*2.3. Visualization of Microstructures*

The morphologies of hydrogels were observed upon freeze-drying using a field emission scanning electron microscope (FESEM; JSM-7610F, Jeol, Tokyo, Japan). The samples were prepared as above described by incubating the mixtures in tight-sealed vials at 37 ◦C until achieving the complete gelation. The samples were immediately frozen using liquid nitrogen for 30 min and at −80 ◦C overnight before lyophilization. Flash freezing the samples in liquid nitrogen was performed to preserve the microstructure of the hydrogels.

To visualize the micromorphological structure using FESEM, the freeze-dried samples were cut and coated with platinum. The FESEM was operated with an acceleration voltage of 5 kV.

#### *2.4. Quantitative Determination of Secondary Structures*

The structures of freeze-dried hydrogels were determined using Fourier-transform infrared (FTIR) spectroscopy in an attenuated total reflection (ATR) mode (Nicolet™ iS™ 5, Thermo Fisher Scientific, Waltham, MA, USA). The absorbance spectra within the range of 4000 to 800 cm−<sup>1</sup> were collected with 1.0 cm−<sup>1</sup> resolution. The secondary structures were quantified using Fourier self-deconvolution (FSD) and curve-fitting techniques according to the established protocol [15]. In brief, the deconvolution of amide I spectrum (1725–1575 cm−1) was obtained using Omnic 8.0 software (Thermo Fisher Scientific, Waltham, MA, USA) by fitting the Voigt line shape with a half-bandwidth of 25 cm−<sup>1</sup> and an enhancement factor of 2.5. Subsequently, the deconvoluted spectrum was fitted with the Gaussian function using Origin Pro 9.0 software (OriginLab, MA, USA). The content of beta sheet structure was obtained from the peak area between 1616–1637 cm−1. Other structures, namely random coil, alpha-helix and beta turn, were determined from the peaks at 1638–1655, 1656–1662 and 1663–1696 cm<sup>−</sup>1, respectively [16].

#### *2.5. Protein Adsorptivity of Blended Hydrogels*

Fetal bovine serum (FBS), a mixture of soluble proteins which is widely used in in vitro biological experiments, was selected to study the protein adsorptivity of the SF:eADF4(C16) hydrogels. 2% Hydrogels were prepared in a silicone mold, cut into a disc shape with a diameter of 5 mm and a thickness of 1 mm, and immersed in 10% FBS in PBS buffer (pH 7.4) at 37 ◦C for a particular period. After that, the hydrogels were washed with PBS to remove

redundant proteins and incubated in 1 mL of PBS at 4 ◦C overnight with gentle shaking to extract the protein. Supernatants obtained from the samples immersed in PBS overnight without FBS were used as blanks. Protein content in the extracted solution was determined using Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer's instruction. 80 μL of the supernatant was mixed with 20 μL of the dye, incubated at room temperature for 15 min, and the absorbance was measured at 595 nm.
