Article pubs.acs.org/cm
Synthesis and Characterization of PEGylated Luminescent Gold Nanoclusters Doped with Silver and Other Metals Eunkeu Oh,*,†,∥ James B. Delehanty,‡ Lauren D. Field,‡,⊥ Antti J. Mak̈ inen,† Ramasis Goswami,§ Alan L. Huston,† and Igor L. Medintz*,‡ †
Optical Sciences Division, Code 5600, ‡Center for Bio/Molecular Science and Engineering, Code 6900, §Multifunctional Materials, Code 6355, U.S. Naval Research Laboratory, Washington, D.C. 20375, United States ∥ Sotera Defense Solutions, Columbia, Maryland 21046, United States ⊥ Fischell Department of Bioengineering, University of Maryland, College Park, Maryland 20742, United States S Supporting Information *
ABSTRACT: Doping of fluorescent noble metal nanoclusters is being pursued to manipulate the structure of such materials along with improving physicochemical characteristics such as long-term stability and photoluminescence quantum yield. Here, we synthesize metal-doped and alloyed ultrasmall gold nanoclusters (AuNCs) directly in water using a facile one-step coreduction reaction with bidentate dithiolane PEGylated ligands that terminate in different functional groups including a methoxy, carboxy, amine, and azide. Two primary types of cluster materials were the focus of synthesis and characterization: first, a series of doped/alloyed Ag-doped AuNCs, where the ratio of Au:Ag was varied across a wide range including 99:1, 98:2, 90:10, 80:20, 50:50, 20:80, 10:90, and 2:98 along with pure AuNC and AgNC controls; second, doped Au:D NCs, where D included Pt, Cu, Zn, and Cd. Physical characterization of the modified AuNCs included TEM analysis of size, XPS/EDX analysis of dopant content, and a detailed analysis of photophysical properties including absorption and photoluminescence profiles, quantum yields over time, photoluminescence lifetimes, and examination of energy levels for selected materials. The addition of just a few Ag dopant atoms per AuNC yielded significant enhancement in quantum yield along with improving long-term photostability especially in comparison to materials with a very high Ag content. Preliminary cell imaging applications of the Ag-doped AuNCs were also investigated. Facilitated cellular uptake by mammalian cells via endocytosis following modification with cell penetrating peptides was confirmed by colabeling with specific cellular markers. Long-term intracellular photostability and lack of aggregation were confirmed with microinjection studies, and cytoviability assays showed the doped clusters to be minimally toxic.
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INTRODUCTION Metal doping in nanoparticles (NPs) is a process by which specific atoms are intentionally added during NP synthesis to provide new physicochemical properties and/or alter the structure of the product.1 Decades of research have focused on optimizing the properties of colloidal metal NPs using this approach for applications such as catalysis, for example.2,3 However, far less progress has been made on applying this same strategy to improving the qualities of small luminescent noble metal nanoclusters (NCs).4 Similar to semiconductor quantum dots (QDs), noble metal NC-materials exhibit unique quantum confinement effects that can lead to size-dependent optical spectra and tuning of photoluminescence (PL) across the ultraviolet, visible and near-infrared portions of the spectrum.3,5,6 Because metal NCs and especially those synthesized from gold (AuNCs) are far smaller in size than QDs and other luminescent NPs and tend to have higher long-term photostability than many molecular fluorophores, they have been suggested as strong candidates for developing solar cells, solidstate lighting, cellular probes, and for multiphoton imaging applications.3,7−10 However, their generally lower PL quantum © XXXX American Chemical Society
yield (QY) as compared to organic dyes or QDs continues to be a major roadblock for expanding their applications especially into the biological realm. Some representative examples highlighting typical QYs of this nature include: 830 nm emitting Au25 with a QY ≈ 0.025% in hexane,11 700 nm emitting 1.5 nm AuNC synthesized by etching QY ≈ 3.5% in methanol/1.8% in water (pH 9),12 800 nm glutathionestabilized Au28 QY ≈ 0.015% in water,13 and 530 nm emitting mercaptoundecanoic acid-coated AuNCs QY ≈ 3.1% in water.14 Various approaches are potentially available to improve the QY of NCs, including both size optimization and surface control through the use of different discrete ligands.9,12,13,15−21 Recently, the synthesis of bimetallic Au/Ag clusters along with Ag-doping of AuNCs have been reported as alternative ways to improve intrinsic QY and other desirable properties.22−29 For example, Udayabhaskararao et al. synthesized bimetallic NCs Received: September 9, 2016 Revised: October 26, 2016
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DOI: 10.1021/acs.chemmater.6b03838 Chem. Mater. XXXX, XXX, XXX−XXX
Article
Chemistry of Materials
Figure 1. Doped gold nanocluster schematic. (a) Schematic diagram of Au:D NCs surrounded by the different PEGylated surface ligands used to synthesize them in this study (not to scale). TA-PEG ligands terminating in different functional groups; NH2, amine; COOH, carboxyl; N3, azide; OCH3, methoxy. (b) Equal concentrations of TA-PEG-NH2 functionalized colloidal AuNCs (undoped, 100% Au) and Au:Ag 98:2 NCs excited with a 405 nm laser. The Ag-doped NCs show ∼2× brighter emission compared to the undoped AuNCs, see also Table 1.
cluster surface, which mitigates the high dynamic off-rates commonly encountered with monothiolated ligands.35 Several types of biomolecular conjugation reactions, biosensing assays, and both one- and two-photon cellular imaging were demonstrated with the AuNC materials as well; however, their QYs of 4−8% in water are still quite modest compared to those of QDs (>20% in water).36,37 Herein, we describe the synthesis of ultrasmall AuNCs doped with different metals including Ag, Cu, Pt, Zn, and Cd. The metal-doped AuNCs (Au:D NC, where D = Ag, Cu, Pt, Zn, or Cd) were coreduced directly in water at room temperature from metal-precursors in the presence of bidentate-PEG dithiolane ligands that terminated in one of several different functional groups (methoxy, amine, carboxy, or azide group). We systematically altered the doping ratio of metal for Au:Ag NCs along with making a series of Cu-, Pt-, Zn-, and Cd-doped AuNCs. Extensive characterization of the physical and optical properties of selected representative NC materials were undertaken along with examining their biological utility in preliminary cellular labeling experiments. We find that the presence of even small amounts of Ag dopants per AuNC can significantly enhance long-term physicochemical stability and improve the QY to almost twice that of pure AuNCs synthesized without metal dopants. Also we observed that the emission wavelength can be systematically tuned between 670 and 820 nm by changing the doping rate.
from a mercaptosuccinic acid-protected Ag7,8 core with Au alloying yielding a QY of 3.5%.22 Mohanty et al. reported on a bovine serum albumin protein-templated AuAg with a QY of ∼2.8%.23 Le Guevel et al. reported a high QY of ∼16% for a glutathione-protected AuNC core that underwent a later doping step with Ag.24 Wang et al. reported that Au12Ag13, derived from presynthesized triphenylphosphine-capped AuNCs which were then alloyed with an alkanethiol-Ag, showed a dramatic increase in the QY to ∼40% in organic solvent.25 Beyond the multistep schemes utilized above, other approaches to synthesizing such bimetallic NCs include laser ablation and galvanic exchange.30,31 To continue developing fluorescent NCs for biological applications in particular, far more facile and widely accessible synthetic schemes are still critically needed. Ideally, these chemistries should provide small, low-polydispersity NCs that have colloidal stability across many ionic buffers and a broad pH range. Moreover, these cluster materials should display different functional groups and be amenable to chemical modification with other biomolecules such as proteins or peptides, be nonfouling such that they are stable and do not undergo aggregation in biological media including, for example, the intracellular environment, and, most importantly, demonstrate an improved QY. Previously we, along with others, have reported synthetic methods for obtaining biocompatible, near-infrared luminescent metal (Au and Ag) NCs directly in water by using poly(ethylene glycol)(PEG) − dithiolane ligands.9,32 We successfully synthesized ∼1.5 nm diameter AuNCs with an emission centered at ∼820 nm, excellent long-term colloidal stability over several years, high buffer/salt stability, and strong resistance to denaturing agents.9,33,34 The latter properties are believed to arise from the bidentate nature of the dithiols to the
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MATERIALS AND METHODS
TA-PEG Ligands. PEG-modified thioctic acid ligands terminating in an amine (TA-PEG-NH2), carboxyl (TA-PEG-COOH), azide (TAPEG-N3), and methoxy (TA-PEG-OCH3) were synthesized, purified, and characterized following the procedures detailed in refs 9, 38, and B
DOI: 10.1021/acs.chemmater.6b03838 Chem. Mater. XXXX, XXX, XXX−XXX
Article
Chemistry of Materials 39. For the −NH2, −COOH, and −N3 terminated ligands, the average MW of PEG was ∼600 corresponding to ∼12 ethylene oxides, while for the −OCH 3 ligands the average PEG MW was ∼550 corresponding to ∼10 ethylene oxides. See Figure 1 for the chemical structures. Synthesis of AuNCs with Metal Dopants. PEGylated Au:D NCs with specific dopants were synthesized by directly reducing the metal (Au and D) precursors with sodium borohydride (NaBH4) in the presence of the dithiolated PEG ligand; this method is similar to the previous synthesis of PEGylated AuNCs except with two metal precursors used simultaneously.9 First, 140 μL (2.8 × 10−4 mol) of 2 M NaOH and 188 μL (2.5 × 10−5 mol) of 133 mM aqueous stock solution of the TA-PEG ligands (with one of the different functional groups: TA-PEG-NH2, TA-PEG-N3, TA-PEG-COOH, TA-PEGOCH3) were dissolved in 50 mL of deionized water. 100 μL (1.0 × 10−5 mol) of metal precursors mixture with a specific ratio, made from 100 mM tetrachloroauric (III) acid (HAuCl4·3H2O) aqueous stock solution and 100 mM dopant stock solution, was then added to the ligand solution under stirring at room temperature. Dopant stock solution was constituted from either silver nitrate (AgNO3), zinc acetate (Zn(O2CCH3)2·2H2O), copper acetate (Cu(CO2CH3)2· XH2O), platinum chloride (PtCl4·5H2O), or cadmium acetate (Cd(CO2CH3)2·2H2O). Following 10 min of stirring of mixture, 700 μL (7 × 10−5 mol) of 0.1 M NaBH4 stock solution freshly prepared in deionized water was added dropwise to the reaction with vigorous stirring. Following addition of the reducing agent, the color of the reaction mixture usually turned light yellow-brown from transparent. The reaction was then left stirring for at least 3 h. For ripening, the mixture was kept for another one day at room temperature without stirring. The dispersion was then purified from free ligands and concentrated to ∼20 μM by three cycles of centrifugation using a centrifugal membrane filtration device (10K MW cutoff, Millipore Corporation, Billerica, MA) and redispersed in water. AuNC concentration (μM NC) was determined as described previously.9,33,34 The same method was used to prepare AuNCs with mixed ligand surfaces, which were added in the desired ratios to the synthetic reaction. UV−vis Spectroscopy. Electronic absorption spectra were recorded using an HP 8453 diode array spectrophotometer (Agilent Technologies, Santa Clara, CA). The NC concentrations of NC-PEG samples were adjusted to
