrofluidic probe for lysis and capture of the contents of selected single adherent cells from standard tissue culture platforms by creating a small lysis zone at its tip using hydrodynamic confinement. The single cell lysate is then divided and mixed with different substrates and confined in small chambers for fluorimetric assays. An integrated nanofluidic concentrator enables further concentration-enhancement. We demonstrate the ability to measure, from selected single cells, the activity of kinases: Akt, MAPKAPK2, PKA and a metabolic enzyme, GAPDH - separately or simultaneously. This assay platform can correlate single cell phenotype or extracellular context to intracellular biochemical state. We present preliminary explorations of the correlation of cell morphology or local cell population density to kinase activity.en_US
dc.description.statementofresponsibilityby Aniruddh Sarkar.en_US
dc.format.extent91 p.en_US
dc.language.isoengen_US
dc.publisherMassachusetts Institute of Technologyen_US
dc.rightsM.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.en_US
dc.rights.urihttp://dspace.mit.edu/handle/1721.1/7582en_US
dc.subjectElectrical Engineering and Computer Science.en_US
dc.titleMicrofluidic concentration-enhanced single cell enzyme activity assayen_US
dc.typeThesisen_US
dc.description.degreePh.D.en_US
dc.contributor.departmentMassachusetts Institute of Technology. Department of Electrical Engineering and Computer Science.en_US
dc.identifier.oclc844761440en_US


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