Structural phases in Ca²⁺-triggered alginate assembly and gelation: circular dichroism-guided multimodal analysis

This study investigates the assembly and gelation behaviour of sodium alginate (SA) induced by calcium ions (Ca²⁺), using circular dichroism (CD), FTIR, SAXS, and AFM analyses. CD spectroscopy proved to be a highly sensitive and straightforward technique for detecting conformational changes and enabled construction of SA-Ca²⁺ phase diagram, revealing three distinct phases: nucleation, intermediate, and gelation. A red shift in the CD spectra in the 200-220 nm range indicated structural changes and the formation of "egg-box" structures. The CD spectra precisely pinpointed the nucleation-to-intermediate transition points across SA concentrations (1.0-6.0 mg mL^-1) and Ca²⁺ levels (1.0-10 mM), such as at 2.59 ± 0.50 mM, 4.33 ± 0.41 mM, and 5.07 ± 0.33 mM Ca²⁺ for 2.0 mg mL^-1, 4.0 mg mL^-1, and 6.0 mg mL^-1 SA, respectively. These results were consistent with FTIR, SAXS, and AFM data. The FTIR spectra showed red shifts and splitting of the carboxylate (-COO^-) band around 1600 cm^-1, signifying progressive cross-linking into the intermediate stage prior to gel formation. SAXS analysis revealed fiber aggregation at critical Ca²⁺ concentrations, with a sharp increase in the cylinder radius observed between 2.0-4.0 mM (2.0 mg mL^-1 SA) and 4.0-6.0 mM (4.0 and 6.0 mg mL^-1 SA). AFM imaging confirmed the three-phase model: early-stage nucleation with fibril formation at low Ca²⁺, aggregation into dense fibers at intermediate Ca²⁺ concentrations, and the development of a 3D entangled network in the gelation phase (SA > 6.0 mg mL^-1, Ca²⁺ > 6.0 mM). Overall, CD spectroscopy proved to be a highly sensitive and easy technique for detecting early assembly transitions in the non-gel phase and effectively complemented FTIR, SAXS, and AFM in mapping the SA-Ca²⁺ assembly process.