Rf 5301pc fluorescence spectrometer

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.

The transmission electron microscopy (TEM) image was taken by a H-7650 electron microscope (Hitachi, Japan) at an accelerating voltage of 80 kV. The high resolution transmission electron microscopy (HRTEM) image was observed with a Talos F200C electron microscope (FEI, USA) at an accelerating voltage of 200 kV. The scanning electron microscopy (SEM) image was obtained using a SU8010 electron microscope (Hitachi, Japan). The elemental analysis was measured with a vario EL III elemental analyzer (Elementar, Germany). The X-ray diffraction (XRD) pattern was performed using a Rigaku D/max-2500 diffractometer with a filtered Cu Kα radiation. Fourier transform infrared (FT-IR) spectroscopy were conducted on a NEXUS 670 FTIR spectrometer, ranging from 400 to 4000 cm⁻¹. The X-ray photoelectron spectroscopy (XPS) result of the sample was collected using an ESCALab 250Xi XPS instrument (Thermo Scientific). Ultraviolet-visible (UV-vis) absorption spectra were recorded by a Shimadzu 2450 UV-vis spectrophotometer (Shimadzu, Japan). All fluorescence spectra were measured by a RF-5301PC fluorescence spectrometer (Shimadzu, Japan) with 5 nm slit width for the excitation and emission.

Circular RCA template was prepared in 20 μL reaction mixture containing 1 × T4 DNA ligase buffer (50 mM Tris-HCl, 10 mM MgCl₂, 1 mM ATP, pH 7.5), 500 nM linear padlock probe and different concentrations of target DNA-containing oligonucleotide. To ensure that padlock probe can fully hybridize with target DNA, the mixture was heated at 95 °C for 5 min, and then cooled to 37 °C and incubated at this temperature for 0.5 h. After addition of 20 U of T4 DNA ligase, the mixture was allowed to incubate at 16 °C for 4 h to ensure that the 5′-phosphate and 3′-hydroxyl ends of linear padlock probe can be ligated to form a circular template. Above mixture was prepared in 1 × Phi29 DNA polymerase buffer (50 mM Tris-HCl, 10 mM (NH₄)₂SO₄, 4 mM DTT, pH 7.5), 3 µg/mL of BSA, 250 μM each dNTP, 5 U Phi29 DNA polymerase, 5 U Nb.BbvCI and deionized water. The obtained 100 μL reaction mixture was incubated at 30 °C for 5 h to perform RCA reaction. Then, 8 μM ThT (final concentration) was added and sufficiently mixed. Corresponding fluorescence signal of the mixture was measured on a Shimadzu RF-5301pc fluorescence spectrometer (Shimadzu Ltd., Japan). The emission spectra were collected from 450 nm to 620 nm using 425 nm as the excitation wavelength, and the fluorescence signal intensity at 485 nm was used for quantitative analysis of target DNA-containing DNA fragments.