Gold nanoisland substrates for SERS characterization of cultured cells

Abstract

We demonstrate a simple approach for fabricating cell-compatible SERS substrates, using repeated gold deposition and thermal annealing. The substrates exhibit SERS enhancement up to six orders of magnitude and high uniformity. We have carried out Raman imaging of fixed mesenchymal stromal cells cultured directly on the substrates. Results of viability assays confirm that the substrates are highly biocompatible and Raman imaging confirms that cell attachment to the substrates is sufficient to realize significant SERS enhancement of cellular components. Using the SERS substrates as an in vitro sensing platform allowed us to identify multiple characteristic molecular fingerprints of the cells, providing a promising avenue towards non-invasive chemical characterization of biological samples.

Fig. 1.

Schematic illustrations of the fabrication process involving repeated gold deposition (A, C, E) and subsequent post-deposition thermal annealing (B, D, F) resulting in nanoparticle aggregation.

Fig. 2.

A) Scanning electron micrographs of SERS substrates at different stages of fabrication. Upper images correspond to 1^st, 2^nd and 3^rd gold deposition and lower images correspond to 1^st, 2^nd and 3^rd thermal annealing. All scale bars represent 100 nm. B) Particle size distributions corresponding to each step of sample fabrication after annealing. C) AFM images corresponding to sequential steps of deposition and post-deposition annealing (step 1, 2 and 3).

Fig. 3.

A) FDTD simulations showing the product of field intensities at an excitation wavelength of 785 nm and a Raman scattered wavelength of 830 nm (|E|²_exc·|E|²_Stokes), which relates to the SERS enhancement in the experiment. The calculations were performed for structures modelled according to the gold particle geometry following steps 1, 2 and 3 of substrate fabrication (Fig. 2A). The direction of polarization of the incident light is indicated with the white arrow. B) Maximum product of field intensities at excitation wavelength 785 nm and Stokes wavelength 830 nm for different heights above the glass surface. C) Maximum field enhancement factor obtained by FDTD simulations for sequential fabrication steps, averaged over the simulated range of heights and Stokes wavelengths.

Fig. 4.

A) Representative single SERS spectrum of crystal violet (c = 10⁻⁴ M). The signal at 1618 cm⁻¹ (marked in green) was used to estimate the enhancement factor (excitation: 785 nm, intensity: 2.0 × 10⁵ W·cm⁻², acquisition time: 1 s). B) Experimentally determined SERS enhancement factor for each step of substrate fabrication. Inset: Schematic distribution of enhancement factors at positions (x,y) on a substrate after three deposition and annealing steps. Data points are separated by 10 µm in x and y and the diameter of the probed spot was 1 µm (not to scale in the schematic).

Fig. 5.

A) Results for PrestoBlue cell proliferation assay for BM-MSCs cultured on glass controls and on SERS substrates (n = 3), error bars represent the standard deviation. B) Results for LDH cell cytotoxicity assay for BM-MSCs cultured on glass controls and SERS substrates (n = 3), error bars represent the standard deviation. C) Representative confocal image of focal adhesion plaques (green), actin cytoskeleton (red) and nuclei of mesenchymal stromal cells (blue) grown on a SERS substrate and D) on a conventional culture slide as control. Scale bars: 20 µm.

Fig. 6.

Representative Raman spectra obtained on mesenchymal stromal cell (BM-MSC) grown on a SERS substrate described in the text (red line) and on a glass cover slip (black line), respectively. The same excitation and collection conditions were used in both cases (excitation: 785 nm, intensity: 2.0 × 10⁵ W·cm⁻², acquisition time: 3 s).

Fig. 7.

Representative SERS spectra extracted from the mapping datasets of two different mesenchymal stromal cells on two different SERS substrates (excitation: 785 nm, intensity: 2.0 × 10⁵ W·cm⁻², acquisition time: 3 s, step size: 2 µm).

Fig. 8.

Raman maps showing the distribution of SERS signals in two different BM-MSCs on two different SERS substrates, and their overlay with microscope images. Raman maps are generated by mapping intensities at 418 cm⁻¹ assigned to cholesterol (green), 1140 cm⁻¹ to phospholipid alkyl chains (red), 1440 cm⁻¹ to CH₂ deformation in lipids (red) and 1270 cm⁻¹ to C = C groups in unsaturated fatty acids (green), 524 cm⁻¹ to S-S disulfide stretching in proteins (blue), 1002 cm⁻¹ to phenylalanine symmetric C-C stretching (blue), 1552 cm⁻¹ to tryptophan C = C stretching (blue) and 842 cm⁻¹ to polysaccharides (yellow). Scale bars: 10 µm.

Fig. 9.

Chemical image displaying the distribution of the band intensity at 418 cm⁻¹, assigned to cholesterol, followed by example spectra extracted from the maps at two different points labeled in the panel. The SERS spectra represent two different intensities of the 418 cm⁻¹ band (excitation: 785 nm, intensity: 2.0 × 10⁵ W·cm⁻², acquisition time: 3 s, step size: 2 µm). Scale bar: 10 µm.