Development and Application of Technologies for Liquid Manipulation on Droplet Microarrays

This doctoral thesis investigates the development and application of strategies for liquid manipulation on Droplet Microarrays (DMAs), a platform that offers a distinctive approach for confining stationary liquids in a planar, parallel, and miniaturized format. Unlike other microfluidic systems, which typically involve continuous flow, closed compartmentalization, or the use of sophisticated components like pumps, valves and actuators, DMAs provide a simpler, open-format system. This system positions samples as nanoliter droplets in a static, easily indexable 2D array format, resembling the familiar format of well plates but with the capacity to conduct from 0.4 to 31.8 experiments per square centimeter. This similarity facilitates the integration and adaptation of both standard and new laboratory protocols into a miniaturized setting.
This work is driven by the growing demand for miniaturization, parallelization, and sustainability in scientific research, particularly in fields such as drug discovery, cell-based high-throughput screening, and omics. By developing and refining strategies for DMAs, this
thesis aims to expand their use in these areas, providing a practical approach to integrating traditional laboratory protocols into a miniaturized format.
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The thesis begins by introducing foundational concepts for understanding and controlling droplet evaporation on planar patterned arrays used in cell culture. A mathematical model, based on the Cahn-Hilliard phase-field approach, predicts evaporation dynamics on droplet arrays. The simulations reveal critical interactions between humidity, temperature, and neighboring droplets due to the shielding effect. These findings, validated by experimental data, enabled the creation of a digital twin of the DMA, allowing for the early prediction of high-risk evaporation events during biological assays, particularly those involving sample incubation or heat treatment. This work highlights the importance of optimizing wettability and patterning, the dynamics of droplet positioning and spacing within the array, and precise control of environmental parameters to ensure the reproducibility of miniaturized experiments,
particularly in applications such as high-throughput screening.
The second part of this work presents the further development of a strategy called the “sandwiching method” for manipulating nanoliter-scale droplets. This technique facilitates parallel medium replenishment and cell manipulation, enabling long-term culture of cell spheroids on-chip. It also allows for the non-destructive and non-invasive transfer and merging of these cellular structures between DMAs, moving hundreds of them simultaneously in a single step. This method opens new experimental possibilities, including seamless collection, transfer, harvesting and sampling, all without interrupting ongoing experiments.
The final section describes the design and development of the Automated Nanoliter Droplet Selection and Collection Device (ANDeS), which integrates DMAs with non-optical analytical instruments. ANDeS is designed to address the challenges of handling and transferring nanoliter-sized droplets, minimizing sample loss while allowing for efficient sample transport.
This device shows great potential in miniaturized applications, enabling the collection and transfer of biological samples contained in nanoliter droplets into fused silica capillaries for subsequent analysis. Experimental results demonstrate that ANDeS significantly enhances the versatility of the DMA platform by enabling automated retrieval of nanoliter samples, a task not feasible manually at the individual droplet level. This advancement marks a major step towards integrating DMAs with existing analytical pipelines, thereby facilitating a broader range of experimental readouts.
Throughout the thesis, the underlying theme is the transformation of laboratory practices towards miniaturization and parallelization. Advancements in DMA technologies promise to significantly reduce resource consumption, increase experimental throughput, and accelerate the development of biological and chemical assays. By addressing key challenges and proposing innovative solutions, this work offers new on-chip experimental strategies and methodologies, ultimately bridging the gap between miniaturized assays and broader experimental applications.