Mechanical characterization of alginate hydrogels

The linear viscoelasticity of alginate hydrogels was probed with a torque controlled rotational rheometer (model AR-G2, TA instruments) using 20 mm stainless steel parallel plate geometry on temperature controlled Peltier system at 25 °C. Alginate disks were prepared by adding alginate-calcium-EDTA solution in custom-made latex rubber molds (~3.5 mm thick) (catalog no. 1374N35, McMaster) adhered to hydrophobic surface followed by addition of acetic acid (12.73 µl acetic acid/ml alginate solution, the same ratio used for droplet gelation). After 4 h of gelation, rubber molds were cut and removed, and cylindrical gel samples were kept in deionized water from 2 to 36 days. Alginate disks were transferred to the rheometer with a 60 grit, adhesive-back sand paper (Norton Abrasives) adhered to the contact surfaces to prevent slipping. To ensure contact initiation, the head was slowly lowered until reaching the normal force of ~30 mN. The disks were trimmed to the size of the flat plate and coated with mineral oil on the exposed edge to minimize solvent evaporation. Strain sweeps were performed to determine linear stress-strain regimes. Subsequently, the frequency sweeps at 1% strain was performed to probe time-dependent mechanical properties. For each concentration, six repeat experiments were performed. To probe the rheology in the nonlinear regime, a separated motor-transducer rheometer (MCR702 from Anton Paar) was used. Raw data were analyzed in MITLAOS⁵² software to obtain Lissajous curves.