Microfluidic Engineering for Ultrasensitive Molecular Analysis of cells

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dc.contributor.authorCao, Zhenningen
dc.contributor.committeechairLu, Chang-Tienen
dc.contributor.committeememberAgah, Masouden
dc.contributor.committeememberLi, Liwuen
dc.contributor.committeememberDavalos, Rafael V.en
dc.contributor.committeememberJung, Sunghwanen
dc.contributor.departmentBiomedical Engineeringen
dc.date.accessioned2017-03-29T06:00:20Zen
dc.date.available2017-03-29T06:00:20Zen
dc.date.issued2015-10-05en
dc.description.abstractThe main focus of this research was the development of microfluidic technology for ultrasensitive and fast molecular analysis of cells. Chromatin immunoprecipitation (ChIP) assay followed by next generation sequencing serves as the primary technique to characterize the genomic locations associated with histone modifications. However, conventional ChIP-seq assay requires large numbers of cells. We demonstrate a novel microfluidics-based ChIP-seq assay which dramatically reduced the required cell number. Coupled with next generation sequencing, the assay permitted the analysis of histone modifications at the whole genome from as few as ~100 cells. Using the same device, we demonstrated that MeDIP-seq with tiny amount of DNA (<5ng) generated high quality genome-wide profiles of DNA methylation. Off-chip sonication often leads to sample loss due to multiple tube transferring. In addition, conventional sonicators are not able to manipulate samples with small volume. We developed a novel microfluidic sonicator, which is able to achieve on-chip DNA/chromatin shearing into ideal fragment size (100~600bp) for both chromatin immunoprecipitation (ChIP) and methylated DNA immunoprecipitation (MeDIP). The integrated on-chip sonication followed by immunoprecipitation (IP) reaction can significantly reduce sample loss and contamination. Simple and accessible detection methods that can rapidly screen a large cell population with single cell resolution have been seriously lacking. We demonstrate a simple protocol for detecting translocation of native proteins using a common flow cytometer which detects fluorescence intensity without imaging. Using our approach, we successfully detected the translocation of native NF-kappa B (an important transcription factor) at its native expression level and examine the temporal dynamics in the process. Droplets with encapsulated beads and cells have been increasingly used for studying molecular and cellular biology. However, a mixed population of droplets with an uneven number or type of encapsulated particles is resulted and used for screening. We developed a fluorescence-activated microfluidic droplet sorter that integrated a simple deflection mechanism. By passing droplets through a narrow interrogation channel, the encapsulated particles were detected individually. The microcontroller conducted the computation to determine the number and type of encapsulated particles in each droplet and made the sorting decision. Our results showed high efficiency and accuracy for sorting and enrichment.en
dc.description.degreePh. D.en
dc.format.mediumETDen
dc.identifier.othervt_gsexam:6089en
dc.identifier.urihttp://hdl.handle.net/10919/76721en
dc.publisherVirginia Techen
dc.rightsIn Copyrighten
dc.rights.urihttp://rightsstatements.org/vocab/InC/1.0/en
dc.subjectchromatin immunoprecipitation(ChIP)en
dc.subjectnext generation sequencingen
dc.subjectmicrofluidicsen
dc.subjectdroplet sortingen
dc.subjectprotein translocationen
dc.subjectsonicationen
dc.titleMicrofluidic Engineering for Ultrasensitive Molecular Analysis of cellsen
dc.typeDissertationen
thesis.degree.disciplineBiomedical Engineeringen
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen
thesis.degree.leveldoctoralen
thesis.degree.namePh. D.en

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