Figure 1 A-C) Transmission electron microscopy images of AuNP50, AuNP70 and AuNS respectively; D) Extinction spectra of GNPs normalised to 1 at their maximum; E) Size distribution measured by Nanoparticles Tracking Analysis (NTA)
2.2 Protein-induced stabilization of GNPs in media with high ionic strength
To test the nanoparticles’ stability in high ionic strength media, GNPs (concentration adjusted to 1010 particles/mL for all samples) were either centrifuged and resuspended in 1× PBS (NaCl: 137 mM, KCl: 2.7 mM, Na2HPO4: 10 mM, KH2PO4: 1.8 mM) or incubated for 30 min with (9 mg/mL) bovine serum albumin (BSA) [31] and then centrifuged and resuspended in PBS. The presence of a large amount of salts in the PBS caused GNPs (without protein) aggregation as indicated in the extinction spectra by a loss of peak sharpness and extinction intensity (Figure 2 A, B and C ). By way of contrast, nanoparticles coated with protein prior to resuspension in PBS, remained stable. The LSPR bands of GNPs (with protein) remained sharp with extinction intensities similar to those of the original GNPs. A small red-shift of the LSPR bands of GNPs (with protein) is observed due to the change of the refractive index of the media near the nanoparticles’ surface upon protein absorption.[32,33] GNPs salt-induced aggregation or protein-induced stabilization were also confirmed by nanoparticles size analysis – the mean size and size standard deviation of nanoparticles increased 2 times (seeSupporting Information Table S1 ).
It is accepted that the high ionic strength medium reduces the nanoparticle citrate electrostatic repulsion leading to aggregation whereas the PC displaces the citrate but stabilizes nanoparticles by acting as a “complex” surfactant and protecting the GNPs.[25] That knowledge is, however, based on tests only with spherical particles. In other publications that discuss PC formation on anisotropic nanoparticles, the adsorption sites and protein-induced stabilization are either not discussed or assumed to be the same as for spherical nanoparticles – that is uniform protein coverage. However, we had previously observed that AuNS samples had reduced BSA fouling – i.e. not full PC coverage – and retained the ability of AuNS to enhance Raman intensities of small molecules.[31] We, therefore, expected to see some effect of ionic strength (aggregation) even in the presence of BSA and were surprised to see the nanostar data of Figure 2 which imply that the nanostars and spherical particles behave in the same way (retaining their stability after pre-incubation with BSA). We are left with the question of how GNPs with complex shapes have a protein-mediated stabilization mechanism which prevents aggregation, while having plasmonic “hot-spots”, areas of high curvature with tightly confined plasmons, that create large electric filed enhancement, accessible to analytes (Figure 2 D ).[34]