pH-Responsive Near-Infrared Emitting Gold Nanoclusters

: Near-infrared (NIR) fluorophores with pH-responsive properties suggest merits in biological analyses. This work establishes a general and effective method to obtain pH-responsive NIR emissive gold nanoclusters by introducing aliphatic tertiary amine (TA) groups into the ligands. Computational study suggests that the pH-responsive NIR emission is associated with electronic structure change upon protonation and deprotonation of TA groups. Photo-induced electron transfer between deprotonated TA groups and the surface Au-S motifs of gold nanoclusters can disrupt the radiative transitions and thereby decrease the photo-luminescence intensity in basic environments (pH = 7– 11). By contrast, protonated TA groups curb the electron transfer and restore the photoluminescence intensity in acidic environments (pH = 4–7). The pH-responsive NIR-emitting gold nanoclusters serve as a specific and sensitive probe for the lysosomes in the cells, offering non-invasive emissions without interferences from intracellular autofluorescence.

One fascinating feature of fluorescent AuNCs is the accessible engineering of their photoluminescence (PL) properties.[23][24] Therefore, the molecular design of the ligands is a potential toolbox to allow AuNCs with desired properties.For instance, the PL intensity of AuNCs can be increased by prolonging the ligand alkyl chain, [25] introducing benzene functional groups, [26] or increasing the amount of gold-affinitive thiol groups in the ligands. [27]To date, major attention has been directed to PL efficiency enhancement, while the exploration of NIR emissions and pH responsiveness has been overlooked.
In this work, the relevant role of the TA groups in the pH-responsive PL properties is demonstrated through density functional theory (DFT) and time-dependent DFT (TD-DFT) study.It is the charge transfer between the TA groups and the Au-S motifs that affects the emission intensity at different pH.Upon pH changes, the TA groups are protonated/deprotonated, altering the electronic structures of PDMAEMA-AuNCs.Deprotonating TA groups facilitates the nonradiative charge transfer between TA groups and AuNCs, leading to decreased PL intensity.Protonation, on the other hand, curbs the PL-unfavorable charge transfer and restores the radiative transitions to increase the PL intensity.Experimentally, AuNCs capped by other quaternary and tertiary amine-rich ligands show similar NIR emissions.Remarkably, TA groups can lead to the pH-responsive PL, suggesting their crucial effect on the properties.
[32] PDMAEMA, the polymer ligand of PDMAEMA-AuNCs, has been extensively used in gene/ drug deliveries [33] because of TA groups that show promising lysosome-targeting features. [34]Capped by these ligands, PDMAEMA-AuNCs can enter the cells and be easily directed to lysosomes, whose acidic environments stimulate the PL enhancement of PDMAEMA-AuNCs, allowing bioimaging.This work presents PDMAEMA-AuNCs as a promising biocompatible, pH-responsive, and NIR-emitting probe with great potential in bio-analytical applications.

Results and Discussion
In this work, the TA-rich polymer ligand, PDMAEMA, is terminated with a thiol group to increase its chemical affinity to gold (Figure S1).Besides TA and thiol groups, it also embraces carboxylic and nitrile groups, which do not contribute to the observed pH-responsiveness of the nanoclusters, thus warranting a denotation simply as PDMAE-MA to emphasize the functionality(for details, see Supporting Information).PDMAEMA-AuNCs are produced following the modified Brust-Schiffrin route (Scheme 1), which is a typical two-step process: (a) the generation of intermediates, Au(I)-SR oligomers, and (b) the formation of Au(I)-SR motif-capped AuNCs. [29]Firstly, the thiol-terminated PDMAEMA (PDMAEMA-SH) is mixed with HAuCl 4 in water.The mild reducibility of the thiol group allows an incomplete reduction of Au(III) to Au(I), forming Au(I)-S(PDMAEMA) oligomers as the intermediates. [20]ubsequent addition of NaBH 4 partially reduces Au(I) to Au(0) upon controlling the NaBH 4 amount, forming the Au(0) kernels that are capped by the Au(I)-S(PDMAEMA) oligomers.Consequently, the AuNCs stabilized by the TArich thiolate ligands are synthesized.
As indicated by Fourier-transform infrared spectroscopy (FTIR), AuNCs are well encapsulated in the polymer scaffolds.The FTIR spectra of PDMAEMA-AuNCs and the pure polymer ligand, PDMAEMA, are almost identical (Figure 1a).The transmittance peaks at 2989 and 2928 cm À 1 are ascribed to the stretching vibration of CÀ H, and 1149 cm À 1 corresponds to the bending vibration of À CH 2 while the peak at 1149 cm À 1 is assigned to the stretching vibration of CÀ N and 1716 cm À 1 the stretching vibration of C=O, [35] suggesting the presence of TA groups.High-angle annular dark-field scanning transmission electron microscope (HAADF-STEM) reveals the microscopic morphology of PDMAEMA-AuNCs, which can efficiently exclude the interferences from the polymer ligand and show heavier atoms like Au.As shown in Figure 1b, PDMAEMA-AuNCs appear as ultra-small particles with an average diameter of � 1.2 nm.The size of PDMAEMA-AuNCs is in accordance with most fluorescent AuNCs (d < 3 nm). [1]The few-atom Scheme 1.The synthetic process of PDMAEMA-AuNCs following the modified one-phase Brust-Schiffrin strategy. [28,29]For clarity, the schematics show only the contributing groups, i.e., TA and thiol groups.The exact structure of a ligand is shown in Figure S3 in the Supporting Information.The TA-rich ligands are protonated and deprotonated in acidic and basic environments, respectively.structures can be observed in a zoomed-in view (Figures 1c  and 1d).
PDMAEMA-AuNCs show a NIR emission upon excitation in the far-red region, as shown in Figure 1e.The excitation peak is centered at 680 nm ( � 1.83 eV), and the emission peak is at 792 nm ( � 1.57 eV), exhibiting a Stokes shift of 112 nm (0.26 eV).The average PL lifetime of PDMAEMA-AuNCs is 3.6 μs (Figure 1f).The triplet emission feature suggests the PL stems from the ligand-gold charge transfer processes. [26]The charge transfer-induced PL is less size-dependent in terms of emission energy, as compared with that originating from the gold kernel. [6,9]This is manifested in the PL properties of PDMAEMA-AuNCs composed of different numbers of gold atoms, which can be tuned by changing the molar ratio of ligand to gold in the synthesis (Figure S5).As shown in Figure 1g, PDMAEMA-AuNCs with numbers of gold atoms differing by � 10 give out almost the same emission at � 790 nm ( � 1.57 eV).It means that the emission energy is gold-atom-numberindependent in PDMAEMA-AuNCs, which is unlikely for the emissions from the gold kernel. [6]On the other hand, the size-independent PL of PDMAEMA-AuNCs is consistent with other surface-emissive AuNCs, whose PL originates from the radiative S!Au charge transfer. [11]n addition, the effects of PDMAEMA on the PL properties of PDMAEMA-AuNCs are also investigated, since hyperbranched or linear polymer structures with TA groups can generate fluorescent centers, allowing intrinsic blue emission. [36,37]In this work, PDMAEMA is nonemissive, and NIR emissions cannot be observed without gold precursor in the synthesis, suggesting the indispensable role of gold in the PL properties of PDMAEMA-AuNCs (Figure S6).All the results indicate the PL of PDMAEMA-AuNCs is derived from their surface Au(I)-S(PDMAEMA) oligomeric motifs, where the S-Au charge transfer is essential.Radiative transitions between low-potential Au-S d orbitals and high-potential Au-S/Au-Au sp orbitals result in the PL emission (Figure 1h). [38]

Reversible pH-responsive properties
The PL intensity of PDMAEMA-AuNCs exhibits an interesting pH-responsive behavior.As shown in Figures 2a  and 2b, PDMAEMA-AuNCs show enhanced emission in an acidic environment (pH 4-7), reaching the maximum at pH = 4.4.The intensity drops sharply when the pH rises from 8 and reaches the minimum at pH = 10.5.In contrast, the emission wavelength does not show major pH dependence.The results suggest that the intensity change is related to the change in the number of emissive centers, which varies with different pH values.The basic environment reduces the number of PL centers and thereby decreases the PL intensity.Figure 2c presents the fluorescence QYs of PDMAEMA-AuNCs at different pH values.Notably, PDMAEMA-AuNCs have a QY of � 3.5 % at pH = 4.1, using indocyanine green as the reference. [39]This QY value significantly exceeds the QY value of 0.2 % typically seen in water-soluble NIR-emitting AuNCs.The QYs also exhibit pH-dependent changes, mirroring the trend observed in the PL intensity.When the pH reaches 11.0, the QY decreases to 0.84 %.Both the QY and the PL intensity start to decrease significantly with deprotonation of TA groups (pK a = 8.4). Figure 2d shows the PL intensity of PDMAE-MA-AuNCs undergoing a few cycles of acid-base condition.The pH-responsiveness of the PL intensity is reversible in the pH range of 4 and 11.The PL intensity change is protonselective and highly associated with the pH-induced protonation and deprotonation of TA groups.The absorption peaks at � 315 nm and � 400 nm are assigned to the internal charge-transfer transitions that occur upon deprotonation of TA groups. [40,41]The absorbance increases with the rising pH as a result of increased amine deprotonation.Cryogenic transmission electron microscopy (cryo-TEM) is employed to investigate the dispersion state of PDMAEMA-AuNCs in water at different pH values (Figure 2g and Figure S7).The Cryo-TEM results indicate that no aggregation of PDMAEMA-AuNCs occurs within the pH range of 4 to 11, except when approaching the isoelectric point, which leads to surface charge removal and subsequent precipitation.These observations rule out the possibility of aggregation induced emission enhancement, which could have contributed to the increase in PL intensity under acidic conditions.All these results demonstrate that the protonation and deprotonation of PDMAEMA-AuNCs' ligands at different pH values affect the PL intensity.

Computational study on the pH-responsive PL properties
DFT and TD-DFT calculations are employed to investigate the underlying mechanism of pH-dependent PL.As discussed above, the PL emission of PDMAEMA-AuNCs stems from the surface Au(I)-S(PDMAEMA) scaffold.For simplicity, the constitutive unit of Au(I)-S(PDMAEMA) is considered in the computational study (Figure 3a).Given that multiple TA groups are included in one PDMAEMA ligand, the study starts with one TA group.Ligand-S-Au structures with an increasing number of TA groups are investigated to reveal the effect of multiple TA groups on electronic properties.In this work, structures with one, two, and three TA groups are studied, and they are denoted as 1DMAEMA-S-Au 2 , 2DMAEMA-S-Au 2 , and 3DMAEMA-S-Au 2 , respectively.
Figure 3b shows the major molecular orbitals of the 1DMAEMA-S-Au 2 structure in both deprotonated and protonated states.Note that the lowest unoccupied molecular orbital (LUMO) does not change noticeably despite the protonation state of the TA group, which is always located at the Au-S motif.It is the highest occupied molecular orbital (HOMO) that changes in the process.The sp orbital of the deprotonated TA group has a relatively low energy level, placing itself at a position between the Au-S sp and d orbitals.Therefore, HOMO is distributed at the TA group when considering the structure as a whole.For the TA + group, protonation increases the energy level of its molec-ular orbitals above that of the LUMO, restoring the highest Au-S d orbitals to the HOMO.
Investigations on the neighboring orbitals further demonstrate the HOMO change.The HOMO-1 of 1DMAEMA-S-Au 2 exhibits close spatial distribution and energy level with the HOMO of 1DMA + EMA-S-Au 2 .The same sit-uation is observed between the HOMO-2 of 1DMAEMA-S-Au 2 and the HOMO-1 of 1DMA + EMA-S-Au 2 .This is because the sp orbitals of the TA group replace the Au-S d orbital as the HOMO upon deprotonation.On the other hand, the orbital of the TA + group can be found at the LUMO + 5 of 1DMA + EMA-S-Au 2 , which is close to a The same situation can be observed in the 2DMAEMA-S-Au 2 , where two TA groups are included in the structure.Upon deprotonation, the HOMO is spatially distributed at the TA groups, and the LUMO at the Au-S motifs, while the HOMO reverts to the highest Au-S orbital as the TA groups are protonated (Figure S8).Competitive behavior among TA groups emerges when there are more than 2 TA groups in the ligand, as demonstrated by the electronic structures of 3DMAEMA-S-Au 2 (Figure 3c).Nevertheless, at the deprotonated state, the HOMO and its neighboring orbitals are distributed exclusively at the TA groups, and the protonation returns the HOMO to the Au-S motifs.The LUMOs do not exhibit obvious changes upon protonation and deprotonation of the TA groups regardless of their number in the ligand.Thus, it is reasonable to expect that the number of TA groups in the ligand does not affect the HOMO-LUMO structure.
Table 1 summarizes the calculated excitation energies and oscillator strengths for the HOMO-LUMO transitions in different protonated and deprotonated structures.The HOMO-LUMO transitions of the deprotonated structures, namely -methyl-N-dimethyl!Au-S (sp), are almost nonradiative, as indicated by their near-zero oscillator strengths.It means that the charge transfer between TA groups and Au-S motifs is non-radiative.The oscillator strength of the HOMO-LUMO transition in the protonated structures is one order of magnitude higher than that in the deprotonated structures, if non-zero.This result suggests that the d-sp transitions within the Au-S motifs are radiative, further supporting that PDMAEMA-AuNCs are surface-emissive, as discussed above.Protonating the TA groups curbs the PL-unfavorable charge transfers to keep the radiative transitions within the Au-S motifs.The energy for the radiative transition is � 1.82 eV, showing little dependency on the number of TA groups.The computational results can be validated experimentally.The PL intensity of PDMAE-MA-AuNCs at pH 4.5 is 6-10 times larger than that at pH 10.5.The excitation energy of PDMAEMA-AuNCs is � 1.83 eV, in good accordance with that of the radiative HOMO-LUMO transitions in the Au-S motifs.
Scheme 2 illustrates the mechanism of pH-responsiveness of PDMAEMA-AuNCs.Upon excitation, electrons and holes are generated within the Au-S motifs.In a decay process, the unpaired electrons from the deprotonated TA group transfer to the Au-S motifs to recombine with the holes, whereas the excited electrons at high-energy sp orbitals transfer to the TA groups (Scheme 2a).Clearly, the radiative transition is disrupted by the non-radiative charge transfers, decreasing the PL intensity in the basic environment.On the other hand, protonation lifts the orbitals of the TA + group out of the HOMO-LUMO gap (Scheme 2b).Radiative S!Au transitions are thereby restored, increasing the PL intensity in the acidic environment.It is the charge transfers between the Au-S motifs and TA groups that determine the PL intensity at different pH values, and this process is affected by the protonation degree of the TA-rich ligand.
To further validate the effect of TA groups, AuNCs capped by different amine-rich/free ligands are synthesized through the same synthetic route (Table 2).The presence of  both quaternary and tertiary amine groups leads to a NIR emission ( � 1.55 eV) with similar excitation energy ( � 1.80 eV), whereas AuNCs stabilized by amine-free ligands do not show NIR emissions.Interestingly, the presence of TA groups can result in reversible pHresponsive PL properties, whereas this is not observed for quaternary amine groups.This is ascribed to the protonation and deprotonation of TA groups.All of the results indicate that TA groups are important for preparing pH-responsive and NIR-emitting AuNCs.

In-vitro NIR imaging of lysosomes
The features of long PL-lifetime, large Stokes-shift, far-red exciting, and NIR-emitting make PDMAEMA-AuNCs favorable for bioanalytical applications.The amine-rich surface of PDMAEMA-AuNCs allows an easy cellular uptake and lysosome-targeting properties. [42]Lysosomes are common organelles in mammalian cells with the important feature of maintaining a weakly acidic (pH 4.5-5.5)environment for enzymatic reactions, which allows the optimized PL emission of PDMAEMA-AuNCs.Figures 4a and 4b show the confocal microscopy images of NIH/3T3 murine fibroblast cells and S180 sarcoma cells with PDMAEMA-AuNCs.The fluorescence images are obtained with an excitation at 640 nm (power level 20 %, exposure time 100 ms).Signals with emission wavelength beyond 655 nm are collected with a long-pass filter.Fluorescent dots with a size of 1-5 microns being observed in those cells are assigned to the lysosomes stained by PDMAEMA-AuNCs (see more in Figures S10 and S11).The cytotoxicity evaluation results reveal that PDMAEMA-AuNCs exhibit low cytotoxicity to NIH/3T3 and S180 cells at concentrations below 700 μg/mL, as shown in Figure 4c.Despite the reported association of cytotoxicity with the presence of TA groups in the ligands of PDMAEMA-AuNCs, the relatively low polymerization degree (25-60) and the copolymer nature of the ligand, have helped to minimize the negative impact of PDMAEMA-AuNCs on cells. [33] commercially available lysosome-staining dye, Lyso Tracker (Green), is adopted as a reference to verify the location of lysosomes in the S180 cells.As revealed in Figure 4d, most PL signals from PDMAEMA-AuNCs are located in lysosomes indicated by Lyso Tracker upon excitation at 470 nm.These results confirm that PDMAE-MA-AuNCs can effectively image the lysosomes in the living cells due to the lysosome-specific TA groups and the PL-favorable acidic environment in those organelles.Compared to visible-fluorescent lysosome-specific organic dyes, NIR-emitting PDMAEMA-AuNCs offer more non-invasive emissions whilst fewer interferences from intracellular autofluorescence (Figure S12).

Conclusion
In summary, the introduction of TA groups into the ligands leads to the pH-responsive NIR emission of AuNCs, which is relatively rare for fluorescent AuNCs and useful in biological analyses.The deprotonation of the TA groups changes the electronic structures of AuNCs, mainly the position of HOMO, and thereby affects the PL emission intensity.In basic environments, the deprotonated TA groups have a low-potential molecular orbital, making electron transfer between TA groups and the emissive Au-S scaffold thermodynamically favorable upon excitation.The PL intensity is decreased because of the non-radiative charge transfers.But the protonated TA groups do not transfer electrons to the fluorescent Au-S motifs because of their high-potential molecular orbitals.Non-radiative charge transfers are thermodynamically unfavorable.Thus, the PL intensity of AuNCs is restored in the acidic environment.This work demonstrates the effects of aliphatic amine groups on the PL properties of AuNCs, establishing a general ligand engineering strategy for synthesizing functional gold nanoclusters that promise great bio-application potential.

Figure 1 .
Figure 1.(a) FTIR spectra of PDMAEMA-AuNCs and the polymer ligand, PDMAEMA.High similarity in terms of the peak positions indicates similar surface composition between PDMAEMA-AuNCs and PDMAEMA.(b-d) HAADF-STEM images of PDMAEMA-AuNCs, which have an average diameter of 1.2 nm (b, inset: the size distribution of PDMAEMA-AuNCs).The few-atom structures can be observed (c, d).(e) Normalized excitation and emission spectra of PDMAEMA-AuNCs.(f) Time-resolved PL spectrum of PDMAEMA-AuNCs.The average PL lifetime reaches 3.6 μs, indicating a phosphorescence nature.(g) Matrix-assisted laser desorption/ionization-time of flight mass spectra of PDMAEMA-AuNCs with different molecular masses, namely PDMAEMA-AuNCs 1 and PDMAEMA-AuNCs 2. The mass difference indicates a molecular mass of � 10 gold atoms.Inset: normalized PL spectra of PDMAEMA-AuNCs 1 and 2. (h) Schematic illustration of the PL origin of PDMAEMA-AuNCs.The surface Au-S charge transfer gives rise to the PL emission.Radiative transitions take place between the d and sp orbitals.

Figures
Figures 2e-f reveal the effects of protonation and deprotonation of the polymer ligand, PDMAEMA, in PDMAEMA-AuNCs.As shown in Figure2e, the zeta potential of PDMAEMA-AuNCs decreases monotonously within the pH range of 4 to 11.The slipping plane of PDMAEMA-AuNCs is negatively charged at pH = 11.5, which is changing with the protonation of TA groups.The zeta potential rises from À 31 mV at pH = 11.4,to + 27 mV at pH = 4.8, due to the accumulation of protonated TA groups in PDMAEMA.Figure2fshows the UV/Vis absorption spectra of PDMAEMA-AuNCs as a function of pH.The absorption peaks at � 315 nm and � 400 nm are assigned to the internal charge-transfer transitions that occur upon deprotonation of TA groups.[40,41]The absorbance increases with the rising pH as a result of increased amine deprotonation.Cryogenic transmission electron microscopy (cryo-TEM) is employed to investigate the dispersion state of PDMAEMA-AuNCs in water at different pH values (Figure2gand FigureS7).The Cryo-TEM results indicate that no aggregation of PDMAEMA-AuNCs occurs within the pH range of 4 to 11, except when approaching the isoelectric point, which leads to surface charge removal and subsequent precipitation.These observations rule out the possibility of aggregation induced emission enhancement, which could have contributed to the increase in PL intensity under acidic conditions.All these results demonstrate that the protonation and deprotonation of PDMAEMA-AuNCs' ligands at different pH values affect the PL intensity.

Figure 2 .
Figure 2. (a-b) The PL emission spectra (a) and the peak intensity at 792 nm (b) of PDMAEMA-AuNCs at different pH values.Inset: photographs of the PDMAEMA-AuNCs solution at different pH values.PDMAEMA-AuNCs remain dispersible under both acidic and basic conditions.(c) The fluorescence QYs of PDMAEMA-AuNCs at different pH values.(d) The PL emission intensity of PDMAEMA-AuNCs at cyclic acidic and basic environments.(e) Zeta potentials of PDMAEMA-AuNCs over pH values.The zeta potential increases monotonously when pH decreases.(f) The UV/Vis absorption spectra of PDMAEMA-AuNCs at different pH values.(g) Cryo-TEM images of PDMAEMA-AuNCs at pH 4.3 (up) and 11.2 (down), respectively.No aggregation can be observed.

Scheme 2 .
Scheme 2. The mechanism of the pH-responsiveness of PDMAEMA-AuNCs.The electronic structure of PDMAEMA-AuNCs varies upon deprotonation and protonation of TA groups, affecting the PLunfavorable charge transfer between TA groups and the emissive Au-S scaffold.

Table 2 :*
Excitation energies, emission energies, and pH-responsive properties of AuNCs capped by different aliphatic amine-rich/free ligands.All the AuNCs are synthesized through the same modified Brust-Schiffrin method.For detailed structure please see FigureS3.

Figure 4 .
Figure 4. In-vitro NIR imaging of NIH/3T3 and S180 cells with PDMAEMA-AuNCs.(a 1 -a 3 ) Confocal microscopy images of NIH/3T3 cells in the transmitted light mode (a 1 ), the fluorescence mode (a 2 ), and the combined mode of the transmitted light and fluorescence channels (a 3 ).Fluorescent structures are marked in red in the combined mode.(b 1 -b 3 ) Confocal microscopy images of S180 cells in the transmitted light mode (b 1 ), the fluorescence mode (b 2 ), and the combined mode of the transmitted light and fluorescence channels (b 3 ).(c) The cell viability of NIH/3T3 and S180 cell lines after incubation with varying concentrations of PDMAEMA-AuNCs for 24 h.(d 1 -d 3 ) Colocalization imaging results of S180 cells.(d 1 ) The red channel only shows the fluorescence signal from the PDMAEMA-AuNCs (excitation at 640 nm).(d 2 ) The blue channel only shows the fluorescence signal from Lyso Tracker (excitation at 470 nm).(d 3 ) The merged image shows both the signals from PDMAEMA-AuNCs (red) and Lyso Tracker (blue).Scale bars 20 μm.

Table 1 :
TD-DFT calculation results.The energy for the HOMO-LUMO transition and the corresponding oscillator strength.