Hybrid lipid-AuNP clusters as highly efficient SERS substrates for biomedical applications

Abstract

Although Surface Enhanced Raman Scattering (SERS) is widely applied for ultrasensitive diagnostics and imaging, its potential is largely limited by the difficult preparation of SERS tags, typically metallic nanoparticles (NPs) functionalized with Raman-active molecules (RRs), whose production often involves complex synthetic approaches, low colloidal stability and poor reproducibility. Here, we introduce LipoGold Tags, a simple platform where gold NPs (AuNPs) clusters form via self-assembly on lipid vesicle. RRs embedded in the lipid bilayer experience enhanced electromagnetic field, significantly increasing their Raman signals. We modulate RRs and lipid vesicle concentrations to achieve optimal SERS enhancement and we provide robust structural characterization. We further demonstrate the versatility of LipoGold Tags by functionalizing them with biomolecular probes, including antibodies. As proof of concept, we successfully detect intracellular GM1 alterations, distinguishing healthy donors from patients with infantile GM1 gangliosidosis, showcasing LipoGold Tags as advancement in SERS probes production.

Fig. 1. Self-assembly of AuNPs on liposomes with different membrane rigidity.

a Graphical representation of the proposed approach to form LipoGold SERS tags; (b) UV-visible spectra of 6 nM AuNPs containing 1.2 nM (DOPC(MBA) black, POPC(MBA) red, DPPC(MBA) blue, and DSPC(MBA) green) liposomes collected after 30 min of incubation; Cryo-EM images showing (c) AuNPs-DOPC and (d) AuNPs-DPPC adducts. Scale bar is 200 nm. Cryo-TEM representative experiments have been repeated 2 times. See SI for additional images. Source data are provided as a Source Data file.

Fig. 2. SERS signal as a function of membrane rigidity of liposomes.

Evaluation of the Raman-SERS and plasmonic properties of LipoGold Tags prepared with four different liposomes’ types (DSPC, DPPC, POPC and DOPC). a Raman-SERS spectra acquired for different kinds of liposomes (DSPC, DPPC, POPC, and DOPC) with 4-MBA molecules embedded in the bilayer. Excitation wavelength: 638 nm; accumulation time: 60 s; number of acquisitions: 2; objective magnification: 10x. b Comparison between the Raman Intensity at 1060 cm^-1 and UV-Vis signal at 600 nm. c Graphical representation of the different interactions between AuNPs and various types of liposomes (DOPC, POPC, DPPC, DSPC). d Average enhancement factors (EFs) for clusters of colloidal AuNPs conjugated with 4-MBA (red shaded area) from the literature^,,); cluster of colloidal AuNPs conjugated with 4-MBA and immobilized onto different substrates (green shaded area) from the literature^,,); and cluster of colloidal AuNPs interacting with liposome DOPC(MBA) (blue shaded area) presented in this work. Source data are provided as a Source Data file.

Fig. 3. Effect of RR concentration on SERS signal and reproducibility of LipoGold tags.

Optimization of the synthetic parameters in terms of Raman-SERS performances, reproducibility, colloidal, and functional stability. a Raman-SERS spectra of AuNPs interacting with liposome dispersion of DOPC(MBA) at different concentrations; (b) Raman-SERS spectra of AuNPs interacting with DOPC liposome dispersion with different MBA concentrations; (c) Raman-SERS spectra of six different batches of LipoGold tags; (d) Batch-to-batch Raman intensity variation for the 1069 cm⁻¹ and 1577 cm⁻¹ peaks calculated from the Raman-SERS spectra reported in (c). Excitation wavelength: 638 nm; accumulation time: 60 s; number of acquisitions: 2; objective magnification: 10x. Source data are provided as a Source Data file.

Fig. 4. Temporal evolution of LipoGold tags.

a Time evolution of the Raman and (b) UV-visible spectra of DOPC(MBA)-AuNPs LipoGold tags aqueous dispersions; (c) Comparison between the time evolution of Raman Intensity at 1060 cm⁻¹ and UV-vis signal at 600 nm; (d) Time evolution of the DLS autocorrelation functions of DOPC(MBA)-AuNPs LipoGold tags. Excitation wavelength: 638 nm; accumulation time: 60 s; number of acquisitions: 2; objective magnification: 10x. Source data are provided as a Source Data file.

Fig. 5. Multiplexing properties of LipoGold Tags.

a Graphical model representing the three SERS probes constituted by three different RRs; (b) Raman-SERS spectra of DOPC(RR)-AuNPs with (colored curves) and without (black curves) the presence of AuNPs. Spectra backgrounds are removed by subtracting the profile of water samples with a 638 nm excitation laser at the same power and same acquisition parameters. Excitation wavelength: 638 nm; accumulation time: 60 s; number of acquisitions: 2; objective magnification: 10x. (c) UV-Visible spectra of DOPC(RR)-AuNPs after mixing; (d) Dynamic light scattering (DLS) curves obtained by measuring DOPC(RR)-AuNPs after 30 min. of incubation. Source data are provided as a Source Data file.

Fig. 6. Bioconjugation of LipoGold tags.

a Graphical scheme reporting the functionalization protocol of liposomes-AuNPs surface with antibodies, exploiting the EDC/NHS coupling mechanism; (b) Fluorescence correlation spectroscopy (FCS) measurements of antibodies (Ab(I)) and DOPC-AuNPs@antibody conjugates; (c) Graphical scheme reporting the principle of coupling between antibodies(II) attached to the glass slide and antibodies(I) conjugated to the AuNPs-liposome surface (Ab(I)-DOPC(MBA)-PEG-AuNPs); (d) Mean Raman-SERS spectra of liposomes with 0%, 1%, and 5% PEG molecules with (red curves) and without (gray curves) antibodies attached to the surface of the glass slide through a sandwich-like immunoassay. Excitation wavelength: 532 nm; accumulation time: 60 s; number of acquisitions: 2; objective magnification: 10x. Data are presented as mean values ± SD. e Raman intensity at 1577 cm⁻¹ for each sample at different quantities of PEG, with and without antibodies. Data are presented as mean values ± SD. (N_total is the number of the replicates (=3); a two-sided student’s t-test was performed, with NS > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001). Source data are provided as a Source Data file.

Fig. 7. In vitro targeting of cells with LipoGold tags.

a Representative confocal microscope images of SHSY-5Y cells incubated with free CT-B, CT-B- LipoGold, and bare LipoGold. Scale bar is 50  μm in all cases; (b) Left panel shows the bright field image of the analyzed SH-SY5Y cells treated with LipoGold(RR6) for 90 min. The green rectangle represents the scanned area with Raman confocal microscopy. Right panel shows the SERS map generated exploiting the intensity at 2210 cm⁻¹. Scalebar is 10 μm in all cases. Measurements were performed with the following parameters: laser 638 nm, 20 mW laser power, 30 s acquisition time, 1 accumulation, 272 data points, Y step 2  μm and X step 3 μm, objective magnification: 60x. c Representative SERS spectra acquired from cells in (b). Yellow spectrum corresponds to x = −7.5 μm, y = −5  μm (yellow dot); blue spectrum corresponds to x = −15 um, y = 22 μm (blue dot); green spectrum corresponds to x = 10 um; y = 0 μm (green dot). The representative microscopy experiments have been repeated two times. Source data are provided as a Source Data file.

Fig. 8. Detection of GM1 alterations using LipoGold tags.

a FACS analysis of lymphocytes from WT and from patient affected by juvenile GM1 gangliosidosis. Lymphocytes were fixed and incubated for 45 min with 5 µg/mL of CT-B or 5 µg/mL of CT-B LipoGold, previously dissolved in distilled H₂O. FACS analysis showed a significant variation in the SSC-A of WT cells labeled with CT-B-LipoGold compared with WT marked with CT-B. b MFI/MFIWT mean values are obtained by dividing the MFI of a distribution by the mean MFI obtained from the controls. MFI/MFIWT mean values increase significantly in patients compared to WT control. N_total is the number of replicates (=3), 1000 cells were analyzed. Data are presented as mean values ± SD. A two-sided student’s t-test was performed with NS > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001. Median: the data value located halfway between the smallest and largest value. Upper Quartile: the data value located halfway between the median and the largest data value. Lower Quartile: the data value located halfway between the median and the smallest data value. Interquartile Distance (IQD): the distance between the Upper and the Lower Quartiles. Max: maximum value. Min: minimum value. c Averaged Raman-SERS spectra acquired for WT in the presence of CT-B (green line, n = 4) and CT-B-LipoGold (red line, n = 9), and averaged Raman-SERS spectra acquired for patient sample in the presence of CT-B (blue line, n = 5) and CT-B-LipoGold (gray line, n = 9). Data are presented as mean values ± SD. Excitation wavelength: 638 nm; accumulation time: 120 s; number of acquisitions: 2; objective magnification: 10x. d Histogram showing the quantitative values of Raman intensity at 1060 cm⁻¹. Error bars are standard deviations (n = 4, 9, 5, 9, respectively; a two-sided student’s t test was performed, with NS > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001). Source data are provided as a Source Data file.