3.2. Synthesis of Fluorescein Isothiocyanate-Labeled Chitosan Biopolymer (FITC-CHT)

The synthesis of FITC-labeled CHT was carried out following a previous report [62]. Briefly, purified CHT (200 mg) was dissolved overnight in 0.1 M acetic acid (20 mL). Then, anhydrous methanol (20 mL) was added to the CHT solution with continuous stirring for 3 h. Afterwards, FITC (10 mL) dissolved in anhydrous methanol at 2 mg/mL was added to the CHT solution. The reaction between the isothiocyanate group of FITC and the primary amino group of the d-glucosamine residue proceeded for 4 h in the dark at room temperature. Then, the FITC-labeled CHT (FITC-CHT) was precipitated in 0.2 M NaOH and washed with 70% methanol until the supernatant was free of fluorescence. FITC-CHT was dissolved in 0.1 M acetic acid and dialyzed against DI water for 7 days (Standard RC tubing with a MWCO = 6–8 kDa, Spectrum Laboratories Inc., Los Angeles, CA, USA). The whole reaction and dialysis purification process were protected from light. Finally, FITC-CHT was freeze-dried and stored at 4 °C until further use.