Surface-Engineered Gold Nanoclusters with Biological Assembly

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Surfaces, Interfaces, and Catalysis; Physical Properties of Nanomaterials and Materials

Surface-Engineered Gold Nanoclusters with Biological Assembly-Amplified Emission for Multi-Mode Imaging Xiaofeng Jiang, Xiaoyu Wang, Chuang Yao, Shuxian Zhu, Lu Liu, Ronghua Liu, and Lidong Li J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.9b02046 • Publication Date (Web): 23 Aug 2019 Downloaded from pubs.acs.org on August 23, 2019

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The Journal of Physical Chemistry Letters

Surface-Engineered Gold Nanoclusters with Biological Assembly-Amplified Emission for MultiMode Imaging Xiaofeng Jiang,§,† Xiaoyu Wang,§, †,* Chuang Yao,‡ Shuxian Zhu,† Lu Liu,† Ronghua Liu,† and Lidong Li†,* †State

Key Laboratory for Advanced Metals and Materials, School of Materials Science and

Engineering, University of Science and Technology Beijing, Beijing 100083, P. R. China ‡Key

Laboratory of Extraordinary Bond Engineering and Advance Materials Technology

(EBEAM) of Chongqing, Yangtze Normal University, Chongqing 408100, People’s Republic of China AUTHOR INFORMATION Corresponding Author *E-mail: [email protected] (X.W.). *E-mail: [email protected] (L.L.).

ACS Paragon Plus Environment

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ABSTRACT: Here, we develop bifunctional ligand-engineered gold nanoclusters (AuNCs) as signal amplifying reporters for multi-mode imaging. Modified streptavidin (SA) and biotin alkyl acid-based ligands were applied to AuNCs to form AuNC-SA and AuNC-biotin. The zwitterionic ligands promoted bio-assembly and avoided non-specific adsorption. The AuNCs resisted aggregation-induced quenching and showed strong emission benefited from biological self-assembly. The engineered AuNCs featured stable emission, a large two-photon absorption cross section, long fluorescence lifetime, and good biocompatibility. Thus, cell-expressed antigen-induced protein-binding events were effectively converted into signals from the biological assemble of AuNCs. We performed a comprehensive assay of specific antigens and the cell structure, through one-photon imaging, two-photon imaging, and fluorescence lifetime imaging of AuNCs in a simple, sensitive, and reliable way.

TOC GRAPHICS

KEYWORDS: gold nanoclusters, fluorescence, dual-ligand, enhanced emission, bioimaging


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Ultra-small gold nanoclusters (AuNCs) protected by thiolate ligands are different from larger gold nanoparticles, and have stimulated great interest in optical imaging owing to their unique luminescence properties.1-5 These unique features are attributed to the small dimensions of the AuNCs (of the order of the Fermi wavelength of electrons) and the presence of discrete energy levels.6,7 Hence, electronic transitions within the AuNCs generate optical signals. Ligand-tometal charge transfer,8,9 which occurs in the luminescence process of AuNCs can impart a long fluorescence lifetime, which might enable the influence of auto-fluorescence to be eliminated from the background to improve sensitivity. Furthermore, gold-based nanomaterials have excellent biocompatibility.10-16 AuNCs have unique luminescence properties and long fluorescence lifetimes, which has led to their use as novel fluorescence imaging agents. Recently, gold nanorods with high two-photon absorption cross sections [~2000 Goeppert−Mayer (GM)] have been used to absorb tissue-penetrating near-infrared (NIR) light and shown particular utility in bio-imaging.17,18 Furthermore, AuNCs have been confirmed to possess even higher twophoton absorption cross sections of ~105 GM.19 Therefore, the use of AuNCs as imaging agents offers great potential for multi-mode imaging with excellent performance. In bio-imaging, obtaining effective optical signals is based on interactions between the AuNCs and biological systems. Maintaining the unique luminescence properties of the AuNCs and installing functionalities to precisely control their interactions with biological systems remains challenging.

However,

biological

surface

engineering,

particularly

biomarker-based

engineering20-22 of AuNCs, allows for fine tuning of the interactions. In an assembly of AuNCs having an appropriate nanoformulation for a target, the optical signals of the NCs might be promoted and even amplified. Thus, multi-mode imaging of AuNCs offers the ability to provide comprehensive information on biological systems at molecular, structural, and functional levels.


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Herein, we designed the surface structures of the AuNCs to achieve controllable agglomeration, by designing bifunctional-ligand modified AuNCs. A zwitterionic ligand (ZW) and 11-mercaptoundecanoic acid (MUA) were decorated onto the AuNCs, which are denoted as AuNC@ZW/MUA (Scheme 1a). This ZW overcomes nonspecific adsorption and maintains colloidal stability.23,24 The surface MUA ligand provides multiple sites for biomarker conjugation.25 Streptavidin (SA) and biotin, which are specific binding motifs,26,27 were conjugated with MUA to prepare AuNC-SA and AuNC-biotin to enable biological assembly of the AuNCs. Owing to the minimal non-specific adsorption, protein binding events induced by the cell-expressed antigen were effectively converted to amplified emission signals from the biological assemble of AuNCs (Scheme 1b).


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Scheme 1. (a) Schematic diagram of structures of AuNC@ZW/MUA, AuNC-SA, and AuNCbiotin. (b) Schematic representations of biological assemblies of AuNC-SA and AuNC-biotin on biotinylated cells. The ZW and MUA modified AuNCs (denoted AuNC@ZW/MUA) were prepared through a typical etching process.28 The synthesis process is detailed in the Supporting Information. Highresolution transmission electron microscope (HRTEM) imaging (Figure 1a) showed that the synthesized AuNC@ZW/MUA were monodisperse and approximately 2.19 nm in size. Furthermore, the lattice fringe spacing was measured to be 2.3 Å (Figure S1), which is consistent


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with the (111) lattice spacing of face-centered cubic metallic gold.29 We performed X-ray photoelectron spectroscopy (XPS) to determine the surface composition of AuNC@ZW/MUA (Figure 1b) and confirmed the presence of the elements O, C, Au, and S. We attribute a weak peak centered at 400 eV to the N 1s signal. High-resolution XPS spectra (Figure 1c) show the presence of a N 1s signal in the ZW ligand. As a control sample, we prepared MUA modified AuNC (denoted AuNC@MUA) with an average diameter of 2.26 nm (Figure S2a). No signals were detected from N in the XPS spectrum (Figure S2b). Thus, the detected O, C, Au, S, and N signals correspond to the constituent elements of AuNC@ZW/MUA. The weak peak of N 1s is attributed to the low molar ratio of ZW to MUA used in the controlled in the preparation of AuNC@ZW/MUA. High-resolution XPS spectra of the Au 4f region, as shown in Figure 1d, indicate that the binding energies of Au 4f5/2 and Au 4f7/2 were 88.1 and 84.4 eV, respectively. The two peaks were fitted as the Au 4f5/2 (87.9 eV) and Au 4f7/2 (84.3 eV) signals of metallic gold (Au0) and Au 4f5/2 (88.4 eV) and Au 4f7/2 (84.7 eV) signals of Au+. Hence, the chemical state of Au in AuNC@ZW/MUA varied between 0 and 1, which is consistent with previous reports on XPS of thiolate ligand protected AuNCs.30 The peaks appearing in Figure 1e were fitted into three peaks, which indicated the presence of different chemical states of S. The peak centered at 162.9 eV originated from the Au-S bond, whereas the weak peak at 164 eV suggested that ZW was anchored to Au through a single S atom.31 Remarkably, the existing S in the oxidized form (at 167.9 eV) was from a sulfonic acid group in ZW.32 These results confirmed that the AuNCs were formed and protected by ZW. We also measured Fourier transform infrared (FTIR) spectra of AuNC@ZW/MUA (Figure S3). Compared with MUA, the disappearance of the characteristic peak of –SH at 2543 cm-1 indicating the formation of Au-S bond in AuNC@ZW/MUA. Besides, AuNC@ZW/MUA showed a characteristic peak of –COOH at


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1700 cm−1 from MUA as well as characteristic peaks of the ZW ligand at 1550, 1205, 1040, and 605 cm−1 assigned to amide N-H, sulfonate S=O, aliphatic amine C-N, and sulfonate S-O bonds, respectively. These data suggest that the AuNC@ZW/MUA was successfully modified with both ZW and MUA ligands.

Figure 1. (a) HRTEM image of AuNC@ZW/MUA, scale bar: 10 nm. Inset shows the crystal structure of a single AuNC@ZW/MUA, scale bar: 2 nm. XPS survey spectrum (b) and highresolution scans of N (c), Au (d), and S (e) regions of AuNC@ZW/MUA. (f) UV-vis absorption (black line) and fluorescence emission (red line) spectra of AuNC@ZW/MUA, excitation wavelength was 370 nm. (g) Linear relationship between integrated fluorescence intensity of AuNC@ZW/MUA and excitation power, two-photon excitation wavelength was 800 nm. (h) Two-photon cross sections of AuNC@ZW/MUA in the excitation wavelength range of 750–810 nm. To gain insight into the AuNC@ZW/MUA formation, we calculated the Au/S atomic concentration ratio from the XPS spectra. Under the same conditions, the Au/S atomic concentration ratios were 0.34 and 0.27 for AuNC@ZW/MUA and AuNC@MUA, respectively. We subsequently simulated the interaction between the Au atoms and ZW and that between Au


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atom and MUA. As shown in Figure 2, one ZW molecule with a disulfide group could bind three Au atoms with a strong binding energy of 1.97 eV, whereas the MUA molecule with a sulfide group could only bind one Au atom with a binding energy of 0.73 eV. Thus, ZW likely has a stronger ability of affinity with Au atoms than that of MUA. Meanwhile, smaller nanoparticles with a higher curvature allow different ligands to approach the surface without surface shielding.33 In the etching process, both ZW and MUA etched the surface Au atoms to reduce the size of Au nanoparticles and form AuNCs. Thus, the increased Au/S atomic concentration ratio of AuNC@ZW/MUA is mainly attributed to the strong interactions between ZW and Au atoms. By adding ZW and MUA, it was easier to obtain AuNCs protected by both ligands.

Figure 2. Simulation of the binding of ZW and MUA to Au atoms, respectively. We next examined the optical properties of AuNC@ZW/MUA. As shown in Figure 1f, the AuNC@ZW/MUA exhibited a maximum absorption at 370 nm and an emission at 515 nm. Compared with the absorption and emission peaks of AuNC@MUA, which located at 374 nm and 519 nm respectively (Figure S4), there reflects a considerable blue shift for AuNC@ZW/MUA. The luminescent properties of AuNC are size-dependent; hence, the blue shifted emission might be attributed to the smaller particles that have a larger energy gap.34,35 In terms of nonlinear optical properties, two-photon absorption (TPA) is a process whereby a molecule adsorbs two near-infrared photons and transitions to a higher energy states.36 The TPA


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cross section is a measure of a molecule’s TPA ability. By varying the excitation power of 800 nm from 60 to 150 Hz, the two-photon fluorescence intensity of AuNC@ZW/MUA gradually increased with increasing laser power (Figure 1g). The logarithm of the laser power has a linear relationship with the logarithm of the integrated fluorescence intensity with a slope of ~2, indicating a two-photon excitation process. Through the method of two-photon excitation fluorescence (TPEF), the TPA cross sections of AuNC@ZW/MUA at different excitation wavelengths were measured and calculated. As shown in Figure 1h, AuNC@ZW/MUA had a maximum TPA cross section of ~2.27×105 GM at approximately 770 nm, which was higher than that of glutathione protected AuNCs (~1.90×105 GM).37 This large TPA cross section permits AuNC@ZW/MUA to be applied in two-photon imaging.38 In biological imaging, stability is crucial for imaging performance. As shown in Figure 3a, the fluorescence intensities of AuNC@ZW/MUA were essentially unchanged over a pH range from 4 to 10. Stable green fluorescence was observed for all AuNC@ZW/MUA over a wide pH range (Figure 3b). However, aggregates obviously appeared in the AuNC@MUA solution at pH 4 and 5 (Figure 3c and 3d). Due to the acid-base equilibrium, the H+ ions interact with –COO- of MUA to form –COOH in the acid conditions.39 As the charges of MUA are shielded, the electrostatic repulsion among AuNC@MUA becomes weak. It results to the appearance of aggregation of AuNC@MUA. The ZW of AuNC@ZW/MUA has both positively charged tertiary amine and negative charged sulfonic acid groups. The enhanced colloid stability of AuNC@ZW/MUA to the weakened protonation of MUA owing to protection by ZW. Furthermore, the emission of AuNC@ZW/MUA was investigated in salt solution. As shown in Figure S5a, the fluorescence intensities of AuNC@ZW/MUA were same as the NaCl concentration varied from 0 to 500 mM. As the temperature was increased from 25 °C to 60 °C,


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the fluorescence intensities of AuNC@ZW/MUA changed only slightly (Figure S5b). Thus, the emission of AuNC@ZW/MUA was stable in the salt solution and were not influenced by temperature. Under UV-irradiation (1 mW/cm2) for 10 min, AuNC@ZW/MUA retains 70% of the original intensity (Figure S6), which shows their good photobleaching resistance. On the basis of this good stability, we further engineered AuNC@ZW/MUA with a SA-biotin motif by conjugating amino groups with carboxyl groups of MUA. This modification was directly confirmed by electrophoresis analysis. As shown in Figure S7, AuNC-biotin and AuNC-SA moved more slowly than AuNC@ZW/MUA, which indicated that AuNC-biotin and AuNC-SA have higher molecular weights than AuNC@ZW/MUA after modification with biotin and SA. The fluorescence intensities of AuNC-SA and AuNC-biotin had a relatively same intensity to that of AuNC@ZW/MUA (Figure 3e). The quantum yields of AuNC@ZW/MUA, AuNC-SA, and AuNC-biotin were 1.63%, 1.53%, and 1.65%, respectively (Figure 3f). As HRTEM images displayed in Figure S8, the average size of AuNC-SA and AuNC-biotin were 2.27 nm and 2.20 nm, respectively. These results indicate that engineering AuNC@ZW/MUA with SA-biotin does not affect the ligand-to-metal charge transfer or emission properties. Moreover, the ZW of AuNC@ZW/MUA can bind water molecules to form a hydration layer through electrostatic interactions, which limits nonspecific adsorption and promotes high specific SA-biotin interactions between AuNC-SA and AuNC-biotin. Considering the pH value of a physiologically environment, fluorescence intensities of AuNC-SA and AuNC-biotin were detected over the pH range from 4 to 8. As displayed in Figure S9, both AuNC-SA and AuNC-biotin show stable emission in different pH solutions. We conducted a layer-by-layer bio-assembly of AuNC-SA and AuNC-biotin on biotinylated quartz slides. The biotin modified quartz slide was examined by observing the fluorescence emission at 520 nm from fluorescein conjugated avidin (FITC-A),


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as shown in Figure S10. By alternative incubation of AuNC-SA and AuNC-biotin, the fluorescence intensities of AuNC-SA, AuNC-SA/AuNC-biotin/AuNC-SA, and (AuNCSA/AuNC-biotin)2/AuNC-SA gradually increased (Figure 3g), which confirmed a self-assembly process based on specific SA-biotin interactions. More importantly, the radiative decay rates of the AuNC-SA, AuNC-SA/AuNC-biotin/AuNC-SA, and (AuNC-SA/AuNC-biotin)2/AuNC-SA layers were almost the same (Figure 3h). The calculated average lifetimes were all relatively long at approximately 118.56, 116.83, and 116.42 ns for AuNC-SA, AuNC-SA/AuNCbiotin/AuNC-SA, and (AuNC-SA/AuNC-biotin)2/AuNC-SA, respectively. The result indicates that the radiative transitions of AuNCs in our system were not affected by assembly induced aggregation. Since the emission of AuNCs is largely influenced by the ligand-to-metal charge transfer, the self-assembly process was achieved by specific recognition between modified streptavidin and biotin on the AuNCs. During this process, the charge transfers between the ligand and the gold core was not affected, so the fluorescence quenching was resisted. The fluorescence signals were increased by bio-assembly of AuNC-SA and AuNC-biotin without any aggregation-induced quenching interactions.


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Figure 3. (a) Fluorescence spectra and (b) corresponding fluorescence images of AuNC@ZW/MUA at different pH. (c) Fluorescence spectra and (d) corresponding fluorescence images of AuNC@MUA at different pH. Excitation wavelengths were 380 and 390 nm. (e) Fluorescence spectra and (f) quantum yields of AuNC@ZW/MUA, AuNC-SA, and AuNCbiotin. (g) Fluorescence spectra and (h) fluorescence intensity decay of AuNC-SA, AuNCSA/AuNC-biotin/AuNC-SA, and (AuNC-SA/AuNC-biotin)2/AuNC-SA on biotinylated quartz slides. In cancer cells, antigens overexpressed on the cell membrane are often used as identifying biomarkers.40 On the basis of the specific antibody-antigen interactions, fluorescence labelled protein-binding events induced by the cell-expressed antigen can produce signals for detection.41 However, the imaging mode that relies on the signal of a commercial fluorescence labelled protein is single and the emission cannot be amplified. To confirm signal amplification and multi-mode imaging of cancer cells, AuNC-SA and AuNC-biotin were assembled on breast cancer cell (MCF-7). MCF-7 cells, which overexpress epithelial cell adhesion molecule (EpCAM) on their membrane, were chosen.42 First, the biocompatibility of AuNC-SA and AuNC-biotin was confirmed with a 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay. As shown in Figure S11, when the concentration of Au changed from 1×10−7 to 1×10−6 mol/L, more than 90% of the MCF-7 cells were alive after incubation with AuNC-SA and AuNC-biotin. Thus, both AuNC-SA and AuNC-biotin have good biocompatibility. The MCF-7 cells were then treated with biotinylated anti-EpCAM to achieve the assembly. After incubationwith FITC-A, the cells showed green emission of FITC-A from its membrane, indicating that biotinylated anti-EpCAM was immobilized on the MCF-7 cell by specific antigen-antibody interactions (Figure S12). Through alternate assembly of AuNC-SA and


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AuNC-biotin, the (AuNC-SA/AuNC-biotin)2/AuNC-SA showed more intense green fluorescence than that of AuNC-SA/AuNC-biotin/AuNC-SA, which indicated signal amplification from the biological assemble of AuNC-SA and AuNC-biotin (Figure 4a). Owing to the biotinylated membrane of the MCF-7 cell, the AuNCs were localized on the surface. Furthermore, the presence of Au on the surface of the MCF-7 cells was confirmed through elemental mapping analysis, which could indicating the successful assembly of AuNCs on the cell membrane (Figure S13). The cell membranes of MCF-7 were gradually visualized and the over-expressed EpCAM was imaged from the fluorescence signals. However, for the MCF-7 cells untreated with biotinylated anti-EpCAM, biotin-streptavidin interaction cannot be carried out. There were no fluorescence signals. Thus, AuNC-SA and AuNC-biotin show strong specific interactions and minimal nonspecific adsorption, which improved the signal reliability and sensitivity. Compared with one-photon excitation, two-photon excitation in the near-infrared region can minimize autofluorescence and improve spatial resolution.43 Taking advantage of the large TPA cross section of AuNC@ZW/MUA, we performed two-photon imaging. As shown in Figure 4b, green fluorescence was mostly observed at the membrane of MCF-7 cells, and background autofluorescence was eliminated to some extent, compared with that in Figure 4a. Only a small portion of the AuNCs entered the cell through endocytosis mediated by the antigen-antibody interaction. As AuNCs shows a long lifetime which is not affected by the assembly (Figure 3h), additional information may be provided by fluorescence lifetime imaging. Combined with the results in Figure 4a and 4b, the lifetime of the AuNCs located on the membrane (~8 ns) was longer than that in cytosol (~5 ns) (Figure 4c). However, both the lifetimes in the cells decreased to be approximately 5% that measured from the quartz plate, resulting from quenching effects in the complex biological environment. Nevertheless, both the lifetimes of AuNCs were longer than


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the autofluorescence (occurring on the 2–3 ns time scale).44 The lifetime differences of the AuNCs on the membrane and those in the cytosol might indicate differences of the environment inside and outside the cells. Through the three imaging modes, detailed information on cellexpressed EpCAM antigen in the membrane can be acquired through biological assembly of AuNC-SA and AuNC-biotin.


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Figure 4. (a) Bright field, fluorescence, and merged images of biotinylated MCF-7 cells assembled with AuNC-SA/AuNC-biotin/AuNC-SA and (AuNC-SA/AuNC-biotin)2/AuNC-SA, respectively, and no-biotinylated MCF-7 cells assembled with AuNC-SA. Scale bar: 20 μm. (b) Two-photon fluorescence images of biotinylated MCF-7 cells assembled with AuNC-SA/AuNCbiotin/AuNC-SA and (AuNC-SA/AuNC-biotin)2/AuNC-SA. Excitation wavelength was 760 nm. Scale bar: 20 μm. (c) Fluorescence lifetime image of MCF-7 cells assembled with (AuNCSA/AuNC-biotin)2/AuNC-SA, scale bar: 10 μm. In conclusion, we prepared AuNCs engineered with two ligands, namely ZW and MUA, for targeted multi-modal imaging. The prepared AuNCs exhibited stable emission over a wide pH range and had large TPA cross sections and long fluorescence lifetime. After conjugating SA and biotin onto MUA, the AuNC-SA and AuNC-biotin maintained the optical properties of AuNCs. The AuNC-SA and AuNC-biotin pair featured a zwitterionic surface, which as designed to bioassemble without nonspecific adsorption. Notably, assembly-amplified emission of AuNC-SA and AuNC-biotin against aggregation induced quenching were achieved. The systems had good biocompatibility and MCF-7 cells over-expressing EpCAM were effectively imaged from the amplified emission from biological assemblies of AuNCs. The signals of the AuNCs allowed clear one-photon, two-photon, and fluorescence lifetime imaging of the cell structures. We hope that these engineered AuNCs will be useful for simple and sensitive multi-modal analysis of disease related biomarkers. ASSOCIATED CONTENT Supporting Information


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Experimental section. Computational methods for the binding energy of two ligands with Au atom separately. HRTEM image and XPS spectrum of N in AuNC@MUA. FTIR spectra of MUA, ZW, and AuNC@ZW/MUA. UV-vis absorption and fluorescence emission spectra of AuNC@MUA. Gel electrophoresis of AuNC@ZW/MUA, AuNC-SA, and AuNC-biotin. Fluorescence spectra of FITC-Avidin assembled on quartz plate. Cell viability of MCF-7 cells after incubated with different concentrations of AuNC-SA and AuNC-biotin. Confocal images of MCF-7 cells assembled with FITC-A. Energy spectrum analysis of MCF-7 cells assembled with AuNCs. AUTHOR INFORMATION ORCID Xiaoyu Wang: 0000-0002-2139-3152 Lidong Li: 0000-0003-0797-2518 Author Contributions §X.J.

and X.W. contributed equally to this study.

Notes The authors declare no competing financial interests. ACKNOWLEDGMENT This research was supported by the National Natural Science Foundation of China (51673022, 51703009), the State Key Laboratory for Advanced Metals and Materials (2018Z-18). We acknowledge the assistance of Imaging Core Facility, Technology Center for Protein Sciences, Tsinghua University for measurements in imaging sections.


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REFERENCES (1) Tao, Y.; Li, M.; Ren, J.; Qu, X. Metal Nanoclusters: Novel Probes for Diagnostic and Therapeutic Applications. Chem. Soc. Rev. 2015, 44, 8636-8663. (2) Chen, T.; Hu, Y.; Cen, Y.; Chu, X.; Lu, Y. A Dual-Emission Fluorescent Nanocomplex of Gold-Cluster-Decorated Silica Particles for Live Cell Imaging of Highly Reactive Oxygen Species. J. Am. Chem. Soc. 2013, 135, 11595-11602. (3) Chen, D.; Luo, Z.; Li, N.; Lee, J. Y.; Xie, J.; Lu, J. Amphiphilic Polymeric Nanocarriers with Luminescent Gold Nanoclusters for Concurrent Bioimaging and Controlled Drug Release. Adv. Funct. Mater. 2013, 23, 4324-4331. (4) Wang, H.-H.; Lin, C.-A. J.; Lee, C.-H.; Lin, Y.-C.; Tseng, Y.-M.; Hsieh, C.-L.; Chen, C.H.; Tsai, C.-H.; Hsieh, C.-T.; Shen, J.-L.; Chan, W.-H.; Chang, W. H.; Yeh, H.-I Fluorescent Gold Nanoclusters as a Biocompatible Marker for In Vitro and In Vivo Tracking of Endothelial Cells. ACS Nano 2011, 5, 4337-4344. (5) Vankayala, R.; Kuo, C.-L.; Nuthalapati, K.; Chiang, C.-S.; Hwang, K.C. Nucleus-Targeting Gold Nanoclusters for Simultaneous In Vivo Fluorescence Imaging, Gene Delivery, and NIRLight Activated Photodynamic Therapy. Adv. Funct. Mater. 2015, 25, 5934-5945. (6) Jia, X.; Li, J.; Zhang, X.; Wang, E. Controlling the Synthesis and Assembly of Fluorescent Au/Ag Alloy Nanoclusters. Chem. Commun. 2015, 51, 17417-17419. (7) Shi, J.; Cooper, J. K.; Lindley, S.; Williams, O.; Kliger, D. S.; Xu, Y.; Bao, Y.; Zhang, J. The Excited State Dynamics of Protein-Encapsulated Au Nanoclusters. Chem. Phys. Lett. 2014, 610, 125-130.


17


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(8) Wu, Z.; Jin, R. On the Ligand’s Role in the Fluorescence of Gold Nanoclusters. Nano Lett. 2010, 10, 2568-2573. (9) Yahia-Ammar, A.; Sierra, D.; Mérola, F.; Hildebrandt, N.; Guével, X. L. Self-Assembled Gold Nanoclusters for Bright Fluorescence Imaging and Enhanced Drug Delivery. ACS Nano 2016, 10, 2591-2599. (10) Xu, H.; Li, Q.; Wang, L.; He, Y.; Shi, J.; Tang, B.; Fan, C. Nanoscale Optical Probes for Cellular Imaging. Chem. Soc. Rev. 2014, 43, 2650-2661. (11) Malola, S.; Lehtovaara, L.; Häkkinen, H. A DFT Study of Linear Gold-Thiolate Superclusters Absorbing in the Therapeutic NIR Window. J. Phys. Chem. Lett. 2014, 5, 13291334. (12) Xia, J.; Wang, X.; Zhu, S.; Liu, L.; Li, L. Gold Nanocluster-Decorated Nanocomposites with Enhanced Emission and Reactive Oxygen Species Generation. ACS Appl. Mater. Interfaces 2019, 11, 7369-7378. (13) Cui, Q.; He, F.; Li, L.; Möhwald, H. Controllable Metal-Enhanced Fluorescence in Organized Films and Colloidal System. Adv. Colloid Interface Sci. 2014, 207, 164-177. (14) Xing, R.; Liu, K.; Jiao, T.; Zhang, N.; Ma, K.; Zhang, R.; Zou, Q.; Ma, G.; Yan, X. An Injectable Self-Assembling Collagen-Gold Hybrid Hydrogel for Combinatorial Antitumor Photothermal/Photodynamic Therapy. Adv. Mater. 2016, 28, 3669-3676. (15) García, I.; Henriksen-Lacey, M.; Sánchez-Iglesias, A.; Grzelczak, M.; Penadés, S.; LizMarzán, L. M. Residual CTAB Ligands as Mass Spectrometry Labels to Monitor Cellular Uptake of Au Nanorods. J. Phys. Chem. Lett. 2015, 6, 2003-2008.


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(16) Cheng, L.-C.; Chen, H. M.; Lai, T.-C.; Chan, Y.-C.; Liu, R.-S.; Sung, J. C.; Hsiao, M.; Chen, C.-H.; Her, L.-J.; Tsai, D. P. Targeting Polymeric Fluorescent Nanodiamond-Gold/Silver Multi-Functional Nanoparticles as a Light-Transforming Hyperthermia Reagent for Cancer Cells. Nanoscale 2013, 5, 3931-3940. (17) Durr, N. J.; Larson, T.; Smith, D. K.; Korgel, B. A.; Sokolov, K.; Ben-Yakar, A. TwoPhoton Luminescence Imaging of Cancer Cells Using Molecularly Targeted Gold Nanorods. Nano Lett. 2007, 7, 941-945. (18) Weng, H.; Huff, T. B.; Zweifel, D. A.; He, W.; Low, P. S.; Wei, A.; Cheng, J.-X. In Vitro and In Vivo Two-Photon Luminescence Imaging of Single Gold Nanorods. Proc. Natl. Acad. Sci. U. S. A. 2005, 102, 15752-15756. (19) Ramakrishna, G.; Vamavski, O.; Kim, J.; Lee, D.; Goodson, T. Quantum-Sized Gold Clusters as Efficient Two-Photon Absorbers. J. Am. Chem. Soc. 2008, 130, 5032-5033. (20) Zheng, K.; Setyawati, M. I.; Leong, D. T.; Xie, J. Surface Ligand Chemistry of Gold Nanoclusters Determines Their Antimicrobial Ability. Chem. Mater. 2018, 30, 2800-2808. (21) Park, J.; Park, Y.; Kim, S.; Signal Amplification via Biological Self-Assembly of SurfaceEngineered Quantum Dots for Multiplexed Subattomolar Immunoassays and Apoptosis Imaging. ACS Nano 2013, 7, 9416-9427. (22) Cui, Q.; Wang, X.; Yang, Y.; Li, S.; Li, L.; Wang, S. Binding-Directed Energy Transfer of Conjugated Polymer Materials for Dual-Color Imaging of Cell Membrane. Chem. Mater. 2016, 28, 4661-4669.


19


Page 20 of 23

(23) Shao, Q.; Jiang, S.; Molecular Understanding and Design of Zwitterionic Materials. Adv. Mater. 2015, 27, 15-26. (24) Krieg, F.; Ochsenbein, S. T.; Yakunin, S.; Brinck, S. t.; Aellen , P.; Süess, A.; Clerc, B.; Guggisberg, D.; Nazarenko, O.; Shynkarenko, Y.; Kumar, S.; Shih, C.-J.; Infante, I.; Kovalenko, M. V. Colloidal CsPbX3 (X= Cl, Br, I) Nanocrystals 2.0: Zwitterionic Capping Ligands for Improved Durability and Stability. ACS Energy Lett. 2018, 3, 641-646. (25) Hu, X.; Guiseppi-Elie, A.; Dinu, C. Z. Biomolecular Interfaces Based on Self-Assembly and Self-Recognition Form Biosensors Capable of Recording Molecular Binding and Release. Nanoscale 2019, 11, 4987-4998. (26) Wang, X.; Jiang, X.; Zhu, S.; Liu, L.; Xia, J.; Li, L. Preparation of Optical Functional Composite Films and Their Application in Protein Detection. Colloids Surf., A 2017, 535, 69-74. (27) Chan, M. S.; Landig, R.; Choi, J.; Zhou, H.; Liao, X.; Lukin, M. D.; Park, H.; Lo, P. K. Stepwise Ligand-Induced Self-Assembly for Facile Fabrication of Nanodiamond-Gold Nanoparticle Dimers via Non-Covalent Biotin-Streptavidin Interactions. Nano Lett. 2019, 19, 2020-2026. (28) Tao, Y.; Li, M.; Auguste, D. T. Pattern-Based Sensing of Triple Negative Breast Cancer Cells with Dual-Ligand Cofunctionalized Gold Nanoclusters. Biomaterials 2017, 116, 21-33. (29) Wang, X.; Xia, J.; Wang, C.; Liu, L.; Zhu, S.; Feng, W.; Li, L. Preparation of Novel Fluorescent Nanocomposites Based on Au Nanoclusters and Their Application in Targeted Detection of Cancer Cells. ACS Appl. Mater. Interfaces 2017, 9, 44856-44863.


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Page 21 of 23 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(30) Zhu, H.; Goswami, N.; Yao, Q.; Chen, T.; Liu, Y.; Xu, Q.; Chen, D.; Lu, J.; Xie, J. Cyclodextrin-Gold Nanoclusters on TiO2 Enhances Photocatalytic Decomposition of Organic Pollutants. J. Mater. Chem. A 2018, 6, 1102-1108. (31) Shang, L.; Azadfar, N.; Stockmar, F.; Send, W.; Trouillet, V.; Bruns, M.; Gerthsen, D.; Nienhaus, G. U. One-Pot Synthesis of Near-Infrared Fluorescent Gold Clusters for Cellular Fluorescence Lifetime Imaging. Small 2011, 7, 2614-2620. (32) Moulder, J. F.; Stickle, W. F.; Sobol, P. E.; Bomben, K. D. Handbook of X-Ray Photoelectron Spectroscopy; Perkin-Elmer Corporation: Eden Prairie, MN, 1992. (33) Mei, B. C.; Oh, E.; Susumu, K.; Farrell, D.; Mountziaris, T.J.; Mattoussi, H. Effects of Ligand Coordination Number and Surface Curvature on the Stability of Gold Nanoparticles in Aqueous Solutions. Langmuir 2009, 25, 10604-10611. (34) Shang, L.; Dong, S.; Nienhaus, G. U. Ultra-Small Fluorescent Metal Nanoclusters: Synthesis and Biological Applications. Nano Today 2011, 6, 401-418. (35) Chen, Z.; Liu, C.; Cao, F.; Ren, J.; Qu, X. DNA Metallization: Principles, Methods, Structures, and Applications. Chem. Soc. Rev. 2018, 47, 4017-4072. (36) Pawlicki, M.; Collins, H. A.; Denning, R. G.; Anderson, H. L. Two-Photon Absorption and the Design of Two-Photon Dyes. Angew. Chem. Int. Ed. 2009, 48, 3244-3266. (37) Polavarapu, L.; Manna, M.; Xu, Q.-H. Biocompatible Glutathione Capped Gold Clusters as One- and Two-Photon Excitation Fluorescence Contrast Agents for Live Cells Imaging. Nanoscale 2011, 3, 429-434.


21


Page 22 of 23

(38) Pramanik, A.; Chavva, S. R.; Fan, Z.; Sinha, S. S.; Nellore, B. P. V.; Ray, P. C. Extremely High Two-Photon Absorbing Graphene Oxide for Imaging of Tumor Cells in the Second Biological Window. J. Phys. Chem. Lett. 2014, 5, 2150-2154. (39) Wang, D.; Nap, R. J.; Lagzi, I.; Kowalczyk, B.; Han, S.; Grzybowski, B. A.; Szleifer, I. How and Why Nanoparticle’s Curvature Regulates the Apparent pKa of the Coating Ligands. J. Am. Chem. Soc. 2011, 133, 2192–2197. (40) Wang, X.; Li, S.; Zhang, P.; Lv, F.; Liu, L.; Li, L.; Wang, S. An Optical Nanoruler Based on a Conjugated Polymer-Silver Nanoprism Pair for Label-Free Protein Detection. Adv. Mater. 2015, 27, 6040-6045. (41) Kuo, C. T.; Thompson, A. M.; Gallina, M. E.; Ye, F.; Johnson, E. S.; Sun, W.; Zhao, M.; Yu, J.; Wu, I-C.; Fujimoto, B.; DuFort, C. C.; Carlson, M. A.; Hingorani, S. R.; Paguirigan, A. L.; Radich, J. P.; Chiu, D. T. Optical Painting and Fluorescence Activated Sorting of Single Adherent Cells Labelled with Photoswitchable Pdots. Nat. Commun. 2016, 7, 11468. (42) Shi, J.; Lyu, J.; Tian, F.; Yang, M. A fluorescence Turn-On Biosensor Based on Graphene Quantum Dots (GQDs) and Molybdenum Disulfide (MoS2) Nanosheets for Epithelial Cell Adhesion Molecule (EpCAM) Detection. Biosens. Bioelectron. 2017, 93, 182-188. (43) Shin, J.; Verwilst, P.; Choi, H.; Kang, S.; Han, J.; Kim, N. H.; Choi, J. G.; Oh, M. S.; Hwang, J. S.; Kim, D.; Mook-Jung, I.; Kim, J. S. Harnessing Intramolecular Rotation to Enhance Two-Photon Imaging of Aβ Plaques through Minimizing Background Fluorescence. Angew. Chem. 2019, 131, 5704-5708.


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(44) Dahan, M.; Laurence, T.; Pinaud, F.; Chemla, D. S. Time-Gated Biological Imaging by Use of Colloidal Quantum Dots. Opt. Lett. 2001, 26, 825-827.


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Surface-Engineered Gold Nanoclusters with Biological Assembly

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