Multispec 1501

The conjugation of fucoidan on the AuNR surface (Fu-AuNRs) was conducted following a previously reported method [22 (link)] based on the electrostatic interaction between the cationic –N⁺(CH₃)₃ group of CTAB and the anionic sulfate group of fucoidan (see the “Supplementary information” for details).
For the imaging of the AuNRs embedded onto solidified PVA, a PVA solution (20 mL) was prepared following a previously described protocol [23 ]. The AuNRs were fixed onto solidified PVA to investigate the scattering intensity bias from the chromatic aberrations of the illumination objective lens (see the “Supplementary information” for details).
In addition, the AuNRs and Fu-AuNRs were characterized by transmission electron microscopy (TEM, 2100F, JEOL Ltd, Tokyo, Japan) [24 (link)], UV–vis spectroscopy (MultiSpec-1501, Shimadzu, Tokyo, Japan), dynamic light scattering (802DLS, Viscotek, Westborough, MA, USA), and Fourier transform infrared (FT-IR) spectroscopy (Perkin Elmer, Inc., Norwalk, CT, USA) [19 (link)] (see the “Supplementary information” for details).

For absorbance spectroscopy, the Arabidopsis phot1 LOV1 + 2 (amino acid residues 180–628) was expressed and purified as described previously using the pCAL-n-EK vector (Agilent) to create an N-terminal calmodulin-binding peptide fusion by calmodulin affinity chromatography (20 (link), 31 (link)). Absorption spectra were measured using a Shimadzu MultiSpec-1501 diode array spectrophotometer at room temperature. The optical path length was 0.5 cm, and protein concentrations were determined by Bradford protein assay (Bio-Rad) using BSA as standard. For CD measurements, a DNA fragment encoding the A'α-LOV2-Jα region of Arabidopsis phot1 (amino acid residues 467–629) was PCR-amplified (supplemental Table S1) and cloned into the pHS vector (61 (link)) by Gibson assembly (New England Biolabs) via NcoI and NotI to create an N-terminal tagged His₇-Strep-SUMO fusion. LOV2 protein was expressed and purified by double affinity chromatography as described previously (61 (link)). CD spectra were recorded with a JASCO J-810 spectropolarimeter at a protein concentration of 0.4 mg ml⁻¹ using a 0.02-cm path length quartz cuvette. Protein samples were irradiated for 5 s (λ_max, 443 nm; half-bandwidth, 10 nm; 0.06 W cm⁻²) with a bluephase 16i dental curing light (Ivoclar Vivadent).

UV-Visible spectra of aqueous dispersions of GNP, GNR, and SNP were measured using a UV-Visible spectrometer (MultiSpec-1501, Shimadzu, Tokyo, Japan). Absorption peaks for GNP, GNR, and SNP were at 577 nm, 663 nm, and 477 nm, respectively.

Folin Ciocalteu reagent was used for the analysis of total phenolic content of the extract and the fractions [3 (link)]. Stock solutions of the ethanol extract and the fractions in methanol (10 mg/ml) were prepared and 0.02 ml of each stock solution was added to 1.58 ml of distilled water in a test tube. Then, 0.1 ml of diluted Folin Ciocalteau reagent was added to the test tube. The mixture was kept at room temperature for 3 min and then, 0.3 ml Na₂CO₃ 7.5 % solution was added. After 30 min, absorbance of the mixture was measured at 765 nm by UV-spectrophotometer (Multispec-1501 Shimadzu). A standard curve was prepared using gallic acid (Merck, Germany). The determinations were carried out in triplicate and the total phenolic content was expressed as gallic acid equivalents (mg of GAE/g of sample) [3 (link)].

The extinction spectra of aqueous dispersions of GNPs, GNRs, and SNPs were measured using a UV-Vis spectrometer (MultiSpec-1501, Shimadzu, Tokyo, Japan). The extinction peaks of GNPs, GNRs, and SNPs (Supplementary Fig. 12) were 577 nm, 663 nm, and 477 nm, respectively. The scattering spectra of 100-nm gold nanospots were described previously54 (link).