Whole gene amplification and protein separation from a few cells.

Despite the growing interest to explore untapped microbial gene and protein diversity, no single platform has been able to acquire both gene and protein information from just a few cells. We present a microfluidic system that simultaneously performs on-chip capillary electrophoresis for protein analysis and whole genome amplification (WGA), and we demonstrate this by doing both for the same cohort of cyanobacterial cells. This technology opens avenues for studying protein profiles of precious environmental microbial samples and simultaneously accessing genomic information based on WGA.

Particle sorting using a porous membrane in a microfluidic device.

Porous membranes have been fabricated based on the development of the perforated membrane mold [Y. Luo and R. N. Zare, Lab Chip, 2008, 8, 1688-1694] to create a single filter that contains multiple pore sizes ranging from 6.4 to 16.6 µm inside a monolithic three-dimensional poly(dimethylsiloxane) microfluidic structure. By overlapping two filters we are able to achieve smaller pore size openings (2.5 to 3.3 µm). This filter operates without any detectable irreversible clogging, which is achieved using a cross-flow placed in front of each filtration section. The utility of a particle-sorting device that contains this filter is demonstrated by separating polystyrene beads of different diameters with an efficiency greater than 99.9%. Additionally, we demonstrate the effectiveness of this particle-sorting device by separating whole blood samples into white blood cells and red blood cells with platelets.

Patterning alginate hydrogels using light-directed release of caged calcium in a microfluidic device.

This paper describes a simple reversible hydrogel patterning method for 3D cell culture. Alginate gel is formed in select regions of a microfluidic device through light-triggered release of caged calcium. In the pre-gelled alginate solution, calcium is chelated by DM-nitrophen (DM-n) to prevent cross-linking of alginate. After sufficient UV exposure the caged calcium is released from DM-n causing alginate to cross-link. The effect of using different concentrations of calcium and chelating agents as well as the duration of UV exposure is described. Since the cross-linking is based on calcium concentration, the cross-linked alginate can easily be dissolved by EDTA. We also demonstrate application of this capability to patterned microscale 3D co-culture using endothelial cells and osteoblastic cells in a microchannel.

Efficient formation of uniform-sized embryoid bodies using a compartmentalized microchannel device.

The formation of spherical aggregates of cells called embryoid bodies (EBs) is an indispensable step in many protocols in which embryonic stem (ES) cells are differentiated to other cell types. Appropriate morphology and embryo size are critical for the sequential developmental stages of naturally conceived embryos. Likewise, regulating the size of EBs and the timing of their formation is crucial for controlling the differentiation of ES cells within the EB. Existing methods of formation of EBs, however, are tedious or provide heterogeneously-sized EBs. Here we describe a microfluidic system for straightforward synchronized formation of uniform-sized EBs, the size of which can be controlled by changing the cross-sectional size of microchannels in the microfluidic device. The device consists of two microchannels separated by a semi-porous polycarbonate membrane treated to be resistant to cell adhesion. ES cells introduced into the upper channel self-aggregate to form uniformly-sized EBs. The semi-porous membrane also allows subsequent treatment of the non-attached EBs with different reagents from the lower channel without the need for wash out because of the compartmentalization afforded by the membrane. This method provides a simple yet robust means to control the formation of EBs and the subsequent differentiation of ES cells in a format compatible for ES cell processing on a chip.

Leakage-free bonding of porous membranes into layered microfluidic array systems.

The integration of semiporous membranes into poly(dimethylsiloxane) (PDMS) microfluidic devices is useful for mass transport control. Several methods such as plasma oxidation and manual application of PDMS prepolymer exist to sandwich such membranes into simple channel structures, but these methods are difficult to implement with reliable sealing and no leakage or clogging for devices with intricate channel features. This paper describes a simple but robust strategy to bond semiporous polyester and polycarbonate membranes between layers of PDMS microchannel structures effectively without channel clogging. A thin layer of PDMS prepolymer, spin-coated on a glass slide, is transferred to PDMS substrates with channel features as well as to the edges of the semiporous membrane by stamping. This thin PDMS prepolymer serves as "mortar" to strongly bond the two PDMS layers and seal off the crevices generated from the thickness of the membranes. This bonding method enabled the fabrication of an 8x12 criss-crossing microfluidic channel array with 96 combinations of fluid interactions. The capability of this device for bioanalysis was demonstrated by measuring responses of cells to different color fluorescent reagents.

Arrays of horizontally-oriented mini-reservoirs generate steady microfluidic flows for continuous perfusion cell culture and gradient generation.

This paper describes the use of arrays of horizontally-oriented reservoirs to deliver liquids through microchannels at a constant flow rate over extended periods of time (hours to days). The horizontal orientation maintains a constant hydraulic pressure drop across microfluidic channels even as the volumes of liquids within the reservoirs change over time. For a given channel-reservoir system, the magnitude of the flow velocity depends linearly on the height difference between reservoirs. The simple structure and operation mechanism make this pumping system versatile. A one-inlet-one-outlet system was used to continuously deliver media for perfusion cell culture, and an array of inlet reservoirs coupled to an outlet reservoir via microchannels was used to drive flows of multiple laminar streams. The parallel pumping scheme conveniently generated various smooth and step concentration gradients, and allowed evaluation of the effect of colchicine on myoblasts. Since the reservoir arrays are configured to be compatible with commercialized multichannel pipettors designed for 96 well plate handling, this simple pumping scheme is envisioned to be broadly useful for medium to high throughput microfluidic perfusion cell culture assays, cell migration assays, multiple laminar flow drug tests, and any other applications needing multiple microfluidic streams.