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]