2.12. Surface hydrophobicity (H0) measurement

Surface hydrophobicity was determined using 1-anilino-8-naphthalenesulfonate (ANS) as a fluorescence probe according to the method of Shigeru and Shuryo [26] with some modifications. Briefly, 4 mL samples (0.5 mg/mL) were well-mixed with 20 µL 8 mM ANS solution (100 mM phosphate buffer, pH 7.0), and then incubated for 15 min at room temperature in the dark. The ANS fluorescence was excited at a wavelength of 390 nm, and the emission spectrum was collected from 400 to 600 nm by an Rf-5301pc fluorescence spectrophotometer (Shimadzu, Japan). Both slit widths used were 2 nm. The fluorescence intensity was labelled as normalized intensity (a.u.), which indicated the surface hydrophobicity. Each emission spectrum represents the average of three scans.