Z 5000

Ingredients, diets and excreta samples were ground through a 1-mm screen and then analyzed for dry matter (DM; AOAC, 2007; method 930.15), crude protein (CP; AOAC, 2007; method 976.05) and ash (AOAC, 2007; method 942.15) [17 ]. The neutral detergent fiber (NDF) and acid detergent fiber (ADF) were determined using fiber analyzer (Ankom Technology, Macedon, NY, USA) according to Van Soest, et al. [18 (link)]. The gross energy (GE) was determined using an automatic adiabatic oxygen bomb calorimeter (Parr 6300 Calorimeter, Moline, IL, USA). The total dietary fiber (TDF) and IDF were analyzed using AOAC (2007) [17 ] methods 985.29 and 991.43, respectively. The SDF was calculated as the difference between TDF and IDF. The chromium (Cr) content was measured using anatomic absorption spectrophotometer (Z-5000; Hitachi, Tokyo, Japan) according to the procedure of Williams et al. [19 (link)]. The apparent total tract digestibility (ATTD) values were calculated by the equation as follows: ATTD (%) = [1 − (Cr_diet × Nutrient_excreta)/(Cr_excreta × Nutrient_diet)] × 100%, where Cr_diet represents the chromium content in diet, and Cr_excreta represents the chromium content in excreta. Nutrient_excreta represents the nutrient content in excreta, and Nutrient_diet represents the nutrient content in diet.

The concentration of Fe₃O₄ nanoparticles in the emulsion was measured by an atomic absorption spectrophotometer (AAS, Hitachi Z-5000, Tokyo, Japan).
GalPLL/M-PFOBNP with different concentration of Fe₃O₄ nanoparticles were placed in Eppendorf tubes of 1 cm in diameter. For T2^∗-weighted imaging, an axial multi-echo fast gradient echo sequence with 6 echoes was performed. The parameters were as follows: TR/TE range, 160/2.7–22.3 ms; slice thickness, 2.5 mm; flip angle 30°. The transverse relaxation rate R2^∗ (1/T2^∗) was measured. All MR studies were performed with a 3-T system (Discovery 750, GE Healthcare, Milwaukee, Wisconsin, USA).
The mean size of GalPLL/M-PFOBNP was determined with a laser light-scattering submicron particle sizer (Malvern Instruments, Malvern, Worcestershire, United Kingdom).

The method quoted by Altaf et al. (2021a) (link) was used to determine the Ni content, samples of roots and leaves were harvested individually. Oven-dried samples were ground and digested at 80°C with HNO₃:HClO₄ (5:1 v/v). The concentration of Ni in leaves and roots was determined using an atomic absorption spectrophotometer (Z-5000, Hitachi, Japan). The procedure described by Altaf et al. (2021c) (link) was used to determine the nutritional element content of pepper seedling roots and leaves. After harvesting, samples of leaves and roots were obtained and washed multiple times with ultrapure water before being oven-dried at a temperature of 65°C. Following that, dried samples (leaves and roots) were digested separately in 3 mL of 1 M HNO₃, then samples were boiled for 10 min at 95°C. Finally, N, P, and K contents were measured using an inductively coupled plasma optical emission spectrometer (ICP-OES; SPS3100, SII Nano Technology, Japan).

For the estimation of Na and K content, the separately harvested root and leaf samples were thoroughly washed with double distilled water to eliminate Na and K ions that might be adhering to the surface. The 0.1 g sample of oven dried (80 °C for 48 h) tissue was ground and digested with a HNO₃:HClO₄ (5:1 v/v) mixture at 80 °C until the yellow colour vanished. The content of Na and K in roots and leaves was analysed by flame atomic absorption spectrophotometry (Z-5000; Hitachi, Japan).

The Se content in wheat leaves and roots was measured by following the methods of Mostofa et al. [13 (link)]. An atomic absorption spectrophotometer (Z-5000; Hitachi, Japan) was used to quantify the Se content. The spectrophotometer was calibrated with a Se standard solution, and the Se contents were calculated by using Mostofa et al. [13 (link)] formula.
Nitric oxide (NO) was quantified according to the Griess reaction, which is based on the spontaneous oxidation of NO to nitrite under physiological conditions according to the method described by Kaya et al. [35 (link)]. A 0.6 g leaf sample was homogenised with 50 mM cold acetic acid (3 mL, pH 3.6) and zinc diacetate (4%). The leaf extract was centrifuged at 10,000× g for 15 min at 4 °C. The upper layer was collected from the extract and turned into a pellet that was washed with the extraction medium (1 mL) after centrifugation. Charcoal (0.1 g) was added to the supernatant and mixed well. The mixture was filtered and vortexed. Then, 1 mL of Griess reagent was added to the mixture, which was left for 30 min at room temperature. The absorbance was taken at 540 nm, and finally, the nitrite derived from NO was quantified.

High-resolution transmission electron microscopy (HR-TEM) observations were carried out in a JEM-2010 electron microscope working at 200 kV for measuring the morphology and microstructure of nanocomposites.
Fourier transform infrared spectra (FTIR) were recorded on a Bio-Rad FTS-40 Fourier transform infrared spectrophotometer in the wavenumber range of 4000-650 cm⁻¹. The spectra were collected at 2 cm-1 resolution with 128 scans by preparing KBr pellets with a 3:100 “sample-to-KBr” ratio.
Thermogravimetry-differential scanning calorimetry (TG/DSC) measurement was performed on an EXSTAR TG/DTA 6300 instrument (Seiko, Japan) to quantify protein content in nanocomposites.
The real time analysis was performed according to the crystalline phase and Zn/Cd ratio of nanocomposites by powder X-ray diffraction (XRD, D & Advance, Bruker, Germany) and atomic absorption spectroscopy (AAS, Z-5000, Hitachi, Japan). To determine the crystalline phase, XRD measurements were performed on a Bruker D & Advance X-ray powder diffractometer with graphite monochromatized Cu/Kα (γ = 0.15406 nm). A scanning rate of 0.05 deg/s was applied to record the pattern in the 2θ range of 10–80°. At the same time, the samples were also digested by nitric acid and the Zn and Cd concentrations were quantified by AAS test.

Each bed sediment sample was air dried, ground with a mortar, and passed through a 100-mesh sieve. Then, 1 g of pretreated sediment sample was digested with HClO₄-HNO₃-HF [17 ,18 ], and the concentrations of Zn, Cr, Pb, Ni, and Cu in the extracts were determined using an atom absorption spectrophotometer (Z-5000, HITACHI, Tokyo, Japan).
The values of sediment pH were measured (sediment:water 1:2.5 dry weight/volume) using a pH-meter (pHS-3B, Leici, Shanghai, China). Organic matter contents were determined by the Walkey-Black method [19 ].