Invia qontor micro raman spectrometer

Raman measurements were performed in a Renishaw inVia Qontor micro-Raman spectrometer equipped with an air-cooled charge-coupled device (CCD) as detector and a He–Ne laser operating at 32 mW of 632.81 nm. For the SERS-immunotags in solution (300 μL), and the final immunoassay performed on the membrane placed onto a microscope slide, the laser beam was focused with 5× (n.a. 0.12) and long-distance 50× (n.a. 0.5) respectively. An integration of 10 scans of 20 s each was used for all SERS-immunotags measurements. The intensity of the incident laser beam was 3.2 mW. Raman images of sandwich immunocomplexes on the RCH were obtained using a Raman point mapping method (scan of 21 × 21 μm, 1 μm steps). All spectra were obtained in triplicate, generating a total of 1233 points (pixels) per sample. Between different Raman sessions, the spectrograph was calibrated using the Raman line at 520.7 cm⁻¹ of an internal crystalline Si sample. All SERS spectra were recorded at room temperature. All raw data were collected digitally with Wire 5.0 software. Noise reduction, available on the software, was used to estimate and remove the noise through principal component analysis (PCA). Baseline correction using a polynomial fitting (11^th order) was then performed, taking care to ensure minimal alteration of raw data.

Raman measurements were performed in a Renishaw inVia Qontor micro-Raman spectrometer equipped with an air-cooled charge-coupled device (CCD) detector and an He–Ne laser operating at 32 mW of 633 nm laser excitation. The spectral resolution of the spectroscopic system is 0.3 cm⁻¹. The laser beam was focused with a 5× Leica objective lens (N Plan EPI) with a numerical aperture of 0.12. An integration time of 10 scans of 20 s each was used for all measurements to reduce the random background noise induced by the detector, without significantly increasing the acquisition time. The intensity of the incident laser was 3.2 mW. Triplicates were taken of all spectra. Between different Raman sessions, the spectrograph was calibrated using the Raman line at 521 cm⁻¹ of an internal Si wafer for reducing possible fluctuations of the Raman system. A volume of 300 µL of each sample was deposited on a multi-well (n = 96) plate and the objective was focused inside the well. All SERS spectra were recorded at room temperature. All the raw data were collected digitally with Wire 5.0 software for processing. Vibrational line areas were determined with the aid of the Wire 5.0 software for all spectra.