Colloidal crystal based plasma polymer patterning to control Pseudomonas aeruginosa attachment to surfaces

Abstract

Event Abstract Back to Event Colloidal crystal based plasma polymer patterning to control Pseudomonas aeruginosa attachment to surfaces Hitesh Pingle1, Peng-Yuan Wang1, 2, Helmut Thissen2, Sally Mcarthur1 and Peter Kingshott1 1 Swinburne University Of Technology,Hawthorn, Department of Chemistry and Biotechnology, Australia 2 CSIRO Manufacturing Flagship, Bay view Avenue, Clayton,, Australia Introduction: Infections from biofilm formation on medical devices is a global problem with risks to human health and huge costs to health care worldwide[1],[2]. Bacteria have unique capabilities to generate resistance against antimicrobial agents. Recent studies have shown that extra-cellular DNA is a key molecule that helps biofilms form[3],[4], but the precise mechanisms of how bacteria and DNA interact at surfaces remains unknown. It has been shown that micro- and nanotopography and chemistry on surfaces can influence initial biomolecule adsorption, and subsequent bacterial adhesion[5]-[7]. However, there are fewer reports using such approaches for selectively grafting of DNA to understand the mechanisms of initial bacterial attachment responsible for biofilm formation. Material and Methods: Colloidal self-assembly was used to pattern surfaces with colloidal crystals to use as masks against allylamine plasma polymer (AAMpp) and acrylic acid plasma (AACpp) deposition to generate highly ordered patterns from the micro- to the nanoscale. PEG-aldehyde was grafted to the plasma regions via ‘cloud point’ grafting to prevent the attachment of bacteria on the plasma patterned surface regions, thereby controlling the adhesive sites by choice of the colloidal crystal morphology. Salmon sperm DNA was physically or covalently attached on both patterned and flat surfaces. P. aeruginosa was chosen to study the bacterial interactions with these chemically patterned surfaces. Scanning electron microscope (SEM), x-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), and Epi-fluorescence microscopy were used for pattern characterization, surface chemical analysis, and imaging of attached bacteria. Results: The AAMpp, AACpp, PEG and eDNA patterns were developed using the colloidal lithography approach. XPS results confirm the successful grafting of PEG and eDNA on the plasma coated surfaces. The AAMpp surface showed increased bacterial attachment compared to control while PEG and eDNA coated surface showed reductions in bacterial attachment as highlighted in Figures 1 and 2. FIG. 1. Epifluorescence images of DAPI stained P. aeruginosa attachment to (a) a 5 µm AAMpp pattern (b) a 5 µm AAMpp-DNA pattern (c) a 5 µm AAMpp_PEG pattern (d) a 5 µm AAMpp-PEG_DNA pattern. All scale bars: 50µm. FIG. 2. SEM images of P. aeruginosa attachment onto (a) a 5 µm AAMpp pattern (b) a 5 µm AAMpp-DNA pattern (c) a 5 µm AAMpp-PEG pattern (d) a 5 µm AAMpp-PEG-DNA pattern All scale bars: 10µm. Discussion: The bacterial attachment increase on the AAMpp surface is most likely due to the amine groups displaying a positive charge that attract the negatively charged extra-cellular polysaccharides on the bacterial cell surfaces. AACpp and DNA both contain negatively charged groups thus repulsion of bacteria is the most likely cause for the reduced bacterial attachment. Micron sized plasma patterns were able to trap bacteria due to the different chemistries presented. Different sized plasma patterns were generated through the use of different sized colloidal particles used as masks for plasma deposition. Conclusion: Surface charge plays an essential role in initial attachment of bacteria. Different chemical patterns made from colloidal crystal masking are easy to fabricate and could be useful in further applications in biomaterials research. The results showed that PEG and DNA patterns can be easily fabricated with exquisite control of pattern size and spacing and this is a useful tool to help understand how bacterial attach to surfaces. The Australian Research Council is acknowledged for funding a PhD scholarship for Hitesh Pingle through the Discovery.; The Scientific Industrial Endowment Fund (SIEF) is acknowledged for providing a John Stocker Postdoctoral Research Fellowship for Peng Yuan Wang.; This work was supported in part at both the Bio-interface Engineering Hub at Swinburne and the Melbourne Centre for Nanofabrication as part of the Victorian Node of the Australian National Fabrication Facility, a company established under the National Collaborative Research Infrastructure Strategy to provide nano and micro-fabrication facilities for Australia’s researchers.