Functionalized Gold Nanoparticles as Hypersensitive Biosensors for Monitoring Cellular Uptake and for Cancer Diagnostics

Noble metallic nanostructures exhibit a phenomenon known as surface-enhanced Raman scattering (SERS) in which the scattering cross sections are dramatically enhanced for molecules adsorbed thereon. In recent years, SERS has been established as one of the emerging analytical techniques, with sensitivity up to the single molecule. The present contribution reports on the preparation, characterization and functionalization of colloidal gold nanoparticles (AuNPs) to be used as a biosensing platform for cellular uptake studies and for diagnostic purposes in early cancer detection. The main tool to characterize the AuNPs morphology is Transmission Electron Microscopy (TEM). In Fig. 1 is shown a micrograph of a typical colloidal solution, highlighting the absence of aggregates; different shapes are produced (spheres, nanorods, triangles). Image analysis (inset of Fig. 1) detects a bimodal distribution: the main population (74 %) displays a spherical shape (roundness above 0.8) and a mean diameter of 31 nm. The remaining fraction (26 %) is made of nanorods (roundness less than 0.6) and has a mean diameter of 43 nm. The size distribution and shape homogeneity can be further improved by post- processing (ultracentrifugation). In general, shape multiplicity does not reduce the SERS response. Once prepared, the gold nanoparticles have been functionalized with 4- mercaptobenzoic acid (4-MBA) to form a Self-Assembled Monolayer (SAM); the resulting colloidal solution is stable. The bare nanoparticle has no Raman activity, while strong SERS signals are produced by the functionalized nanoparticles due to the chemisorbed 4-MBA. The enhancement factor (EF) in solution can be evaluated in a number of ways. The present AuNPs provide EF values ranging between 106 and 108, resulting in highly efficient substrates for biosensing applications. The enhancement factor depends on particle size, with the maximum occurring for diameters of around 50 nm. However, even for smaller sizes (30 and 20 nm) SERS signaling remains efficient, which adds to the versatility of the approach. Hyperspectral Raman imaging represents an emerging technique to investigate the cellular environment at a molecular level [1, 2]. It provides several advantages among which a high contrast, originating from the specificity of the Raman spectrum, molecular level information, appropriate spatial resolution, versatile sampling. Fluorescence emission may be occasionally problematic. We have investigated normal and tumor lines of human prostatic cells by SERS spectroscopy. We employed the gold nanoparticles functionalized with 4-MBA as molecular reporters. These were added as colloidal solution to a water dispersion of the cells, incubated overnight and then deposited on a quartz substrate for Raman inspection. In the case of healthy cells, we found that the AuNPs aggregate around the cell membrane, possibly through a molecular interaction between the carboxyl groups of 4-MBA and the polar groups on the membrane surface (see Fig. 2A). Thus, an undamaged membrane prevents the nanoparticles from entering the cell body. For the tumor cells, strong SERS signaling is observed in the whole cell area (see Fig. 2B). In the latter case, SERS imaging demonstrates a conspicuous uptake of nanoparticles, which cross the more porous cellular membrane. This effect is evidenced by the spectra collected within the cell body, which are featureless for the normal cell and are very intense (more than in the periphery) for the tumor cell. These results may have important implications for diagnostic purposes. They also demonstrate the sensitivity and the specificity of SERS imaging for studies of nanoparticle uptake in cells and tissues, which are becoming very relevant to test cytotoxicity, selective uptake of drug-carriers, and more.