3.1. Microscopic Investigation
The colloid obtained by laser ablation of an iron target in water has a zeta potential value of +20.0 mV, which is lowered to +13.9 mV when a gold target is also ablated. This lowering is due to the adsorption of negative ions deriving from the water environment on the (positive) surface of the gold nanoparticles. However, the zeta potential is sufficient to provide stability to the bimetallic colloid, with no precipitate visible a week after preparation. The zeta potential data are reported in Figure S1
The bimetallic colloid presents a red color and exhibits magnetic properties, as shown in Figure S2
. When approaching a magnet, the colloidal nanoparticles aggregate, until they appear as a dark red precipitate visible to the naked eye.
Observing the TEM images (see Figure 1
), the colloid consists of spheroidal particles with dimensions ranging from a few nanometers to almost 20 nm in diameter. Based on the contrast, two kind of nanoparticles, with weaker contrast (low contrast, LC) and stronger contrast (high contrast, HC), can be distinguished. LC particles are mainly particles of a few nanometers, whereas the HC particles have two size classes: particles of a few nanometers and particles with a diameter of 10–20 nm. From the point of view of the metallic composition, the sample contains Fe and Au (in addition to O). The large HC particles are substantially composed of Au. Large LC particles are composed of Fe. The small particles, for which it is not possible to make EDX measurements on single individuals, show both Au and Fe. The EDX analyses of typical HC and LC nanoparticles are reported in Figure S3
SAED on an enlarged field of the sample (Figure 1
) shows interplanar distances consistent with magnetite (Fe3
) and metallic gold. In particular, the ring around 2.36 Å is quite strong and must be attributed to the 111 reflection of gold [39
]. Analysis of the high-resolution microscopic images (HRTEM) (Figure 2
) shows interplanar distances typical of metallic Au for HC particles, both large and small. Crystalline growth in HC particles is observed as icosahedrons. LC particles show interplanar distances typical of magnetite [40
], but also the presence of some small particles with amorphous characteristics. In conclusion, the gold nanoparticles appear to be embedded in a mucilaginous matrix consisting of magnetite in the form of nanoparticles of various sizes, with scarce tendency to aggregation, which can be separated from the aqueous environment under the action of a magnetic field.
3.5. Surface-Enhanced Raman Scattering
The evidence of the surface plasmon band of nanosized gold particles (see Figure 5
) suggests the possibility of SERS activity of this bimetallic system. However, the molecular ligand needs to be effectively adsorbed to provide a reliable Raman enhancement. Hence, in order to find confirmation of our hypothesis, we checked the SERS response of the bimetallic colloid in the presence of 10−4
M 2,2′-bipyridine (bpy). By activation with NaCl, we observed a satisfactory SERS spectrum of bpy in the bimetallic colloid (Figure 6
), with results quite similar to those reported in the literature for the adsorption of bpy on pure gold colloidal nanoparticles [44
]; those frequencies are reported in Table 1
for comparison. This similarity indicates that our SERS spectrum is attributable to 2,2′-bipyridine bound to the gold nanoparticles present in the Fe3
colloidal matrix, which does not impair the ligand adsorption on gold. In Table 1
the IR and Raman frequencies of solid bpy [45
] are also reported.
The addition of NaCl was necessary to obtain a satisfactory SERS spectrum of bpy. The presence of chloride anions, which strongly adsorb on the surface of the gold nanoparticles, has double validity because it can promote both the nanoparticle aggregation necessary for an efficient SERS response and the formation of active sites capable of strongly binding ligand molecules, similar to what occurs with silver nanoparticles activated by chloride anions [46
]. In practice, in our bimetallic suspension it was not possible to obtain a valid SERS spectrum of 2,2′-bipyridine, even at 10−4
M concentration, unless we added NaCl. To induce particle aggregation or concentration, magnetic attraction could be employed, instead of adding chloride anions, in order to improve the SERS signal of the adsorbed ligands. In the future, this method will be tested by also evaluating the occurrence of possible problems in colloidal stability. In the present work, we used chloride activation to obtain an effective SERS response in a stable aqueous suspension in order to evaluate the possible use of these nanoparticles in biomedical applications.
However, one last problem remains to be solved: what kind of active site on the surface of the gold nanoparticles is involved in the interaction with the molecule, given that the XPS spectrum also showed the presence of ionized gold such as Au(I)? DFT calculations on the molecule linked to a neutral or a positively charged gold adatom can help in this purpose.
3.6. DFT Calculations
In Table 1
the experimental SERS frequencies of bpy are compared with those calculated for bpy/gold model complexes, along with the IR and Raman frequencies of solid bpy [45
], whose molecules present a trans-planar structure. We observe that the prominent SERS bands (at 353, 651, 764, 1016, 1059, 1179, 1306, and 1485 cm−1
) correspond to the bpy Raman bands of Ag
symmetry species. For the simulation of the SERS spectra of the adsorbed bpy, we used the functional B3LYP, along with the Lanl2DZ basis set.
The choice to use this basis set was justified by the following considerations.
This basis set has been widely employed in many literature articles to successfully reproduce both the structural and vibrational properties of different molecules. Here we report only a few very recent examples [49
Core electrons can be treated in an approximate way via effective core potentials (ECPs). This treatment includes scalar relativistic effects, which are important for the proper description of the geometric, electronic, and spectroscopic properties of heavy atoms. The LanL2DZ basis set is the best known basis set for molecular systems containing these atoms and for the efficient simulation of the Raman spectra of complexes with transition metals and the SERS spectra of molecules adsorbed on silver or gold nanoparticles, as demonstrated by many recent papers (for example, [38
We also tested the reliability of this basis set by examining the free 2,2′-bipyridine molecule in its typical trans
conformation and comparing our DFT results with those reported in the literature [48
] for the same molecule, with the same functional but with a different basis, 6-31+G*. The Lanl2DZ basis set used by us provided results generally comparable with those reported in the literature, as shown in the Supplementary Materials
, regarding both structural parameters (Table S1
) and vibrational frequencies (Table S2
DFT calculations were performed for two gold complexes, where the bpy molecule in cis
conformation is linked by means of the nitrogen atoms to a neutral Au atom or to a gold cation, Au+
. The complex bpy/Au+
better reproduces the observed SERS frequencies than the complex bpy/Au°. In the first case, the average error between the calculated and observed frequencies is 7.75 cm−1
; in the second one, the average error is significantly larger at 13.27 cm−1
. In addition, the interaction of the molecule with a neutral atom is quite weak, in comparison with the interaction with Au+
, as shown by the bpy→gold electronic charge transfers and the N–gold bond distances reported in Table 2
, with |e
| being the unsigned electron charge. The Mulliken partial charges are reported in Table S1
. Hence, it is possible to conclude that the ligand molecules, when they adsorb on gold, strongly interact with positively charged active sites of the nanoparticle surface. Figure S4
shows the calculated normal modes of the bpy/Au+
complex relative to the prominent SERS bands. All these correspond to in-plane vibrations of the bpy molecule, in particular, the bands observed at 356, 651, 764, and 1016 cm−1
correspond to ring deformations, and those at 1306 and 1485 cm−1
to H bending modes.
To better quantify the charge transfer, we also employed a descriptor (called DCT
, charge transfer distance) [56
] that was mainly proposed to describe electron–hole displacement in optical excitations (Sn
= 1, 2…, with S being singlet electronic states). The DCT
version adopted here is based on a partial charge (namely Mulliken’s) approach, using the spreadsheet reported in the Supplementary Materials
of reference [56
] and already employed with success for electronic transitions [37
]. With the DCT
scheme, the difference between the electronic density of the ground state (S0
) and the excited state of interest (Sn
) gives rise to a charge separation that can be modeled in a dipolar fashion due to a barycenter of reduced electronic charge (Q+
here) and a barycenter of increased electronic charge (Q
− here). The vector connecting the two points gives a straightforward depiction of the direction and magnitude of the overall charge movement and allows for calculating the amount of charge transferred. While this powerful yet easy approach was mainly developed to model different electronic states of the same system, it can, in principle, also be adopted for ground states of systems with different components (as long as the geometry of common moieties of the relaxed systems does not change significantly); this is discussed in more detail in the Supplementary Materials
To the best of our knowledge, this is the first time the DCT
index has been adopted to describe charge rearrangements due to surface effects and not to light excitations, and it is reported in Figure 7
With this approach, the computed charge transfer distance is about ~1.95 Å and the amount of charge moving is ~1.1 |e|, higher than that estimated from just the increase of electron charge on the Au atom; this is due to the fact that the DCT takes into account the charge displacement over the whole system.
Finally, it is appropriate to define the limits of the DFT calculation model used by us, based on the chemical interaction between a bpy molecule and a single (positively charged) metal adatom. This complex correctly reproduces the positions of the SERS bands, because it is able to predict how the structure and, therefore, the force constants of the molecule change due to interaction with the metal. However, our model fails to satisfactorily reproduce the observed SERS intensities, as shown in the simulated SERS spectrum reported in Figure S5
. Actually, in the case of 2,2′-bipyridine, which is linked to gold in a bidentate way by means of the lone pairs of the nitrogen atoms, our model cannot simulate the effect that the gold nanoparticles have on the polarizability of the adsorbed molecule and, therefore, on the intensities of the observed SERS spectrum.