We present the fabrication, characterization and cell culture results of a microfluidic device for generating steep gradient interfaces of small molecules (<1 kDa) across cell culture with no convective shear stresses applied to the cells. We use a novel streamline of two fluids to generate stable and uniform gradient interfaces/boundaries by confronting one fluid with the other. We separate a gradient generation channel and a cell culture channel by a polyester membrane so that viscous shear stress by the bottom channel flow does not convectively disturb the chemical environment of cultured cells seeded on the membrane in the top channel. Using two-component dyes to characterize the steepness of the diffusional interface, we demonstrate 50 μm wide steps for about 400 Da molecules. Using BCECF, a 689 Da pH-sensitive diffusible dye which is actively taken up by living cells, we demonstrate gradient boundaries narrower than five cell diameters in HeLa culture. We also demonstrate steep gradients of pH across cells in the same device. This work should be of interest to researchers attempting to generate gradients of small, rapidly diffusing molecules for studies in cellular differentiation and signaling.

We present the design, modeling, fabrication and testing of a microsystem for the electrolytic patterning and sensing of oxidative microgradients within 1 x 1 mm2 area during cell culture. The system employs an array of microfabricated electrodes (3-40 microm in width) embedded in gas-permeable microchannels to generate precise doses of dissolved oxygen (ranging from 10 fmol O2 mm(-2) s(-1) to 100 nmol O2 mm(-2) s(-1)) via electrolysis. The microgradients generated by different microelectrodes in the array can be superimposed to pattern multi-dimensional oxygen profiles not possible with other methods. We demonstrate the patterning, sensing and quantification of dissolved oxygen microgradients in the 0 to 40% dO2 range using this microsystem. Reactive oxygen species generation and dosing is also quantified. Lastly, we demonstrate how the microtechnology enables new types of experiments in three different cell culture models: localized hyperoxia-induced apoptosis in C2C12 myoblasts, dynamic aerotaxis assays of Bacillus subtilis, and studies of calcium release in an ischemia/re-oxygenation myoblast model.

We present a new microfluidic method for dynamically assaying bacterial aerotaxis and controlling taxis-based bacterial self-assembly.The method utilizes an electrochemical oxygen-generating technology previously developed by our group [1]. This device uses electrolysis at many microelectrodes to generate controlled doses of dissolved oxygen; these doses can be superimposed to pattern microscale gradients with precise spatial and temporal resolution.We demonstrate our method by performing aerotaxis experiments with B.subtilis, an obligatory aerobe. We show how this method of generating oxygen gradients with microscale resolution can be used to perform: 1) dynamic response assays of traditional bacterial suspensions not possible with existing methods, and 2) studies on oxygen responses of single bacterium. We also show that the basic premise only functions in a microscale environment. Lastly, we demonstrate how this method can be used to self-assemble large-density and small numbers of bacteria into pre-determined patterns using aerotaxis.