Average and single SERS spectra of BSA and HSA

The SERS spectra of BSA and HSA were collected under identical experimental conditions. After the 0.01 g/mL protein solutions were respectively mixed with the gold nanoparticles in 1:10 volume ratio, the sample pH was ~4.5 for both proteins. At this pH, both BSA and HSA possess a net positive surface charge, given their respective pI’s of 5.60 and 5.67 calculated using the ‘Compute pI/Mw tool’ of ExPASy Bioinformatics Resource Portal [231]. At this pH, BSA was shown to remain in its native folded structure [232], and due to their high structural similarity, it is expected that major pH-induced changes in the folding of HSA also do not occur. The samples were respectively transferred onto a CaF2 slide, and SERS spectra were recorded as described in Sections 4.4 and 4.6.

Figure 6.2 shows the averages of ~100 background corrected and vectornormalized BSA and HSA SERS spectra obtained with gold nanoparticles in solution. Table 6.2 provides the assignments of important bands in the spectra. In accordance with previous work [199, 233, 234], the spectra are dominated by amide II and amide III vibrations [104, 216] at 1559 cm^-1 and ~1250 cm^-1, respectively, and by the bands assigned to vibrations of aromatic amino acid side chains that have high Raman cross-sections, such as their ring vibrations at ~680 cm^-1, ~840 cm^-1, ~860 cm^-1, or ~890 cm^-1 [105, 219].

The presence of the stretching vibrations of S-S bonds, at 523 cm^-1 and 533 cm^-1 [105] in HSA and BSA, respectively, suggests that the protein molecules maintain their disulfide bridges as important elements of their secondary structure upon interaction with the nanoparticle surface. This is different from the interaction of other proteins with the surface of silver nanoparticles, where cleavage of disulfide bonds is possible [39, 235].

71 Figure 6.2. Averages of ~100 BSA and ~100 HSA SERS spectra obtained with citrate-stabilized gold nanoparticles. Excitation wavelength: 785 nm, acquisition time: 1 s, excitation intensity of 5.7×10⁵ W/cm². The spectra were stacked for clarity.

The average spectra show differences in the bands 865 cm^-1, 840 cm^-1, and 642 cm^-1 assigned to different vibrations of tyrosine (see Table 6.2). Contrary to normal Raman experiments in bulk, the confinement of SERS to the immediate proximity to the nanoparticle enables the probing of those parts of the protein that interact with the surface [26-28, 233, 234]. When the primary structures of BSA and HSA are compared, it can be observed that BSA has two more tyrosine residues at the positions 155-156 than HSA [180, 181], which could potentially account for the slight spectral differences. This could be facilitated by their location in the three-dimensional protein structure: as observed in the crystallographic data of BSA [180], the two tyrosine residues at positions 155 and 156 are partially revealed on the surface of the molecule with the aromatic functional group pointing away from the protein structure. Residues closer to the protein-nanoparticle interface are expected to contribute more to the SERS signal than residues hidden in the folded structure.

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Even though vectornormalization prevents the average spectra from being dominated by a few high-intensity individual spectra, and those bands that frequently occur in the individual spectra are present in the average spectra, some band shifts and bands that occur rarely or have low intensity may disappear upon averaging. Therefore, it is useful to analyze individual spectra, and also their variation (see Figure 6.3 and Figure 6.5 below). In Figure 6.3, representative BSA and HSA single spectra from the data sets are shown, the respective band assignments are also given in Table 6.2.

Figure 6.3. Representative single SERS spectra of BSA and HSA obtained with citrate-stabilized gold nanoparticles. Excitation wavelength: 785 nm, excitation intensity:

5.7×10⁵ W/cm², acquisition time: 1 s. The spectra are stacked for clarity.

73 Table 6.2. Tentative assignments of the bands in the SERS spectra of BSA and HSA shown in Figures 6.2 and 6.3, based on refs.[40, 104-106, 215-219]. Abbreviations: ν stretching, δ deformation, br breathing, symm symmetric, wag wagging, sciss scissoring, bend bending, rock rocking, tor torsion, R benzene ring, r pyrrole ring.

Raman bands

The BSA spectrum shows pronounced signals of the -NH3+ deformation vibration of lysine residues at 1523 cm^-1 [215], of the amide III band at 1253 cm^-1 [104], of a ring

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vibration of tryptophan at 897 cm^-1 [105], and of the C-C and/or Cα-N stretching vibration at 838 cm^-1 [219]. Interestingly, the band of lysine [215] is not pronounced in the normal Raman spectrum of BSA reported, e.g., in refs.[104, 199] (Figure 6.1 and Table 6.1).

Nevertheless, its strong contribution to the SERS spectrum despite its small Raman cross-section suggests that the lysine residues must be in very close proximity to the gold surface. A direct lysine-citrate interaction has been discussed in previous work using other approaches [24, 25, 28, 63, 236], and the SERS spectra of BSA support the hypothesis that binding takes place by such an electrostatic interaction [24, 25, 28, 63, 236]. The strong amide III band at 1253 cm^-1 and the band at 838 cm^-1 assigned to vibrations of both the protein backbone and tyrosine in the BSA SERS spectrum suggest that the peptide backbone must be very close to the nanoparticle surface as well.

Interestingly, the usually very strong ring vibration of phenylalanine and tryptophan at 1004 cm^-1 [104] does not appear in the spectrum, excluding the proximity of aromatic side chains to the nanoparticle surface. Since the nonpolar amino acid side chains are mostly located inside the folded structure [180, 182], hidden from the hydrophilic environment, the absence of the 1004 cm^-1 band suggests that the probed BSA molecules preserved their secondary structure in so far as these residues do not become exposed.

In addition, the single SERS spectrum of HSA (Figure 6.3) displays selective enhancement of bands that are not particularly prominent in the normal Raman spectrum of the molecule (Figure 6.1 and Table 6.1) [237, 238]. Examples are the pronounced signals at 1550 cm^-1 of the ring stretching of tryptophan [219] or the deformation vibrations of the -NH2 group at 1512 cm^-1, at 1058 cm^-1, as well as at 1070 cm^-1 [219].

The latter two bands also contain contributions from vibrations of Cα-N and C-CH2 bonds in the peptide backbone, in accord with a distinct signal of the C-N stretching band at 1170 cm^-1 [104]. The enhancement of the SERS signals from the -NH2 groups and of the protein backbone indicate the proximity of the basic amino acid residues and of the peptide backbone, respectively, to the nanoparticle. The basic NH2 groups can be expected to interact with citrate ions in an acid-base equilibrium more than with the net positive surface of the bare nanoparticles [239] that can be revealed after the desorption of citrate ions since upon their protonation, -NH3+ groups would be electrostatically repelled from the positively charged bare gold surface. Therefore, it can be concluded that just like the BSA spectrum, also the HSA spectrum suggests the interaction of the

75 protein based on the electrostatic binding hypothesis [22, 24, 25, 63]. In the complete datasets of BSA and HSA, the 1523 cm^-1 band appears in 80 and 62, and the 1512 cm^-1 band appears in 74 and 56 spectra, respectively. This means that the interaction with the surface lysine groups has a substantial contribution to the whole dataset, which indicates the general proximity of the -NH3+ and -NH2 groups to the nanoparticles’ surface. The schematic illustration of the interaction of lysine residues on the surface of HSA and the citrate layer of the nanoparticles is shown in Figure 6.4.

As seen in the case of the SERS spectra of BSA, the 1004 cm^-1 band is absent, which suggests that the native structure of the adsorbed HSA molecules is at least partially intact.

There are several bands in both the BSA and HSA SERS spectra assigned to aromatic and aliphatic vibrations (Table 6.2), indicating their proximity to the nanoparticle surface, which could also point toward a hydrophobic interaction with the nanoparticles.

Figure 6.4. Schematic representation of the expected interaction between the citrate layer on the gold nanoparticle surface and the lysine residues of HSA (marked with green).

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