Supplementary Information

Effect of Quantity and Configuration of Attached Bacteria on Bacterial Propulsion of Micro-Beads

Bahareh Behkam and Metin Sitti

NanoRobotics Laboratory, Department of Mechanical Engineering,

CarnegieMellonUniversity, Pittsburgh, PA15213, USA

1. Fabrication of Unpatterned Polystyrene Micro-Beads

The 10 µm pure polystyrene (PS) micro-beads (72986, Sigma-Aldrich, St. Louis, MO) used in this research work were electrostatically stabilized with an alkyl sulfonate. It was observed that the surfactant interfered with the particle-protein binding leading to weak adhesion of bacteria at very small densities (approximately 1 bacterium/100 µm2).

Unpatterned beads were prepared by first removing their surfactant coating. 10% polystyrene beads were diluted in deionized (DI) water 5 times. The suspension was then centrifuged at 800g and resuspened in 1:1 DI water/Isopropyl alcohol (IPA). The wash procedure was repeated five times to ensure removal of substantial amount of the adsorbed surfactant, thus, causing the bacteria to uniformly coat the surface of the unpatterned beads at an adhesion density of approximately 1 bacterium/7 µm2 (Fig.S1(a)).

2. Fabrication of Patterned Polystyrene Micro-Beads

A plasma-based patterning technique was developed to prepare the patterned micro-beads. Depending on plasma power, process time, and the type of gas used, exposure of polystyrene to gas plasma leads to etching of up to few nanometers from the surface.S1,S2Moreover, plasma treatment provides a rich variety of chemical functionalities at the surface, leading to attraction of proteins to the surface by electrostatic or dipolar interactions. 10% polystyrene bead suspension was diluted in DI water 10 times and the diluted suspension was washed 3 times.For plasma etching, a monolayer of PS beads, approximately 4 mm in diameter was prepared on a glass coverslip, usinga previously developed particle self-assemblymethod.S3 Briefly, a 3 mm thick layer of polydimethylsiloxane (PDMS) (Sylgard 184, Dow Corning, Midland, MI) was prepared according to the manufacturer's instruction. A circle, approximately 3 mm in diameter was cut out of the PDMS layer. The perimeter of the circle was cut slantwise to allow wetting of the walls and to provide a concave air-water interface. The PDMS ring was then placed on a glass slide and an aliquot portion of the 1% PS bead suspension (the volume was calculated to provide a dense monolayer of the beads after evaporation of water) was deposited within the circle. The assembly was placed in a 10 cm Petri dish and covered to reduce the evaporation rate, thus allowing for the formation of the monolayer. After 24 hours, the glass slide was taken out and the PDMS ring was removed. To pattern the beads, the monolayer bead sample was placed in an air plasma cleaner (PDC-32G, Harrick Plasma, Ithaca, NY) at 18 W RF coil power for 2.5 minutes. This process etched the portion of the micro-bead that was not masked by the underlying glass slide or the adjacent beads, revealing a functionalized hydrophilic surface. Afterwards, the sample was immediately placed in motility medium (0.01 M potassium phosphate, 0.067 M sodium chloride, 10-4 M EDTA, 0.01 M glucose, and 0.002% Tween-20, pH 7.0) S4 and sonicated for 2.5 minutes to release the beads from the glass slide. Bacteria attach to the plasma treated regions of the patterned beads at a density of approximately 1 bacterium/12 µm2; while very few or none adhere to the untreated area (Fig.S1(b)).

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