Nano-engineered Sanitation Surfaces for Prevention of Bacterial Adhesion

Introduction: The bacterial adhesion mechanism is considerably complex and many factors can affect adhesion scenarios. Although many techniques have been tried to minimize the surface contamination resulting from bacterial adhesion, the effect of nanoscale surface patterns with modulated surface wettability to the bacterial adhesion was still under investigation. Cell adhesion in vivo is a three-dimensional (3D) phenomenon that is distinct from the interaction on two-dimensional (2D) surfaces in vitro. The attachment of bacterial cells is associated with various cell-surface interactions including hydrodynamic force, porosity, surface energy, and roughness. Recent advancements in fabrication have made it possible to create well-organized nanofeatures (i.e. nanoporous and nanopillared) uniformly over a large surface area of a metal specimen. However, it has not yet been studied systematically how such well-regulated nanofeatures affect the adhesion of bacteria and the formation of biofilms in various surface wetting conditions.

Purpose: This study was aimed to explore how nanoscale surface patterns with modulated surface wettability would affect the bacteria adhesion and potentially prevent biofilm formation.

Methods: Nanosmooth (control) and nanoporous stainless steel surfaces were fabricated by anodizing the degreased specimen in a 5% vol. of perchloric acid in anhydrous ethylene glycol. At this step, the back side (unpolished surface) of the specimen was covered with a water resistant tape to prevent the effect of surface irregularities on the intensity and direction of the electric field. Thereafter, the taped specimen was served as an anode and a platinum foil was used as a cathode. The applied voltage and anodization time were varied to obtain different pore diameters. In order to achieve 50 and 80 nm in pore diameters, the anodizing voltage and time combinations were 40V for 10 min and 50V for 35 min, respectively. A field emission scanning electron microscope and atomic force microscope (AFMs) with silicon tip coated with reflective aluminum coating were used to visualize the micro/nanoscale surface morphologies and determine surface roughness of the developed surfaces. For bacterial quantification, sample plates were dried in the air then a droplet of fluorescence stain namely 4,6-diamidino-2-phenylindole (DAPI) and a glass cover slip were placed on the surfaces in order to make bacterial cells easily observed under the fluorescence microscope.

Results: The presences of 50 and 80 nm nanoporous patterns significantly inhibited the adhesion of L. monocytogenesby 2.0 to 2.3 log-cycles, depending on the pore diameter. However, an increase in the pore diameter from 50 to 80 nm did not significantly increase the anti-adhesion effect of the nanoporous surface; hence, the 50 nm pore size which required less power consumption and time to fabricate (40V 10 min) was preferable. Both of the nanoporous surfaces were fabricated to have three-dimensional porous structures which had ‘peak to valley’ distances of 27 to 33 nm, depending on the pore sizes. Therefore, during adhesion test the bacterial cells were limited to contact and interact with only the peak areas of the nanofeatures. As a result, the attractive interactions between the cells and substratum, which were maximum at the cell-substratum distance of less than 15 nm, were limited and most of the cells which were not stably anchor to the surface could possibly remain in a planktonic state in the aqueous suspension.

Significance: The anodization technique developed in this study is simple, scalable, and adaptable to various types of materials so that the 3D nanopillared substrates would be of great significance in many anti-microbial applications, such as water supplies, biomaterials, and food processing, where the biofilm formation could be widespread public health problems.