Agilent 725 es

Five samples of each group were prepared to investigate the degradation performance. Each set of scaffolds was weighed after drying and recorded as M₁. After weighing, the scaffold was immersed in 10 mL of phosphate buffer solution (PBS, pH = 7.4) and placed in a 37 °C incubator for degradation for six months. The PBS was changed at 1, 2, 4, 8, 12, 16 and 24 weeks. Then the scaffold was washed, dried and weighed, denoted as M₂. The degradation rate (DR) at each time point was calculated and the degradation rate curve was drawn. The degradation rate of the scaffold was estimated using the following equation:^{28 (link)}
Each sample was immersed in 10 mL of deionized water for 1, 3 and 6 months, respectively. At the given time points, the extracts were replaced of the fresh 10 mL deionized water and detected. Calcium and phosphorus ion release from scaffolds were measured by inductively coupled plasma spectrometer (ICP-AES, Agilent 725-ES). The fluoride ion concentration was determined by fluoride ion-selective electrode connected to an expandable ion analyzer. The cumulative release concentrations were obtained by adding up the concentration at each time point. Five parallel samples were tested in each group.

The UV- visible absorption spectra of purified enzymes before and after incubation with Fe²⁺ (0.012 mM–0.112 mM) was recorded using a UV-2700 UV/vis spectrophotometer (Shimadzu, Kyoto, Japan). All experiments were carried out at room temperature with 0.6 U/mg enzyme in 20 mM Tris-HCl buffer, pH 7.5.
The fluorescent measurements of the enzyme before and after incubation with Fe²⁺ (0.01–0.47 mM) were determined by RF-5301PC fluorescence spectrometer (Shimadzu, Kyoto, Japan). All experiments were carried out at room temperature with 0.6 U/mg enzyme in 20 mM Tris-HCl buffer, pH 7.5.
The metal content of purified enzyme was measured on an ICP-OES instrument (Agilent 725-ES, Agilent, Palo Alto, CA, USA). Protein samples (1 mg/mL) were mixed with ultra-pure nitric acid (67–70%) and nitrated at 200 °C for 15 min. After that, the nitrated sample was diluted to 50 mL with 2% nitric acid for metal content analysis.
Electron paramagnetic resonance (EPR) spectra of purified laccase were recorded by a Bruker Elexsys E500 spectrometer (Bruker, Karlsruhe, Germany) at 9.5 GHz, with a microwave power of 5.0 mW, a modulation frequency of 100 kHz, and amplitude modulation of 2 G. The sweep time used in the experiments was 120 s. The probe temperature was regulated by means of a liquid nitrogen cryostat equipped with a temperature control unit and kept at 100 K.

After harvesting, 0.2 g of the plant tissue (roots and shoots) was dried at 60 °C in a Yamato Scientific DX 602C oven (Santa Clara, CA, USA) for 72 h. The resulting material was crushed and subjected to acid digestion in a mixture of perchloric acid and nitric acid [26 ]. Zn concentrations in the tissues were carried out using the acid digestion extract using an ICP-AES Agilent 725-ES atomic emission induction plasma coupled spectrometer (Santa Clara, CA, USA).