F 4600 fluorescence spectrometer

The concentration calculations for PC were based on the excitation wavelength at 590 nm and the maximum emission wavelength between 600–750 nm. The slit width was fixed to 10 nm. Fluorescence spectra of PC@Mg-CaP before and after the heat treatment were acquired with a F-4600 Fluorescence spectrometer (Hitachi, Tokyo, Japan). The relative concentration (C_R) was calculated, and the specific calculation method was shown in Equation (4) [30 (link)] as follows: $${\mathrm{C}}_{\mathrm{R}}(\%)=\frac{{\mathrm{C}}_{\mathrm{remaining}}}{{\mathrm{C}}_{\mathrm{initial}}}\times 100\%$$
where C_remaining and C_initial are the concentrations of PC before and after the heat treatment, respectively. To evaluate the significance of thermal stability of samples with different treatments, all measurements were repeated three times, and the data were analyzed using the analysis of variance (ANOVA).

TEM images were acquired on a Hitachi (Japan) TEM‐HT7700 at 100 kV accelerating voltage. The UV–vis‐NIR spectra were measured on a Shimadzu UV‐2600 spectrometer. DLS study was carried out on a Nano‐ZS Zetasizer (Malvern, U.K.). Fluorescence spectra were measured on the Hitachi F‐4600 fluorescence spectrometer. In vivo NIR‐II fluorescence imaging was conducted on a Series III 900/1700‐D NIR‐II imaging system (Yingrui, Suzhou). In vivo photoacoustic imaging was acquired using a commercial ORPAM system (NIR‐VIS‐50, PAOMTek Inc.). Flow cytometry analysis for cell apoptosis was performed on a FACSCanto Analyzer. The 808 and 660 nm laser was purchased from Changchun Laser Technology Co., Ltd (Changchun, China) and the infrared thermal images were recorded by FLIR E6 thermal imagers. Fluorescence confocal imaging was conducted on a Leica SP8 confocal microscopy.

The prepared Cu/Au BNCs solution were incubated at different temperatures for 45 min before a fluorescence test. The temperatures ranged from 20 to 70 °C. The fluorescence spectra were performed on F-4600 fluorescence spectrometer (Hitachi, Japan), with the slit widths of excitation and emission being 10 nm.

The indexes of surface hydrophobicity (H₀) of the free OVA and OVA-CMC solutions with different heating duration were analyzed by using ANS as probe to interact with hydrophobic moieties on the surface of OVA to give a fluorescent signal^{12 (link)}. The OVA-CMC samples were diluted from 0.3125 to 5 g/L, respectively. To study the thermal aggregation of free OVA, OVA samples were diluted to lower concentrations ranging from 0.3125 to 1.25 g/L. Twenty microliters of 8 mM ANS solution (PBS, pH 7.0, 50 mM) were mixed into 4 mL OVA-CMC solutions and kept it in the dark for 15 min. The emission fluorescence intensities were measured by a F4600 fluorescence spectrometer (HITACHI, Tokyo, Japan) with the same emission and excitation slits as 5 nm at a voltage of 500 V, 25 °C. The results were reported as averages of three times. Scatter diagram with maximum fluorescence intensity as ordinate and concentration as abscissa were plotted to make a linear fitting of the graph. H₀ value is the slope (K) of the line.

The shape and detailed microstructure of the prepared product were characterized by field-emission scanning electron microscopy (FE-SEM; S-4800, Hitachi, Ltd., Tokyo, Japan) operated at an acceleration voltage of 5.0 kV, and the corresponding crystalline structure was analyzed using an X-ray diffractometer (XRD; D/MAX-RB, Rigaku Co., Tokyo, Japan) with Cu Kα radiation (λ) 1.5418 Å. Transmission electron microscopy (TEM) and high-resolution transmission electron microscopy (HRTEM) observations were carried out on a JEM-2100F (JEOL Ltd., Tokyo, Japan). UV-vis absorption spectra were studied and recorded on a UV-Vis spectrometer (UV-2550, Shimadzu Co., Kyoto, Japan) equipped with an integrating sphere. The N₂ adsorption–desorption isotherm and Barrett–Joyner–Halenda (BJH) pore size distribution were analyzed on a Micromeritics ASAP 2020 nitrogen adsorption instrument (Micromeritics Instrument Co., Norcross, GR, USA). The photoluminescence (PL) spectra were investigated at room temperature with a Hitachi (Hitachi, Ltd., Tokyo, Japan) F-4600 fluorescence spectrometer. The electron spin resonance (ESR) spectra of the obtained samples were recorded on an ESR spectrometer (JEOL JES-FA 200, JEOL Ltd., Tokyo, Japan).