Doubling growth of egg-box structure during Calcium-mediated molecular assembly of alginate

Ca²⁺-mediated molecular assembly of alginate underpins its wide range of applications in foods, pharmaceutics, biomedicines, tissue engineering and environmental treatments. The mode of growth of egg-box structure of alginate in the presence of Ca²⁺ is a long-standing fundamental problem to be concluded. In this work, we investigate the Ca-induced structural evolution of alginate in dilute solution using atomic force microscopy and dilute solution viscometry. It is demonstrated that the structural evolution follows the three critical steps of monocomplexation, dimerization and multimerization, upon binding with Ca²⁺. Interestingly, the alginate single chains grow into dimers and multimers via a doubling mode, i.e., successive emerging of dimer, tetramer, octamer, and hexadecamer. Compared with lower guluronate (G) alginate, higher G alginate exhibits a more pronounced multimerization process occurring at a lower ratio of Ca/G. A mechanistic model depicting the evolution of egg-box structure is proposed. The results would add new knowledge to the current egg-box model regarding the molecular assembly and gelation of an important biopolymer alginate, and provide fundamental basis for molecular engineering of alginate for more advanced applications.