Xplora

TERS measurements were performed on a NanoRaman system consisting of an atomic force microscope (OmegaScope, formerly AIST-NT, now HORIBA Scientific) combined with a Raman spectrometer (XploRA, HORIBA Scientific, France) in side illumination geometry as schematically shown in Fig. 1. A 638 nm excitation laser was focussed on the sample at an angle of 60° using a ×100, 0.7 NA, 20 mm long working distance objective lens (Mitutoyo, Japan). TERS spectra were measured using a spectrometer grating with 600 lines/mm and an electron-multiplying charged-coupled device detector (Andor, Ireland) with a laser power of 100 µW at the sample.
For fast and efficient TERS mapping, TERS measurements were conducted in SpecTop™ TERS mapping mode in which TERS spectrum at a particular pixel in the TERS map is measured when the tip is in direct contact with the surface, with a typical interaction force of 2–10 nN and integration time of 0.05 s–0.5 s. Transition between the pixels of the TERS map is performed in semi-contact mode, which preserves both the sharpness and plasmonic enhancement of the tip eliminating lateral forces that might otherwise sweep aside or pick up loosely attached contaminants from the sample surface. All TERS measurements were performed using Au coated AFM TERS tips (k = 7 N/m, f = 150 kHz, formerly AIST-NT, now HORIBA Scientific).

For the measurements of the excitation-dependent fluorescence spectra, CDs suspension were measured using a FluoroLog®-3 spectrofluorometer (HORIBA, Kyoto, Japan) equipped with a xenon lamp (CW 450 W), a detector photomultiplier (model R928P) and using an excitation wavelength ranging from 310 to 532 nm. The CDs suspension was externally heated at 303 and 453 K. After solubilize, the fluorescence was measured for both cases at room temperature.
For the measurements of the temperature-dependent fluorescence spectra, CDs film were recorded using a Xplora (HORIBA, Kyoto, Japan) equipped with a laser of 532 nm, lens 10× objective (NA = 0.3 and WD = 17.5 mm). A heater (INSTEC hot and cold stage) was coupled to the Xplora. The spectra were collected during sample heating from 303 K to 453 K, varying of 30k. After the first cycle heating, the sample was cooled to room temperature.

These tested samples are all industrial, commercial materials that were obtained from five different suppliers. They include a total of 22 GBMs: 15 GNPs and 7 RGOs. Their specific surface area was determined with the BET technique (adsorption of nitrogen, with degassing system Micromeritics, Norcross, GA, USA). Their surface oxidation was determined with XPS (X-ray Photo Spectroscopy, Quantera Scanning XPS microprobe, Physical Electronics, Chanhassen, MN, USA). Lateral size was determined with electronic microscopy (Field Emission Scanning Electron Microscope, from JEOL, Tokyo, Japan). Surface defects were estimated with the ID/IG intensity ratio, calculated with Raman spectroscopy (XploRA, Horiba Scientific, Kyoto, Japan). For comparison, we also tested two samples of carbon black and one sample of amorphous silica.

Spontaneous Raman scattering spectra are acquired on an upright confocal Raman microspectrometer (Xplora, Horiba Jobin Yvon) with 532 nm diode laser source and 1800 lines per millimeter grating at room temperature. The excitation power is ~40 mW after passing through a 50× air objective (MPlan N, 0.75 N.A., Olympus), and 40 s acquisition time accumulated by 22 was used to collect Raman spectra of all samples at a single point under identical conditions. For cultured cells, the Raman background of water and cover glass is removed from all cell spectra by subtracting the signal at empty space from the signals collected from cells.

Structural characterization of diatom valves as well as of the ultrathin gold layer was performed with a Hitachi SU8030 scanning electron microscope, supported with a secondary electron detector.
For SERS measurements, about 7 µL of 1 mM Rhodamine 6G (R6G) in ethanol was dropped on top of the gold and left to dry at ambient conditions. SERS measurements and mapping were carried out with a backscattering configuration on a Horiba XploRA confocal Raman instrument equipped with a charge-coupled device (CCD) detector. The spectra acquisition was carried out using an excitation laser wavelength of 638 nm of ca. 40 mW power, in a spectral range of 500–2100 cm⁻¹, with an integration time of 15 s per spectrum and averaged over 5 accumulations. Raman mapping was performed with a 1 μm step in the case of Aula and a 0.5 μm step in the cases of Cosc and Gomp, with an integration time of 1 s per step. For focusing the light, the 100x objective (NA = 0.9) was used, giving a beam size of approximately 0.5 μm. Grating was set to 1200 grooves/mm.