3D Microfluidics for Environmental Pathogen Detection and Single-cell Phenotype-to-Genotype Analysis

Citation

Zhu, Yanzhe (2020) 3D Microfluidics for Environmental Pathogen Detection and Single-cell Phenotype-to-Genotype Analysis. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/vk3d-7212. https://resolver.caltech.edu/CaltechTHESIS:06082020-172255300

Abstract

The emergence of microfluidic technologies has enabled the miniaturization of cell analysis processes, including nucleic acid analysis, single cell phenotypic analysis, single cell DNA and RNA sequencing, etc. Traditional chip fabrication via soft lithography cost thousands of dollars just in personnel training and capital cost. The design of these systems is also confined to two dimensions limited by their fabrication. To address the needs of smooth transition from technology to adoption by end-users, less complexity is urgently needed for microfluidics to be applied in pathogen detection under low-resource settings and more powerful integration of analyses to understand single cells. This dissertation presents my explorations in 3D microfluidics involving simulation-aided design of pretreatment devices for pathogen detection, fabrication through 3D printing, utilization of alternative commercial parts, and the combination with hydrogel material to link phenotypic analysis with in situ molecular detection for single cells. The main outputs of this dissertation are as follows:

1) COMSOL Multiphysics® was used to aid the design and understanding of microfluidic systems for environmental pathogen detection. In the development of an asymmetric membrane for concentration and digital detection of bacteria, the quantification requires Poisson distribution of cells into membrane pores; the flow field and particle trajectories were simulated to validate the cell distribution in capturing pores. In electrochemical bacterial DNA extraction, the hydroxide ion generation, species diffusion, and cation exchange were modeled to understand the pH gradient within the chamber. To address the overestimated risk by polymerase chain reactions (PCR) that detects all target nucleic acids regardless of cell viability, we developed a microfluidic device to carry out on-chip propidium monoazide (PMA) pretreatment. The design utilizes split-and-recombine (SAR) mixers for initial PMA-sample mixing and a serpentine flow channel containing herringbone structures for dark and light incubation. Ten SAR mixers were employed based on fluid flow and diffusion simulation. High-resolution 3D printing was used for prototyping. On-chip PMA pretreatment to differentiate live and dead bacterial cells in buffer and natural pond water samples was experimentally demonstrated.

2) Water-in-oil droplet-based microfluidic platforms for digital nucleic acid analysis eliminates the need for calibration that is required for qPCR-based environmental pathogen detection. However, utilizing droplet microfluidics generally requires fabrication of sub-100 µm channels and complicated operation of multiple syringe pumps, thus hindering the wide adoption of this powerful tool. We designed a disposable centrifugal droplet generation device made simply from needles and microcentrifuge tubes. The aqueous phase was added into the Luer-Lock of the commercial needle, with the oil at the bottom of the tube. The average droplet size was tunable from 96 μm to 334 μm and the coefficient of variance (CV) was minimized to 5%. For droplets of a diameter of 175 μm, each standard 20 μL reaction could produce ~10⁴ droplets. Based on this calculated compartmentalization, the dynamic range is theoretically from 0.5 to 3×10³ target copies or cells per μL, and the detection limit is 0.1 copies or cells per μL.

3) Based on the disposable droplet generation device, we further developed a novel platform that enables both high-throughput digital molecular detection and single-cell phenotypic analysis, utilizing nanoliter-sized biocompatible polyethylene glycol (PEG) hydrogel beads. The crosslinked hydrogel network in aqueous phase adds additional robustness to droplet microfluidics by allowing reagent exchange. The hydrogel beads demonstrated enhanced thermal stability, and achieved uncompromised efficiencies in digital PCR, digital loop-mediated isothermal amplification (dLAMP), and single cell phenotyping. The crosslinked hydrogel network highlights the prospective linkage of various subsequent molecular analyses to address the genotypic differences between cellular subpopulations exhibiting distinct phenotypes. This platform has the potential to advance the understanding of single cell genotype-to-phenotype correlations.

4) For effective sorting of the hydrogel beads after single cell phenotyping, a gravity-driven acoustic fluorescence-based hydrogel beads sorter was developed. The design involves a 3D-printed microfluidic tube, two sequential photodetectors, acoustic actuator, and a control system. Instead of bulky syringe pumps used in traditional cell or droplet sorting, this invention drives beads suspended in heavier fluorinated oil simply by buoyancy force to have the beads float through a vertical channel. Along the channel, sequential photodetectors quantify the bead acceleration and inform the action of downstream acoustic actuator. Hydrogel beads with different fluorescence intensity level were led into different collection chambers. The developed sorter promises cheap instrumentation, easy operation, and low contamination for beads sorting, and thus the full establishment of the single cell phenotype-genotype link.

In summary, the work in this dissertation established a) the simulation-aided design and 3D printing to reduce the complexity of microfluidics, and thus lowered its barrier for environmental applications, b) a simple and disposable device using cheap commercial components to produce monodispersed water-in-oil droplets to enable easy adoption of droplet microfluidics by non-specialized labs, c) a hydrogel bead-based analysis platform that links single-cell phenotype and genotype to open new research avenues, and d) a gravity-driven portable bead sorting system that may extend to a broader application of hydrogel microfluidics to point of care and point of sample collection. These simple-for-end-user solutions are envisioned to open new research avenues to tackle problems in antibiotic heteroresistance, environmental microbial ecology, and other related fundamental problems.

Thesis Files