Droplet-based systems for analysis of single biological targets

Abstract

The stochastic expression of genes, proteins and metabolites generate a complex and diverse reservoir of biological targets, including eukaryotic cells and viruses. Yet, traditional population-based analytical assays hinge on the measurement and analysis of averaged signals, failing to probe the information held on a minor subpopulation or individuals. Despite the enabled handling of single targets by some emerging tools, the time-consuming operations and poorly controlled processing significantly limit their widespread use. Consequently, many fundamental problems regarding the specificity of biological systems have yet to be elucidated owing to the lack of approaches for efficient isolation and processing of single cells and single viruses. By confining samples within segmented compartments, droplet microfluidics has demonstrated unique advantages in isolating and manipulating individual biological targets. The rapid generation enables high-efficiency loading of single targets, while the functional manipulations allow independent processing of the samples within the compartments. Despite the superiority in handling single targets, however, some bottlenecks remain to pose a barrier between the droplet microfluidics and in-depth analysis of single targets. First, the carrier oil, which serves as a natural barrier to prevent sample diffusion, tends to decrease the biocompatibility and prevent physical access to the droplet contents. Second, the droplets after microfluidic processing are normally converged and broken in one reservoir, hampering retrieval and off-chip analysis of single targets. Third, the low-throughput of droplet barcoding and indexing limit comprehensive analysis of the diversity and specificity of biological systems. In this thesis, a series of droplet-based systems are developed to bridge the gap between droplet microfluidics and in-depth analysis of single cells and single viruses. First, to increase the biocompatibility, an integrated system to solidify the droplets is presented. The biocompatible matrix enables prolonged culture of single cells within the compartments. Second, a high-throughput strategy to generate and sort water-in-water droplets is developed. The all-aqueous environment facilitates physiological study of single cells with high throughput. Third, for analysis of individual cells after droplet processing, an automated droplet collection system is constructed. Through alternate sorting, dispensing and collection of droplets in three branch channels, the droplet number can be precisely controlled down to single one. The precise operations enable capturing single cells into the microplate and studying their heterogeneity upon virus infection. Fourth, to process and analyze single viruses within the droplets, a single-droplet extraction system is presented. Through encapsulation of single viruses followed by amplification and extraction of their genomes, the samples can be sequenced and analyzed at single-virus level. Fifth, to analyze the diversity of B cell receptors, a high-throughput droplet barcoding system is developed. Through barcoding individual B cell receptors and epitopes within the droplets, the interactions between antibodies and antigens can be precisely mapped. To summarize, the presented systems can facilitate more comprehensive understanding of the diversity and specificity of biological systems.