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 FLAGELLATED BACTERIA: MICROFLUIDIC MIXERS AND PUMPS By Dustin Hughes February 24, 2009 In partial fulfillment of the requirements of the course ME497/ CHEM 470 Microfluidics Professor T. Adams Professor D. Morris DEPARTMENT OF MECHANICAL ENGINEERING ROSE‐HULMAN INSTITUTE OF TECHNOLOGY The development of microfluidic devices has led to a variety of different methods of testing, and with these new testing methods comes the difficulty in dealing with such small amounts of fluids. Fluids of the micro scale tend to be laminar, which proves to be problematic when mixing of multiple fluids is desired. One major obstacle faced by microfluidicians is promoting mixing in fluids in the absence of turbulent flow. A solution to this problem has been found by utilizing the flagella located on some bacteria as mixers. Min Jun Kim, of Brown University, investigated the use of flagellated bacteria as microfluidic mixers and pumps in his PhD Thesis, “Bacterial Flows: Mixing and Pumping in Microfluidic Systems Using Flagellated Bacteria.” In particular, he studied the use of Esherichia coli (E.coli) and Serratia Marcescens to increase the diffusion of two fluids, as well as, the flow velocity of fluids in microchannels. His experiments show the effectiveness of bacteria as microfluidic mixers and pumps and the external stimuli which allow for their control. Prior to the testing process, flagellated bacteria is injected into the microchannel and allowed to grow on the walls, creating what is called a “bacteria carpet.” Most of the bacteria deposits separately with the microchannel wall, with only 20% partially overlapping each other. When injecting, the microchannel sometimes needs to be rotated to ensure coverage on all sides of the wall, with a total of approximately 85% of the microchannel wall becoming covered by bacteria when finished. For the flagella, also referred to as cilia, to be utilized appropriately, they need to be oriented within 30o perpendicular to the fluid flow. Figure 1 is a schematic of the system along with a picture of the bacteria. As you can see, the size of the bacteria allows for ease of use within microchannels. For testing procedures, Serratia Marcescens and E. Coli bacteria were used. A basic diagram of flagellated bacteria is shown in Figure 2. The filament represents the flagella. Appendix A shows the process of etching the microchannel (Figure A1), as well as the microchannel layout (Figure A2) and testing equipment (Figure A3). The serratia bacteria were found to more readily adhere to PDMS than a glass substrate. Fluorescence staining is inexpensive, fast, and easy to use. Fluorescence detection made it possible to visualize movement, shape, and size of the bacteria. These carpets may be manipulated by altering its temperature, food, and the specific wavelength of light acting on the carpet in such a way as to perform as a mixer. An additional benefit of these carpets is that the flagella may also act as a pump to propel the fluid down the microchannel, thus reducing the need for bulky power supplies or other expensive equipment. The effectiveness of mixing two fluids can be measured by way of tracking the dispersion of small particles. The diffusion coefficient, diffusion rate, and associated time scale are used to quantify the dispersion of these particles. Table 1, taken from Kim’s Thesis, compares the diffusion numerically between microchannels without bacteria, with non‐motile carpets, and with a live carpet. The larger the numbers for D, α, and τ represent higher diffusion and more mixing. Three external stimuli can be inflicted upon the bacteria to facilitate the diffusion between two fluids. The addition of more food into the system by increasing the amount of glucose in the surrounding buffer increases cellular activity, providing more pumping velocity as well. Raising the temperature of the surrounding buffer boosts motor activity, aiding the mixing process. The ability for the bacteria to interact with a larger percentage of the fluid clearly has a positive impact on the capability to mix and provide pumping power. Thus, narrower channels allow for superior pumping velocity and improved mixing. There are a few disadvantages with the utilization of live bacteria in that they must be obtained and maintained properly in order to ensure the best performance. With the fragility of the bacteria and the sensitivity of being affected so easily by external stimuli, it is essential to have the capabilities to control the testing environment. In addition, performance also largely depends on sufficient bacteria coverage of the microchannel walls. With the expansion of microfluidics and the ability to produce micro‐sized objects efficiently and accurately, investigation has begun in the creation of artificial flagella and flagella‐like appendages. Figure 4 illustrates the use of polymer‐based artificial cilia. The cilia respond to electric and magnetic fields to induce flow and mixing and can be strategically placed to maximize turbulent behavior. Actuation is achieved by applying a voltage difference from the ITO electrode to the Cr layer, which causes the cilium unroll. Elastic recovery ensues upon removal of the applied voltage, and the cilium returns to its coiled position. The ability to effectively and consistently produce mixing and increased fluid flow with minimal effort or equipment gives the field of microfluidics the ability to investigate a wider range of applications. Immobile, mobile, and artificial bacteria allow for the enhanced diffusion of particles between two microfluids, with mobile bacteria having the additional ability to pump fluids as well. References Berger, M. “Live bacteria as mechanical actuators in fluid systems.” Nanowerk LLC. http://www.nanowerk.com/spotlight/spotid=30x.php. January 2008. Kim, M. J. “Bacterial Flows: Mixing and Pumping in Microfluidic Systems Using Flagellated Bacteria,” Thesis. Brown University. May 2005. Meijer, H.E.H., Singh, M.K., Kang, T.G., Toonder, J.M.J., Anderson, P.D. “Passive and Active Mixing in Microfluidic Devices.” Appendix A Figure A1: Photolithography Technique Figure A2: Test geometry of the microchannel including placement of cameras for detection of fluid diffusion. The microchannel was 40 μm deep, 200 μm wide, and 28 mm in length. Photos were taken at 0.5, 4, 8, 12, 16, 20, 24 mm from the Y‐junction. Figure A3: Diagram of testing equipment.