Equinox 55 ftir

Bruker Avance 500 (Bruker Co., USA) was used to record proton NMR spectroscopy of PNS-SS-ADA and CD-mPEG using D₂O as a solvent. FTIR spectra of PNS, PNS-SS-ADA, and PNS-SS-ADA@CD-mPEG were recorded on a Bruker Equinox 55 FTIR (Bruker Co., USA) in order to investigate the presence of SS-ADA and CD-mPEG on the surface of PNS nanoparticles. TGA was carried out using TG Analyzer (Perkin Elmer Pryris 1, USA). N₂ adsorption-desorption isotherms were measured using a NOVA 1000e system (Quantachrome Instruments, USA). The samples were outgassed for 3 h at 150°C before the measurements. Morphology and size of PNS and PNS-SS-ADA@CD-mPEG nanoparticles were imaged by TEM using JEM-1400 (JEOL, Tokyo, Japan) at an accelerating voltage of 300 kV. The samples for TEM observations were prepared by placing a drop of solution in diH₂O (1 mg/mL) onto a carbon-copper grid (300 mesh, Ted Pella, Inc., USA) and air-dried for 10 min.

Prior to sample loading, a ‘96 well’ silicon transmission plate (Bruker, www.bruker.com) was pre-washed in analytical grade methanol three times followed by dH₂O three times, and the plate dried. To 30 mg (±1 mg) of ground leaf tissue 1.5 ml of sterile ultra pure dH₂O was added and the sample thoroughly mixed. Thirty microlitre homogenates of each biological replicate were loaded onto the pre-washed sample plate to generate technical replicates, and three readings were taken from each sample spot to serve as analytical replicates. The plate was oven dried at 50 °C until samples were completely dry prior to loading into the motorised high-throughput stage (HTS-XT; Bruker) attached to a Bruker Equinox 55 FT-IR (Winder et al., 2004, 2006 ). The FT-IR transmission mode protocol was based precisely on the method previously described by Harrigan et al. (2004) (link). Spectra were collected over the wavelength range of 4000–600 cm⁻¹ with a resolution of 4 cm⁻¹. To improve signal-to-noise ratio, the resulting spectra were co-added and averaged. Spectra were displayed in terms of absorbance as calculated using Opus 4 software, which uses the background spectrum of the reference well subtracted from the spectra recorded from the sample wells.

All chemicals, reagents and solvents used were purchased from Sigma‐Aldrich (St Louis, MO, USA). For FTIR spectroscopy analysis, fully expanded leaves were excised under growth light conditions using a razor blade, flash‐frozen and lyophilized overnight, after which the leaves were ground and transferred to −80 °C storage prior to extraction. Leaf tissue [30 mg dry weight (DW)] was homogenized in 1.5 mL of HPLC‐grade water, 15 μL of homogenate was directly loaded on to the wells of a silicon 96‐well IR target plate (Bruker, Coventry, UK) and dried.
Sample homogenates were loaded onto the IR target plate in duplicate and two readings were taken from each sample well to serve as analytical replicates. The plate was loaded onto a motorized high‐throughput stage (HTR‐XT; Bruker, Billerica, MA, USA) attached to a Bruker Equinox 55 FTIR and run in transmission mode according to Winder et al. (2004).