(143g) A Multiplexed Microfluidic Platform Utilizing Normally Closed Valves for Investigation of Microbial Gene Expression

In this work, we describe a microfluidic platform that utilizes normally closed valves to enable quantitative biological measurements of gene expression and stimulus driven responses of living cells such as E. coli and P. aeruginosa. Multiplexing on-chip is achieved by having multiple cell and reagent loading compartments on the same device, which also enables single cell analysis [1]. We built a 4x6 microfluidic chip capable of high-throughput screening and on-chip mixing within each sub-chamber in combinatorial format. The device permits controlled exposure of cells in ~50 pL volume compartments to different chemical stimuli loaded in contiguous compartments. The device design is convenient for biological assays due to increased detection sensitivity since the analysis is done at microscale. In addition, the device facilitates portability and exhibits multiple advantages as a miniaturized biological assay, including low sample and reagent volumes for analysis and integration of assay steps in an “on-chip” format.

Using this device in a proof-of-concept experiment, we study bistability in gene expression associated with positive regulation in a bacterial genetic network, specifically the gene network mediating microbial growth on a lactose carbon source. Stimulation of E. coli bacterial cells in the device with varying concentrations of a lactose analog in the different compartments initiates a bistable response [2]. The cells display mixed ON-OFF response, and at high concentrations of inducer, the cells are uniformly either ON or OFF. Overall, this result emphasizes the utility of the microfluidic platform for single cell measurements over a wide range of stimuli on a single integrated chip. In ongoing work, we are using the multiplexed microfluidic device to study the effects of antibiotics on mediating cell death as a function of the growth stage of cell cycle. Overall, this research demonstrates the utility of a microfluidic platform for single cell analysis in variable environmental conditions, including antibiotics, hormones, toxins and growth factors.

References:

[1] Boedicker, J., Vincent, M., and Ismagilov, R. (2009), Microfluidic Confinement of Single Cells of Bacteria in Small Volumes Initiates High-Density Behavior of Quorum Sensing and Growth and Reveals Its Variability. Angewandte Chemie International Edition, 48: 5908–5911.

[2]Ertugrul M. Ozbudak, Mukund Thattai, Han N Lim, Boris I. Shraiman, and Alexander van Oudenaarden (2004), Multistability in the lactose utilization network of Escherichia coli. Nature, 427