Abstract
A microfluidic laminar flow cell (LFC) forms an indispensable component in single-molecule experiments, enabling different substances to be delivered directly to the point under observation and thereby tightly controlling the biochemical environment immediately surrounding single molecules. Despite substantial progress in the production of such components, the process remains relatively inefficient, inaccurate and time-consuming. Here we address challenges and limitations in the routines, materials and the designs that have been commonly employed in the field, and introduce a new generation of LFCs designed for single-molecule experiments and assembled using additive manufacturing. We present single- and multi-channel, as well as reservoir-based LFCs produced by 3D printing to perform single-molecule experiments. Using these flow cells along with optical tweezers, we show compatibility with single-molecule experiments including the isolation and manipulation of single DNA molecules either attached to the surface of a coverslip or as freely movable DNA dumbbells, as well as direct observation of protein-DNA interactions. Using additive manufacturing to produce LFCs with versatility of design and ease of production allow experimentalists to optimize the flow cells to their biological experiments and provide considerable potential for performing multi-component single-molecule experiments.
Highlights
A microfluidic laminar flow cell (LFC) forms an indispensable component in single-molecule experiments, enabling different substances to be delivered directly to the point under observation and thereby tightly controlling the biochemical environment immediately surrounding single molecules
We introduce reservoir-based LFC designs using 3D printing, addressing the challenges related to the general idea of multi-channel LFCs when a flow-free environment is necessary for carrying out biological experiments, and/or separation of nano-scale particles is essential to the experiment
We demonstrate that different types of microscopic imaging techniques including bright-field transmission, single-molecule fluorescence detection, and optical trapping are applicable when using 3D-printed LFCs
Summary
A microfluidic laminar flow cell (LFC) forms an indispensable component in single-molecule experiments, enabling different substances to be delivered directly to the point under observation and thereby tightly controlling the biochemical environment immediately surrounding single molecules. We present single- and multi-channel, as well as reservoir-based LFCs produced by 3D printing to perform single-molecule experiments Using these flow cells along with optical tweezers, we show compatibility with single-molecule experiments including the isolation and manipulation of single DNA molecules either attached to the surface of a coverslip or as freely movable DNA dumbbells, as well as direct observation of protein-DNA interactions. Trappable components may be rapidly collected in one stream and moved across the interface to a neighboring stream with a different environment This approach is used extensively to construct DNA dumbbells, which are in turn very useful for both the mechanical characterization of DNA22 and the observation of protein-DNA interactions[34]. Single-molecule experimentalists are obliged to adjust their experimental plans to be compatible with a limited number of available LFC designs
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