Afm5500m

Samples for SEM and Atomic Force Microscopy (AFM) were immersed in 25% glutaraldehyde overnight, afterwards dipped ten times in 50% EtOH [50 (link),52 (link)] and dried in a vacuum desiccator for at least 2 h. Morphology of the samples was studied using a scanning electron microscope (FE-SEM SU5000, Hitachi, Japan), at an acceleration voltage of 10–15 kV. Samples were sputtered with ~10 nm of gold. Surface topography was acquired on an atomic force microscope (AFM5500M, Hitachi, Japan) in semi-contact mode with a silicon tip and at different scanning areas at a scan rate of 1 Hz. The experiments were conducted in air and at room temperature.

The morphology of NG gel was observed by transmission electron microscopy (TEM, JEM‐2100, Hitachi, Tokyo, Japan). The sample solution was evenly dropped onto a copper grid, stained with 2% phosphotungstic acid and air‐dried at room temperature for TEM observation immediately at 100 kV. Further, scanning electron microscope (SEM, JSM‐7800F, Hitachi, Tokyo, Japan) and atomic force microscopy (AFM‐5500 M, Hitachi, Tokyo, Japan) were applied to observe the microstructure of NG gel. The structural characteristics of samples were scanned by Fourier transform infrared (FT‐IR) spectrometer (Perkin Elmer, Waltham, Ma, USA) in the range of 4000–400 cm⁻¹. Circular dichroism (CD, J‐1500, JASCO, Tokyo, Japan) was used for secondary structure analysis of the sample. All scans were performed in the range of 200–400 nm with an average of 3 scans across all spectra.

The molecular weight of FCDs was measured by matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF-MS, Autoflex III, Bruker Daltonics, Bremen, Germany), using 2, 5-dihydroxybenzoic acid as a matrix. Transmission electron microscope (TEM) analysis was used a TEM (JEM-2100, JEOL, Tokyo, Japan) at a voltage of 200 kV. The ultrasonic particle size analyzer (DT1200) was used to measure the ζ-potential of the samples. UV-Vis spectrophotometer (Lambda 35, PerkinElmer, Norwalk, CT, USA) was used to record the absorption spectra. The fluorescence spectra were performed with the F-2700 spectrofluorometer (Hitachi, Tokyo, Japan). The fluorescence lifetime was ascertained by an FLS980 spectrometer (Edinburgh Instruments, Edinburgh, UK). FTIR was conducted by a PerkinElmer spectrometer (Frontier, Norwalk, CT, USA). CD spectra were performed by a JASCO circular dichroism spectrometer (J-1500, Tokyo, Japan). Atomic force microscope (AFM) was used to measure the two/three-dimensional morphology and height of the samples (AFM-5500M, Hitachi, Tokyo, Japan). Unless otherwise stated, the sample concentration was 1 mg/mL. All samples were dissolved in PBS buffer (0.01 M, pH 7.2–7.4). Before the test, the FCDs and protein mixture liquid was incubated in a water bath at 37 °C for 10 min.