Multimodal characterization of fluorescently tagged inulin: FITC-based and fluoresceinamine-based approaches

Fluorescent labeling of long-chain inulin (FXL) is essential for tracking its spatiotemporal dynamics in gastrointestinal systems, yet conventional methods risk altering its native properties. This study systematically compared three labeling strategies: FITC-DB (dibutyltin dilaurate-catalyzed hydroxyl conjugation), FITC-RA (reductive amination), and FA (fluoresceinamine coupling). Multimodal characterization revealed that FITC-DB achieved the highest substitution degree (DS = 0.42 at 50 mg FITC), though fluorescence quenching occurred at elevated loadings. FITC-RA induced significant aggregation (peak size at 50 mg), while FA labeling exhibited minimal structural perturbation but low efficiency (DS < 0.20). Spectroscopic analyses confirmed covalent thiourea bond formation (1720 cm⁻¹ FTIR peak) and aggregation-dependent optical shifts. Crucially, FITC-DB at 50 mg balanced optimal labeling efficiency, colloidal stability (highest zeta potential), and preserved macromolecular architecture. These findings establish design principles for high-fidelity FXL probes in biological tracking applications.