Microfluidic Technologies for Bioanalytical Chemistry: Advancing Epigenetic Profiling via Chromatin Immunoprecipitation in Droplets

Abstract

Beyond the linear sequence of the genome, the epigenome dynamically controls chromatin remodeling and transcriptional poise without inducing changes in DNA sequence. These epigenetic mechanisms, particularly modifications to the histone proteins which mediate chromatin compaction, play critical roles in health and disease. To empower further successes in epigenetic therapies, current tools for understanding these systems must be improved. In particular, Chromatin Immunoprecipitation (ChIP) offers the gold standard technique for probing DNA associations with modified histones. This protocol isolates chromatin complexes (nucleosomes) for affinity purification of only those displaying the modification of interest. Final analysis of captured DNA can identify novel pathways or provide a quantitative view of current transcriptional state. Unfortunately, this assay’s large sample requirements and laborious, user-dependent protocol have generally proven prohibitive to widespread clinical deployment.

This doctoral dissertation presents the development of a semi-automated, miniaturized platform for performing the ChIP assay using droplet microfluidics, capitalizing on an assay format in which pL-scale encapsulation in immiscible oil limits sample loss and accelerates mass transfer. A wide array of supporting technologies empower dynamic control over droplet composition and reaction conditions, making each droplet analogous to a reaction vessel (but for handling up to thousands of discrete volumes every second). Finally, these platforms serially process dynamic samples sizes by adjusting the total number of droplets handled, not individual reagent conditions, suggesting the potential for dynamic scalability of ChIP across a range of sample sizes.

First, the dissertation informs the direction of the work in the context of epigenetic and microfluidic challenges. Chapter I motivates opportunities and hindrances in the analytical characterization of the epigenome from the perspective of precision medicine, providing a survey of both conventional methods and emerging microfluidic techniques. Then, it outlines strategies to address technical difficulties of the ChIP protocol using droplet-based methods. Importantly, it identifies points-of-need in droplet technologies, especially for affinity purification capabilities essential to ChIP (and to other important bioassays).

Next, the dissertation describes fundamental advances in droplet microfluidic technologies. Chapter II describes the K-channel, a multifunctional, switchable, and scalable approach to improve droplet handling and chemical manipulation. K-channels performed reagent injection (0-100% of droplet volume) and magnetically biased droplet splitting (1:1 daughter droplet ratio, 96% magnetic particles retained), among other operations. Chapter III presents translation of droplet techniques into mass manufacturable thermoplastic materials, anticipating future needs in practically deploying these technologies. Chapter IV describes the Counter-Current Continuous Phase Extraction module for determinant control over droplet packing fraction (50 - 85%) and applies it with the K-channel for high temporal resolution analysis (~100 ms) of flow behavior for confined droplets (example incubation time dispersions reduced by up to 50%).

Lastly, the dissertation describes a critical innovation in droplet microfluidic purifications and, empowered by it, the full development and characterization of the droplet-based ChIP technology. Chapter V introduces the Coalesce-Attract-Resegment Washing (CAR-Wash) platform for efficient droplet-mediated particle washing (greater than 100-fold dilutions achieved at 500 Hz processing with 98% particle capture) and demonstrates its efficacy in the bioanalytical context of affinity separations. Chapter VI fully realizes the droplet microfluidic ChIP platform with automated cell lysis, chromatin digestion, immunoprecipitation, and particle washing, achieving successful application to two modified histone targets (H3K4me3 and H3K27me3). Finally, Chapter VII concludes the work and offers future directions in both technical improvements and general directions for droplet bioassay development.