Fluorescent Detection of Chromatin using Functionalized Magnetic Beads on a Digital Microfluidic Device

Abstract

<p> Epigenetics is the study of inheritable mechanisms and factors that regulate gene expression. Although the underlying genetic sequence is the same in every cell, it is the epigenome that controls the expression of these genes and accounts for differences in phenotype. Epigenetic controls have clinical ramifications from cancer to autoimmune disorders to psychiatric pathologies. The main tool to study epigenetics is chromatin immunoprecipitation (ChIP), which probes the relationship between the underlying DNA and its structural histone proteins. Standard benchtop ChIP has five major drawbacks: (1) it requires a large input volume of cells, (2) it is very time consuming, work intensive, and low throughput, (3) it suffers from poor chromatin yield and sensitivity, (4) ChIP antibodies can be non-specific, vary by batch, and have low sensitivity, (5) and ChIP performs bulk tissue analysis which loses the granularity necessary to detect cell-to-cell variations. Digital microfluidic biochips (DMFBs) have proven successful at utilizing small volumes of reagents and samples to perform high throughput analyses using a variety of assaying techniques, making them an ideal platform for ChIP adaptation. Droplet manipulation using electrowetting-on-dielectric, in conjunction with magnetic bead control using magnetic field gradients generated by a current running through a wire on the device, provide all the necessary functionality to successfully run ChIP more efficiently on a DMFB. Translation of the benchtop ChIP protocol onto a DMFB addresses the issues facing epigenetic study workflow. The smaller volumes reduce reaction time, decrease reagent and sample use, and increase sensitivity and granularity towards single-cell resolution. Automation makes ChIP less labor consuming. DMFB platforms can be expanded for parallel operation and multiplexing thus increasing throughput. Finally, streamlining all the steps of ChIP onto one device greatly reduces sample loss, thereby expanding the type of studies possible. Herein, specifically modified nucleosomes and human chromatin were detected in a new semi-quantitative fluorescent immunoassay on a DMFB. Furthermore, chromatin was immunoprecipitated using a new targeted biotinylated technique. Successful chromatin capture and detection is a powerful tool for ChIP protocol development. This approach provides a rapid method to screen for antibody specificity and sensitivity as well as a confirmatory check point in the overall ChIP protocol to ensure that the target analyte has been isolated prior to any downstream analyses. Finally, a new modified ‘pull-through’ DMFB design was introduced to enhance the capture and detection of analyte-bound magnetic beads. The contributions from the studies described in this dissertation have provided the first steps towards ChIP implementation on a DMFB: 1) Developed new fluorescent confirmatory chromatin and nucleosome immunoprecipitation assays.</p><p>2) Demonstrated that the immunoprecipitation assays were detectible on-chip without any complex downstream analyses nor specialized fluoroscopy instrumentation.</p><p>3) Demonstrated that the immunoprecipitation assays performed at higher sensitivity than traditional benchtop ChIP.</p><p>4) Developed a single-channel pixel intensity measurement system for semi-quantitative analysis of chromatin and post-translationally modified nucleosomes directly on-chip.</p><p>5) Designed a new DMFB for improved capture of magnetic beads with twice the measured signal intensity using a new pull-through droplet scan method with on-chip embedded magnetic controls.</p>