Figure 4

Evidence of a rapid and robust adsorption of Teic to TiO₂. (A) TiO₂ powder (50 mg) was treated with 500 µl of 500 µg/ml Teic in 50 mM HEPES (pH 7.4) and incubated at room temperature for 5, 15 and 30 min. At each time point, samples were centrifuged at 11,300 × g for 2 min and 25 µlof each supernatant was combined to 500 µl of BCA reagent. Samples were incubated for 30 min at 60 °C and the absorbances read at 540 nm against an incubation control (Teic only). Within 5 min there is evidence of antibiotic adsorption to TiO₂. (B) To confirm Teic-TiO₂ adsorption, the supernatant was decanted, and samples were washed twice by centrifugation and resuspended in 500 µl HEPES buffer. A 25 µl aliquot of each powder sample was added to 500 µl of BCA reagent and incubated for 30 min at 60 °C. Once incubated, powder samples were centrifuged at 11,300 × g for 2 min and 100 µl of each sample were transferred to a 96-well plate and measured in the same manner as above. (C) TiO₂ powder (50 mg) were similarly treated with 500 µl of Teic and incubated at room temperature for 2 h. Once incubated samples were centrifuged at 11,300 × g for 2 min, the supernatants were discarded, and the pellets were washed up to 10 times with HEPES buffer (500 µl). Aliquots (25 µl) of each wash were subject to the BCA assay to ascertain antibiotic leaching. (D)After each set of washes the remaining TiO₂ pellets were resuspended in fresh buffer (500 µl) and samples containing the powder (25 µl) taken for the BCA assay. All data (mean ± SD; N = 20) represent pooled quadruplicate runs from 5 independent experiments.