High Aspect Ratio Nanostructures Kill Bacteria via Storage and Release of Mechanical Energy

Abstract

The threat of a global rise in the number of untreatable infections caused by antibiotic-resistant bacteria calls for the design and fabrication of a new generation of bactericidal materials. Here, we report a concept for the design of antibacterial surfaces, whereby cell death results from the ability of the nanofeatures to deflect when in contact with attaching cells. We show, using three-dimensional transmission electron microscopy, that the exceptionally high aspect ratio (100-3000) of vertically aligned carbon nanotubes (VACNTs) imparts extreme flexibility, which enhances the elastic energy storage in CNTs as they bend in contact with bacteria. Our experimental and theoretical analyses demonstrate that, for high aspect ratio structures, the bending energy stored in the CNTs is a substantial factor for the physical rupturing of both Gram-positive and Gram-negative bacteria. The highest bactericidal rates (99.3% for Pseudomonas aeruginosa and 84.9% for Staphylococcus aureus) were obtained by modifying the length of the VACNTs, allowing us to identify the optimal substratum properties to kill different types of bacteria efficiently. This work highlights that the bactericidal activity of high aspect ratio nanofeatures can outperform both natural bactericidal surfaces and other synthetic nanostructured multifunctional surfaces reported in previous studies. The present systems exhibit the highest bactericidal activity of a CNT-based substratum against a Gram-negative bacterium reported to date, suggesting the possibility of achieving close to 100% bacterial inactivation on VACNT-based substrata.

<p>Correlative ex situ and Liquid-Cell TEM Observation of Bacterial Cell Membrane Damage Induced by Rough Surface Topology</p>

“…29 Linklater et al proposed another mechanism, in which the bacteria induce mechanical forces on the surface of nanopillars upon binding. 30 These forces then deflect the nanopillars, which then induce strain on the bacteria upon relaxation of the pillars. 30 This strain damages the cell wall and induces the observed cell death.…”

“…30 These forces then deflect the nanopillars, which then induce strain on the bacteria upon relaxation of the pillars. 30 This strain damages the cell wall and induces the observed cell death. 30 While nanopillar surfaces were experimentally shown via traditional fluorescent light microscopy and scanning electron microcopy (SEM) to induce bacterial death, the mechanism of bactericidal activity was mathematically inferred in both Linklater et al and Pogodin et al rather than demonstrated experimentally.…”

“…30 While nanopillar surfaces were experimentally shown via traditional fluorescent light microscopy and scanning electron microcopy (SEM) to induce bacterial death, the mechanism of bactericidal activity was mathematically inferred in both Linklater et al and Pogodin et al rather than demonstrated experimentally. 26,30 In another mechanism, Michalska et al proposed that black silicon nanopillars directly penetrate the cell wall of the bacterium. 31 However, Linklater et al later suggested that the bacterial death observed in Michalska et al occurred via the strain mechanism.…”

Mechano-bactericidal nanopillars require external forces to effectively kill bacteria

“…Mechanical damage caused by nanopillars has been attributed by some researchers to adhesion forces which deform cells upon contact with the nanopillars 8,19 . Alternatively, bacteria may be killed by shear forces generated when attached cells attempt to move laterally on nanopillars [23][24] , or when bending energy stored in high aspect ratio nanopillars is released 25 .…”

“…This has been challenged by others who argued that in many cases, adhesion forces are not of sufficient magnitude to kill bacteria; thereby proposing additional contributions of other external forces 23,[42][43] . Other forces suggested to date include gravity 42 , motility forces 23 , and released bending energy from flexible nanopillars 25 . In our study, we encountered an experimental condition where none of the above forces seems to be dominant -as evidenced by the absence of bactericidal effect in fully immersed conditions.…”