Formation of Au 25 pMBA 18 nanoclusters and their attachment to BSA. Au25 clusters protected by 18 mercaptobenzoic acid (para isomer) ligands were synthesized as described in a previous study.39 The best results were obtained when a slight excess of Au25pMBA18 over BSA (ratio 1.2:1) was used, but the synthesis was also performed with an excess of protein (ratio 1:3). Upon gentle and continuous stirring for 3 days, the Au25 nanocluster was incorporated into the BSA protein via ligand exchange between the sulfur groups from pMBA and the sulfur group from cysteine-containing amino acid residues in the BSA. A schematic representation of the proposed reaction mechanism is shown in Figure 1. These formed Au25-BSA conjugates were purified using 10% acrylamide SDS–PAGE gel separation. The gel resulted in two bands, which were attributed to Au25pMBA18 and BSA-Au25, in contrast with the single band observed for the BSA control (Fig. S1). The slight difference in the positions of the BSA and BSA-Au25 bands in the gel indicates that both the charge and overall shape of the protein subtly changed due to the incorporation of the preformed Au25 NCs into the BSA protein. We noticed that Au25pMBA18 could not bind to BSA even when we used an excess of protein (Fig. S1). This result could suggest that Au25pMBA18 is not stuck to the surface but fixed inside the protein and cannot react anymore.
Characterization of Au 25 -BSA conjugates. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to characterize the BSA-Au NCs (Figure 2a). In addition to the signature peak of the BSA protein at 66 kDa, the MALDI spectrum shows an additional peak at higher molecular weight. The molecular weight difference of this peak from BSA was 7.3 kDa, which indicates the attachment of only one gold nanocluster to the protein, as the mass for one Au25pMBA18 nanocluster is 7.68 kDa. However, the observed mass shift of the Au25-BSA was lower than expected. This discrepancy can be explained by the removal of 2-3 pMBA surface ligands during incorporation into BSA and the replacement of intrinsically available sulfur groups from the cysteine residues in the BSA. The first ligand exchange may preferentially occur with only the free cysteine residue of the BSA (Cys34), and then 1 or 2 cysteines in the disulfide bridge may become available to bind the Au25 NCs in the BSA (see Fig. S2).
Secondary structural changes in the BSA upon Au25 NC incorporation were measured by circular dichroism (CD) spectroscopy, which is shown in Figure S3. The CD spectra of the BSA and BSA-Au25 are very similar, suggesting that the BSA-Au25 retained the α-helicity to a high degree. This observation also confirms the low perturbation of the protein secondary structure content. These results are in stark contrast to the results of Xie’s classical method, which was performed under alkaline conditions, and they show the advantage of the synthetic methodology developed here.21
Small-angle X-ray scattering (SAXS) is a widely used technique for the structural and dynamic characterization of biomolecules in aqueous solutions.40, 41, 42 To obtain the scattering profiles of BSA and the BSA-Au25 complex, inline size-exclusion chromatography coupled to SAXS (SEC-SAXS) was used, as it enables the separation of the major peak corresponding to the BSA monomer from those of the BSA oligomers (Fig. S4a). Consistent with its larger molecular weight, BSA-Au25 eluted earlier than native BSA. SEC-SAXS frames corresponding to the monomer were averaged using standard procedures for subsequent structural analyses. The SAXS profiles of BSA and BSA-Au25 present different features along the momentum transfer range measured, indicating that the presence of the Au25 NC strongly modifies the scattering properties of the protein (Fig. 2b). As expected, BSA-Au25 presents a slightly larger radius of gyration, Rg, than free BSA, 28.16 ± 0.05 Å and 27.95 ± 0.02 and, respectively. When computing the pairwise distance distribution, p(r), we found that the unmodified BSA had a Dmax of 82.1 Å, similar to the value identified in earlier studies43, and a symmetric p(r) profile typical of globular proteins. In contrast, BSA-Au25 had a significantly larger Dmax of 90.85 Å and had a shoulder at small distances, indicating the presence of additional scattering centers in the protein.
Next, the capacity of the crystallographic structure of BSA (PDB entry 3v03) was evaluated to describe both SAXS curves shown in Fig. 2b. While the structure was in excellent agreement with the BSA profile (χ2 = 2.2) (Fig. 2b), unsurprisingly, it was unable the describe the curve measured for BSA-Au25 (χ2 = 24.6). To achieve a better fit between the experimental data and the theoretical structure of the BSA-Au25, we modeled the Au25 NC (see Computational for Au25 NC details in the SI) on the surface of the BSA. we selected Cys34 for this model, as it is the only free cysteine in the protein, and we placed its side chain in the position of one of the pMBA molecules of the external protective shell of the Au NC (Fig. 2c). Although the incorporation of the Au25 NC into the BSA structure improved the agreement with the experimental BSA-Au25 SAXS curve (χ2 = 6.55), systematic deviations throughout the momentum transfer range were still observed. A closer inspection of the BSA structure showed a region of the protein (residues 55-119), consisting of a long unstructured loop (residues 105-119), that could be structurally impacted in the presence of the NC. To test this hypothesis, we performed a normal mode SAXS refinement of the BSA-Au25 structure using the program SREFLEX,44 allowing movement in this region while maintaining the rest of the protein and keeping the NC fixed in place (Fig. 2c).
To show the selectivity of NC binding, we generated seven different structures with the Au25 NC attached to other cysteines of the BSA (Fig. 2d). Note that these cysteines are engaged in disulfide bonds in the native structure. For six of the BSA-Au25 complexes, the resulting structures displayed worse χ2 values than the model built using Cys34 as the anchoring point (Fig. 2e). Even after normal mode refinement, all the resulting structures provide worse agreement with the experimental data than the Cys34-anchored structure. In short, the SAXS analyses strongly indicate that the Au25 NC selectively binds to free Cys34 and that this binding induces structural perturbations to accommodate the metallic cluster.
Optical properties of the Au 25 pMBA 18 and BSA-Au25 NCs. Figure 3a shows the absorption spectra of Au25pMBA18 and BSA-Au25 NCs dispersed in water, with both clusters presenting the “fingerprint” band at ~680 nm, which is characteristic of [Au25(SR)18]− NCs.45 The absorption spectra of Au25pMBA18 and BSA-Au25 clusters are similar to the 25-Au-atom cluster (Au25(SCH2CH2Ph)18) with a known crystallographic structure.46 This result suggests that they all have similar structural features, although UV–vis absorption is not the method of choice to determine or compare structures. The attachment of Au25 to BSA leads to a significant enhancement of the PL signal (upon excitation at 700 nm) at both 850 nm and 1050 nm, as seen in Figure 3b, with a 4.7-fold increase in QY (see Figure 3d). A similar trend is observed for excitation at 400 nm (see Fig. 3c). To confirm that this effect is not only due to simple physisorption of Au25 NCs onto BSA, steady-state and time-resolved PL measurements (Figure 3e) of Au25pMBA18 and BSA-Au25 were performed in water, and Au25pMBA18 was analyzed in the presence of a high BSA concentration (50 mg/mL). Although the addition of BSA resulted in a PL intensity enhancement, the overall effect was much lower than that of direct incorporation. Better insight into the influence of BSA on the fluorescence kinetics of Au NCs can be obtained by time-resolved measurements. The addition of BSA to the Au NC solution resulted in an increase in the average amplitude weighted lifetime from 72.1 ns to 396.3 ns. However, the Au25-BSA complex possessed a PL lifetime of 936.1 ns, which indicates that the local environment of the Au25 NCs in BSA-Au25 is more rigid than that of free Au25pMBA18 dispersed in water or in the presence of unbound protein. Therefore, the tight entrapment of Au25 in the protein scaffold of BSA-Au25 is prone to multiple energy transfers associated with intersystem crossings (vide infra).47, 48
The absence of any changes in the PL intensity and lifetime of the Au25-BSA complex when added to a concentrated BSA solution is additional evidence for the successful incorporation of the cluster in the BSA protein and thus protection from its environmental surroundings (Figure S5), indicating the presence of a strong protective shell around the Au25 in this labeled protein.
In summary, by attaching a single Au25 NC to BSA through ligand exchange between protecting pMBA ligands and natural sulfur-containing BSA cysteine residues, we managed to tailor the NIR-II signal while keeping the structure of Au25 intact. Interestingly, the ratio between the relative intensity at 850 nm and 1050 nm strongly depends on the excitation wavelength (compare 𝜆exc. = 400 nm and 𝜆exc. = 700 nm, see Fig. 3 and Table S1). The I1050/I850nm ratio is also dependent on the nature of the ligands protecting the Au25 (for a comparison of Au25pMBA18 and Au25-BSA, see Table S1). To better understand the PL properties of the Au25 nanoclusters that attach to BSA more favorably via cysteine residues, which was suggested by the SAXS/MALDI results, we synthesized mixed liganded nanoclusters, Au25pMBA(18-x)Cysx with x= 2, 5, 18, through ligand exchange. Optical measurements and density functional theory allowed us to determine the key role of the metallic-ligand interface in the photophysical pathways.
Insight into the relationship between the structure and optical properties and the origin of the NIR-II emission. The PL of metal nanoclusters originates from a subtle interplay between excitations arising within the metal core and from charge transfer between the metal core and surface ligands.49, 50 A recent study also suggests the influence of structural distortion accompanied by electron redistribution in photoexcited gold nanoclusters, which induces controllable dual PL emission.51 However, the detailed PL mechanism of gold nanoclusters is still under debate.52 The high stability and detailed structure determination of Au25(SR)18 (gold nanoclusters protected by SR thiolate ligands) enabled extensive investigation of the relationship between the structure and optical properties; in particular, it enabled the study of its PL properties. Upon visible excitation, both red (700−800 nm) and near-infrared emissions (approximately and above 1000 nm) have been observed in Au25(SR)18 by different groups.53, 54, 55 On the basis of time-resolved emission and nanosecond transient absorption spectroscopy analyses, Meng Zhou and Yongbo Song proposed a simplified model,55 where visible and near-infrared emissions have different lifetimes and arise from the core−interface charge transfer state and the Au13 core state, respectively.
When Au25 is incorporated into BSA, at least one ligand exchange with the protein occurs via a cysteine residue, which significantly modifies the PL profile (Fig. 3b). To reveal the key role of the metallic-ligand interface in the photophysical pathways of BSA-Au NCs, we produced Au25pMBA(18-x)Cysx NCs with x=0, 2, 5, and 18 by ligand exchange (between pMBA and cysteine) from the Au25pMBA18 precursor nanocluster. These NCs were fully characterized by ESI-MS (see Fig. S6). Au25pMBA(18-x)Cysx with x=0, <2>, <5> and 18 presents the typical absorption features of Au25 NCs with the characteristic band centered at 680 nm and a tail band above 780 nm (Figure 4a).56 The PL profile of these Au25 NCs dispersed in water exhibits two main broad bands at 800-950 nm and 1050-1250 nm (Fig. 4b and c). When the number of Cys molecules on the Au NC surface increased from 2 to 5, an increase in the PL band at 1050 nm was observed (see Fig. 4b and c). To compare the evolution of the two main NIR-II emission bands, we determined the PL ratio of the peak at 1050 nm to that at 920 nm to determine if there is a correlation between the presence of the co-ligand and the PL enhancement at 1050 nm (Table S1). The results indicated a more pronounced effect when 700 nm was used as the excitation wavelength than when 400 nm was used, indicating that the 1050 nm emission probably involves photophysical relaxation pathways taking place within the metal core.55 Indeed, with an excitation wavelength of 700 nm, the lowest energy excited states (S1, S2) are mainly involved in photoexcitation, as confirmed by the time-dependent density functional theory (TD-DFT) linear absorption spectrum (Fig. 4d and 4f, and see computational details in SI). Low-lying states in the NIR are key to obtaining large two-photon absorption cross sections (due to double resonance effects), which would make them ideal labels for multiphoton excited luminescence (see Fig. S7). The lowest excited states in the absorption spectrum mainly belong to the “core” in Nature, primarily arising from transitions from the occupied P orbitals into the first and second sets of D orbitals (see Fig. 4e and Table S2). For low-lying excited states, some contributions of the ligands to the transitions are more readily observed in Au25pMBA16Cys2 than in Au25pMBA18 (Table S2). Under 400 nm excitation, the nature of excited states is both “interface-like” and “core-like.” The characteristic of such excited states is mainly “interface-like”57 for which the contribution from the Au-S interface in orbitals is more pronounced, particularly in the cysteine-containing Au NCs (Table S2). Although pMBA and cysteine are rich electron donors, cysteine is more flexible due to the Cα-Cβ bond and is better able to interact with the surface of the gold core than pMBA, as its carboxylic group points outward toward the surface of the gold core. Such electron-rich donor groups may contribute to the “interface-like” excited states and thus may increase the contribution of the 850 nm band.52
This phenomenon is particularly true for Au25Cys18, which mainly displays a strong emission at 850 nm. On the basis of these experimental and theoretical results, we propose the following mechanism to explain the relative contribution of emission bands (at 850 and 1050 nm) of the Au25-BSA conjugates (Figure S8): first, the local environment of Au25 NCs attached to BSA is more rigid than free Au25pMBA18 and prone to multiple energy transfers associated with intersystem crossing (reinforced ISC), which could explain the overall enhancement in PL emission and the longer PL lifetimes. Second, the nature of ligands, and in particular their capability to interact through electron-rich donor groups with the surface of the Au core, may increase the number of surface states involved in excitation (specifically at 400 nm).