3.1 Synthesis and characterization of L-AuNP@HTT
L-AuNP@HTT were synthesised by a photochemical method by irradiating a reaction solution containing gold precursor (HAuCl4), surface ligands (HTT) and an organic solvent mixture to accelerate the process. In general, the Au(I)-HTT complex formed first, and the L-AuNPs were generated in the presence of the reducing agent EtOH[40]. As shown in Fig. S1, the HTT ligand showed most intense p-p* transition absorption at 323 nm based on the 1,3,4-thiadiazole heterocyclic system [41-44], L-AuNP@HTT and Au(I)-HTT complex displayed weak p-p* transition absorption at 323 nm and a new strong p-p* transition absorption band at 350-400 nm, arising from a new delocalized π system. Additionally, new absorptions of Au(I)-Au(I) at 279 nm and S-Au at 420 nm [16, 37] were also observed. In contrast, the n-p* transition absorption at 242 nm [45] remained almost unchanged. These observations suggest that the wavelength of the irradiation light may have a considerable effect on the rate of photochemical reactions, as well as the optical properties of L-AuNP@HTT. Further analysis, as shown in Fig. S2, revealed that when exposed to the light of 390 nm (or 365 nm/350 nm), the PL intensity of the reaction system was significantly amplified, indicating that the delocalized π system of the Au(I)-HTT complex plays a crucial role in the photochemical reduction process. The subsequent synthesis reaction utilised the 390 nm light, taking into account its penetration in solution. Conversely, 420 nm or 323 nm only resulted in a faint luminescent emission from AuNPs, suggesting that the π-π* transition of HTT or the S-Au bond has minimal influence on emission. Furthermore, the PL intensity did not increase under continuous excitation of 279 nm or 242 nm, indicating that the n-π* transition or Au(I)-Au(I) bond has little contribution to emission. These results collectively indicate that the formation of the Au(I)-HTT complex with a newly delocalized π system is a critical prerequisite for initiating photochemical reduction in the presence of electron donors.
Table 1 Photochemical synthesis of AuNPs under different solution conditions.
Sample
|
Solvent
|
Final product
|
Emission Peak (nm)
|
FWHM
(nm)
|
Relative
PL Intensity
|
1
|
TCM/EtOH (V/V=9:1)
with NaOH (4 μM)
|
Luminescent AuNPs
|
528
|
49
|
100%
|
2
|
TCM/EtOH (V/V=7.5:1)
with NaOH (4 μM)
|
Luminescent AuNPs
|
525
|
58
|
~ 25%
|
3
|
TCM/EtOH (V/V=9:1)
without NaOH
|
Luminescent AuNPs
|
526
|
61
|
~ 6%
|
4
|
TCM
without NaOH
|
-
|
N
|
N
|
N
|
5
|
EtOH
without NaOH
|
Luminescent AuNPs
|
540
|
90
|
~ 21%
|
6
|
THF/EtOH (V/V=4:1) without NaOH
|
deep red colloidal solution
|
N
|
N
|
N
|
Different organic solvents were subsequently used to prepare the L-AuNP@HTT. As shown in Table 1 and Fig. S3, highly luminescent L-AuNP@HTT could be obtained only in an alkaline trichloromethane/ethanol (TCM/EtOH) solution containing NaOH (4 μM). For comparison, similar experiments without the addition of NaOH in EtOH or TCM/EtOH solution yielded L-AuNPs with weak emission. Additionally, luminescent L-AuNP@HTT was not observed in the TCM or THF or THF/EtOH reaction solution.
Under the condition of an alkaline TCM/EtOH solution containing NaOH (4 μM), the dynamic profiles of optical and size characteristics as determined by TEM at different reaction time points (Fig. 1). The intensity of luminescence in the solution increased gradually as the reaction time was delayed, but the position of the maximum luminescence peak did not change significantly (Fig. 1a). When the creation time reached 60 hours, the photoluminescence intensity reached its maximum. Fig. 1b also depicted a linear correlation between PL intensity and time, indicating a zero-order kinetics process-a characteristic feature of photochemical reduction [46, 47]. The time-dependent UV-vis spectra shown in Fig. 1c revealed the presence of an isosbestic band in the range of 355-375 nm. This band indicates the gradual conversion of the Au(I)-HTT complex to L-AuNP@HTT as the reaction proceeds. No SPR peak was observed during the entire reaction process when 390 nm was used as the irradiation light. However, under dark or incandescent light conditions, SPR of 550 nm occurred as the reaction extended, indicating big gold nanoparticles generated (Fig. S4). The size of L-AuNP@HTT increased from 2.15 nm at 6 hours to 3.19 nm at 60 hours (Figure 1d-g).
Under optimal conditions, L-AuNP@HTT was finally prepared using an alkaline TCM/EtOH solution, 390 nm irradiation light, and 60 hours. The resulting L-AuNP@HTT exhibited a bright green emission with a peak at 528 nm and an FWHM of 49 nm (Fig. 2a and Fig. S5). This FWHM is notably narrower than previously reported luminescent AuNPs [22, 27, 29]. Furthermore, the quantum yield (QY) of L-AuNP@HTT was calculated to be 12.9% (Fig. S6), which is higher than other luminescent AuNPs (Table S1) [22, 24, 27, 32]. The absorption spectrum of L-AuNP@HTT (Fig. 2a) displays strong UV absorption and multiple discrete excitation bands. However, the typical SPR band at around 520 nm, characteristic of AuNPs, was not observed. These discrete absorption bands can be attributed to distinct electronic states and unique HOMO-LUMO transitions inherent within the gold core and between the ligand and the surface of the AuNPs. Moreover, these L-AuNP@HTT exhibit a large Stokes shift of 108 nm (Fig. 2a). This phenomenon, commonly associated with luminescent AuNPs [48] and heteroatoms-rich (mainly S, N) ligands, is likely due to an LMCT transition from the ligand to the gold(I) core [37, 49]. In addition, such a large Stokes shift is highly favorable for multicolor bioimaging and high-sensitivity biosensors, as it minimizes cross-talk between excitation and PL emission.
The photostability of L-AuNP@HTT was also demonstrated in Fig. 2b, which showed that their PL intensity remained almost constant (< 2%) under a continuous excitation at 405 nm for 1800 s. Moreover, their luminescence lifetimes were also measured (Fig. 2c), with an averaged lifetime τmean up to 0.85 μs (τ1 = 0.27 μs, weight 43%; τ2 = 1.21 μs, weight 57%). Such long luminescence lifetimes are often observed in luminescent AuNPs, and attributed to triplet luminescence through LMCT process [37, 49]. Furthermore, L-AuNP@HTT exhibited exceptional two-photon optical characteristics. As shown in Fig. S7, the emission of L-AuNP@HTT when excited by a 790 nm fs laser was comparable to that observed with 405 nm excitation. The emission signal was found to be directly proportional to the square of the excitation intensity at 790 nm (Fig. 2d), thus confirming that the measured photoluminescence (PL) emission resulted from a two-photon absorption (TPA) process. The cross-section (σ2) of L-AuNP@HTT was determined to be approximately 8×104 GM at 740 nm (Fig. 2e). This value is significantly higher than that of CdSe/ZnS QDs (~ 4.7×104 GM) and most organic chromophores (generally<102 GM) [50].
HRTEM imaging of L-AuNP@HTT revealed that the lattice spacings of 2.35 Å and 2.03 Å correspond to {111} and {200} planes of Au, respectively (Fig. 2f). Thermal gravimetric analysis (TGA) of L-AuNP@HTT was conducted in Fig. S8 and the average molecular formula of L-AuNP@HTT was determined to be Au1002HTT780. To further investigate the gold valence state, X-ray photoelectron spectroscopy (XPS) analysis was conducted (Fig. 2g). Two distinct peaks at 84.4 and 85.0 eV were detected, which were attributed to Au(0) and Au(I) states respectively, based on previous reports [37, 51]. The XPS analysis indicated that 27.9% of the gold atoms were present in Au(I) state. This suggests that surface HTT, acting as an electron transfer system in the alkaline TCM/EtOH solution, was partially responsible for the reduction of gold and it also served as a protective surface ligand during the synthesis of L-AuNP@HTT.
The underlying photochemical processes are presented in Fig. 3. Initially, an Au(I)-HTT complex is formed by mixing HTT and HAuCl4. Upon light irradiation, the photosensitive Au(I)-HTT complex decomposes to form a mixture of gold nanoclusters (AuNCs) of varying sizes. These resulting AuNCs finally grow into single size particles, forming luminescent L-AuNPs. Ethanol is pivotal in the photochemical reduction process, which is attributed to providing electrons to the photosensitive Au(I)-HTT complex. The presence of an alkali solution further increases the reducibility.
3.2 The effect of Au(I)-HTT complex on the optical properties of L-AuNP@HTT
To investigate the underlying mechanism of the superior optical emission, the influence of ligands was first examined. Surface ligands are essential for ligand-protected AuNPs as the interface chemistry between ligands and gold atoms can significantly affect both the structures and physical-chemical properties of AuNPs [52]. L-AuNP@HTT displayed an average size of 3.19 nm, but showed no SPR absorption. A ligand exchange experiment was carried out to elucidate the source of this aberrant behavior. As displayed in Fig. 4a and Fig. 4b, after exchange with dihydro lipoic acid (DHLA), DHLA-coated AuNPs (L-AuNP@DHLA) had a diameter of 2.94 ± 0.36 nm (N = 173), a slight decrease compared to the L-AuNP@HTT (3.19 ± 0.42 nm). Nevertheless, a visible SPR absorption at 520 nm was observed for the L-AuNP@DHLA (Fig. 4c). Concurrently, total PL intensity was obliterated after the undertaken ligand exchange (Fig. 4d). Similar phenomena were also observed for (5-mercapto-1,3,4-thiadiazole-2-ylthio) acetic acid (MTY) coated AuNPs (L-AuNP@MTY) (Fig. S9). These results above collectively demonstrated that the surface ligands played critical roles in both high bright emission and SPR formation of AuNPs.
The 1,3,4-thiadiazole thiols derivatives of HTT and MTY, as shown in Fig. S10, are typically conjugated π system ligands [53], which can hybridize and overlap their orbitals with the Au atoms, similar to LMCT [37], to participate in PL emission. This electron-withdrawing property of the ligands could decrease the electron density surrounding the AuNPs and, therefore, not enough free electrons on the surface of the AuNPs would be available to generate the SPR absorption band [41, 54].
Besides the surface ligand, the valence states of surface gold atoms also play an important role in the optical properties of AuNPs. Both L-AuNP@HTT and L-AuNP@MTY were observed to have Au(0) and Au(I) states (Fig. S11). To more deeply understand the effect of Au(I) on the PL emission of AuNPs, L-AuNP@MTY were carefully reduced using NaBH4. Interestingly, after NaBH4 treatment, the size of L-AuNP@MTY only changed slightly, from 2.66 ± 0.56 nm to 2.75 ± 0.47 nm (Fig. 5a). XPS spectra also confirmed that the Au(I) on the surface were completely reduced to Au (0), indicated by the Au 4f7/2 binding energy shift from 84.5 eV to 84.1 eV (Fig. 5b). The characteristic absorption peak near 420 nm weakened significantly, accompanied by the distinct appearance of a SPR absorption band at 520 nm (Fig. 5c). And the PL almost completely disappeared after NaBH4 treatment (Fig. 5d). These results demonstrated the close relationship between Au(I) and the PL emission, as well as the effect on SPR formation along the surface of AuNPs. It is noted that the high content of Au(I) species (~27.9%) in AuNPs lead to a limited number of free electrons on the gold surface, which not only affects the emission of PL but also hinders the formation of SPR absorption bands [55].
3.3 The synthesis of p-AuNPs and applications for bioimaging
The hydrophobic nature of the prepared L-AuNP@HTT poses a challenge for its potential biomedical applications, as it does not disperse well in aqueous solutions. To address this issue, we prepared PMMA-co-MAA copolymer-encapsulated AuNPs (p-AuNPs) using a method previously reported4. In comparison to L-AuNP@HTT (φ ~12%), p-AuNPs exhibited a higher QY of approximately 15% (Fig. S12) and long luminescence lifetime of 1.4 μs (Fig. S13). TEM analysis revealed that p-AuNPs had an average diameter of 42.19 nm (Fig. 6a and Fig. S14). Given that L-AuNP@HTT had a diameter of 3.19 nm, it can be estimated that each p-AuNPs contained approximately 2000 L-AuNP@HTT nanoparticles. Fig. 6b shows the absorption and PL spectra of p-AuNPs under 800 nm femtosecond laser irradiation. The maximum of the PL emission was near 534 nm, which represents a red shift of 6 nm compared to L-AuNP@HTT (Fig. 6b). Fig. 6c showed the quadratic relationship between the PL intensity of the p-AuNPs aqueous solution and various excitation laser powers at 800 nm, indicating TPE PL of p-AuNPs. Furthermore, the TPA cross-sections (σ) were above 107 GM per p-AuNPs within the excitation wavelengths range of 720-860 nm, with σ reaching up to 1.1 × 108 GM at 750 nm (Fig. 6d). The TPE brightness (σ × φ, φ is the QY) was determined to be 1.6 × 107 GM per p-AuNPs. This huge TPE brightness value is 3 orders of magnitude higher than that of CdSe/ZnS QDs determined using a similar method (~ 4.7 × 104 GM) [50]. With these p-AuNPs, high-quality subcellular TPE imaging with a high signal-to-background (SBR) and tissue imaging with a substantial penetration depth can be expected. In addition, TEM and mapping images of HRTEM in Fig. 6e and energy dispersive spectroscopy (EDS) in Fig. 6f showed that the polymer was covered on the surface of p-AuNPs particles and the Au and S elements were uniformly distributed, suggesting that the shape of p-AuNPs was nearly spherical and the distribution of L-AuNP@HTT was uniform.
The optical properties of p-AuNPs show superior stability in solutions with high salt concentrations (Fig. S15) and over a wide pH range (Fig. S16). In addition, these prepared p-AuNPs do not induce the generation of reactive oxygen species (ROS) under 365 nm irradiation (Fig. S17). These attractive properties favour p-AuNPs as versatile probes for both in vitro and in vivo bioimaging. The biocompatibility of p-AuNPs was further assessed using HepG2 cells through a growth curve analysis. As shown in Fig. S18, the growth curve of the cells indicated revealed no significant difference between the p-AuNPs group and the control group, up to a concentration of 0.2 mg mL-1 p-AuNPs. This excellent biocompatibility is highly advantageous for potential biomedical applications.
Further investigations revealed that p-AuNPs possess a robust ability to target mitochondria. As shown in Fig. 7a-c, the intracellular localization of p-AuNPs in live cells was examined through co-staining experiments in conjunction with a commercially available MitoTracker Red. Cells were co-incubated with p-AuNPs (0.2 mg mL-1) for 2 hours and MitoTracker Red (50 nM) for 30 min at 37 °C. The green luminescence emitted by p-AuNPs exhibited significant colocalization with the red luminescence from the MitoTracker dye, as evidenced by a Pearson's correlation coefficient of 0.86. These results confirmed that p-AuNPs serve as organelle-specific probes, targeting mitochondria and being retained within the organelle similar to the commercial MitoTracker dye.
Due to the outstanding biocompatibility and superior TP optical properties of p-AuNPs, high-quality two-photon mitochondria-targeting imaging have been achieved (Fig. 8a). The achieved SBR reached up to 10, indicating the clarity and precision of the imaging. Furthermore, emission spectra of the internalized p-AuNPs were acquired, conclusively confirming that the green signal indeed originated from the p-AuNPs (Fig. 8b). These results not only validate the stability of the p-AuNPs' structure but also highlight their ability to remain intact within a cellular environment.
In addition, a photostability experiment revealed that, in contrast to MitoTracker Red, p-AuNPs displayed significantly superior photostability. The photostability of p-AuNPs was assessed by continuous irradiation with confocal lasers. As shown in Fig. 8a and Fig. 8c, the luminescence intensities of p-AuNPs showed minimal signal loss after 20 scans (approximately 10% reduction), whereas the luminescence signals of MitoTracker Red decreased significantly (by approximately 70%). These results clearly demonstrate that p-AuNPs have remarkable resistance to photobleaching, indicating their suitability for long-term mitochondrial tracking application
To fully demonstrate the advantage of superior two-photon optical properties, as well as the extremely long luminescence lifetime, p-AuNPs are further utilized for deep-tissue bioimaging with the time-gated imaging (TGI), which is used for improving the contrast of luminescence images via complete removal of autofluorescence [56]. The TGI setup is similar to that used in a previous report [57]. As shown in Fig. 9a and Fig. 9b, based on p-AuNPs, high quality bioimaging with a SBR of up to 90 has been achieved, when the delay time was set to 100 ns. Furthermore, we demonstrated an incredibly large imaging depth of approximately 2000 μm using intralipid as the optical tissue phantom, as shown in Fig. 9c and Fig. 9d. These results collectively highlight the potential of p-AuNPs as promising nanoprobes for in vivo deep tissue imaging applications.
The feasibility of p-AuNPs as a contrast agent for in vivo CT imaging has been demonstrated via the intratumoral injection p-AuNPs in BALB/c mouse. As shown in Fig. 10, with intratumoral injection of p-AuNPs, transverse (x-axis) and longitudinal planes (y-axis and z-axis) CT images of the tumor provided apparent contrast and exhibited the outline against the background. Therefore, the attractive p-AuNPs would serve as a prospective probe to guide further diagnosis and treatment of tumors.