INTRODUCTION: The Silanization Reaction in the Design of Antimicrobial Surfaces The chemisorption of organosilanes to oxide surfaces (i.e., quartz, SiO2, or TiO2) by means of silanization is an important method of binding biologically active molecules to a solid surface. 1, 2 The original study reported back in 1972 describing antibacterial contact active surfaces was based on a quaternary ammonia biocide covalently bound to biomaterial surfaces. 3 Two reports in the literature on titanium modified surfaces with bactericidal activity have been reported. Originally, Nikawa et al. (2005) immobilized an organosilicon quaternary compound, 3-(trimethoxysilyl)-propyldimethyl-octadecyl ammonium chloride (Si-QAC) on titanium metal via a silanization reaction between the metal surface and the organosilane (Ti-O-Si-QAC). 4 Similarly Gabriel et al. (2007) covalently immobilized an antimicrobial peptide (AMP) cathelin LL37 reponsible for the disruption of microbial membranes on to Titanium via an organo silane (Ti-O-Si-AMP). Note surface pretreatement required sanding, degreasing, and curing with heat. The type and condition of the metallic surface dictates the quality of bonding interaction of the silane coupling agents with the metals. To obtain a stong and even coating the metal substrates are first degreased ultrasonically in various solvents such as hexane, ethanol, and acetone for 3-5 min, and then cleaned in a diluted alkaline cleaner (AC1055, provided by Brent P L C, Lakebluff, I L, USA) at 60-70ºC for 3-5 min, after which they are rinsed with deionized (DI) water and air dried. 5 A typical Silanization Reaction on Titanium plates from the literature is given below: Ti platelets of 10 10 mm 2 size were cut from 2 mm thick foil. One side of each platelet was ground with 600- and 1200-grid sandpaper and cleaned by etching in a solution of 9% NH4F HF (w/v), 8.5% H2SO4 (v/v), and 0.5% urotropin (w/v) in deionized water for 2 min at room temper- ature. Passivation was accomplished by treatment with a 1:1 mixture of 30% H2O2 and concentrated H2SO4 (Piranha solution) for 1 h. Ti specimens were silanized with a solution of 2% 3-aminopropyl 1
triethoxysilane (amino silane) and glycidyloxy- propyl triethoxysilane (epoxy silane), respectively, in dry toluene for 18 h at room temperature. After being washed with toluene and methanol, the silanized samples were cured at 80 C for 1 h. 6 References 1. Rezania, A., Johnson, R., Lefkow, A. R. & Healy, K. E. Bioactivation of Metal Oxide Surfaces. 1. Surface Characterization and Cell Response. Langmuir 15, 6931-6939 (1999). 2. Nanci, A. et al. Chemical modification of titanium surfaces for covalent attachment of biological molecules. J. Biomed. Mater. Res. 40, 324-335 (1998). 3. Isquith, A. J., Abbott, E. A., and Walters, P. A. Surface- Surface bonded antimicrobial activity of an organosilicon quaternary ammonium chloride. Appl. Microbiol. Appl. Microbiol 2, 859 (1972). 4. Nikawa, H. Ishida, K. Hamada, T. Satoda, T. Murayama, T. Takemoto, T. Tamamoto, M. Tajima, H. Shimoe, S. Fujimoto, H. Immobilization of Octadecyl Ammonium Chloride on the Surface of Titanium and Its Effect on Microbial Colonization In Vitro. Dent. Mater. J. 24, 570 (2005). 5. van Ooij, W. J. et al. Corrosion Protection Properties of Organofunctional Silanes An Overview. Tsinghua Science & Technology 10, 639-664 (2005). 6. Gabriel, M., Nazmi, K., Veerman, E. C., Nieuw Amerongen, A. V. & Zentner, A. Preparation of LL-37-Grafted Titanium Surfaces with Bactericidal Activity. Bioconjug. Chem. 17, 548-550 (2006). 2
DETERMINATION OF ADHESION AND COVERAGE OF AN ANTIMICROBIAL AEROSOL WATER-BASED COATING ON POROUS AND NON-POROUS SURFACES RESULTS: LEACHING, or desorption of QAC The general trend in terms of surface coverage = porous surfaces >>> non-porous surfaces. Nonporous surfaces (wood, cloth) show a significantly larger amount of BSD uptake vs. non-porous surfaces (s.steel, glass, plastic). The difference is around 87-97% more BSD bound to non-porous surfaces per square inch. SG S G 9 0 SURFACE GUARD 90 electrosprayed wood and laundered bedsheets (porous surfaces) resulted in strong adhesion of the molecule to the surface with no excess removed into the solution. Non-porous surfaces showed poor adhesion with the majority of SURFACE GUARD 90 electrosprayed removed during the photometric analysis*: For example spraying with 0.5% BSD for 3 sec/ side resulted in >30ug/inch 2 application of BSD on the surface. Excess BSD was removed and complexed by DCF in solution (solution turned pink, shift in absorption due to the complex). Removing or decomplexing excess pink stain from the surface (DCF exchanged by anionic surfactant soap) revealed the actual amount of BSD bound to the surface. Note: excess complex in solution can bind the surface. Titrating the water from the overnight soak with tetraphenylborate reveals the amount of SURFACE GUARD 90 removed from the surface and correlates well with the spectrophotometic value. Destaining the soaked samples confirms the results * Note: (i) Stainless Steel contains a lower density of hydroxyl groups compared to titanium oxide (TiO2), (ii) the silanization pre-treatment only employed a degreasing wash with ethanol excluding sanding and curing steps (iii) the amount of 3
SURFACE GUARD 90 on steel, glass and plastic are averages only after one wash whereas the bedsheet amounts include the average from a five and ten wash/dry laundered samples. POROUS SURFACES: BEDSHEET (CLOTH): SurfaceGuard90 S G 9 0 - The controls showed no changes in absorbance (no complex formation) - The 1-wash and dry cycle (1A) bedsheets have the lowest amount of SURFACE GUARD 90 and show irregular SURFACE GUARD 90 application (patches of DCF or BPB staining), see appendix I. This is a result of too many sheets in the machine's capacity specifically some sheets were folded or creased and SURFACE GUARD 90 could effectively reach. - The results indicate when a sheet is exposed the SURFACE GUARD 90 the binding is strong and maintained even after 10 wash and dry cycles. 4
5) WOOD: Wood sample were sprayed half way from the nozzle and the recommended spray distance (the tip) due to stronger BPB staining. The higher concentration of BSD sprayed the more is applied. Wood samples took up the most of the molecule per inch squared due to the large non-porous surface area. 5
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