Three-Dimensional Chemical Profile Manipulation Using Two-Dimensional Autonomous Microfluidic Control YongTae Kim, † Kerem Pekkan, †,‡ William C. Messner,* ,† and Philip R. LeDuc* ,†,‡,§ Departments of Mechanical Engineering, Biomedical Engineering, and Biological Sciences, Carnegie Mellon UniVersity, 5000 Forbes AVenue, Pittsburgh, PennsylVania 15213-3890 Received September 22, 2009; E-mail: prleduc@cmu.edu; bmessner@andrew.cmu.edu Abstract: The ability to specify or control spatiotemporal chemical environments is critical for controlling diverse processes from chemical synthesis to cellular responses. When established by microfluidics methods, this chemical control has largely been limited to two dimensions and by the need for using complex approaches. The ability to create three-dimensional (3D) chemical patterns is becoming more critical as microfluidics is beginning to have novel applications at larger millifluidic scales, including model organism behavior, embryonic development, and optofluidics. Here, we present a simple approach to create 3D chemical patterns that can be controlled in space and time via two-dimensional (2D), single-layer fluidic modules. Not only can we employ autonomous flow in a 2D fluidic configuration to produce a 3D pattern, but with very simple changes in the 2D configuration, the chemical pattern can be “focused and defocused” within the 3D cross section. We also show that these chemical patterns can be predicted by computational fluid dynamics simulations with high experimental correlation. These simulations allow analyses of the characteristics of interface behaviors with respect to three basic yet critical parameters that need to be thoroughly considered in scaling-up from microfluidic to millifluidic research: Reynolds number (Re), inlet geometry, and channel height. The findings not only indicate proof of concept for 3D pattern creation but also reveal that a number of fluidic experiments may have inherent limitations resulting from unrecognized 3D profiles that depend on these parameter choices. These results will be useful for research areas including embryonic development, cellular stimulation, and chemical fabrication approaches. Introduction Spatiotemporal manipulation of three-dimensional (3D) chemi- cal patterns requires highly integrated microdevices that have proven successful in diverse fields ranging from biological response to chemical interface applications. 1-5 Microfabrication approaches have enabled high-throughput microcomponents (e.g., sensors, mixers, valves, pumps) to be coupled together into multilayer microfluidic devices. 6 However, focusing on simple principles to generate complex responses can yield significant technological advances. Thus, rather than continuing to miniaturize and integrate many complex elements, one may derive advantages from basic principles that have been previ- ously overlooked. A diversity of two-dimensional (2D) microf- luidic studies have been published that rely on 2D visualization of the microfluidic interfaces with conventional inverted optical microscopes to characterize the chemical distributions and patterns. 7 This simple procedure may have led to a misunder- standing of real 3D chemical profiles, especially as the scale of the fluidics transitions from the micro- to the millifluidic range. These profiles can be sensitive to parameters derived from the flow conditions and channel geometries. 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