2.6. In vitro bioadhesion of mucin to chitosan and the threechitosan conjugates
2.6.1. Mucin determination by the periodic acid Schiff (PAS)method The periodic acid Schiff (PAS) colorimetric method was used tomeasure the concentration of free mucin. The Schiff reagent was prepared as previously reported (Kafedjiiski, Kraul and, H offer, & Bernkop-Schnourch, 2005). To 100 mL of 1% (w/v) basic fuchsin (pararosaniline) in deionized water was added 20 mL of 1 M HCland 1.67% (w/v) sodium metabisufite and incubated at 37◦C until pale yellow. The periodic acid reagent was freshly prepared by the addition of 10 L of 50% (v/v) periodic acid in water to 7 mL of 7%(v/v) acetic acid solution. Standard calibration curves of mucin were prepared from mucin standard solutions in the range of 0.2–1.0 mg/mL. First, 100 L of periodic acid reagent was added to 20 L of sample solution and then incubated at 37◦C for 2 h before 100 L of Schiff reagent wasadded and incubated at RT for 30 min. Finally, the absorbance was measured at a wavelength of 555 nm. The mucin content was thencalculated by reference to the standard calibration curve.2.6.2. Adsorption of mucin on chitosan and the three chitosan conjugates Mucin was prepared in 0.1 M phosphate buffer pH 1.2, pH 4.0 and pH 6.4 at 0.5% (w/v). Suspensions of sample stock solution were mixed with type II mucin (1 mg/mL), vortexed, and incubated at 37◦C for 2 h. After that the dispersions were centrifuged at 12,000 rpm for 2 min and the residual free mucin was measured in the supernatant using the PAS assay. The mucin concentration was calculated by reference to the calibration curve and the amount of mucin adsorbed to the polymer was calculated as the difference between the total amount of mucin added and the free mucincontent in the supernatant